Pocket Guide to

Bacterial Infections
 POCKET GUIDES TO
BIOMEDICAL SCIENCES
Series Editor
Lijuan Yuan
Virginia Polytechnic Institute and State University

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Editorial Board
Sascha Al-Dahouk, Federal Institute for Risk Assessment, Germany
Frank Austin, Mississippi State University
K. Balamurugan, Alagappa University, India
Rossana de Aguiar Cordeiro, Universidade Federal do Ceará, Brazil
Lisa Gorski, U. S. Department of Agriculture
Baochuan Lin, U. S. Naval Research Laboratory
Russell Paterson, University of Minho, Portugal
Yiqun Zhou, Roche Pharmaceuticals

Science Publisher
Charles R. Crumly, CRC Press/Taylor & Francis Group
 Pocket Guide to
Bacterial Infections

Edited by
K. Balamurugan

Co-editor
U. Prithika
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Library of Congress Cataloging‑in‑Publication Data

Names: Balamurugan, K. (Krishnaswamy), editor.

Title: Pocket guide to bacterial infections / editor(s), K. Balamurugan.
Other titles: Pocket guides to biomedical sciences.
Description: Boca Raton : Taylor & Francis, 2019. | Series: Pocket guides to
biomedical sciences | Includes bibliographical references.
Identifiers: LCCN 2018043418| ISBN 9781138054899 (paperback : alk. paper) |
ISBN 9781138054912 (hardback : alk. paper) | ISBN 9781315166377 (general)
| ISBN 9781351679817 (pdf) | ISBN 9781351679800 (epub) | ISBN
9781351679794 (mobi/kindle)
Subjects: | MESH: Bacterial Infections--etiology | Bacterial
Infections--pathology | Bacteria--pathogenicity | Bacteria--cytology
Classification: LCC RC115 | NLM WC 200 | DDC 616.9/2--dc23
LC record available at https://lccn.loc.gov/2018043418

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Contents

Series Preface vii

Editors ix
Contributors xi

1 Bacteria Causing Gastrointestinal Infections: Overview of

Types of Bacteria Causing Gastrointestinal Infections, Mode
of Infection, Challenges in Diagnostic Methods, and Treatment 1
B. Vinoth, M. Krishna Raja, and B. Agieshkumar

2 Gateways of Pathogenic Bacterial Entry into Host

Cells—Salmonella 59
Balakrishnan Senthilkumar, Duraisamy Senbagam, Chidambaram
Prahalathan, and Kumarasamy Anbarasu

3 Prevalence of Bacterial Infections in Respiratory Tract 79

Boopathi Balasubramaniam, U. Prithika, and K. Balamurugan

4 Oral Health: A Delicate Balance between Colonization

and Infection 103
Ana Moura Teles and José Manuel Cabeda

5 Bacterial Infections in Atherosclerosis: Atherosclerosis

Microbiome 135
Emil Kozarov and Ann Progulske-Fox

6 Neonatal Bacterial Infection: Insights into Pathogenic

Strategy and Onset of Meningitis and Sepsis 147
Koilmani Emmanuvel Rajan and Christopher Karen

7 Bacterial Infections of the Oral Cavity: Bacterial Profile,

Diagnostic Characteristics, and Treatment Strategies 167
P. S. Manoharan and Praveen Rajesh

v
vi    Contents

8 Prognosis and Impact of Recurrent Uveitis, the Ophthalmic

Infection Caused by Leptospira Spp. 203
Charles Solomon Akino Mercy and Kalimuthusamy
Natarajaseenivasan

9 Beneficial Lactic Acid Bacteria: Use of Lactic Acid Bacteria in

Production of Probiotics 225
Galina Novik and Victoria Savich

10 Role of Bacteria in Dermatological Infections 279

Thirukannamangai Krishnan Swetha and Shunmugiah
Karutha Pandian

11 Bacteriology of Ophthalmic Infections 319

Arumugam Priya and Shunmugiah Karutha Pandian

12 Role of Bacteria in Urinary Tract Infections 365

Gnanasekaran JebaMercy, Kannan Balaji, and K. Balamurugan

13 Role of Bacteria in Blood Infections 375

Kannan Balaji, Gnanasekaran JebaMercy, and K. Balamurugan

Index 391
Series Preface

Dramatic breakthroughs and nonstop discoveries have rendered biomedi-

cine increasingly relevant to everyday life. Keeping pace with all these
advances is a daunting task, even for active researchers. There is an obvi-
ous demand for succinct reviews and synthetic summaries of biomedical
topics for graduate students, undergraduates, faculty, biomedical research-
ers, medical professionals, science policy makers, and the general public.
Recognizing this pressing need, CRC Press established the Pocket Guides
to Biomedical Science series, with the main goal being to provide state-
of-the-art, authoritative reviews of far ranging subjects using short, read-
able f­ormats intended for a broad audience. The volumes in this series will
address and integrate the principles and concepts of the natural sciences
and liberal arts, especially those relating to biomedicine and human well-
being. Future volumes will come from biochemistry, bioethics, cell biology,
genetics, immunology, microbiology, molecular biology, neuroscience,
oncology, parasitology, pathology, virology, and other related disciplines.

This current volume addresses the use of open-access data as a source for
cutting-edge scholarship, especially for biomedical research. Over the past
three decades, since the formation of the internet in 1991, a large ­number
of government agencies, research institutions, and academic ­institutions
across the world have created databases in various fields of science and
technology. Today, many of these databases are freely available on the
World Wide Web through the individual portals and websites of these
organizations. Open-access data, such as clinical trial metadata, genome,
proteome, microbiome, and metabolome from many repositories, provide
ever-increasing opportunities for using these data for research. This con-
cise volume provides practical guidance for how to use open-access data in
research, from generating research ideas to publishing in referred journals.
Both young researchers and well-established scholars can use this book
to upgrade their skills with respect to data sources, accessing open data,
ethical considerations, analyzing data to reach conclusions, writing manu-
scripts, publishing, and even post-publishing promotion.

vii
viii    Series Preface

The goal of this volume is the same as the goal for the series—to sim-
plify, summarize, and synthesize a complex topic so that readers can reach
to the core of the matter without the necessity of carrying out their own
time-consuming literature searches. We welcome suggestions and recom-
mendations from readers and members of the biomedical community for
future topics in the series and urge other experts to serve as future volume
authors/editors.

Lijuan Yuan
Blacksburg, Virginia, U.S.A
Editors

K. Balamurugan is endowed professor at the Department of

Biotechnology, Alagappa University, Karaikudi, India. His research aims at
understanding host-pathogen interaction using Caenorhabditis elegans as
an in vivo model system, a popular and efficient alternative to animal mod-
els. The extensive areas of his research using C. elegans as a model s­ ystem
include genomic and proteomic level analysis of various pathogens medi-
ated immune (both cellular and neuronal) response, characterization of
anti-aging genes, identification of post-translational modifications during
host-pathogen interactions, and investigation of wound healing. He has
published widely, with 83 peer-reviewed journal publications, 11 patents,
numerous book chapters, and hundreds of conference proceedings in his
field of research including in the area of host-pathogen interaction using
C. elegans as the model.

U. Prithika currently focuses her research on e­ xploring and enhancing

the stability of different probiotic bacteria present in fermented foods.
She is also investigating the effects of probiotics on irritable bowel syn-
drome. Her previous research ­experience, which includes working under
the mentorship of Prof. K. Balamurugan on model system Caenorhabditis
elegans, has built a strong foundation in molecular and immunobiology.
She has been actively involved in understanding the role of specific genes/
proteins during heat-shock and immune-­compromised host conditions.

ix
Contributors

B. Agieshkumar José Manuel Cabeda

Senior Scientist Health Sciences Faculty
Central Inter Disciplinary Research Fernando Pessoa University
Facility (CIDRF) and
Sri Balaji Vidyapeeth Deemed Fernando Pessoa Energy
University Environment and Health Research
Puducherry, India Unit (FP-ENAS)
Porto, Portugal
Charles Solomon Akino Mercy
Medical Microbiology Laboratory Gnanasekaran JebaMercy
(MML) Department of Biotechnology
Department of Microbiology Alagappa University
Centre of Excellence in Life Sciences Karaikudi, India
Bharathidasan University
Christopher Karen
Tiruchirappalli, India
Behavioural Neuroscience
Kumarasamy Anbarasu Laboratory
Department of Marine Department of Animal Science
Biotechnology School of Life Sciences
Bharathidasan University Bharathidasan University
Tiruchirappalli, India Tiruchirappalli, India

Kannan Balaji Shunmugiah Karutha Pandian

Department of Biotechnology Department of Biotechnology
Alagappa University Alagappa University
Karaikudi, India Karaikudi, India

K. Balamurugan Emil Kozarov

Department of Biotechnology Center for Molecular Microbiology
Alagappa University Department of Oral Biology
Karaikudi, India University of Florida Health
Science Center
Boopathi Balasubramaniam School of Dentistry
Department of Biotechnology Gainesville, Florida
Alagappa University
Karaikudi, India

xi
xii    Contributors

M. Krishna Raja U. Prithika

Department of Biochemistry Department of Biotechnology
JIPMER Alagappa University
Puducherry, India Karaikudi, India

P. S. Manoharan Arumugam Priya

Department of Prosthodontics and Department of Biotechnology
Crown & Bridge Alagappa University
Indira Gandhi Institute of Dental Karaikudi, India
Sciences
Puducherry, India Ann Progulske-Fox
Center for Molecular Microbiology
Kalimuthusamy Department of Oral Biology
Natarajaseenivasan University of Florida Health
Medical Microbiology Laboratory Science Center
(MML) School of Dentistry
Department of Microbiology Gainesville, Florida
Centre of Excellence in Life
Sciences Koilmani Emmanuvel Rajan
Bharathidasan University Behavioural Neuroscience
Tiruchirappalli, India Laboratory
Department of Animal Science
and
School of Life Sciences
Neuroscience Bharathidasan University
Lewis Katz School of Medicine Tiruchirappalli, India
Temple University
Philadelphia, Pennsylvania Praveen Rajesh
Department of Conservative
Galina Novik Dentistry and Endodontics
Belarusian Collection of Indira Gandhi Institute of Dental
Microorganisms Sciences
Institute of Microbiology Puducherry, India
National Academy of Sciences of
Belarus Victoria Savich
Minsk, Republic of Belarus Belarusian Collection of
Microorganisms
Chidambaram Prahalathan
Institute of Microbiology
Department of Biochemistry
National Academy of Sciences
Bharathidasan University
of Belarus
Tiruchirappalli, India
Minsk, Republic of Belarus
 Contributors     xiii

Duraisamy Senbagam Ana Moura Teles

Department of Marine Health Sciences Faculty
Biotechnology Fernando Pessoa University
Bharathidasan University and
Tiruchirappalli, India Fernando Pessoa Energy
Environment and Health Research
Balakrishnan Senthilkumar Unit (FP-ENAS)
Department of Medical Porto, Portugal
Microbiology
College of Health and Medical B. Vinoth
Sciences Associate professor
Haramaya University Department of Medical
Harar, Ethiopia Gastroenterology
Aarupadai Veedu Medical College
Thirukannamangai Krishnan Puducherry, India
Swetha
Department of Biotechnology
Alagappa University
Karaikudi, India
1
Bacteria Causing
Gastrointestinal Infections
Overview of Types of Bacteria
Causing Gastrointestinal Infections,
Mode of Infection, Challenges in
Diagnostic Methods, and Treatment
B. Vinoth, M. Krishna Raja, and B. Agieshkumar

Contents

1.1 Bacterial infections of the gut 3

1.1.1 Introduction 3
1.2 Escherichia coli 5
1.2.1 Enteropathogenic E. coli (EPEC) 5
1.2.1.1 Pathogenesis 7
1.2.1.2 Clinical features 7
1.2.1.3 Diagnosis 7
1.2.1.4 Treatment 7
1.2.2 Enterotoxigenic E. coli (ETEC) 7
1.2.2.1 Pathogenesis 8
1.2.2.2 Clinical features 8
1.2.2.3 Diagnosis 8
1.2.2.4 Treatment 8
1.2.3 Entero hemorrhagic E. coli (EHEC) (STEC/VTEC) 9
1.2.3.1 Pathogenesis 9
1.2.3.2 Clinical features 10
1.2.3.3 Diagnosis 10
1.2.3.4 Treatment 11
1.2.4 Enteroinvasive E. coli (EIEC) 11
1.2.5 Enteroaggregative E. coli (EAEC) 11
1.2.6 Diffusely Adherent E. coli (DAEC) 12
1.3 Shigella 12
1.3.1 Pathogenesis 13
1.3.2 Clinical features 13

1
2    Pocket Guide to Bacterial Infections

1.3.3 Diagnosis 15
1.3.4 Treatment 16
1.3.5 Prevention 16
1.4 Salmonella 16
1.4.1 Nontyphoidal salmonellosis 16
1.4.2 Salmonella typhi 17
1.4.2.1  Clinical features 18
1.4.2.2 Diagnosis 18
1.4.2.3  Treatment 19
1.5 Campylobacter 19
1.5.1 Pathogenesis 19
1.5.2 Clinical features 20
1.5.3 Diagnosis 21
1.5.4 Treatment 21
1.5.5 Prevention 22
1.6 Yersinia 22
1.6.1 Pathogenesis 22
1.6.2 Clinical features 23
1.6.3 Diagnosis 23
1.6.4 Treatment 24
1.7 Clostridium difficile 24
1.7.1 Incidence 25
1.7.2 Clinical and microbiological properties 25
1.7.3 Clinical features 25
1.7.4 Pathogenesis 26
1.7.5 Diagnosis 26
1.7.6 Non-laboratory-based tests 26
1.7.7 Treatment 27
1.8 Clostridium perfringens 27
1.8.1 General microbiology 27
1.8.2 Incidence 28
1.8.3 Symptoms 28
1.8.4 Pathogenesis 28
1.8.5 Diagnosis 29
1.8.6 Treatment 29
1.9 Vibrio 29
1.10 Aeromonas 31
1.11 Plesiomonas 31
1.12 Bacteriodes fragilis 32
1.13 Helicobacter pylori 32
1.13.1 Pathogenesis 33
1.13.2 Symptoms 33
 Bacteria Causing Gastrointestinal Infections     3

1.13.3 Diagnosis 34
1.13.4 Treatment 35
1.14 Foodborne illness and food poisoning 35
1.14.1 Introduction 35
1.14.2 Staphylococcus aureus 35
1.14.3 Bacillus cereus 36
1.14.3.1 Clinical symptoms 38
1.14.3.2 Laboratory test 39
1.14.4.3 Treatment 39
1.14.4 Listeria monocytogenes 39
1.14.5 Vibrio vulnificus 40
1.14.6 Cronobacter sakazaki 40
1.14.7 Mycobacterium tuberculosis 40
1.15 Future perspective 43
References 44

Gastrointestinal (GI) infections are bacterial, parasitic, or viral infections

that cause gastroenteritis, an inflammation of the GI tract. A wide range
of GI diseases are caused by bacteria, when bacteria or its associated
toxins are ingested through contaminated food or water. Though most
bacterial GI illness is short lived and self-limiting, it can be fatal, if not
treated properly.

This remains a common problem in both primary care and emergency cen-
ters in the developing world. Some of the major bacteria that causes GI ill-
ness include Escherichia coli, Salmonella spp., Shigella, Campylobacter spp.,
Clostridium spp., Yersinia, and Bacillus cereus. This chapter discusses dif-
ferent types of causative bacteria, mode of infection, and mechanism that
contributes to pathophysiology of the disease. This review also adds a note
on various methods and challenges in diagnosis and treatment of bacteria
causing GI infections.

1.1  Bacterial infections of the gut

1.1.1 Introduction

GI infection caused by bacteria is common throughout the global popula-

tion, causing significant morbidity and mortality (Ternhag et  al. 2008). The
disease burden is to such an extent that it stands second in mortality of chil-
dren younger than 5 years of age. Epidemic outbreak of some bacteria often
resulted in high mortality and liability to the community. For instance, around
0.7 million children younger than age of 5 lost their lives among the 1.7 billion
4    Pocket Guide to Bacterial Infections

diarrheal episodes in the year 2010–2011 (Walker et al. 2013). A changing bur-
den of GI infections around the world is being reported with several newly rec-
ognized organisms causing GI disease in the last three decades (Eslick 2010).

The gastrointestinal tract (GIT) is the home for several commensal bac-
teria living in harmony with the host. This is however being disrupted
by some organisms like Escherichia coli, Clostridium perfringens, and
Clostridium ­ difficile, which can cause infection of the GIT. Apart from
these, many pathogenic bacteria can enter the GIT through contaminated
food (i.e., food ­poisoning) or through some other route (i.e., ­tuberculosis)
and cause infection. Foodborne diarrheal illness is one of the leading
causes of acute diarrheal illness worldwide and commonly seen even in
­developed c­ ountries. Bacteria causing GIT infections hence can be grouped
into two types (i)  ­primary enteropathogens, which includes bacteria that
are ­gram-­negative with specific affinity to enterocytes and has specific
pathogenic factors against intestinal epithelial cells. (ii) The second group
are those which cause gastroenteritis as a part of foodborne illness with
many of them ­having affinity and pathogenic targets toward other organ
systems in addition to GIT. The GI infections manifest either primarily with
­symptoms like nausea, vomiting, abdominal pain, diarrhea, and fever or
along with systemic ­symptoms like arthritis, hepatitis, renal failure, and so
on, depending upon the severity (Shaheen et al. 2006; Goh 2007).

Based on the pathogenesis, these organisms produce either watery or

bloody diarrhea. Most of the infections are mild and are associated with
only watery diarrhea; however, few organisms are known to produce
bloody diarrhea like, Shigella, enterohemorrhagic E. coli (EHEC), Salmonella,
Campylobacter, and Yersinia. Gut infections are mostly foodborne, and
the leading cause is Salmonella, followed by Campylobacter, Shigella, and
enterotoxigenic E. coli (ETEC) according to an US data in 2014. The type of
food associated with these infections is also different for different organ-
isms. The frequencies of this infection differ between countries and depend
on the health status of the community, weather conditions, food habits,
and so on. Despite these differences, it is practically difficult to diagnose a
specific organism as a cause primarily based on clinical grounds. The usual
way of diagnosing a specific infection is only by isolating the organism in
most of these cases. But isolation of certain organisms in this group is dif-
ficult and involves either a transport medium or a highly selective medium
or application of special techniques. In addition certain organisms like E. coli
requires molecular methods for knowing the subtypes which are not rou-
tinely available and are restricted to reference laboratories. Hence, evaluat-
ing patients with diarrheal illness is cumbersome.
 Bacteria Causing Gastrointestinal Infections     5

Most of these illness are self-limiting lasting only for few days and do not
require antibiotics. Only severe and complicated cases require antibiotics,
and in fact in some cases, antibiotics might even be harmful as with EHEC.
Bacteria developing resistance toward antibiotics makes it mandatory to
know the sensitivity pattern before selecting an antibiotic. This makes the
isolation of organism even more important. As mentioned before, there
has also been a changing trend of bacteria with falling incidence of well-
known older infections (i.e., EHEC O157, Vibrio cholerae) and increasing
incidence of other organisms (i.e., Campylobacter, Yersinia) with few main-
taining a constant trend (i.e., Salmonella), and finally with lots of emerging
organisms (i.e., Arcobacteria, Edwardsiella tarda, Plesiomonas shigelloides,
Aeromonas hydrophila, Listeria monocytogenes, and Laribacter hongkon-
gensis). This chapter discusses the general biology and transmission means
of more common and emerging bacterial pathogens causing GI infection
and describes various diagnostic and treatment methods. The order of the
pathogens represents their contribution toward disease severity.

1.2  Escherichia coli

E.  coli is the foremost gram-negative facultative anaerobic bacilli resid-

ing in the intestines of humans and other mammals. Most of the strains
remain harmless, but few cause mild to severe diarrheal illness. Six major
types of E.  coli based on their pathogenesis are ETEC, enteropathogenic
E. coli (EPEC), enteroinvasive E. coli (EIEC), EHEC, or Shiga toxin producing
E. coli (STEC), enteroaggregative E. coli (EAEC), and diffusely enteroadher-
ent E. coli (DAEC). A comprehensive overview of these groups of E. coli is
presented in Table 1.1.

Diagnosing the causative agent in individuals is challenging (other than con-

ditions like epidemic outbreaks) because the disease is mostly self-limiting
within days and the procedures for detecting the toxins, pathogenic genes,
and cytotoxicity through immunoassays, DNA hybridization, cell culture,
and adherence assays are done mostly for severe cases.

1.2.1 Enteropathogenic E. coli (EPEC)

EPEC is one of the most important causes of acute diarrheal illness in infants
and children worldwide. EPEC was found to be a major cause of pediatric
diarrhea both in community and hospitalized patients (Lanata et al. 2002).
The bacteria spread through fecal-oral route, either directly or indirectly, by
consuming contaminated food or water. The infective dose is low and is less
than 10,000 colony-forming units (cfu).
Table 1.1  Comprehensive Overview of the Clinical Profiles of Each type of Escherichia coli
Types Site Involved Pathogenesis Population Affected Type of Diarrhea Antibiotic
EPEC Small intestine Attaching effacing lesions Children Osmotic Indicated in
I: Typical – Bundle forming pilus, (watery, persistent traveler’s diarrhea
attachment effacement lesion diarrhea in few)
II: Atypical – atypical adherence
pattern
ETEC Small intestine LT, ST, and cell adherence Children, travelers Secretory (watery) Not indicated
EIEC Large intestine Cell invasion Children and adults Inflammatory Not indicated
(watery, bloody)
EHEC/STEC Large intestine ST1 and ST2 Children and adults Inflammatory Not indicated
6    Pocket Guide to Bacterial Infections

a. O157:H7 (watery, bloody) Might precipitate

b. Non O157 HUS
c. O154:H4
EAEC Large intestine Aggregative adherence to Hep Children Inflammatory Indicated
2 cells (watery, persistent
Stacked brick appearance diarrhea in few)
DAEC Large intestine Diffuse adherence to Hep 2 cells Children Inflammatory Not indicated
(watery, persistent
diarrhea in few)
DAEC, diffusely enteroadherent E. coli; EAEC, enteroaggregative E. coli; EHEC, enterohemorrhagic E. coli; EIEC, enteroinvasive E. coli; EPEC, entero-
pathogenic Escherichia coli; ETEC, enterotoxigenic E. coli; HUS, hemolytic uremic syndrome; LT, labile toxin; ST, stable toxin; STEC, Shiga toxin pro-
ducing E. coli.
 Bacteria Causing Gastrointestinal Infections     7

1.2.1.1 Pathogenesis – EPEC possesses a localized adherence pattern

with the help of their fimbriae. This property is governed by a unique patho-
genicity associated island (PAI) termed as locus for enterocytes effacement
(LEE). Activation of LEE during contact with enterocytes forms type III sec-
retary system (T3SS) through which bacterial effector molecules is injected
into the enterocytes via a pilus-like structure. These effector molecules
then initiate a cascade of events like disruption of gap junction, release of
interleukin-8 (IL-8), adenosine and disruption of sodium chloride (NaCl)-
mediated transport mechanisms leading to diarrhea. Based on the patho-
genesis, they are classified into two groups namely typical EPEC (tEPEC) and
atypical EPEC (aEPEC). tEPEC has classical pilus formation with attaching
effacing lesions, whereas the aEPEC form has atypical adherence pattern.
Atypical form appears to be more important and common pathogen than
the typical form (Nguyen et al. 2006).

1.2.1.2 Clinical features – EPEC causes watery diarrhea with vomiting

and dehydration in infants and children. The incubation period is usually
1–2 days. The diarrhea is often self-limiting, but EPEC are strongly associ-
ated with severe and persistent diarrhea in some cases.

1.2.1.3  Diagnosis – Typically targeting the virulence genes by polymerase

chain reaction (PCR) could detect EPEC. DNA probing or adherence assays
are required to diagnose these E. coli types, and it can be done only in labs
that harbors molecular biological facilities.

1.2.1.4  Treatment – As most cases recover spontaneously without major

complication, antibiotics are not routinely recommended, and the main-
stay of management is supportive care in the form of rehydration therapy
because it is extremely important in pediatric population. The role of antibi-
otics in persistent diarrhea is not clearly known (Ochoa and Contreras 2011).
Effective breastfeeding have declined the incidence of EPEC and could be
preventive against it.

1.2.2 Enterotoxigenic E. coli (ETEC)

These organisms were first described from Calcutta in India by De et  al.
in the year 1956 and were later reported from other areas of the world.
In developing countries, they are one of the leading cause of diarrhea
in children younger than 5  years of age, with an estimated annual inci-
dence of 280  million cases with 380,000  deaths (Wennerås and Erling
2004; Ochoa and Contreras 2011). They were the most common cause
of ­community-acquired diarrhea (14.1%) and was associated with 9.5%
of ­hospital-acquired diarrhea according to data between 1990 and 2000
(Lanata et al. 2002). It is also the leading cause of traveler’s diarrhea.
8    Pocket Guide to Bacterial Infections

1.2.2.1  Pathogenesis – The infection is acquired by consuming contami-

nated food and the infective dose is 106 cfu. They reach the small intestine
and colon and adhere to the enterocytes through adhesive fimbriae or pili
antigens called colonization factor antigens (CFA) and are now renamed as
coli surface antigens. There is no penetration of the enterocytes, and the
diarrhea is mediated through the production of enterotoxins. ETEC pro-
duces two types of toxins namely a heat labile toxin (LT) and a heat stable
toxin (ST). There is marked variation in expression of CFA, LT, and ST among
ETEC isolated from various parts of the world. CFA/I (17%) expression is
more commonly seen among the CFA types and LT expression (60%) is
more common than ST.

The LT is structurally and functionally similar to the cholera toxin and is

destroyed by heat and acid. It is a combination of A subunit and pentameric
ring of 5B subunits. The B subunit binds to GM1 gangliosides of enterocytes
and the A subunit causes G protein coupled activation of adenylate cyclase
inside the enterocytes, leading to increased production of cyclic adenosine
monophosphate (cAMP). These result in increased chloride secretion via
cystic fibrosis transmembrane conductance regulator (CFTR) from the intes-
tinal crypt cells of the small intestine resulting in voluminous diarrhea. The
ST is not destroyed by heating even at 100°C, and their action is mediated
through activation of guanylate kinase resulting in an increase of cyclic gua-
nosine monophosphate (cGMP), leading to increased intestinal secretion
from both small and large intestines.

1.2.2.2 Clinical features – ETEC causes large volume watery diarrhea,

leading to severe dehydration. The incubation period is 1–2 days, and ill-
ness usually lasts for 1–5 days. Fever is rare; however, vomiting is seen in
children. In patients with malnutrition, the illness can last up to 3 weeks.
Dehydration is more common in adults than children. In children in devel-
oping countries, recurrent episodes of diarrhea can cause malnutrition and
growth retardation.

1.2.2.3  Diagnosis – Diagnosing ETEC requires DNA hybridization study or

an enzyme immune assay (EIA) or cell culture assay for detecting its viru-
lence factor like CFA, LT, and ST.

1.2.2.4 Treatment – The mainstay of treatment is fluid resuscitation

because patients can develop severe dehydration following large volume
diarrhea. Antibiotics have shown to be effective in reducing the duration
of illness in traveler’s diarrhea. The antibiotics are chosen based on sus-
ceptibility pattern and the preferred drugs are fluoroquinolones, azithro-
mycin, and rifaximin. Because of the disease burden, vaccination is being
 Bacteria Causing Gastrointestinal Infections     9

tried to prevent traveler’s diarrhea. An oral inactivated whole-cell vaccine

combined with B subunit of cholera toxin (Duckoral) is being used, with
modest response of 67% reported from Bangladesh and 52% from Morocco
(Clemens et al. 1988; Peltola et al. 1991).

1.2.3  Entero hemorrhagic E. coli (EHEC) (STEC/VTEC)

They were first reported from diarrheal outbreaks that occurred in

Michigan and Ohio during 1982 (Riley et al. 1983). Among the dysentery
cases in emergency departments in the United States, EHEC accounted for
2.6% of the cases (Talan et al. 2001). In the year 2011, a large outbreak of
3842 cases were reported in Germany with a new strain O154:H4. EHEC
are associated with hemolytic uremic syndrome (HUS) and hemorrhagic
colitis. Cattle, sheep, goat, dog, cat, and pig are the common animal
reservoir for EHEC. Consumption of undercooked meat, unpasteurized
milk, or open water contamination are some of the ways it is transmitted
from animals. Person-to-person transmission is quite common in nursing
homes, daycare centers, and so on, and precaution is required because
even 100 cfu is enough to be an infective dose. EHEC is also known as
Shiga toxin-producing E. coli (STEC) or verotoxin producing E. coli (VTEC)
due to the production of Shiga toxin (Stx) or verotoxin. EHEC is classi-
fied as O157 and Non O157  group-based on the serotype. O154:H4 a
newer serotype originated from enteroaggregative E. coli (EAEC) and has
acquired the Shiga toxin gene (Stx2a). Centers for Disease Control and
Prevention (CDC) reports in the year 2010 showed that the O157  con-
tributed to 25 outbreaks, and 9 outbreaks were by Non O157 serotypes.
(“FOOD Tool | CDC” 2017).

1.2.3.1  Pathogenesis – EHEC pathogenesis is mainly by their toxin pro-

duction and its effects. Stx (AB5 toxin) has one A subunit and five B subunit
The B subunit binds to the receptor in the enterocytes (globotriaosylce-
ramide) and is then internalized along with the A subunit by endocytosis.
Then the A subunit which has catalytic activity leads to cascade of events
and causes cytotoxicity by inhibiting protein synthesis. The destruction of
enterocytes impairs intestinal absorption contributing to diarrhea. Stx are
particularly toxic to the endothelial cells, and in the submucosa, they cause
microvasculature damage, leading to platelet aggregation and microvascu-
lar fibrin thrombi formation mimicking ischemic colitis. After entering the
circulation, Stx can damage renal endothelial cells in a similar manner and
can cause HUS similar to Shigella infection (Bennish et  al. 2006; Popoff
2011). EHEC share similar pathogenic profile with EPEC by possessing LEE
for adherence and also production of attachment effacement lesions.
10    Pocket Guide to Bacterial Infections

1.2.3.2  Clinical features – The incubation period varies between 1 and

14 days, with watery diarrhea after about 1–3 days, later becoming bloody.
In patients who are infected with O157, bloody diarrhea develops in 80%
of the cases. The other symptoms include nausea, vomiting, and abdominal
pain. The abdominal pain may be severe and can precede diarrhea. EHEC
causes segmental colitis, and the most common part affected is ascending
colon. The involved intestine shows characteristic thumbprint appearance
on radiograph due to severe inflammation of mucosa with mucosal edema.
Colonoscopy will reveal friable inflamed mucosa with patchy erythema,
edema, and superficial ulcerations, mimicking ischemic colitis.

The most important clinical manifestation of EHEC is HUS developing in

6%–9% of cases according to different outbreaks (Bell et al. 1994; Boyce
et  al. 1995; Bender et  al. 1997; Tarr et  al. 2005). Children younger than
5 years of age are commonly affected (15%). It is the most common cause
of HUS in pediatric population accounting for 90% of the cases (Ardissino
et al. 2016). The highest incidence of HUS among EHEC infection was seen
in the German epidemic of 2011 where 22% of cases developed HUS; inter-
estingly, adults and females were more affected than children, and the
strain was found to be O154:H4 (Frank et al. 2011). The German outbreak
resulted from consumption of contaminated sprouts. The classical triad of
HUS is microangiopathic hemolytic anemia, thrombocytopenia, and acute
renal failure. The patients start developing features of HUS between 5 and
13 days after the onset of diarrhea, and the initial clue is thrombocytopenia
that occurs by day 8 or 9 of the illness. The extraintestinal complications
commonly occur in patients with HUS and include appendicitis, intussus-
ceptions, intestinal perforation, pancreatitis, stroke, coma, seizures, intesti-
nal strictures, and sudden death. Usually renal function improves following
the hematological improvement, and patients recover in 1–2  weeks. The
mortality in patients with HUS is about 5%. Some of the patients develop
irritable bowel syndrome (IBS) following an episode of HUS.

1.2.3.3 Diagnosis – Bloody stool and elevated leucocytes in the stool

should raise the suspicion of EHEC infection. However, the diarrhea is most
often watery in the initial stages. The CDC recommends combination of a
culture method for O157 and a nonculture toxin assay (i.e., EIA) for iden-
tifying Shiga toxin to diagnose EHEC infections. O157  strains do not fer-
ment sorbitol, and hence, sorbitol MacConkey agar is used for screening this
organism. Serotyping is done using O157:H7 antisera and simultaneously the
colonies should be sent to research laboratories for confirmation. The yield
in the culture is better when the stools are tested between 2 and 7 days;
however, the fecal shedding might continue for 17 to 29 days. A positive EIA
for the toxins in the absence of O157 culture should be confirmed by further
 Bacteria Causing Gastrointestinal Infections     11

isolation of the organism due to possibility of false-positive results. PCR-

based Shiga toxin tests are also available for detecting these toxins.

1.2.3.4  Treatment – Duration of diarrhea is 3–8 days, and most recover

with no major complications, except few who develops HUS. Many studies
have shown that the antibiotics do not have any role on the duration of ill-
ness and does not reduce the incidence of HUS. Moreover, few studies have
shown that antibiotic usage is associated with increased risk of developing
HUS (Zhang et al. 2000; Mølbak et al. 2002; Safdar et al. 2002). Hence at
present, antibiotics are not routinely recommended for patients with EHEC
infections. The mainstay of management is supportive care with fluid resus-
citation. EHEC infections are prevented by consuming properly cooked beef,
avoiding unpasteurized milk, and maintaining good hygiene if there is pos-
sibility of contact with farm animals.

1.2.4 Enteroinvasive E. coli (EIEC)

EIEC are very similar to Shigella and possess the same toxin and virulence
factors as that of Shigella. Both EIEC and Shigella appear to have evolved
from nonpathogenic E. coli by acquisition of invasion plasmid (pINV) at dif-
ferent times. The pathogenesis and clinical features are similar to Shigella
infection (Refer Shigella); however, the incidence is less when compared
to Shigella. Children younger than 5  years of age are more commonly
affected. They are transmitted through contaminated food and water and
person-to-person spread also occurs. They are diagnosed by the presence
of nonlactose fermenting colonies on MacConkey agar and later by nucleic
acid hybridization technique and phenotypic assay for identifying the spe-
cific virulence genes and their cytotoxicity pattern. Antibiotics are not rou-
tinely recommended for EIEC infections.

1.2.5 Enteroaggregative E. coli (EAEC)

EAEC were first identified during the evaluation of diarrhea in Chilean

­children in 1987 when E. coli with a specific adherence pattern of stacked-
brick appearance was found when compared to controls. Because of the
aggregative clumping of the organism to the enterocytes, they are named
enteroaggregative E.  coli. EAEC causes acute and persistent diarrhea in
­children and adults in developing and developed countries. They are com-
monly associated with diarrhea in patients with HIV and also causes travel-
er’s diarrhea. The major pathogenic factors are its characteristic adherence,
production of enterotoxins and cytotoxins, and mucosal inflammation.
EAEC adheres to the intestinal epithelial cells by aggregative adherence
fimbriae (AAF) in a stacked-brick pattern. The toxins produced by EAEC are
Shiga enterotoxin 1 (ShET1), enteroaggregative heat-stable toxin 1 (EAST1),
12    Pocket Guide to Bacterial Infections

and plasmid encoded toxin termed Pet. ShET1 and EAST1 causes increased
conductance across enterocytes leading to ion secretion and subsequent
diarrhea. Pet toxin is involved in dissolution of the cytoskeleton of the epi-
thelial cell. EAEC causes mucosal inflammation by inducing the release of
IL-8 from the epithelial cells. Diagnosis can be made only by epithelial cell
adherence assay with Hep 2 cells to study the stacked-brick appearance or
by DNA hybridization or PCR assays to detect specific target genes. The role
of antibiotic in EAEC is not well evaluated because of the rarity of its diag-
nosis. However, antibiotics have been shown to be effective for traveler’s
diarrhea and in patients with HIV. The preferred antibiotics are ciprofloxa-
cin, azithromycin, and rifaximin. Rifaximin are particularly useful in traveler’s
diarrhea. In developing countries, EAEC produces long-term sequelae like
stunted growth and development of IBS.

1.2.6  Diffusely Adherent E. coli (DAEC)

DAEC are named because of their characteristic adherence pattern of dif-

fusely adhering to the intestinal epithelial cells. The exact epidemiologi-
cal pattern, pathogenesis, and their complete clinical profile are not clearly
known and are still emerging. The major virulence factor of DAEC is Dr
Family of fimbrial adhesins that are responsible for binding of these organ-
isms to the epithelial cells. After adhesion, a part of the organism is inter-
nalized by microtubule-dependent pathway, and they lead to cascade of
events resulting in a proinflammatory response. DAEC are associated with
diarrhea in children (especially older than 2 years of age) in a few parts of
both developed and developing countries. They usually cause nonbloody
diarrhea. The diagnosis requires cell adhesion assay and DNA hybridization
for detecting adherence pattern and virulence factor genes. The exact roles
of antibiotics are not clearly known and are not routinely recommended.

1.3  Shigella

Shigella is a gram-negative aerobic bacilli belonging to Enterobacteriaceae

family. Humans are the only known reservoir, despite which it is difficult
to control because of the low infective dose (10–100 cfu), their ability to
resist gastric acid, and because of increasing antibiotic resistance (DuPont
et  al. 1989). They are the leading cause of bloody diarrhea in the world.
Shigella has four major species namely S. dysenteriae, S. flexneri, S. boydii,
and S. sonnei. Each of the species, except S. sonnei, is further divided into
various serotypes according to the on the O antigen. Earlier S. dysenteriae
type I was the leading cause of bloody diarrhea in developing countries with
substantial morbidity and mortality (5%–15%) (Khan et al. 1985), but later
 Bacteria Causing Gastrointestinal Infections     13

S. flexneri appears to be more common. In an Indian study, the predominant

species is S. flexneri (60%) followed by S. sonnei (24%), S. dysenteriae (10%),
and S. sonnei (6%) (Pazhani et al. 2005). S. sonnei outbreaks are occasional,
and it causes sporadic diarrhea. In contrast, the predominant species in the
United States is S. Sonnei (80%) followed by S. flexneri (15%–20%), S. boydii
(1%–2%), and S. dysenteriae (<1%) (Scallan et al. 2011).

The mode of transmission is by fecal-oral route. Shigella is highly contagious

and can spread from person to person through contaminated hands either
directly or by the way of contaminated food and water. Sexual transmission
and transmission through fomites and houseflies also occurs.

1.3.1 Pathogenesis

Shigella exerts their effect through various mechanisms, which include

enterotoxin production, direct invasion, and cytotoxicity. The principal toxins
are the Shiga toxin (Stx) primarily produced by S. dysenteriae type 1 and
Shiga enterotoxin (ShET). ShET 1 is produced only by S. flexneri type 2a and
ShET2 is produced by most of the serotypes, and both are responsible for the
early watery diarrhea of their respective species. The pathogenesis of Stx-
induced diarrhea and HUS is similar to that of EHEC as described previously.
However, the risk is less when compared to EHEC (Bennish et al. 2006).

The direct invasion and cytotoxicity appear to be more important factor of

Shigella infection than their toxin production. This organism first invades
the M cells which are specialized epithelial cells situated over the lymphoid
follicles. Then they reach the gut-associated lymphoid tissue (GALT) in the
terminal ileum and colon. There, they are engulfed by the macrophages and
induce apoptosis of the macrophages releasing IL-1B that recruits neutro-
phils, resulting in an inflammatory response and causing destruction of the
gap junctions. Once the gap junctions are breached, the organisms present
in the lumen enter the subepithelial space and gain access to the basolat-
eral membrane of the enterocytes and eventually invade the enterocytes.
They then multiply inside the enterocytes, and this results in destruction of
the enterocytes by activating proinflammatory transcription factor, NF-κB
and spreads to adjacent enterocytes. The bacterial gene products (approxi-
mately 30) involved in the invasion and cytotoxicity are carried on large
virulence plasmid present in all the Shigella species (Parsot 2005).

1.3.2  Clinical features

The cardinal symptoms of Shigella infection are fever, abdominal pain, diar-
rhea (watery, mucoid or bloody), and vomiting. The incubation period is
1–7 days (average 3 days) (Hui 1994). The first to develop is usually fever
14    Pocket Guide to Bacterial Infections

(48  hours), followed by abdominal pain, diarrhea (72  hours), and dysen-
tery (120–144 hours) (DuPont et al. 1969). Though the classical symptom is
bloody diarrhea, it is seen only in 40%–60% of cases, and most often the
diarrhea is mucoid (50%–99%) and sometimes watery (30%–60%) (Khan
et  al. 2013). Dysentery or bloody diarrhea is more common with S. dys-
entriae type 1 (80%) and is less common with S. sonnei (20%) and other
species (Khan et al. 2013).

The clinical course depends on the serotype and host factors like age and
immune status. In children the nonbloody diarrhea is usually mild and lasts
1–3  days. In adults, the milder forms of illness usually lasts 7  days, and
adults recover without major complications. The more severe cases are
usually seen in children with malnutrition and patients with S. dysenteriae
infection. The severe illness may last for 3–4 weeks and is associated with
complications. In prolonged cases, the disease might be confused with
ulcerative colitis because even the endoscopic findings and the histology
looks similar to ulcerative colitis. HUS is one of the frequent and impor-
tant complications of EHEC; children infected with type 1 S. dysenteriae
younger than 5 years of age are also susceptible. The classical triad of HUS is
microangiopathic hemolytic anemia, thrombocytopenia, and renal failure.
The Stx through its toxicity on the vascular endothelium plays a role in the
pathogenesis of HUS. Early antibiotics appears to prevent HUS in S. dysen-
teriae type 1 infection, but it aggravates the chances of HUS in EHEC infec-
tion (Wong et al. 2000; Zhang et al. 2000; Bennish et al. 2006; Butler 2012).

The intestinal complications of Shigella infection include, proctitis, rectal

prolapse, toxic megacolon (3%), intestinal obstruction (2.5%), and perfora-
tion (1.7%) (Azad et al. 1986; Bennish 1991; Bennish et al. 1991; Khan et al.
2013). Proctitis and rectal prolapse are usually seen in younger children.
Toxic megacolon and obstruction are commonly seen with S. dysenteriae
type 1  infection. Perforation is seen in S. dysenteriae or S. flexneri infec-
tion especially in infants and patients who are malnourished. Bacteremia
is an uncommon complication (<7%) and is associated with increased risk
of death. It is commonly seen in children younger than 5 years and people
older than 65 years (Martin et al. 1983; Struelens et al. 1985; Davies and
Karstaedt 2008). They present with leukocytosis, hypothermia, temperature
above 39.5°C, severe dehydration, and lethargy.

Central nervous systems (CNS) complications are commonly encountered in

Shigella infection. Seizure is the most common CNS manifestation occur-
ring in 10% of patients. Children younger than 15 years are most commonly
affected. The other CNS manifestations include encephalopathy with leth-
argy, confusion, headache, obtundation, and coma (Avraham et al. 1982).
 Bacteria Causing Gastrointestinal Infections     15

Ekiri syndrome is a rare CNS manifestation of S. sonnei infection reported

from Japan in pre-World War II era and was characterized by rapid onset
of seizure and coma with very high fever and fewer diarrhea symptoms but
with very high mortality (15,000 deaths per year) (Bennish 1991). Leukemoid
reaction with a white blood cell (WBC) count of more than 50,000  has
been reported in about 4% of Shigella infection from Bangladesh and are
more commonly seen in children between 2 and 10 years with a high mor-
tality (Butler et  al. 1984). Sterile inflammatory arthritis (reactive arthritis)
is seen in few Shigella infections especially following S. flexneri infection.
They are uncommon (0.5%) and occurs 1–2 weeks following the onset of
dysentery (Porter et al. 2013). Patients positive with HLA B27  are commonly
affected with reactive arthritis (70%). This form of arthritis when associated
with conjunctivitis and urethritis is termed Reiter’s syndrome, which occurs
in 1%–2% of Shigella infections and commonly seen in young men between
20 and 40 years of age with HLA B27 (Simon et al. 1981; Finch et al. 1986;
Keat 2010). The joint involvement is asymmetrical, and lower extremities are
commonly affected. The treatment is usually symptomatic with nonsteroi-
dal anti-inflammatory drugs (NSAIDs). Hyponatremia (29%), hypoglycemia,
and protein-losing enteropathy are some of the other complications seen
with Shigella infection.

1.3.3 Diagnosis

Isolation of the organism by culture is required for making a confirmatory

diagnosis. Because the organisms are fastidious, the stool should be inocu-
lated in to a specific medium at the bed side, or it has to be transported in
a special medium (i.e., Cary-Blair medium or Buffered glycerol saline). The
World Health Organization (WHO) has recommended a diagnostic algo-
rithm for diagnosing Shigella infection according to which the stool sam-
ples are inoculated on MacConkey agar and a more selective xylose lysine
deoxycholate agar (XLD agar). Shigella is nonlactose fermenter and hence
appears pale pink on MacConkey agar and appears pink on XLD medium.
The suspected colonies are then inoculated in to Kligler iron agar (KIA)
and motility indole urea (MIU) medium. Based on the specific characteris-
tic ­features on these medium, they can be differentiated from E. coli (i.e.,
Shigella are nonmotile, produce alkaline slant, indole positive, urease and
oxidase negative with no gas or hydrogen sulfide production, and ferment
glucose). Molecular studies like PCR-based assays are currently available to
detect the invasion-associated locus of specific species and can detect as
few as 10–100 organisms (Avraham et al. 1982; Nataro et al. 1995). The
advantage of culture over the molecular studies is that antibiotic susceptibil-
ity can also be tested, which is important in deciding appropriate antibiot-
ics, especially in the current scenario of growing antibiotic resistance.
16    Pocket Guide to Bacterial Infections

1.3.4 Treatment

The principle management of Shigella-induced diarrheal illness includes

rehydration, correction of electrolyte imbalance, appropriate antibiotic, and
management of complications. Antibiotic therapy reduces the duration of
symptoms, duration of excretion of organisms in stool, and also reduces
complications; hence, appropriate antibiotics are indicated. If Shigella infec-
tion is suspected, early empirical antibiotic may be considered if the patient
is severely ill, immunocompromised, a healthcare provider, food handler, or
a child or adult who is associated with a day care. Drug resistance appears
to be a major problem in deciding appropriate antibiotics. Currently cip-
rofloxacin is the first-line regimen, and pivmecillinam, ceftriaxone, and
azithromycin are the second-line regimens. However, the choice of anti-
biotics should always be decided based on the local drug resistance and
sensitivity pattern.

1.3.5 Prevention

Because the organism is highly contagious, frequent hand washing, proper

sanitation, and adequate stool disposal are important to prevent the spread
of this infection. Unfortunately, there are no vaccines commercially available
for this infection.

1.4  Salmonella

1.4.1  Nontyphoidal salmonellosis

The most common bacteria that causes GI infections globally is the nonty-
phoid Salmonella. A study conducted in 2010 reported that the global burden
of nontyphoid Salmonella gastroenteritis went up to 93.8 million cases with
mortality of around 155,000 (Majowicz et al. 2010; Eng et al. 2015). The aver-
age incidence was estimated to be around 1.14 episodes per 100 person each
year. Out of these, nearly 88% of the cases were reported from Asian coun-
tries. Ingestion of Salmonella through contaminated water or food causes
acute gastroenteritis with an incubation period from 4 to 72 hours. Though
the majority of Salmonella cases occurs through ingestion of contaminated
water or food, Salmonella can also be acquired by contact with animals
and direct person-to-person contact via the fecal-oral route. Salmonellosis
may be self-limited or severe and persistent with fever, bloody diarrhea,
and weight loss. Diarrhea is usually self-limiting, lasting for 3 to 7 days, and
may be grossly bloody. Salmonella is reported to be excreted in feces up to
5 weeks after infection in young children and up to 8 weeks in older children.
Bacteremia occurs in 5%–10% of infected cases, and some may develop focal
infections such as meningitis and bone and joint infections. The mechanism
 Bacteria Causing Gastrointestinal Infections     17

that regulates the disease pathogenicity is not completely understood; how-

ever, the disease severity of Salmonella depends on factors like the serotype
of the infecting bacteria, inoculating dose, and predisposing host factors.
Before gaining access to the small intestine, the bacteria can survive in a
gastric acid barrier, thus children younger than 1 year of age, advanced age,
and patients with reduced gastric acid production were reported to have
higher risk of infection. Prolonged or recurrent infection was also reported
in impaired cellular immunity patients. Nontyphoid Salmonella are able to
induce the production of IL-8 by enterocytes, which is a potent neutrophil
chemotactic cytokine. The neutrophils attracted in this way lead to further
tissue damage by degranulation and release of oxidative toxic substances that
render the bacteria to resist the unfavorable environment and survive.

Salmonella can easily be recovered from fecal samples by direct plating, and
most laboratories use one medium with low selectivity such as MacConkey
agar. Different strategies followed for the laboratory diagnosis of Salmonella
on routine stool culture was reviewed by Humphries and Linscott (2015).
Hektoen enteric agar media is the recommended appropriate selective
media for Salmonella. XLD, Salmonella-Shigella, Hektoen enteric, brilliant
green, or bismuth sulfite medium or a chromogenic medium designed for
the recovery and detection of specific enteropathogens can also be used.
Chromogenic media has been reported to have improved sensitivity and
specificity over traditional selective media. Apart from conventional meth-
ods, techniques such as molecular assays and MALDI-TOF are also reported
to detect Salmonella; however, the latter technique is not optimized yet to
detect the serotypes.

Antibiotics are not routinely recommended for uncomplicated salmonel-

losis and are indicated only in patients who are immunocompromised and
in severe cases with complications (Sirinavin and Garner 2000). Quinolones
are currently the drug of choice for these infections, but there is high level
of antibiotic resistance developing to quinolones; hence, judicious usage of
antibiotics is required.

1.4.2  Salmonella typhi

Salmonella typhi cause illness called typhoid fever or enteric fever. They are
more of a systemic illness rather than a primary GI infection. Humans are
the only known reservoir of S. typhi. The organism penetrates the intes-
tinal epithelial cells and reaches the lymphatics with very little inflamma-
tion; hence, GI symptoms are less in the initial space. They then proliferate
in the reticuloendothelial organs, leading to hepatosplenomegaly and are
released systemically in large numbers in the next phase with resultant
18    Pocket Guide to Bacterial Infections

Figure 1.1  Colonoscopy pictures from a patient with typhoid fever. (a) Large deep
ulcer in the terminal ileum. (b) Multiple ulcers in the caecum and ascending colon
with bleeding from one of the ulcer.

involvement of various systemic organs. During this phase, the GI system

might be exposed with heavy bacteremia especially in the payer’s patches
of terminal ileum. The organisms are also seen abundantly in gall bladder.
The lymphoid follicles in the payer’s patches might then ulcerate and lead
to complications like bleeding and ulcerations, which are seen commonly
by the third week of the illness (Figure 1.1). Patients usually recover by 4
weeks, but a few continue to harbor the organism in the gallbladder or
other organs and become carriers. They continue shedding the bacteria in
feces and cause recurrent outbreaks of infection in the community.

1.4.2.1 Clinical features – The organisms are acquired by ingestion of

contaminated food products or contaminated water. The infection char-
acteristically lasts for 4 weeks and then either recovers or ends with com-
plications. The symptoms usually develop after the 7- to 14-day incubation
period. During the first week. the patient presents with high-grade fever,
myalgia, headache, and abdominal pain. Altered bowel habits are less com-
mon during this phase. The patient becomes sick, and the fever continues
in the second week. In the third week, patient develops symptoms of tox-
emia, and GI manifestations might occur in the form of pea soup diarrhea.
Only during this phase GI complications secondary to development of large
ulcerations like perforation and bleeding occur (Figure 1.1). Altered men-
tal status, often referred to as typhoidal state, happens during this phase.
During the fourth week, the patient gradually improves and recovers from
illness if the prior storming course is survived.

1.4.2.2 Diagnosis – Typhoid illness is often diagnosed by isolating the

organism through culture from the blood, stool, or urine during the first
week, second, or third week, respectively. Serological tests in the form of
 Bacteria Causing Gastrointestinal Infections     19

agglutination test (Widal test) to detect the O and H antigen is usually posi-
tive by the second week. Serial rising titers of O and H antibodies are more
important than an isolated elevation of the titers at a given time. PCR-based
assays are now available for the detection of S. typhi infection.

1.4.2.3 Treatment – Quinolones and third-generation cephalosporins are

currently used for the treatment of S. typhi infections, and they are given for
at least 7–14 days for a better response. However, because of emerging drug
resistance, antibiotics should be decided based on the local sensitivity pattern.

1.5  Campylobacter

Campylobacter are gram-negative, motile, comma-shaped bacilli with polar

flagellum. They were once grouped under Vibrio, and in 1973, they were
named a separate genus. Since then, they have emerged as one of the
leading cause of acute diarrhea and systemic illness worldwide (Vernon
and Chatlain 1973; Skirrow 2006). Along with genus Arcobacter and
Helicobacter, it is phylogenetically grouped in a separate family called “rRNA
superfamily VI.” All these organisms share common features of coloniz-
ing the mucosa of alimentary tract and reproductive tract. Currently there
are 18 species recognized, and the most important species causing human
infections are C. jejuni, C. coli, and C. fetus (in patients who are immu-
nocompromised). The other species, which rarely cause human infection,
includes C. hyointestinalis, C. upsaliensis, and C. laridis. Though C. jejuni
is the most important in this genus; Campylobacter enteritis as a whole is
discussed here and wherever appropriate C. jejuni is mentioned separately.

Campylobacter species are one of the leading causes of foodborne dis-

ease worldwide (Scallan et al. 2011). Children younger than 5 years of age
are commonly affected. Campylobacter has an extensive animal reservoir
including cattle, sheep, pig, birds, and dogs. Human infection occurs from
these animals by taking improperly cooked meats or by consuming contam-
inated foods and water. In the United States, drinking unpasteurized milk
and undercooked poultry are the leading cause of Campylobacter infec-
tion. Person-to-person transmission is relatively low. The infective dose is
relatively higher (9000 bacteria); however, a dose as low as 500 can cause
clinical illness (Robinson 1981; Black et al. 1988).

1.5.1 Pathogenesis

Adhesion and invasion of the intestinal cells are the important features
of Campylobacter infection. Because of their spiral shape and the polar
flagella, they easily penetrate the mucus layer and reach the surface of
20    Pocket Guide to Bacterial Infections

epithelial cells. Large-molecular weight plasmids encoding certain virulence

factors (pVir in C. jejuni) play a role in pathogenesis (Tracz et al. 2005). CadF
adhesion factor is responsible for microtubule-mediated internalization of
these organisms along the basolateral membrane of M cells resulting in
mucosal damage. Though an enterotoxin called cytolethal distending toxin
(CDT) is produced by C. jejuni, which causes cell cycle arrest and DNA dam-
age, its exact role in the pathogenesis is not clearly known (Pickett et al.
1996; Whitehouse et al. 1998).

1.5.2  Clinical features

The incubation period is 1–3  days and can extend up to 10  days. The
­illness typically present with acute onset abdominal pain and diarrhea. The
pain is colic and periumbilical. Diarrhea is the most common symptom of
Campylobacter and is seen in almost 90% of cases. It is bloody in 50% of
cases, and the frequency may be up to 10 or more times a day. Prodromal
illness is seen in one-third of cases, usually lasting 1–3 days before the onset
of abdominal pain and diarrhea and includes fever with rigors, headache,
dizziness, myalgia, lassitude, anorexia, and vomiting. Patients with pro-
drome have a more severe illness. The illness is usually self-limited, and
patients recover within 7 days with a mortality as low as less than 0.1%.
Relapses are seen in 25% of the cases.

Campylobacter infection can mimic acute appendicitis and inflammatory

bowel disease (IBD). The severe abdominal pain secondary to ileocecitis
causes tenderness in the right iliac fossa (RIF) mimicking appendicitis. In
a series of 533 cases with clinical diagnosis of acute pancreatitis, ultra-
sound showed mesenteric adenitis with thickening of ileum and caecum
without visualization of appendix in 61 cases (Puylaert et al. 1989). Out
of these 61 patients, 21 were found to have Yersinia enterocolitica and
15  had C. jejuni infection. Rarely, Campylobacter-induced appendici-
tis has also been reported (Van Spreeuwel et  al. 1987). Some of the
Campylobacter infection present with severe acute colitis with bloody
diarrhea; these cases might be confused with IBD, and one has to depend
on histology wherein there is acute inflammation with no chronic changes
like crypt distortion (Van Spreeuwel et al. 1985). The other complications
seen with Campylobacter infection are erythema nodosum, urticaria,
acute cholecystitis, pancreatitis, septic arthritis, HUS, toxic megaco-
lon, GI hemorrhage, bacteremia, nephritis, meningitis, pericarditis, and
myocarditis. Histology of a few patients with immune proliferative small
intestinal disease has shown evidence of C. jejuni infection; hence, it has
been found to be associated with development of intestinal lymphomas
(Lecuit et al. 2004).
 Bacteria Causing Gastrointestinal Infections     21

The late manifestations of Campylobacter infections include reactive

arthritis and Guillain-Barré syndrome (GBS). The reactive arthritis is similar
to Salmonella, Shigella, and other bacterial diarrheal illness and presents
1–2  weeks after the onset of diarrhea. The ankle, knee, wrist, and small
joints of the hand are usually affected and occur commonly in cases posi-
tive with HLA B27. The prognosis is good and usually responds to NSAID
therapy within 6 months. GBS is one of the common late complications and
in the United States: 1 in 1000 cases of Campylobacter enteritis (especially
C. jejuni) develops GBS (Nachamkin et  al. 1998). GBS occurs 1–2  weeks
following the onset of diarrhea. It has been shown that 30%–40% of
GBS were due to Campylobacter infection and the prognosis of C. jejuni–
induced GBS appears to be worse when compared to others (Rees et  al.
1995; Nachamkin et al. 1998). The pathogenesis is secondary to the devel-
opment of antibodies against the epitopes present in Campylobacter cross
reacting with the GM1 gangliosides of the neurons.

1.5.3 Diagnosis

A rapid presumptive diagnosis can be made by examining fecal smear by

Gram stain on dark field microscopy, which shows these organisms as
faint gram-negative motile rods with a typical seagull wing appearance.
But the gold standard for the diagnosis of Campylobacter infection is
isolation of the organism by culture. These organisms are fastidious and
transport medium, like Cary-Blair medium, should be used for transporta-
tion. Generally a selective antibiotic-containing medium is used and incu-
bated at 42°C under carbon dioxide (CO2) and reduced oxygen conditions.
Rapid diagnostic tests, like a latex agglutination test for detecting specific
Campylobacter antigens and PCR-based test to detect Campylobacter DNA
in stool samples, are available (Kulkarni et al. 2002; Granato et al. 2010).

1.5.4 Treatment

Antibiotics are not routinely needed because most of infections are mild
and recover spontaneously. However, antibiotics are indicated in severe
cases with dysenteric symptoms, systemic infection, and immune defi-
ciency; additionally, they should be used during outbreaks in a daycare set-
ting to prevent spreading. Campylobacter is resistant to many drugs, and
the current choices are ciprofloxacin or azithromycin. In view of increas-
ing fluoroquinolone resistance, azithromycin is the preferred drug (DuPont
2007; Tribble et al. 2007), especially for traveler’s diarrhea. In a heavy sys-
temic infection, a more effective drug in the form of carbapenem or ami-
noglycosides are usually preferred, and an oral macrolide is also used for
eradicating intestinal infection (Lachance et al. 1991; Sjögren et al. 1992).
22    Pocket Guide to Bacterial Infections

1.5.5 Prevention

Effective pasteurization of milk and consumption of purified drinking

water and adequately cooked meat are the important means of preventing
Campylobacter infection.

1.6  Yersinia

The genus Yersinia consists of 11 species out of which 3 are important

causes of human infections, namely Y. enterocolitica, Y. pseudotuberculo-
sis, and Y. pestis. Yersiniosis, a foodborne GI infection is primarily caused
by Y. enterocolitica and less commonly by Y. pseudotuberculosis. Y. pestis
is the causative organism for pulmonic and bubonic plague. Y. enteroco-
litica are gram-negative coccobacilli with peritrichous flagella and are fac-
ultative anaerobes belonging to Enterobacteriaceae family (Bottone 1997).
They are psychrophilic and often require a cold enrichment step for isola-
tion. Infections are common during winter and are frequent in temperate
countries. They have an extensive animal reservoir, and the most frequent
mode of infection is by eating undercooked pork and drinking raw milk
and contaminated water. Yersiniosis is the third-most common zoonosis
in the European Union after Campylobacter and nontyphoidal salmonel-
losis (Hoffmann et al. 2012). Lithuania and France have the highest rate at
12.9 and 9.8 cases per 100,000 population, respectively. Children younger
than 5 years of age are commonly affected. Diabetes, iron overload, blood
transfusion, malnutrition, alcoholism (Rabson et al. 1975; Bouza et al. 1980)
are some of the predisposing factors for Yersiniosis. Iron is required for the
virulence of these organisms.

1.6.1 Pathogenesis

The incubation period is 1–11  days, and they are excreted in the stools
for 14–97  days. The main pathogenesis is cell invasion mediated by vari-
ous virulence factors. The organism adheres by Yersinia adhesion A pro-
tein (YadA) binding to the epithelial cells (M cells). Chromosomal genes
inv and ail encode the ability to invade the M cells. Then through plasmid
(pYV)-encoded T3SS, bacterial virulence products are secreted into the
enterocytes. Only strains carrying the pYV plasmids are virulent and are the
primary virulence factor of Y. enterocolitica. The variants of these plasmids
are also seen in Y. pseudotuberculosis and Y. pestis. One of the virulent prod-
ucts, Yersinia outer membrane proteins (Yops) is important in suppressing
inflammation and phagocytosis, leading to increased survival inside the cells
for long period (Boland and Cornelis 1998; Cornelis 2002; Goebel 2012).
 Bacteria Causing Gastrointestinal Infections     23

Yersinia also has a separate iron uptake system called Yersiniabactin (Ybt)
and is involved in efficient uptake of iron even from iron-deprived sites. The
expression of certain pathogenic genes is thermoregulated and is the rea-
son for its increased prevalence in cold climates. After invasion, they reach
the submucosa and then phagocytosed by the macrophages and taken to
payer’s patches of terminal ileum and mesenteric lymph nodes where they
form microcolonies and subsequently cause systemic disease. Following the
primary infection by an autoimmune mechanism, Yersinia can cause ery-
thema nodosum, reactive arthropathy, glomerulonephritis, and thyroiditis
in few of the cases.

1.6.2  Clinical features

Abdominal pain is the most predominant symptom; fever and diarrhea are
less frequent. Diarrhea is usually watery and bloody and seen in one-fourth
cases. The other symptoms include nausea, vomiting, headache, pharyngi-
tis, arthralgia, oral aphthous ulcers, and erythema nodosum. The involve-
ment of mesenteric lymph nodes (i.e., mesenteric lymphadenitis) causes
pain in the RIF and can mimic appendicitis (pseudo-appendicitis). This form
of mesenteric adenitis with pseudo-appendicitis is commonly seen in teen-
agers and young adults. The other form of enteritis and colitis with diarrhea
is commonly seen with children. Some of the complications with severe
disease include bacteremia, hepatic and splenic abscess, peritonitis, poly-
myositis, and osteomyelitis. Septicemia with septic shock is commonly seen
in infants, patients who are immunocompromised, and iron overload states.
Rapid onset of septic shock has been reported in patients following blood
transfusion with a mortality of 50% (Guinet et al. 2011). The common late
manifestations presenting after 2 weeks due to autoimmune mechanisms
are reactive arthritis and are seen commonly in patients positive with HLA-
B27. The others are glomerulonephritis, thyroiditis, and erythema nodosum.
Y. pseudotuberculosis infections are rare and are associated with granulo-
matous colitis.

1.6.3 Diagnosis

Yersinia should be suspected in any patient with bloody diarrhea or if the

stools have leucocytes (inflammatory diarrhea). The only mode of diag-
nosing Yersinia is by isolating the organism from stool, mesenteric lymph
nodes, pharyngeal exudates, peritoneal fluid, or blood. Bacterial identifi-
cation is difficult, and hence, laboratories should be intimated if Yersinia
infection is suspected. Y. enterocolitica can grow easily in ordinary medium
and are nonlactose fermenters on MacConkey agar. However, isolation of
the organism needs inoculation in MacConkey agar at 25°C–30°C or using
24    Pocket Guide to Bacterial Infections

a selective medium. The selective medium is cefsulodin-irgasan-novobiocin

(CIN) agar. Enzyme-linked immunosorbent assay (ELISA) and immunoblot-
ting can be used to detect IgG, IgA, and IgM class antibodies. Detection
of IgM antibody indicates acute infection and a fourfold increase in the
titer form, acute to convalescence stage increases the probability of diagno-
sis. Agglutination tests and complement fixation tests are also available for
diagnosing Yersinia infection. However, the results of these serological tests
should be interpreted with caution because cross-reactions with antigens of
other organisms are possible leading to false-positive results.

1.6.4 Treatment

Yersiniosis is usually a self-limiting illness and mortality is extremely rare (1.2%)

(Long et  al. 2010). However, antibiotics are indicated in severe infections
and patients who are immunocompromised. The preferred antibiotics are
fluoroquinolones for adults and septran for children for a period of 5 days. In
areas where Yersinia is resistant to fluoroquinolones, the alternative choice
is septran or doxycycline. In septicemia and more severe infections, a com-
bination of third-generation cephalosporin along with an aminoglycoside is
preferred, and the duration of treatment is for 3 weeks. Because Yersinia is
almost foodborne (90%), proper cooking of the meat, adequate pasteuriza-
tion of milk, and drinking safe water can prevent such infections.

1.7  Clostridium difficile

C.  difficile is most commonly implicated in Clostridium difficile infection

(CDI), which is a leading cause of hospital-associated GI illness (Johnson
et  al. 1990). It is noninvasive and present in two forms, an antibiotic-­
resistant latent spore and nonresistant vegetative form. The latter, when in
the human body can prioritize growth over toxin production and colonize
the intestine causing the disease.

They produce two toxins, namely toxin A (TcdA, 205 kDa) and toxin B (TcdB,
308 kDa), which are involved in the destruction of the intestinal cell cyto-
skeleton leading to cell death (Carey-Ann and Carroll 2013). C.  ­difficile,
though a normal commensal, do not cause illness because of its lesser
number and competition from other bacterial commensals to assess direct
contact with the epithelial cells. However with the use of antibiotics and
reduction in the number of other commensals, the condition becomes
favorable for C. ­difficile to cause illness (Wilson 1993). C. difficile infections
may have varying presentations with asymptomatic carriage, mild diarrhea,
or a severe condition called pseudomembranous colitis.
 Bacteria Causing Gastrointestinal Infections     25

1.7.1 Incidence

The incidence of C.  difficile is on the rise due to increasing usage of anti-
biotics. In the United States, the incidence is 346,805 among hospitalized
patients during the year 2010 (Lessa et al. 2015). A meta-analysis including
51 studies from Asia has shown an incidence of 14.8% among all patients
with diarrhea, with incidence higher in hospitalized patients when compared
to daycare patients at a rate of 5.3% per 10,000 patient days (Brazier and
Berriello 2000; Borren et  al. 2017). Ramakrishnan and Sriram (2015) who
worked on CDI and antibiotic abuse in India concluded an incidence of 1.67%
in India, which is similar to the United States.

1.7.2  Clinical and microbiological properties

Morphologically C. difficile is seen as irregular rods with elongated spores,

which are slightly bulged at the terminal end. C. difficile is cultured on cyclo-
serine cefoxitin fructose agar (CCFA) medium and identification on other
culture medium is mentioned in Table 1.2. The colonies have a ground glass
appearance under microscope and exhibit a yellow-green fluorescence
under ultraviolet (UV) illumination. A typical odor of horse manure is also
used to identify the microbe. A gas liquid chromatography profile of large
amounts of butyric and iso-caproic acid is considered the best method for
identification (Fedorko and Williams 1997).

1.7.3  Clinical features

The symptoms of C. difficile infection include watery diarrhea, fever, loss of

appetite, nausea, and abdominal pain or tenderness. Based on the severity
of symptoms, the disease is classified as mild to moderate, severe and com-
plicated disease, and recurrent CDI. Symptomatic illness is characterized by

Table 1.2  Identification of Clostridium difficile on Culture Medium

Media Observation
Gelatin No liquefaction
Agar Minute, flat, opaque discs
Egg yolk agar Surface colonies are dry, irregular, flattened, dry, roughened,
somewhat granular, with little or no color
No precipitate in the agar and no luster on the colony
Coagulated albumin No liquefaction
Blood agar Irregular, flat
Hemolysis absent
Blood serum No liquefaction
Source: Sneath, P.H.A. et al., Bergey’s Manual of Systematic Bacteriology, Lippincott
Williams & Wilkins, Baltimore, MD, 1986.
26    Pocket Guide to Bacterial Infections

watery diarrhea occurring often within 2 days of antibiotic usage; 96% of ill-
ness occurs within 14 days of antibiotic usage and almost all of them within
prior 3 months (Olson et al. 1994). The disease is mild when there are no signs
or symptoms of colitis. In moderate disease, patients present with diarrhea and
colitis, characterized by fever and abdominal cramps in the lower quadrant.

Fulminant C. difficile causes toxic megacolon in which colon is distended

more than 6 cm and is prone for perforation. It occurs in less than 5% of
patients and is characterized by severe abdominal pain, guarding, rigidity,
and high fever with either profuse or absent diarrhea (Bartlett and Gerding
2008; McCollum and Rodriguez 2012).

1.7.4 Pathogenesis

The organism is found freely in soil, in variety of animals, and in raw and
processed food products. They are highly prevalent in healthcare ­facilities,
resulting in horizontal transmission through both environmental s­urface
­contamination and direct contact by hospital workers. The mode of ­infection
is primarily through person-to-person and through the f­ecal-oral route.
There have been studies showing the increase in transmission of C. difficile
during hospitalizations, especially in elderly patients in acute care ­facilities
(McFarland et  al. 1989; Samore et  al. 1994; Chang and Nelson 2000;
Vaishnavi 2010).

1.7.5 Diagnosis

In 2010, guideline for the management of patients with CDI was published
by Infectious Diseases Society of America (IDSA) and Society for Healthcare
Epidemiology of America (SHEA). Based on these guidelines, CDI cases are
defined by stool test positivity for C. difficile toxins, toxigenic C. difficile, or
colonoscopic or histopathologic findings, revealing pseudomembranous
­colitis. Standard reference tests such as cell culture neutralization assay (CCNA)
and toxigenic culture methods have being used for the past 30 years for detec-
tion of CDI (Planche and Wilcox 2011). EIA-based toxin detection methods
are being carried out and they are less expensive, easy, and quick to perform
when compared to CCNA. Currently PCR-based identification is widely pre-
ferred because nucleic acid amplification methods have a greater sensitivity
and reduced turnaround time. Nucleic acid amplification tests (NAATs) are the
most recent methods for detection of C. difficile and have been used since
1990 (Wilcox et al. 2000; Snell et al. 2004; Zheng et al. 2004; Berg et al. 2007).

1.7.6  Non-laboratory-based tests

Endoscopy and computed tomography (CT) scan may be done in certain

cases for diagnosis of pseudomembranous colitis (Kazanowski et al. 2014).
 Bacteria Causing Gastrointestinal Infections     27

Figure 1.2  Colonoscopy picture showing multiple yellowish plaques of pseudo-

membranes distributed along the sigmoid colon in a patient with pseudomembra-
nous colitis.

Colonoscopy will reveal pseudo-membranes that are yellow exudative

plaques of about 2–5 mm (Figure 1.2).

1.7.7 Treatment

The antibiotic, which caused the CDI, has to be stopped, and few of the
patients (15%–25%) respond spontaneously without the need for further
specific antibiotics. Antibiotics are only needed in patients who are critically
ill and those with severe symptoms. The drugs of choice are metronida-
zole and vancomycin with a success of about 87% and 97%, respectively
(Farrell and LaMont 2000; Zar et  al. 2007). Metronidazole may be given
orally or intravenously, but vancomycin can only be given orally to be effec-
tive. Relapse is common after successful treatment and is seen in 20% of
the cases.

1.8  Clostridium perfringens

1.8.1  General microbiology

Clostridium perfringens (C. welchii) is gram-positive, spore-forming, non-

motile, rod-shaped, obligate anaerobic bacterium with an optimum growth
at 43°C–47°C and generation time of less than 10 minutes.
28    Pocket Guide to Bacterial Infections

1.8.2 Incidence

According to the CDC, C. perfringens is the second-most common bacteria

that causes foodborne illnesses in the United States, infecting nearly million
people annually. Outbreaks occur regularly and cause substantial morbidity.

1.8.3 Symptoms

After an incubation period of 8 to 16 hours, the symptoms start and last

for 24 hours. Symptoms include profuse watery diarrhea, abdominal pain,
nausea, and vomiting.

1.8.4 Pathogenesis

C. perfringens are usually involved in food poisoning and open wound

infections. The GI infection varies from mild enteric disease to necrotiz-
ing enteritis. It is an intestinal commensal and remains dormant until their
number increases because of contaminated foods. C. perfringens is clas-
sified into five types (A–E) based on the production of four exotoxins
(i.e., alpha, beta, epsilon, and iota; Table 1.3). In addition to this, several
other toxins including enterotoxin (Cpe) and neuraminidase are also pro-
duced. Chromosomally coded Cpe are highly resistant to food-preservation
procedures compared to the plasmid-encoded isolates (Li  and McClane
2006). Because it could not synthesize vital amino acids, it depends on
the breakdown of the host tissue for survival. This damage to the intesti-
nal cells results in diarrhea, flushing out the spores and toxins to prevent

Table 1.3  Clostridium perfringens Toxins and Its Illness

Types of C. perfringens
Toxins Diseases
A Gas gangrene (myonecrosis), foodborne illness, and
infectious diarrhea in humans; enterotoxemia of
lambs, cattle, goats, horses, dogs, alpacas, and others;
necrotic enteritis in fowl; equine intestinal clostridiosis;
acute gastric dilation in nonhuman primates, various
animal species, and humans
B Lamb dysentery; sheep and goat enterotoxemia
(Europe, Middle East); guinea pig enterotoxemia
C Darmbrand (Germany) and pig-bel (New Guinea) in
humans; “struck” in sheep; enterotoxemia in lambs
and pigs; necrotic enteritis in fowl
D Enterotoxemia of sheep; pulpy kidney disease of lambs
E Enterotoxemia in calves; lamb dysentery; guinea pig
enterotoxemia; rabbit “iota” enterotoxemia
 Bacteria Causing Gastrointestinal Infections     29

severity of infection (Doyle et  al. 2007). Heat-resistant spores ingested

from the contaminated food survive, proliferate, and produce toxins, lead-
ing to the disease.

Alpha toxin is produced by type A strain and present frequently in humans.

Zinc activates the toxin after which it interacts with the host cell recep-
tors, and through a series of pathways, the permeability in blood vessels
is increased and blood supply is reduced to tissues. Beta toxin is lethal
and produced by type B and type C strains. Necrotizing enteritis (enteritis
necroticans or pigbel), which is rare, is caused by toxins of type C strains
and is often obtained by ingestion of undercooked pork. Epsilon toxin is
most commonly isolated from animals. which are produced by type B and
type D strains. Potassium ions and fluid leakage occurs due to perfora-
tion in tissues. Iota toxin, known as AB toxin, is produced by type E strain.
B component interacts with the host cell surface receptor to facilitate the
uptake of the toxin, while A component inhibits actin polymerization,
thereby breaking down the cytoskeleton. Bacteremia and clostridial sepsis
is uncommon, but both are fatal occurring with an infection of the uterus,
colon, or biliary tract.

1.8.5 Diagnosis

Culture identification is used for diagnosis with notable colony characteris-

tics like double-zone hemolysis or circular opaque zone on McClung-Toabe
egg yolk agar. PCR-based identification of toxin-producing genes, colori-
metric assay detecting lecithinase, (Dave 2017), and reverse passive latex
agglutination (RPLA) test are available for enterotoxin identification.

1.8.6 Treatment

No specific treatment is needed for milder causes, and even in necrotizing

enteritis (Pigbel), only symptomatic and supportive treatment is required.

1.9  Vibrio

Vibrio cholerae is a comma-shaped gram-negative bacterium that causes

an acute GIT infection known as cholera. Outbreaks of cholera are mainly
reported from the underdeveloped and developing world where there
are no proper sources for clean drinking water and sewage disposals.
However, people can get infected with the bacteria through ingestion of
contaminated shellfish or seafood products containing higher concentra-
tion of V. cholerae. WHO reports that more than 1.4 billion people are at
risk of developing cholera every year with over 130,000 deaths worldwide.
30    Pocket Guide to Bacterial Infections

Currently the highest incidence of cholera is reported in Africa and south

Asian countries. The bacterium V. cholerae that causes infections only in
humans is a free-living inhabitant of freshwater. More than 200 serotypes of
V. cholerae have been reported, with each serogroup showing distinct anti-
genicity based on its O-side chains. V. cholerae O1 subgroup and O139 are
the strains commonly associated with disease epidemics. Serogroups other
than O1 and O139 often produce self-limiting gastroenteritis because they
do not have the cholera toxin gene. The cholera toxin gene encodes for the
cholera toxin which acts by stimulating the adenylate cyclase system of the
GI tract and causes life-threatening secretory diarrhea. Numerous factors
contribute to bacterial colonization and disease progression. However, the
important part is the ADP-ribosylating cholera toxin, which is not directly
required for bacterial colonization, but accounts for the secretory diarrhea
as mentioned previously (Sánchez and Holmgren 2008; Millet et al. 2014).

Laboratory diagnosis for V. cholerae involves biochemical or serological tests

for the identification of the presence of O1 serogroup antigens. The sub-
typing of O1 serogroup as Inaba, Ogawa, and Hikojima could be done by
agglutination test. Slide agglutination test is done by treating the cultures
grown on heart infusion agar, Kligler’s iron agar, and triple sugar iron agar
with the antiserum to detect the specific O antigen. Other than this, some
of the biochemical tests like oxidase test, ring test, triple sugar iron test,
carbohydrate test, decarboxylase test, and Voges-proskauer test are done
infrequently based on the necessity. The hemolytic activity of V.  ­cholerae
is used to distinguish the biotypes such as classical and E1T; classical types
show negative hemolytic activity, whereas E1T or of Australian or the US gulf
coast strain shows strong positive hemolytic activity.

Clinical symptoms begin with the sudden onset of painless watery diarrhea
after a 24- to 48-hour incubation period. In most of the cases, the diarrhea
may quickly become voluminous and is often followed by vomiting with or
without abdominal cramps. Most V. cholerae infections are asymptomatic
with mild to moderate diarrhea, which may not be clinically distinguishable
from other causes of gastroenteritis. An estimated 5% of infected patients
will develop severe watery diarrhea, vomiting, and dehydration. Stool vol-
ume during cholera is more than that of any other infectious diarrhea. The
characteristic cholera stool is an opaque white liquid, and it resembles the
remnant water that has been used to cooking of rice (i.e., rice water diar-
rhea). If untreated, the diarrhea and vomiting due to bacterial infection lead
to isotonic dehydration, which can lead to acute tubular necrosis and renal
failure. Patients with severe disease may develop vascular collapse shock,
leading to death.
 Bacteria Causing Gastrointestinal Infections     31

Intravenous and oral hydration remains the main treatment for cholera.
However, antibiotics have also been used as an adjunct to hydration therapy.
Studies have shown that antibiotic treatment could greatly reduce the ­volume
of stool output, duration of diarrhea, and duration of positivity for bacterial
culture (Roy et al. 1998; Kaushik et al. 2010). Tetracyclines, ­quinolones, and
septran are usually given for the treatment of severe cholera cases.

1.10  Aeromonas

Aeromonas are gram-negative ubiquitous bacteria distributed in fresh and

brackish water. Based on their temperature preference, they are classified
into two groups (Murray and Baron 2007). The psychrophilic species grows
at 22°C–25°C are non-motile and cause infection only in fishes. The meso-
philic species grows at 35°C–37°C, are motile, and causes disease in humans.
Currently, there are eight species in mesophilic group that are known to cause
human infection and the common species include A. hydrophilia, A. caviae,
and A. veronii. They cause acute diarrheal illness (i.e., most common manifes-
tation) after ingestion of contaminated drinking water or foods and wound
infections following swimming in fresh or brackish waters. Children younger
than 2  years of age and adults older than 80  years of age are commonly
affected. Though they have been isolated from few of the outbreaks, some
studies have failed to show its exact role in gastroenteritis.

The Aeromonas adhere to the intestinal epithelial cells and secrete various
toxins like heat labile enterotoxin, hemolysin, and cytotoxins. The other viru-
lence factors include VacB, enolase, and the ability to produce Type VI secre-
tory system. Cell invasion also occurs and leads to colitis and dysentery. The
affected persons usually present with watery diarrhea (75%–89%), but it
may be bloody in few cases. The diarrhea usually lasts for 3 to 10 days. Fever
and abdominal pain are less common. Rare complications include segmental
colitis, ischemic colitis, and HUS. The treatment is often conservative in the
form of rehydration therapy because the illness is self-limited and does not
require routine antibiotics. Antibiotics are only indicated in chronic diarrhea
and in patients with systemic complications. The preferred antibiotics include
fluoroquinolones or a third-generation cephalosporin or a carbapenem.

1.11  Plesiomonas

Plesiomonas are gram-negative facultative anaerobic rods belonging to

Enterobacteriaceae family and present in freshwater (MacDonell and
Colwell 1985). They are acquired by drinking contaminated water, by eating
32    Pocket Guide to Bacterial Infections

raw seafoods (Oysters and shellfish), or by contact with reptiles. Acute diar-
rheal illness is the usual manifestation of Plesiomonas infections, but they
also cause cellulitis and skin abscess during trauma in freshwater as with
Aeromonas. They also cause traveler’s diarrhea in tropical and subtropical
countries. Few diarrheal outbreaks have been reported in Japan, China,
Cameroon, and Bangladesh (Hori et al. 1966; Tsukamoto et al. 1978; Rutala
et  al. 1982; Bai et  al. 2004; Wouafo et  al. 2006). They produce several
toxins, including a cholera-like toxin, and they also cause invasion of the
epithelial cells, leading to colitis and bloody diarrhea. After an incuba-
tion period of 24–48 hours, patients develop watery diarrhea with severe
crampy abdominal pain. Sometimes patients develop severe colitis and can
present with bloody diarrhea. Pain is severe and is the prominent symptom
of Plesiomonas colitis; the other symptoms are fever and vomiting. The diar-
rheal illness is usually self-limiting and recovers without major complications
in healthy individuals; however, in some patients, the illness might last from
2 weeks to 3 months. Complications are seen in patients who are immu-
nocompromised and in children and includes septicemia, meningoencepha-
litis, pneumonia, and so on (Terpeluk et al. 1992; Schneider et al. 2009).
Antibiotics are not routinely given, and they are indicated only in chronic
diarrhea and for extraintestinal complications. The antibiotics currently used
are fluoroquinolones, third-generation cephalosporin, or carbapenem.

1.12  Bacteriodes fragilis

B. fragilis are anaerobic commensal bacteria found in human intestine.

Only during 1980s, it was found to be associated with diarrheal illness in
humans. They produce an enterotoxin called B. fragilis enterotoxin (BFT),
also called as fragilysin, which causes secretion of IL-8 and leads to inflam-
mation. They also cause increased intestinal permeability. The usual clinical
feature is development of nonbloody diarrhea in children and adults. It is
diagnosed by selective culture under anaerobic condition. Genetic and bio-
logical assays are required to diagnose the presence of the enterotoxin and
are not routinely available. Because the diagnosis is rarely done, the exact
role of antibiotics is not clearly known.

1.13  Helicobacter pylori

They are gram-negative, microaerophilic, helical-shaped, motile

(Lophotrichous flagella) organisms. Nearly two-third of the global popula-
tion is infected with H. pylori; however, they are common in developing
nations. They are acquired by ingestion of contaminated water because of
 Bacteria Causing Gastrointestinal Infections     33

poor sanitation and hygiene. Most often, the infection remains asymptom-
atic, but few people develop symptomatic acute gastritis or chronic gastritis
leading to peptic ulcer disease; they are also associated with development
of mucosa-associated lymphoid tissue (MALT) lymphomas and adenocarci-
nomas primarily of the stomach and rarely of the duodenum.

1.13.1 Pathogenesis

These organisms possess certain special features to survive in the gastric acidic
pH. They have urease enzymes that can make the pH alkaline by producing
ammonium ions and form an ammonia cloud that protects the organisms
from acidic pH. Secondly with the help of their flagella, they move toward the
less acidic mucous layer and come in close proximity to the epithelial layers.
And lastly, they have various virulence genes that encodes for various virulence
factors responsible for causing inflammation. Adhesin called BabA helps in
binding to the Lewis b antigen on gastric epithelial cells and SabA to the sialyl-
Lewis x antigen on gastric mucosa. Immune responses are triggered by HcpA
one of the Helicobacter cysteine-rich proteins, cag Pathogenicity Island (PAI).
and peptidoglycan causing inflammation (Viala et  al. 2004; Dumrese et  al.
2009). Expression of vacuolating toxin A (VacA) and the cytotoxin-associated
gene A (CagA) proteins causes severe damage to the host cell, which may also
pave way for ulceration and gastric cancer (Miehlke et al. 2000; Dossumbekova
et al. 2006). CagA is a cag PAI-encoded protein (120–140 kDa), which is also
a potential carcinogen that disrupts adherence to adjacent cells, cytoskeleton,
cell polarity, intracellular signaling, and other cellular activities (Backert and
Selbach 2008). Type IV secretion system helps in injecting the CagA into the
gastric epithelial cells. Tyrosine residues of the CagA at four distinct glutamate-
proline-isoleucine-tyrosine-alanine (EPIYA) motifs are phosphorylated by the
host cell membrane associated Abl and Src kinases, leading to the activation
of proto-oncogene Shp2 (Hatakeyama 2004). Signal transduction and gene
expression of the host are altered by the activation of epidermal growth fac-
tor receptor (EGFR). In perigenetic mechanism, tumor necrosis factor-alpha
(TNF-α) alters gastric epithelial cell adhesion, leading to dispersion and migra-
tion of mutated epithelial cells. Peptidoglycan triggers NF-κB-dependent pro-­
inflammatory pathway and IL-8 cytokine secretion by binding with NodI (Viala
et al. 2004). Peptidoglycan also initiates PI3K-Akt pathway leading to migra-
tion, cell proliferation, and prevention of apoptosis (Nagy et al. 2009).

1.13.2 Symptoms
Symptoms include abdominal pain (epigastric region), diarrhea (bloody
or black), burping, bloating, halitosis, heartburn, and weight loss; less
common symptoms are vomiting, nausea, and anorexia. The pain is often
34    Pocket Guide to Bacterial Infections

related to food and is usually relieved by food in case of duodenal ulcers

and aggravated in case of gastric ulcers.

1.13.3 Diagnosis

Noninvasive modalities of diagnosing H. pylori include IgG antibody detec-

tion, antigen detection in stool sample, and urea breath test. Serology is not
useful because it does not identify an active infection. The invasive method
of diagnosing H. pylori is by performing an endoscopy and taking biopsies
from the stomach, and by subjecting the biopsy specimen for rapid urease
test (RUT), histopathological examination (HPE), culture and sensitivity, or
PCR study. Classical endoscopic finding in patient with H. pylori infection
is the presence of benign-appearing ulcers either in the stomach, usually
along the lesser curve or in the first part of the duodenum (Figure 1.3).
Duodenal ulcers are more common than gastric ulcers. Often special stains
are used to demonstrate H. pylori on HPE (Figure 1.3). The common method

Figure 1.3  Single active ulcer (a) and two active ulcers (b) in the first part of
­duodenum, (c) an active ulcer in the prepyloric region of stomach causing ­obstruction,
(d) Alcian blue stain of the biopsy from stomach showing shaped Helicobacter pylori
in the mucous layer of stomach.
 Bacteria Causing Gastrointestinal Infections     35

of diagnosing H. pylori in clinical practice is by endoscopy and biopsy fol-

lowed by rapid urease test and HPE.

1.13.4 Treatment
All patients with symptomatic ulcer disease need anti–H. pylori therapy.
Eradication of H. pylori requires combination therapy with at least two anti-
biotics and one proton pump indicator (PPI; i.e., triple therapy) given for
10–14 days. The success rate is about 70% to 85%. The most commonly used
combination is one PPI with clarithromycin and amoxicillin. Because of the
development of resistance, the antibiotic regimens keep changing, and it is
better to select antibiotics based on the local sensitivity pattern. Quadruple
therapy with a PPI, bismuth, tetracycline, and metronidazole is also used for
difficult cases, and it has a success for about 75% to 90%. Sequential therapy
in the form of PPI plus amoxicillin for 5–7 days followed by PPI plus clarithro-
mycin and tinidazole for 5–7 days has a success rate of about 75% to 95%.

1.14  Foodborne illness and food poisoning

1.14.1 Introduction

Foodborne illness is common in developed and developing countries and con-

tributes to significant burden to the society even in developed countries. The
most common organisms responsible for foodborne disease vary among dif-
ferent parts of the world. In South Korea, E. coli, including EHEC, is the most
common organism followed by nontyphoidal Salmonella and S. aureus (Park
et al. 2015). In Japan, the most common organism is Campylobacter followed
by Salmonella species and EHEC (Kumagai et al. 2015), and in United States,
the most common is nontyphoidal Salmonella followed by Campylobacter
and Shigella (Crim et al. 2015). In most of the developing countries, the exact
epidemiology and the etiologic organisms of food poisoning are not avail-
able because of improper reporting and difficulties faced in diagnosing the
organisms. In the United States, 48 million out of 350 million diarrheal ­illness
is as a result of food poisoning and accounts for 125,000 hospitalization and
3000  deaths annually (Mead et  al. 1999; Scharff 2012). Common organ-
isms associated with food poisoning are shown in Tables 1.4 and 1.5, and
most of the important organisms causing food poisoning have already been
described. The other organisms are briefly described here.

1.14.2  Staphylococcus aureus

S. aureus are acquired by eating foods, like dairy products, meats, egg,
and salads, prepared by food handlers (Balaban and Rasooly 2000; Centers
for Disease Control and Prevention (CDC) 2013). The organisms multiply
36    Pocket Guide to Bacterial Infections

Table 1.4  Common Foodborne Illness Causing Bacteria and Its Preliminary
Identification Details
Bacteria Type Disease
Bacillus cereus Gram-positive, rod shaped, Foodborne illness
motile, facultative anaerobic,
spore-forming and beta
hemolytic bacterium
Clostridium difficile Gram-positive, long irregular Colitis
with bulge at the ends, motile,
anaerobic, and spore forming
Clostridium perfringens, or Gram-positive, rod-shaped, Foodborne illness
C. welchii, or Bacillus motile, anaerobic,
welchii spore-forming
Helicobacter pylori or Gram-negative, helix-shaped, Stomach ulcer,
Campylobacter pylori motile, microaerophilic stomach cancer
Salmonella spp. Gram-negative, rod-shaped, Foodborne
motile, facultative anaerobe, illness/
non-spore-forming Salmonellosis
Staphylococcus spp. Gram-positive, round, Foodborne illness
(Staphylococcus aureus) nonmotile, facultative
anaerobes, non-spore forming
Yersinia spp. Gram-negative, rod-shaped, Foodborne
(Yersinia enterocolitica) motile (22°C–29°C) illness/yersiniosis

in the food at room temperature and produce enterotoxins. The toxin is

heat stable, and symptoms occur within 1 to 6 hours after food ingestion.
Patients present with nausea, vomiting, and abdominal cramps. Fever and
diarrhea is less common and occurs only in few cases. Management is usu-
ally conservative and supportive.

1.14.3  Bacillus cereus

Species of Bacillus and related genera remains a large threat because of their
resistant endospores. The food-poisoning episode usually occurs because
spores survive normal cooking or pasteurization and then germinate and
multiply when the food is inadequately refrigerated. B. cereus  is a large,
gram-positive, spore forming, rod-shaped bacteria distributed widely as
spore former or vegetative cell in the environment such as freshwater and
marine water, decaying organic matters, vegetables, and as vegetative cell
in intestinal tract of invertebrates (Scheider et al. 2004; Tewari and Abdullah
2015). On ingestion, the contaminated food or soil products transfer the
viable spore or vegetative cells bacteria, leading to the colonization inside
the human intestine.
 Bacteria Causing Gastrointestinal Infections     37

Table 1.5  Diagnosis, Treatment, and Complications of Common Foodborne

Pathogens
Bacteria Diagnosis Treatment and Complications
Bacillus cereus Isolation of more than Self-limiting rarely septicaemia
105 colonies per g of occurs
food
Clostridium difficile Stool culture, stool ORT, antibiotics: metronidazole,
cytotoxin test. PCR, vancomycin, fidaxomicin.
ELISA, EIA for Fecal microbiota transplantation
presence of causative Ion exchange resin:
bacterial genetic Cholestyramine.
material or toxins or Complications: Sepsis,
glutamate toxic megacolon,
dehydrogenase pseudomembranous colitis, and
enzyme and its perforation of the colon
production.
Flexible
sigmoidoscopy and
colonoscopy help in
visualizing the
pseudo-membranes in
colitis
Clostridium Stool culture test, Prevent dehydration.
perfringens, or C. >106 spores of this Complications: bacteremia,
welchii, or Bacillus bacteria per gram of tissue necrosis, cholecystitis,
welchii stool, Nagler’s emphysematous, and gas
reaction gangrene
Helicobacter pylori or Histological Triple therapy – proton pump
Campylobacter pylori examination, urine inhibitors (lansoprazole,
ELISA, stool antigen esomeprazole, pantoprazole, or
test or urea breath rabeprazole) and the antibiotics
test, blood antibody clarithromycin, metronidazole,
test and amoxicillin
Salmonella spp. Blood and stool Electrolytes replacement.
culture, PCR For typhoid and paratyphoid
fever antibiotics may be
prescribed if needed
Complications: reactive arthritis,
bacteremia
Staphylococcus spp. Culture identification, Antibiotic resistance,
(Staphylococcus PCR biofilm formation,
aureus) osteomyelitis, bacteremia, and
septic arthritis

(Continued)
38    Pocket Guide to Bacterial Infections

Table 1.5 (Continued)  Diagnosis, Treatment, and Complications of Common

Foodborne Pathogens
Bacteria Diagnosis Treatment and Complications
Yersinia spp. Stool culture, tube Self-limiting but for severe cases
(Yersinia agglutination, ELISA, antibiotics may be prescribed.
enterocolitica) radioimmunoassays, Third-generation cephalosporins,
imaging studies, trimethoprim-sulfamethoxazole,
colonoscopy tetracyclines, fluoroquinolones
(adults >18 years old),
aminoglycosides.
Complications: sepsis, focal
infection, bowel necrosis

EIA, enzyme immune assay; ELISA, enzyme-linked immunosorbent assay; ORT, oral
rehydration therapy; PCR, polymerase chain reaction.

Milk and rice are the two most commonly contaminated food p ­ roducts
because of paddy soil bacteria. In addition, food poisoning by
B. cereus can also be contributed to by a variety of contaminated foods
like  meat dishes, frozen food and food products, ready-to-eat chicken
products, and dessert mixers. The organism is also present contaminated
with several spices and food additives. The spores of B. cereus are heat
resistant, and they can be destroyed by steaming under pressure, ­frying,
and grilling. Inactivation of bacterial enteric toxins can be done by heat-
ing for 5  minutes at 133°F, and the emetic toxins can be removed by
heating to 259°F for more than 90  minutes. Because the spores can
strongly adhere to hydrophobic surfaces, it is unlikely to remove the bac-
teria completely, but tracing spores from farmer to package can reduce
the occurrence.

1.14.3.1 Clinical symptoms – The two clinical syndromes caused by

B.  cereus food poisoning are diarrheal form and emetic form. Diarrheal
form is the result of a heat labile enterotoxin produced inside the GIT.
The patient develops diarrhea and abdominal pain about 8 to 16  hours
after consumption of the contaminated food. Foods associated with the
diarrheal forms are meat, vegetables, and sauces. The emetic form is due
to the emetic toxin (cereulide) ingested along with the contaminated
food that induces nausea, vomiting, and abdominal cramps within 1 to
5  hours after ingestion. Fried rice is the most common food associated
with emetic form of illness in the United States. The Emetic syndrome is
more severe and acute compared to diarrheal syndrome; however, with
both forms of illness, patients usually recover within 24  hours with no
major complications.
 Bacteria Causing Gastrointestinal Infections     39

1.14.3.2  Laboratory test – Diagnosis of B. cereus infection causing food

poisoning is usually clinical and most often an attempt to confirm by a
laboratory method is not usually undertaken because the illness is short
lived and self-limiting; further, it is not cost effective and is not available
in routine laboratories. However, when a large outbreak of food poison-
ing occurs, the diagnosis is made using certain special reference labora-
tories. B. cereus can be cultured from stool using special culture medium.
Commercial assays are being used to detect the diarrheal toxin like reverse
passive latex agglutination test (Granum et  al. 1993), immunochromato-
graphic tests, and PCR, which targets the gene itself (Fricker et al. 2007; Das
et al. 2009). PCR assays have also been used for diagnosing emetic food-­​
borne B. cereus infection.

1.14.4.3  Treatment – The treatment of B. cereus food poisoning is usually

supportive care and antibiotics are not routinely recommended.

1.14.4  Listeria monocytogenes

Listeria monocytogenes are gram-negative bacilli, usually causing invasive

illness in neonates, patients who are immunocompromised, older, and
pregnant. Febrile gastroenteritis is another manifestation of L. monocyto-
genes, which is almost always (99%) foodborne. They are responsible for
less than 1% of reported cases of foodborne illness (MacDonell and Colwell
1985). The diarrheal illness is often sporadic, but a few outbreaks have been
reported. The incubation period for gastroenteritis is less (usually 24 hours)
ranging from 1 to 10 days, when compared to invasive illness which is usu-
ally 11 days (i.e., 90% occurs within 28 days). Listeria is seen contaminated
in a wide range of foods including rice, vegetables, chocolate milks, cheese,
smoked trout, corned beef, ham, and delicatessen meat. The infective dose
is about 10,000 organisms per gram of food.

Fever is the prominent characteristic clinical feature, and diarrhea is usu-

ally watery. Only a few people develop bloody diarrhea. Other symptoms
include headache, myalgia, nausea, and vomiting. Most are self-limited
and resolve spontaneously, but few can develop dissemination and lead
to more invasive illness. Diagnosing Listeria gastroenteritis is challenging
because they require a highly selective culture medium. Hence, the labo-
ratories should be intimated if Listeria is suspected. Serological method for
detection of listeriolysin O antigen is available for retrospective diagnosis,
and recently DNA-based methods are also available but are not routinely
used. Antibiotics are not routinely recommended for uncomplicated illness;
however, they are given to patients at high risk (e.g., those who are preg-
nant, immunocompromised, and older) during outbreaks and in cases with
40    Pocket Guide to Bacterial Infections

dissemination. The preferred antibiotics in uncomplicated cases are septran,

ampicillin, or amoxicillin and in complicated cases, are aminoglycoside in
combination with ampicillin or amoxicillin. These infections are best pre-
vented by following strict food-safety measures.

1.14.5  Vibrio vulnificus

Vibrio vulnificus is a gram-negative bacilli belonging to the family

Vibrionaceae. It is highly lethal opportunistic organism affecting patients
who are immunocompromised and those with chronic liver disease. In the
United States, V. vulnificus is a leading cause of seafood-associated fatality.
According to the CDC, there were about 96 cases of V. vulnificus infection
with a 91% hospitalization rate and mortality rate of 34.8% (Scallan et al.
2011). They are acquired by ingestion of raw seafoods causing gastroenteritis
and skin infections requiring amputations. Septicemia and necrotizing fas-
ciitis are the common complications leading to high fatality. Special culture
medium is needed for isolation of these organisms. According to the CDC,
doxycycline and ceftazidime are the drugs of choice in adults, and septran
and an aminoglycoside in children. However, the final decision on selection
of the antibiotic should be based on the local susceptibility pattern.

1.14.6  Cronobacter sakazaki

C. sakazaki is a gram-negative bacilli belonging to enterobacteriaceae family

and is an emerging cause of necrotizing enterocolitis, sepsis, and meningi-
tis in low-birthweight infants and in children younger than 4 years of age
(Bowen and Braden 2006; Hunter et al. 2008). It spreads through ingestion
of dry foods like infant feeding formula. The incidence is rare but has a high
mortality of 40% to 80%. Diagnosis is made from blood culture and cerebro-
spinal fluid (CSF) culture. Antibiotics are generally administered because of
its higher mortality and includes a combination of ampicillin and gentamicin,
but in view of resistance, a combination of carbapenem or a newer cephalo-
sporin (cefepime) with an aminoglycoside is also preferable.

1.14.7  Mycobacterium tuberculosis

M. tuberculosis infection of the GIT, though not common, is often seen in

developing countries where tuberculosis (TB) is endemic, like India, Bangladesh,
and Pakistan. Abdominal TB comprises about 5% of all cases of TB (Sharma
and Mohan 2004). Intestinal TB is one of the forms of abdominal TB, and
the other three are peritoneal, lymph nodal, and solid organ TB. The intesti-
nal form often presents with chronic illness with varying manifestations, and
acute diarrheal illness is rare. The disease is acquired by reactivation of latent
infection or by ingestion of Mycobacterium through intake of unpasteurized
 Bacteria Causing Gastrointestinal Infections     41

milk and undercooked meat. In active pulmonary TB or disseminated TB, it can

spread to the GIT by hematogenous route, directly from adjacent structures,
or through lymphatics. The organism reaches the submucosal lymphoid tis-
sue and causes inflammatory reaction, leading to lymphangitis, endarteritis,
granuloma formation, caseation necrosis, mucosal ulceration, and scarring.

Intestinal TB has two forms, ulcerating or stricturizing form and hypertro-

phic form, and in a few patients, both the forms can coexist (Figure 1.4), The
ulcerative or stricturizing form usually affects jejunum and ileum, and the
ulcers are transverse as when compared to longitudinal ulcers of typhoid
and tend to be circumferential. Ulcers are often surrounded by inflamma-
tory mucosa and can lead to perforation, bleeding, fistula formation, and
strictures upon healing. They present with abdominal pain, distension,
chronic diarrhea, nausea, vomiting, constipation, and bleeding. The hyper-
trophic form commonly affects ileocecal region and presents with abdomi-
nal pain, mass in the abdomen, or with signs of obstruction. In addition,

Figure 1.4  Shows various endoscopic findings of intestinal tuberculosis. (a) Ulcer
involving hepatic flexure, (b) multiple ulcers involving caecum and ileo-cecal (IC)
value with deformed IC value (arrow). (c) Proliferative polypoidal lesions in the
ascending colon. (d) Proliferative lesion causing obstruction in the ascending colon.
42    Pocket Guide to Bacterial Infections

both the forms will have constitutional symptoms like loss of appetite, loss
of weight, evening rise of temperature, and night sweats. Abdominal pain is
the most common presentation of intestinal TB. The frequent complications
are fistula, stricture, and bowel obstruction. Both small and large bowels
are involved, and multiple areas of the bowel may be involved. The ileocecal
region is the most common site involved and is seen in 75% of the patients,
followed in frequency by ascending colon, jejunum, appendix, duodenum,
stomach, esophagus, sigmoid, colon, and rectum (Rathi and Gambhire
2016). RIF mass is seen in 25% to 50% of patients (Marshall 1993; Horvath
and Whelan 1998).

Diagnosing intestinal TB is often challenging, and it is difficult to differenti-

ate it from Crohn disease. This is extremely important in TB-endemic coun-
tries because immunosuppressants given for Crohn disease will aggravate
TB if wrongly treated. The definitive diagnosis of TB is only possible by
demonstrating the acid-fast bacilli in the biopsy specimen (Figure 1.5), from
the resected bowel specimen, or by culturing the organisms from these
specimens. Techniques of NAAT, like nested PCR for identifying a particu-
lar genetic sequence of TB bacilli, is being added as an acceptable modality
of diagnosing TB; however, it will not differentiate active disease from an
older infection. The initial evaluation in any patient with suspected intesti-
nal TB would be an abdominal imaging (i.e., preferably contract-enhanced
computed tomography [CECT] of the abdomen), which gives information as
to the site involved and the presence of complications if any. The samples
are usually obtained by performing colonoscopy when the colon or ileocecal
region is involved. Obtaining samples in cases with small bowel involvement
is challenging and might require enteroscopy. In TB, both the sides of the ileo-
cecal valve is usually involved and is often destroyed, giving an appearance
of fish mouth opening. The usual colonoscopy findings are ulcers, strictures,
nodules, pseudo-polyps, fibrous bands, and fistulas (Alvares et al. 2005).

Figure 1.5  Ziehl Neelson stain showing the acid fast Mycobacterium tuberculosis
bacilli (round arrows).
 Bacteria Causing Gastrointestinal Infections     43

The organisms are commonly seen in the submucosa, and hence, deeper
biopsies are required from both the ulcer and the margins for a better yield.
The characteristic histopathological features of intestinal TB include pres-
ence of caseation granulomas and presence of acid-fast bacilli. The features
of TB granuloma that helps in differentiating them from other etiologies
include confluent granulomas, granulomas >400 micrometers in diameter,
more than five granulomas in biopsies from one segment, and granulo-
mas located in the submucosa or granulation tissue (Pulimood et al. 2005;
Rathi and Gambhire 2016). Since the yield of smear and culture is less, the
diagnosis sometimes depends on the combinations of clinical, radiological,
endoscopic, histopathological findings, and the NAAT results. The other
supporting evidence of pulmonary involvement (i.e., chest X-ray [CXR]), if
present, may be helpful. The Mantoux test and the QuantiFERON gold assay
has only a limited value because they cannot differentiate an active disease
from previous infections.

The treatment of the intestinal TB is the anti-tubercular therapy (ATT)

regimen similar to pulmonary TB (Makharia et al. 2015; Jullien et al. 2016).
Patients show clinical improvement usually after 2 weeks, and complete
resolution of ulcers and erosions on follow-up colonoscopy is seen after
2–3  months of anti-tubercular therapy. Surgery is indicated in patients
with complications like stricture, obstruction, perforation, abscess, fistula,
and bleeding (Aston 1997; Horvath and Whelan 1998; Kapoor 1998).

1.15  Future perspective

Larger number of bacterial organisms are believed to be responsible for GI

infections. Some are true pathogens, whereas others are merely commen-
sal in nature without causing any pathological conditions. The molecular
strategies used by these bacteria to interact with the host can be unique
to specific pathogens or may possess a conserved pattern across several
different species. Effective treatment and key to fight the bacterial disease
rely on the precise and rapid identification and characterization of these
bacteria. Conventional culture-based methods that are widely used in many
laboratories can detect only viable bacteria, but not the ones that are non-
culturables that are metabolically active but not reproducing (Figure 1.6).
Current advancements in molecular biology plays a vital role in clinical diag-
nostic settings by identifying a large number of bacteria directly from stool
samples (e.g., metagenomics allows rapid retrieval of genetic material and
screening directly from environmental sources). Thus, these high-through-
put methods that are described elsewhere may add knowledge to meta-
bolic function of bacteria and their role in causing GI infections.
44    Pocket Guide to Bacterial Infections

Figure 1.6  Different diagnostic methods used in identification of bacteria and its
analytes.

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2
Gateways of Pathogenic Bacterial
Entry into Host Cells—Salmonella
Balakrishnan Senthilkumar, Duraisamy Senbagam,
Chidambaram Prahalathan, and Kumarasamy Anbarasu

Contents

2.1 Introduction 59
2.2 Salmonella 61
2.3 Salmonella—Invasion strategies 62
2.4 Host invasion pathways 63
2.4.1 Type III secretion system (T3SS) dependent invasion
mechanism 63
2.4.1.1 T3SS structure 63
2.4.1.2 Machinery system of T3SS-dependent entry 64
2.4.1.3 Actin cytoskeleton rearrangements mediate
invasion 64
2.4.2 T3SS-independent invasion 65
2.4.2.1 OMP Rck invasion 66
2.4.2.2 OMP PagN invasion 66
2.4.2.3 Role of the hemolysin HlyE 67
2.4.3 Caveolae-mediated invasion 67
2.4.4 Unknown factors influencing invasion 68
2.5 Intracellular survival 69
2.6 Conclusion 70
Acknowledgments 70
References 70

2.1 Introduction

Enteric fever is one of the major tropical bacterial, foodborne disease

caused by Salmonella species, causing high mortality and morbid-
ity (Marks et  al. 2017). The major symptoms include nausea, vomiting,
abdominal cramps, loss of appetite, bloody diarrhea, fever, headache,
and so on. The incubation period varies from individual to individual but

59
60    Pocket Guide to Bacterial Infections

usually is from 6 to 72 hours. The bacteria persists a long time asymp-
tomatically in healthy carriers from months to years in the host organism
bowel and is released every day in their feces, but at the same time, they
are highly invasive (Ilakia et  al. 2015; Prestinaci et  al. 2015). Southeast
Asia and western Pacific countries are in the top list of high morbid-
ity and mortality from enteric fever. In 2015, it was assessed to cause
about 178,000  deaths and 17  million hospitalizations; more than 85%
of all incidences occur in three major countries, Bangladesh, India, and
Pakistan (Wang et  al. 2016). In United States alone, 1.4  million cases
of human salmonellosis, 7000  hospitalizations, and almost 600  deaths
were chronicled each year (Hendriksen et al. 2004; Voetsch et al. 2004).
Globally, Salmonella enterica serovar Typhimurium was the frequently
isolated serovar in most of the enteric fever cases (Leekitcharoenphon
et  al. 2016). Other nontyphoidal Salmonella (NTS) serovars that usually
cause self-limiting diarrhea can also lead to multiple systemic infections
and are generally referred to as invasive NTS (iNTS). Globally iNTS is esti-
mated to cause 3.4 million hospitalizations and 681,000 fatalities annually
(Ao et al. 2015). Although, Salmonella infection contributes substantially
to global morbidity and mortality during the pre-antibiotic era, the per-
centage of case fatality rate has decreased from 30% to less than 1% with
the use of effective antibiotics (Crump et al. 2015). But, recently, there
has been increasing number of Salmonella Typhi/Parathyphi incidents that
were reported because of the emergence of antibiotic-resistance behav-
iors (Kariuki et al. 2015). Multidrug-resistant (MDR) Salmonella serovars
are resistant to first-line antibiotics, ampicillin, tetracycline, trimethoprim,
sulfamethoxazole, and chloramphenicol; however, they are sensitive to
fluoroquinolones. In Southeast Asia and African countries, they are also
susceptible to fluoroquinolones.

The transfer of antimicrobial resistance (AMR) genes among bacteria is

commonly enabled by plasmid or transposon exchange. Salmonella patho-
gens are highly invasive in nature. H58, a dominant haplotype of S. typhi
mostly identified in the high risky regions, are mostly associated with an
IncHI1  plasmid. Such plasmids anchor a composite transposon that can
carry multiple resistance genes, including blaTEM-1 (ampicillin resistance),
dfrA7, sul1, sul2 (trimethoprim-sulfamethoxazole resistance), catA1 (chlor-
amphenicol resistance), and strAB (streptomycin resistance) genes (Klemm
et al. 2018). Since 2016, a large proportion of S. typhi resistant to drugs,
including chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole,
and third-generation cephalosporins, have been reported in Pakistan and
the United Kingdom (Feasey et  al. 2015; Hendriksen et  al. 2015; Wong
et al. 2015). Because of the emergence of these extensively drug-resistant
 Gateways of Pathogenic Bacterial Entry into Host Cells—Salmonella     61

(XDR) isolates, urgent action is needed for the hour to avoid lineages.
Understanding their invasive mechanism and their proliferation within the
host cells could be an efficient approach to control the spreading of such
perilous lineages worldwide. Keeping that in mind, this chapter narrates
the mechanism of Salmonella pathogenicity manipulates in the host cell
using different strategies for successful invasion to establish an intracel-
lular niche.

2.2  Salmonella

Salmonella, a gram-negative, rod-shaped, facultative anaerobic, nonspor-

ing bacteria, belongs to the member of Enterobacteriaceae family, causing
gastrointestinal (GI) disorders and multisystemic fatal infections. Initially the
genus Salmonella consists of two species namely S. bongori and S. enterica
(Porwollik et al. 2004). S. enterica, the enteric pathogen, is further classified
with six subspecies (S. enterica subsp. enterica, S. enterica subsp. salamae,
S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp.
indica, and S. enterica subsp. houtenae) and more than 2600  serovers
(Porwollik et al. 2004; Guibourdenche et al. 2010). Salmonella pathogens
can survive in the GI tract of various animals including human and birds.
However, the enteric fever causing Salmonella serotypes and the disease-
causing pathogens spread from asymptomatic human carriers via feces
and contaminated food and water. Based on agglutination properties of
their outer membrane protein antigens such as somatic O, flagellar H and
capsular Vi, they are commonly classified (Guibourdenche et  al. 2010).
Most of the Salmonella human infections are due to strains of Salmonella
enterica subsp. enterica. Now its nomenclature is established on the basis
of serotypes name belonging to subspecies. In case of Salmonella enterica
subsp. Enterica serotype Typhimurium is edited to Salmonella Typhimurium
(Brenner et al. 2000).

In the view of clinical perspective, Salmonella serotypes are grouped on the

basis of host range and outcome of the disease. For example, Salmonella
Typhi is a human-host specific restricted pathogen causing a septicemic
typhoid syndrome (enteric fever), and Salmonella Gallinarum is restricted to
birds and causes a severe systemic disease called fowl typhoid (Shivaprasad
2000). Broad host range serotypes such as Salmonella Typhimurium and
Salmonella Enteritidis cause majority of GI salmonellosis (Velge et al. 2005),
including in human, domestic livestock, and fowl. In a single host, differ-
ent Salmonella serotypes can bring different pathologies. Oral inoculation
of weaned calves with S. Dublin causes severe systemic infection, whereas
62    Pocket Guide to Bacterial Infections

Salmonella Gallinarum becomes avirulent and S. Typhimurium causes acute

enteritis (Paulin 2002). Finally, pathogenesis is not restricted to dose and
route of inoculation, which is mainly influenced by genesis and immune sta-
tus of the host organism (Calenge et al. 2010).

2.3  Salmonella—Invasion strategies

Salmonella infection is initiated by ingestion of either contaminated food

or water followed by passage of the bacteria from the stomach to the
intestine. Stomach acids are good physiological barrier for the prevention
of salmonellosis; however, patients who are immunocompromised or tak-
ing anti-acidic drugs or antibiotics lost their stomach natural acidity bal-
ance, which leads to the infection. Once they successfully cross the stomach
barriers, they easily adhere to intestinal epithelial cells to invade the stom-
ach, and their passage is initiated by transcytosis (through enterocytes or
M cells at the apical side), migration to the basolateral side, and exocy-
tosis into the interstitial space of the lamina propria (Muller et  al. 2012).
Within the propria, the cells are taken up accidentally by phagocytes and
disseminate quickly via the efferent lymph in mesenteric lymph node and
bloodstream in spleen and liver. But different routes of entry have been
observed in different host and serotypes. In the case of cattle, S. Dublin
passage was observed through the epithelial layer and associates with MHC
class II–positive cells in propria because of their extracellular nature in effer-
ent lymph (Pullinger et al. 2007).

The successful invasion of Salmonella into host cells is known to be criti-

cal for bacterial survival and onset of disease. In general, the intracel-
lular pathogenic bacteria have evolved in two strategies such as trigger
and zipper mechanism. Previously Takeuchi (1967) who first reported the
mechanism of entry via trigger mechanism using Type III secretion system
(T3SS-1); however, recent studies added zipper mechanism (Coombes et al.
2005; Desin et al. 2009; Rosselin et al. 2010). Among them, most of the
Salmonella serovars are using zipper mechanism via the Rck invasin, a pro-
tein encoded by the rck gene located on the large virulence plasmid (Rychlik
et al. 2006; Futagawa-Saito et al. 2010; Rosselin et al. 2010). However, the
other pathogenic enterobacteria such as Shigella flexneri enters via trig-
ger mechanism by secreting T3SS (Schroeder and Hilbi 2008), and Yersinia
pseudotuberculosis uses zipper mechanism. None of these bacteria, other
than Salmonella, has been shown to use both mechanisms to invade their
host cells (Cossart and Sansonetti 2004), which demonstrates its strong
pathogenicity.
 Gateways of Pathogenic Bacterial Entry into Host Cells—Salmonella     63

2.4  Host invasion pathways

2.4.1 Type III secretion system (T3SS)

dependent invasion mechanism

T3SS is one of the best studied entry mechanism of Salmonella. It allows the
bacteria to enter the host nonphagocytic cells via trigger mechanism and
persuades massive actin rearrangements and intense membrane distressing
at that site (Cossart and Sansonetti 2004).

2.4.1.1 T3SS structure – Salmonella serovars contain a large number

of gene clusters, commonly known as Salmonella pathogenicity islands
(SPIs) that usually encode virulence factors. Until recently, SPI-1 is thought
to have genes required for bacterial entry (Galan 1996), whereas SPI-2 is
essential for intracellular survival (Shea et al. 1996). However, both SPI-1
and SPI-2 encode T3SS conserved among many gram-negative pathogens
(Tampakaki et  al. 2004) and consisted more than 20  proteins. Subsets
of these proteins forms a needle-like complex structure that spans both
inner and outer membrane and inserts into the host plasma membrane
(Kubori and Galan 2003) by energy-dependent (i.e., adenosine triphos-
phate [ATP]) manner (Galan and Wolf-Watz 2006). They can cross over the
inner and outer membrane of a host cell and create a pore in the mem-
brane on contact with the host cells. Their structure bears a resemblance
to basal body of flagella, proposing an evolutionary relationship of these
two organelles.

The needle complex of T3SS consists of a multiring base constituted with

SPI-1–encoded proteins such as PrgK, PrgH, and InvG and a slight needle-like
single protein PrgI. The detailed protein system encoded by SPI-1 is tabulated
with their specific activity (Table 2.1). The assembly of all these proceed in
an orderly manner (Schraidt and Marlovits 2011). The needle projects single
protein PrgI arrives from the outer membrane as a filament of 50-nm length,
a complex of three proteins, namely SipB, SipC, and SipD, commonly known
as translocon and located at the edge of needle, which enables it to make a
pore in the target cell and allows the secretion of effector proteins (Mattei
et al. 2011). Hydrophilic domains of SipD make direct interaction with PrgI
needle protein and hydrophobic domains of SipB and SipC directly involved
in pore formation (Miki et al. 2004; Rathinavelan et al. 2011). The interaction
between SipB and cellular cholesterol is essential for effector translocation.
In conclusion, the translocation of T3SS-1 effector proteins involves an appa-
ratus present at the inner membrane and are made up of highly conserved
proteins namely SpaP, SpaQ, SpaR, SpaS, InvA, InvC, and OrgB.
64    Pocket Guide to Bacterial Infections

Table 2.1  Functional System of Salmonella

Pathogenicity Island (SPI-1)
SPI-1 Effector Proteins Functions
SpaP, SpaQ, SpaR, SpaS Exportation apparatus
InvA, InvC (ATPase)
OrgB
PrgH, PrgK Needle complex
InvG, InvH
PrgJ
InvJ
SipB, SipC, SipD Translocon
InvF Regulators
HilA, HilD
SirC, SprB
SipA, SptP, AvrA Effectors
SicA Chaperones
InvB
SicP
OrgA, OrgC, InvE, InvI Unclassified
IacP, IagB, SpaO

2.4.1.2  Machinery system of T3SS-dependent entry – Among the vast

number of SPI-1 effectors, six proteins, SipA, SipC, SopB, SopD, SopE, and
SopE2, are playing a key role to invade the cell, whereas others are con-
tributed in postinvasional process, including host cell survival and modula-
tory inflammatory responses (Patel and Galan 2005). To trigger Salmonella
invasion into host cells, effectors operate actin cytoskeleton either directly or
indirectly and also influence the delivery of vesicles to the entry site to provide
additional membrane and allow the extension and ruffling of the plasma
membrane to promote invasion. Later, membrane fusion occurs to induce the
sealing of the future Salmonella-containing vacuole (SCV) and actin filaments
are depolymerized, enabling the host cell to recover its normal shape.

2.4.1.3 Actin cytoskeleton rearrangements mediate invasion –

Salmonella entry into the host cells is directly or indirectly mediated by actin
cytoskeleton. Drugs that are interrupting actin dynamics completely prevent
bacterial internalization (Finlay et al. 1991). After establishing contact with
epithelial cells, Salmonella induces actin cytoskeletal rearrangements at the
site of bacterial entry that direct bacterial internalization (Finlay et al. 1991;
Francis et al. 1993; Ginocchio et al. 1994).

SipC, one of the components of bacterial translocon, consists of two mem-

brane-spanning domain, s120 amino acid N-terminus and 209 amino acid
 Gateways of Pathogenic Bacterial Entry into Host Cells—Salmonella     65

C-terminus, while the C-terminus extends into the host cytoplasm (Hayward
and Koronakis 1999). C-terminal domain of SipC nucleates the gathering of
actin filaments, resulting in rapid filament growth from the pointed ends
(Hayward and Koronakis 1999). Notably, the SipC C-terminus is also required
for translocation of effector protein, suggesting that it modulates both
translocon assembly and activities. Chang et  al. (2005) has demonstrated
that actin nucleation and effector translocation are detachable. A short
region proximately next to the second transmembrane domain of SipC (resi-
dues 201–220) is responsible for promoting actin nucleation, whereas the
C-terminal 88 residues are accountable for effector translocation. Actin fila-
ment polymerization has been promoted by a second effector protein, SipA;
by sinking the monomer concentration, it is essential for filament assembly
by which improving the filament bundling activity of fimbrin, a host protein
and influencing nucleation function of SipC (Zhou et al. 1999; McGhie et al.
2001). Besides, SipA binds to assembled filaments and prevents depolymer-
ization (McGhie et al. 2004). Topological analysis indicates that SipA acts as
a “molecular staple” using two extended arm domains to truss actin mono-
mers. So, the synergistic activity of SipC and SipA supports the formation of
actin filaments in the vicinity to the attached host bacteria, and they become
stable over these filaments by host regulatory proteins (Lilic et al. 2003).

Once the bacterial invasion is accomplished, host cell cytoskeleton immedi-

ately returns to its basal state, specifically noted in polarized epithelial cells,
in which apical microvilli are totally reassembled. This process is mediated
by effector protein SptP, and it contains two distinct catalytic segments
such as, an N-terminal Rho GAP domain and a C-terminal tyrosine phos-
phatase domain (Kaniga et  al. 1996). GAP domain of SptP clearly mim-
ics eukaryotic RhoGAPs in its overall structure and catalytic mechanism.
Stebbins and Galan (2000) observed a reduced actin assembly once the
host cells are treated with purified SptP. The role of SptP tyrosine phospha-
tase is in cytoskeletal recovery after Salmonella entry. The known targets
of SptP phosphatase activity are tyrosine kinase ACK (activated Cdc42-
associated kinase), intermediate filament protein vimentin, and p130Cas
(Murli et al. 2001). ACK is a downstream effector protein of Cdc42 and has
been involved in stimulation of ERK activation during Salmonella infection
(Ly and Casanova 2007). The p130Cas becomes transitorily tyrosine phos-
phorylated in the course of Salmonella infection (Shi and Casanova 2006).

2.4.2  T3SS-independent invasion

To onset diseases ranging from typhoid fever to gastroenteritis or to asymp-

tomatic carrier state, Salmonella needs to cross several barriers. Until now,
SP-1– and SP-2–mediated invasion has been the focus because of their key
66    Pocket Guide to Bacterial Infections

role in invasion and internalization into various cell types, particularly entero-
cyte cell lines (Agbor and McCormick 2011). Conversely, the role of outer
membrane proteins (OMP) consists of adhesive molecules, and virulence
factors have been demonstrated in a number of pathogenic gram-negative
bacteria. The association of Salmonella OMP with host cells is known to
trigger a variety of biological events that include induction of innate and
adaptive immunity and stimulate cell invasion (Galdiero et al. 2003).

2.4.2.1 OMP Rck invasion – Rck, a 19-kDa outer membrane protein

belongs to Ail/Lom family, consists of five members (Rck, Ail, Lom, OmpX,
and PagC). This rck gene located on large virulence plasmid contributing the
expression of virulence genes such as spvRABCD (Salmonella plasmid viru-
lence), pef (plasmid-encoded fimbriae), srgA (SdiA-regulated gene, putative
disulphide bond oxidoreductase), or mig-5  genes (macrophage-inducible
gene coding for putative carbonic anhydrase) (Rychlik et al. 2006).

In general, rck carries virulence gene (Buisan et  al. 1994); it is highly
conserved and persist only in serovars of Enteritidis and Typhimurium
(Futagawa-Saito et al. 2010), which are frequently associated with human
and domestic animals infections causing pathogens; on the other hand, it
was not detected in the serovars of Choleraesuis, Gallinarum, and Pullorum
(Chu et al. 1999; Rychlik et al. 2006).

Rck is able to promote adhesion and internalization of coated beads (Rosselin

et al. 2010) or of noninvasive Escherichia coli strains (Heffernan et al. 1994).
It was showed that 46 amino acids of Rck were screened as being necessary
for entry process. Their binding to the cell surface is generally inhibited by
soluble Rck and induces distinct membrane rearrangements due to cell sig-
naling (Rosselin et al. 2010). These demonstrate that Rck induces a Zipper-
mode of invasion mechanism, supporting the certainty that Salmonella is
the first bacterium to be demonstrated as able to induce both zipper and
trigger mechanisms for their host cell invasion (Rosselin et al. 2010).

2.4.2.2  OMP PagN invasion – PagN, a 26 KDa outer membrane protein.

has been identified as the important protein involved in Salmonella inva-
sion mechanism (Lambert and Smith 2008). The gene, pagN, is located
on the seventh centisome genomic island and is broadly scattered among
Salmonella enterica serotypes (Folkesson et  al. 1999). The pagN open
reading frame was primarily identified during a TnphoA random-insertion
screening in S. Typhimurium performed to discover PhoP-activated genes
(Belden and Miller 1994). In general, PagN is similar to both the Tia and Hek
invasions of E. coli and represents 39% and 42% similarity in amino acids
with these two invasions, respectively. Tia and Hek are predicted to have
 Gateways of Pathogenic Bacterial Entry into Host Cells—Salmonella     67

eight transmembrane regions, four long exposed extracellular loops, and

three short periplasmic turns (Mammarappallil and Elsinghorst 2000; Fagan
et al. 2008). Thus, PagN probably adopts a similar conformation as that of
Hek and Tia.

The role of PagN in host invasion is supported by the fact that PagN over-
expressed in a noninvasive E.  coli strain-induced cell invasion (Lambert
and Smith 2008). However, they also demonstrated that pagN deletion
in S. Typhimurium leads to a three- to fivefold reduction in invasion of
enterocytes. Thus, PagN might facilitate interactions between Salmonella
and mammalian cells in specific conditions that do not allow SPI-1 expres-
sion (Lambert and Smith 2008). The PagN protein interacts with cell sur-
face heparin sulfate proteoglycans to enter into the mammalian cell line
CHO-K1 (Lambert and Smith 2009). However, because of the inability of
proteoglycans to transduce signaling cascade, they might act as core-
ceptors for invasion and not as the receptor. At the cellular and molec-
ular level, the PagN-mediated invasion mechanism is poorly described;
hence, detailed studies are needed to categorize the PagN receptors at
the molecular level.

2.4.2.3  Role of the hemolysin HlyE – HlyE, a small 2.3-kb pore-forming

hemolysin encoded by SPI-18 island is missing in S. Typhimurium but was
found in S. Typhi and S. Paratyphi A. It shares more than 90% similarity with
E. coli HlyE (ClyA) hemolysin. S. Typhi hlyE mutants are weak in their ability
to invade HEp-2 cells, compared to that of wild-type strain (Fuentes et al.
2008). However, the cellular mechanism of enhancing their invasion is not
yet understood.

2.4.3  Caveolae-mediated invasion

Caveolae, a 21–24 kDa integral membrane flask-shaped projection in the

plasma membrane of endothelial cells, consist of three caveolin proteins
named as caveolin 1, 2, and 3. Caveolin 1 and 2 are expressed together as
a hetero oligomer in the plasma membrane, and caveolin 3 was expressed
only in muscle tissue (Tang et  al. 1996; Smart et  al. 1999). Caveolin-1,
a scaffolding protein expressed within the caveolar membrane, inter-
acts with signaling proteins, such as epidermal growth factor receptor,
G-proteins, Src-like kinases, Ha-Ras, insulin receptors, and integrins for
regulating their activities (Smart et al. 1999). Caveolar endocytosis is an
endocytosis mechanism including nutrients and pathogens in most of
the prokaryotes and parasites via endoplasmic reticulum (ER) → golgi →
­c ytoplasm not fuse with lysosome, letting helps the pathogens to escape
from lysosomal degradation (Schnitzer et al. 1994).
68    Pocket Guide to Bacterial Infections

Salmonella can invade nonphagocytic cells by the effector proteins T3SS,

and all are encoded within SPI-1. These proteins activate signaling cas-
cades inside the host cell, leading to a variety of responses, including the
formation of actin-rich membrane ruffles, which are ultimately responsible
for bacterial internalization. Salmonella could be easily invaded by non-
phagocytic senescent host cells in which caveolin-1  was also increased.
At the time of destruction of caveolae structures by methyl-b-cyclodextrin
or siRNA of caveolin-1  in the senescent cells, Salmonella invasion was
reduced noticeably compared to that in nonsenescent cells (Lim et  al.
2010). Besides, caveolin-1 is highly expressed in Peyer’s patch and spleen
of aged mice that are target sites for Salmonella invasion. This is the evi-
dence supporting that increased level of caveolae and caveolin-1 in aged
cells might be responsible for their increased susceptibility to microbial
infections (Lim et al. 2010).

Lim et al. (2014) described a new cellular and molecular machinery for the
caveolin-1–dependent entry of Salmonella into host cells via the direct regu-
lation of actin reorganization. The caveolae are not able to form Salmonella-
containing vacuoles or endosomes within the host cells. Instead, they
quickly moved to the apical plasma membrane upon actin condensation
during early invasion. The injected bacterial protein SopE counteracted with
Rac1 to regulate actin reorganization, and both proteins are directly inter-
acted with caveolin-1 in caveolae during the early stage. After the complete
internalization of Salmonella, SopE levels decreased both in the caveolae
and in the host cytoplasm; Rac1 activity was also reduced. Downregulation
of caveolin-1 leads to decreased invasion of Salmonella. These results sug-
gested that caveolin-1 might be involved in Salmonella entry through their
interaction with SopE and Rac1 of T3SS-1, leading to better membrane ruf-
fling for phagocytosis into host cells (Lim et al. 2014).

2.4.4  Unknown factors influencing invasion

Besides all these well-documented entry mechanisms, some other unidenti-

fied and unfamiliar factors seems to be involved during cell invasion. Rosselin
et al. (2011) demonstrated that Salmonella serovars that were not expressing
the effectors like Rck, PagN, and T3SS-1 have the ability to invade different
host cells by unknown mechanism. This unknown entry mechanism depends
on the cell type and cell line. For example, 3T3 fibroblasts and MA104 kidney
epithelial cells are most accommodating to these mechanisms to enter inde-
pendently of T3SS-1, PagN, and Rck. But nonpolarized HT29 enterocytes are
not prone to these unknown entry mechanisms. They stated that cytoskel-
etal and membrane rearrangements similar to zipper or trigger machinery
system have been observed during microscopic examination of infected
 Gateways of Pathogenic Bacterial Entry into Host Cells—Salmonella     69

cell types. These factors are found to be involved in inducing the signaling
cascade that mediate the Salmonella entry into human foreskin fibroblasts
(Aiastui et al. 2010). Steffen et al. (2004) and Hanisch et al. (2012) described
a new invasion pathway that could be independent of Arp2/3 complex, and
it depends on the formation of myosin II-rich stress fiber-like structures at
entry sites via the activation of RhoA/Rho kinase signaling pathway.

In fact, a Salmonella mutant does not express any of the known invasion
factors displayed both local and massive actin accumulations and also in
intense membrane rearrangements. These findings showed that invasion
factors other than PagN, Rck, and T3SS-1 apparatus were able to induce
either a zipper or trigger mechanism (Rosselin et al. 2011). Overall and con-
trary to the existing theory, Salmonella can enter cells through a zipper-
like mechanism mediated by Rck and other unknown invasions, in addition
to the trigger mechanism mediated by its T3SS-1  apparatus and other
unknown determinants. In consequence of these findings, avenues are
open for the identification of new and unknown invasion factors.

2.5  Intracellular survival

After host cell invasion, Salmonella are internalized within a membrane-

bound compartment known as small colony variants (SCVs). (Many bacte-
rial pathogens have developed different strategies to neutralize the host
immune response, either by preventing vacuole–lysosome fusion or by
escaping into cytosol.) Salmonella are classified as a vacuolar pathogen;
hence upon invasion, this bacterium resides and multiplies within SCV,
a specialized vacuole, as the only intracellular niche of Salmonella (Bakowski
et  al. 2008), and it undergoes different stages of maturation like macro-
phages. These SCVs consist with markers such as Rab5, EEA1 (early endo-
some antigen 1), and TfR (transferrin receptor) (Steele-Mortimer et  al.
1999; Bakowski et al. 2008). Once the SCV matures and is surrounded by
actin, it migrates toward a perinuclear position and initiates formation of
Salmonella-induced filaments (SIF), which helps transportation of nutrients
to the SCV, thereby facilitating bacterial replication (Knodler and Steele-
Mortimer 2003; Salcedo and Holden 2003).

Due to the defect in SCV maturation, Salmonella are released in nutrient-

rich cytosol and replicated in higher rate termed hyper-replication (Knodler
et al. 2014). These cytosolic Salmonella is sensed by the host epithelial cells,
leading to an inflammatory response characterized by the activation of cas-
pase 1 and caspases 3/7 and the apical release of IL-18, an important cyto-
kine regulator of intestinal inflammation (Monack et al. 2001).
70    Pocket Guide to Bacterial Infections

In the SCV, when Salmonella adapts to the intravascular environment,

the two component regulatory system PhoP/PhoQ suppresses the expres-
sion of SPI-1  genes, which are no longer needed, but through reduction
in hilA transcription another component increases SPI-2 T3SS expression
(Bijlsma and Groisman 2005; Golubeva et al. 2012). Death of internalized
Salmonella could be associated either with the complete SCV–lysosome
fusion (Viboud and Bliska 2001) or autophagy, a capture mechanism of
either cytosol-adapted or vacuolar bacteria that allow them to lysosomal
compartment for killing. So, it is concluded that the net intracellular growth
of Salmonella is the replication in both vacuolar and cytosolic region and of
intracellular bacteria destruction.

Salmonella, in their latent period, is fused with SCV-lysosome to proliferate

with in the host cell without being destroyed. Increase in the SCV number
imbalances in the ratio of number of vacuoles to the number of acidic lyso-
somes. So, cells deficient in lysosomes are helpful for Salmonella by increasing
their survival and proliferation with in the host cell (Eswarappa et al. 2010).

2.6 Conclusion

The detailed traits associated with epidemics of Salmonella serovars are

still not understood. This is specifically evidenced with the recent screening
of SPI-1–deficient Salmonella associated with human enteric disease and
indicating that SPI-1 T3SS is not only the sole effector to cause pathogen-
esis (Boumart et al. 2014). Some new paradigms discussed in this chapter
describe that Salmonella serovars could be entered in nonphagocytic cells
by various invasion strategies that should adapt our vision and helps to
revisit the host-specificity bases. Therefore, further studies are needed to
focus on T3SS-independent mechanisms to identify the signaling pathways
induced and host receptors involved in it.

Acknowledgments

KA thanks to DBT (Government of India) for financial assistance (BT/

PR20721/BBE/117/241/2016) and DS thanks to UGC for Women postdoc-
toral fellowship program.

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3
Prevalence of Bacterial
Infections in Respiratory Tract
Boopathi Balasubramaniam, U. Prithika,
and K. Balamurugan

Contents

3.1 Introduction 79
3.2 An overview of Respiratory Tract Bacterial Infections 80
3.3 Upper Respiratory Tract Bacterial Infections (URTBIs) and
Lower Respiratory Tract Bacterial Infections (LRTBIs) 82
3.3.1 Staphylococcus aureus 84
3.3.1.1 Pathogenesis 84
3.3.1.2 Prophylaxis 86
3.3.2 Streptococcus pneumoniae 86
3.3.2.1 Pathogenesis 87
3.3.2.2 Prophylaxis 88
3.3.3 Haemophilus influenzae 88
3.3.3.1 Pathogenesis 89
3.3.3.2 Prophylaxis 89
3.3.4 Mycobacterium tuberculosis 91
3.3.4.1 Pathogenesis 91
3.3.4.2 Prophylaxis 93
3.3.5 Klebsiella pneumoniae 93
3.3.5.1 Pathogenesis 94
3.3.5.2 Prophylaxis 94
3.4 Conclusion 95
References 96

3.1 Introduction

The respiratory tract is mainly responsible for breathing. It is distributed into

two major portions, the upper respiratory tract (URT) and the lower respi-
ratory tract (LRT). URT comprises of various organs such as sinuses, nasal
cavity, pharynx, and larynx; LRT comprises of trachea, bronchi, lungs, and
diaphragm (Figure 3.1). The foremost function of the human respiratory

79
80    Pocket Guide to Bacterial Infections

Figure 3.1  The representative figure illustrates the internal organs exist in upper
respiratory and lower respiratory tracts of a human being. (Extracted and Modified
from Wikiversity contributors, WikiJournal of Medicine/Medical gallery of Blausen
Medical 2014, Wikiversity, https://en.wikiversity.org/w/index.php?title=WikiJournal_
of_Medicine/Medical_gallery_of_Blausen_Medical_2014&oldid=1753045, 2017.)

system is to passage the inhaled air into lungs and to aid the diffusion of
oxygen molecules into the bloodstream. Respiratory tract infections (RTIs)
represent a common major health problem and also the most frequently
reported infections throughout the world because of their ease of trans-
mission, occurrence, and considerable mortality and morbidity by affecting
people of all ages. Traditionally, they are divided into two classes: lower
respiratory tract infections (LRTIs) and upper respiratory tract infections
(URTIs). Generally, bacterial infections may affect the lower or upper respi-
ratory tract and the pathogens causing these symptoms have a tendency
to localize to one region. Some of these RTIs are mild (acute), some are
chronic (long-lasting), and sometimes some are self-limiting (Richter et al.
2016). The respiratory tract can be infected by various pathogenic bacteria,
both gram-positive and -negative (Felmingham and Gruneberg 2000). But
by chance, most of these infections can be treated by well-known antibi-
otic therapies.

3.2  An overview of Respiratory Tract Bacterial Infections

According to World Lung Foundation’s Acute Respiratory Infections Atlas,

acute respiratory infections are a major cause for 4.25 million deaths every
year and also the third-leading cause of deaths (after heart disease and
stroke) in the world. Pneumonia, a form of acute RTI frequently caused by
both bacteria and viruses, is the sole leading cause of infantile death around
 Prevalence of Bacterial Infections in Respiratory Tract     81

the world. According to the World Health Organization (WHO), pneumonia

accounts for 16% of all deaths in children younger than the age of 5, killing
920,136  children in 2015 (WHO 2016). Tuberculosis, a chronic RTI caused
due to Mycobacterium tuberculosis, had at least 6.3 million new cases (up
from 6.1 million in 2015), which is equivalent to 61% of the estimated inci-
dence of 10.4 million (WHO 2018), leading to 1.5 million deaths (Table 3.1).

Table 3.1  The Prevalence of Respiratory Tract Infections Caused by the

Pathogens
Frequency/
Disease Prevalence Bacteria Reference(s)
Pharyngitis/ 7.5% of people Haemophilus influenzae Jones et al. (2005)
tonsillitis (in any given Corynebacterium
3 months) diptheriae
Streptococcus
pneumoniae
Bordetella pertussis
Mycoplasma
pneumoniae
Laryngitis Common Haemophilus influenzae Wood et al. (2014)
Streptococcus
pneumoniae
Acute otitis 471 million Streptococcus Wood et al. (2014)
media cases (2015) pneumoniae
Staphylococcus aureus
Haemophilus influenzae
β-Haemolytic
streptococci
Acute 10%–30% each Streptococcus Rosenfeld et al.
rhinosinusitis year pneumoniae (2015)
(developed Haemophilus influenzae
world) Staphylococcus aureus
Bronchiolitis ~20% (children Haemophilus influenzae Schroeder and
younger than Corynebacterium Mansbach (2014);
age group 2) diptheriae Friedman et al.
Streptococcus (2014)
pneumoniae
Bordetella pertussis
Mycoplasma
pneumoniae
(Continued)
82    Pocket Guide to Bacterial Infections

Table 3.1 (Continued)  The Prevalence of Respiratory Tract Infections

Caused by the Pathogens
Frequency/
Disease Prevalence Bacteria Reference(s)
Acute ~5% of people/ Haemophilus influenzae Wenzel and Fowler
bronchitis year Corynebacterium (2006)
diptheriae
Streptococcus
pneumoniae
Bordetella pertussis
Mycoplasma
pneumoniae
Pneumonia 450 million Staphylococcus aureus Ruuskanen et al.
cases (7%) Streptococcus (2011); Lodha et al.
per year pneumoniae (2013)
Haemophilus influenzae
Klebsiella pneumoniae
Mycobacterium
tuberculosis
Mycoplasma
pneumoniae
Legionella spp.
Tuberculosis 33% of people Mycobacterium WHO (2017)
tuberculosis

3.3 Upper Respiratory Tract Bacterial Infections

(URTBIs) and Lower Respiratory Tract
Bacterial Infections (LRTBIs)

Upper RTIs are defined as the infections transmitted and localized in the
nasal cavity including, sinuses, pharynx, and larynx. The foremost roles of
the URT are to humidify the exposed heat and filter the respired air through
several compartments (i.e., nasal cavity/meatus, oropharynx, nasopharynx,
and pharynx) before the respired air reaches the lungs (Sahin-Yilmaz and
Naclerio 2011). These compartments usually allow for the passage and colo-
nization of bacterial pathogens (Siegel and Weiser 2015). Differences in
mucus secretion, temperature, and relative oxygen concentration all over
the URT regulate the bacterial colonization (Rigottier-Gois 2003). The lead-
ing causes of URTBIs are Staphylococcus aureus, Streptococcus pneumoniae,
Haemophilus influenzae, β-hemolytic streptococci, Corynebacterium dip-
theriae, Neisseria gonorrhoeae, Mycoplasma hominis, and Mycoplasma
pneumoniae (Figure 3.2). The LRT is called the tracheobronchial tree or
respiratory tree, which starts with the larynx and includes trachea, two
Figure 3.2  The representative figure demonstrates the bacterial infections caused in both the lower respiratory tract and upper respiratory tract.
(Extracted and Modified from Dasaraju, P.V. and Liu, C., Med. Microbial., 4, 1996.)
Prevalence of Bacterial Infections in Respiratory Tract     83
84    Pocket Guide to Bacterial Infections

bronchi that divides from the trachea, and the lungs. The lung is the organ
where gas exchange generally takes place (Wikibooks contributors 2018).
The LTBIs are caused by the colonization of several pathogens such as
H. influenzae, C. diptheriae, S. pneumoniae, Bordetella pertussis, M. pneu-
moniae, S. aureus, Klebsiella pneumoniae, M. tuberculosis, and Legionella
species (Figure 3.2).

3.3.1  Staphylococcus aureus

S. aureus is one of the major commensal bacteria present in human beings

and preferably colonizes the epithelium of the anterior nares (Foster 2004).
S. aureus is a gram-positive bacterium that is capable of causing a multitude
of diseases ranging from mild to chronic severity. Approximately 20% of the
total population are colonized with S. aureus; 60% are periodic, and 20%
population never carry the bacteria (Von Eiff et al. 2001a). The emergence
of methicillin-resistant S. aureus (MRSA) is considered to be a major threat
in the current scenario. The rates of MRSA carriages differ extensively from
3% to 30% (Kitti et al. 2011; Shaw et al. 2013; Jimenez-Truque et al. 2017).
A report by Davis et al. (2004) revealed that 21% of admitted patients were
methicillin-susceptible S. aureus (MSSA) carriers and 3.4% of patients were
MRSA carriers. However, a higher ratio of the carriers of MRSA (19%) devel-
oped invasive staphylococcal disease than MSSA carriers (1.5%).

3.3.1.1  Pathogenesis – The pathogenesis of S. aureus was referred to classi-

cal mode and resembles the pathology of soft-tissue infection and skin (Foster
2005; Tong et  al. 2015). Reports revealed that first-line of defense against
S. aureus infection was the neutrophil response (Figure 3.3). Generally,
S. aureus invades the cells by means of different methods (e.g., chemotaxis of
leukocytes, blocking sequestering host antibodies, resisting destruction after
ingestion by phagocytes, and hiding from detection via capsule or biofilm
formation). The bacterial cell surface receptors present in the cell walls cor-
respond to microbial surface components recognizing adhesive matrix mol-
ecules (MSCRAMMs) or adhesins (Dunyach-Remy et al. 2016). MSCRAMMs
assist the bacterial adhesion to epithelial cells. In the intracellular module,
S. aureus forms small-colony variants (SCVs) (Proctor et al. 2006). So, they
would be able to persist in a metabolically inactive state while conserving the
integrity of the epithelial cells. SCVs retain several phenotypic and metabolic
differences from the usual S. aureus clinical isolates (Von Eiff et al. 2001b;
Tuchscherr et al. 2010). Certainly, they are comparatively resistant to the usual
antibiotics (Baumert et al. 2002; Garcia et al. 2013). Therefore, it is challeng-
ing to eradicate S. aureus with well-known antibiotic therapies (Proctor and
Peters 1998). Furthermore, the synthesis and secretion of glycocalyx matrix
Figure 3.3  Schematic diagram of Staphylococcus aureus pathogenesis and methicillin-resistant S. aureus (MRSA) emergence. S. aureus secretes
several virulence factors that evade host immune defenses. On the other hand, the bacterium expresses adhesins and also secretes many toxins (i.e.,
α-toxins, etc.) and enzymes by the activation of needed chromosomal genes. Methicillin-resistance is attained by the insertion of a DNA element
called SCCmec through horizontal gene transfer mechanism. And also, the mecA gene encodes a novel β-lactam–resistant penicillin binding pro-
tein, PBP2a, which endures to synthesize a new cell wall peptidoglycan moiety even when the penicillin-binding proteins are repressed. (Extracted
and Modified from Parker, D. and Prince, A., Semin. Immunopathol., 34, 281–297, 2012; Foster, T. J., J. Clin. Invest., 114, 1693–1696, 2004.)
Prevalence of Bacterial Infections in Respiratory Tract     85
86    Pocket Guide to Bacterial Infections

with the combination of specific surface adherence mechanisms also plays a

crucial role in the S. aureus virulence. It was reported that S. aureus was able
to secrete hazardous toxins, which can lead to tissue necrosis in the host.
S. aureus toxins also have vital role in the excavating and spreading of the
infection in the patients (Dunyach-Remy et al. 2016).

3.3.1.2  Prophylaxis – Bacterial strains lacking cytotoxins are considered

to be avirulent in a mice model of S. aureus pneumonia; similarly, vaccine-
based therapies that provoke the toxins come up with the protection
against lethal disease. Studies suggest that disrupting the function of the
cytotoxin affords a potent preventive mechanism to treat S. aureus pneu-
monia. Derivatives of β-cyclodextrin are sevenfold symmetrical small mole-
cules that block the assembled α-hemolysin pore, negotiating the functions
of toxin (Ragle et al. 2010). In the current research field, a significant prob-
lem common with S. aureus infections is the rapid progression of antibiotic
resistance. In S. aureus biofilm-associated infections, this may be provoked
by the proliferation of antibiotic minimum inhibitory concentrations (MICs)
compared with planktonic bacteria (Howlin et al. 2015). Vancomycin is the
most commonly prescribed antibiotic for the S. aureus biofilm-associated
infections (Liu et  al. 2011). Conversely, physicians are extremely cautious
about the direction of this antibiotic owing to the tendency of S. aureus to
develop resistance. Evidence for this tendency is the recent development
of vancomycin intermediate S. aureus and vancomycin-resistant S. aureus
(VRSA) strains (Howden et al. 2010). Still, the combined therapy of vanco-
mycin with rifampicin has shown conflicting activities. Also multiple studies
show that this combination might be effective against MSSA rather than
MRSA biofilm infections (Olson et  al. 2010; Salem et  al. 2011; Zimmerli
2014). Daptomycin, a cyclic lipopeptide molecule, is a novel drug that has
been administrated for vancomycin-unresponsive S. aureus conditions. The
mode of actions of daptomycin relies with the disruption of cytoplasmic
membrane resulting in cessation of DNA, RNA, protein synthesis, and rapid
depolarization. Daptomycin was found to be the most effective among the
drugs tested (i.e., clindamycin, linezolid, vancomycin, and tigecycline) in
clearing-off the prevailing biofilm of S. aureus (Bhattacharya et al. 2015).

3.3.2  Streptococcus pneumoniae

S. pneumoniae (Pneumococcus) is a gram-positive, extracellular bacterial

pathogen, and a classic example of highly invasive bacteria. It is reported
to be the leading cause of mortality and morbidity globally, causing more
deaths than any other communicable diseases. At the highest risk, 1 ­million
children (younger than 5  years of age) are dying annually (Centers for
Disease, Control, and Prevention 2008). Pneumococcal diseases vary from
 Prevalence of Bacterial Infections in Respiratory Tract     87

mild RTIs such as sinusitis and otitis media to more chronic diseases such as
septicemia, pneumonia, and meningitis. Though, Pneumococcus can cause
mild to lethal diseases, it is more commonly a dormant colonizer of the URT
where up to 60% of children could carry Pneumococci in the nasal routes
asymptomatically (Henriqus Normark et al. 2003; Nunes et al. 2005).

3.3.2.1  Pathogenesis – Be it in the mucosal surface or blood, the bacte-

rial pathogenesis involves the bacterial adherence to cell surfaces of host
system followed by cellular invasion (Figure 3.4). The successful invasion of
the bacterial cells was due to adherence to the two receptors present on
the surface of various cell types (e.g., the platelet activating factor receptor
(PAFr) and 37/67 kDa laminin receptor [LR]). Orihuela et al. (2009) reported

Figure 3.4  Schematic illustration of Staphylococcus pneumoniae pathogenesis.

S. pneumoniae are generally transmitted through air and the attachment followed
by colonization is mediated through surface proteins, containing unknown methods
of immune evasion. Bacteria dissociate from the matrix formed because of biofilms
and rise into the middle ear and to the lung through the pharynx. The interaction
most commonly leads to serotype-specific immunity, and the disease progression
of otitis media is most common in children. The most severe development in the
S. pneumoniae pathogenesis is meningitis. This series of aggressive steps is shared
by the three major bacterial pathogens (Pneumonococcus, Haemophilus, and
Meningococcus) and is driven by interplay between innate immunity and innate inva-
sion. (Extracted and Modified from Henriques-Normark, B. and Tuomanen, E.I., Cold
Spring Harb. Perspect. Med., 3, a010215, 2013.)
88    Pocket Guide to Bacterial Infections

that the bacterial adhesin protein CbpA primarily bound to endothelial LR.
CbpA surface-exposed loop mediated binding to the polymeric immuno-
globulin receptor was accountable for the Pneumococcus translocation
across the nasopharyngeal epithelium (Zhang et al. 2000). It was also nota-
ble that CbpA binding mediated adherence of the bacterial cells to LR but
not invasion. Significantly, LR also acted as a target for Neisseria menin-
gitidis and H. influenzae pathogenesis. The antibody of CbpA cross-reacted
and blocked the adherence of these meningeal pathogens, representing a
shared binding mechanism. These results suggest that a series of pathogens
target LR as a major step in the host-pathogen interactions.

3.3.2.2  Prophylaxis – Even though successful conjugate polysaccharide

vaccines exist, serotype-independent protein-based pneumococcal vaccines
deal a foremost progression for preventing life-threatening pneumococcal
infections in terms of cost, particularly in developing countries. IL-17A-
secreting CD4+ T cells (TH17) facilitates resistance to the mucosal coloniza-
tion of multiple pathogens including S. pneumoniae. A study by Moffitt
et  al. revealed that screening of expression library containing >96% of
predicted pneumococcal virulence proteins. They have identified antigens
recognized by TH17 cells from mice immune system due to pneumococcal
colonization. The identified antigens also provoked IL-17A secretion from
colonized mice splenocytes and human peripheral blood mononuclear cells
(PBMCs) proposed that comparable responses were clued-up during natu-
ral contact. The report demonstrated the potential of screening through
proteomic approaches to identify the specific antigens for designing the
subunit vaccines against mucosal pathogens by means of harnessing TH17-
mediated immunity (Moffitt et al. 2011). Extensive use of the 7-valent pneu-
mococcal conjugate vaccine (PCV7) in US infants (23 months of age) was
also endorsed for certain children (59 months of age) to prevent the pneu-
mococcal disease distribution. After the PCV7 introduction, vivid decreases
in invasive pneumococcal disease (IPD) were observed in children. A sig-
nificant reduction in adult pneumococcal diseases was also seen. However,
other IPD serotypes have not been comprised in the PCV7, which provoked
the necessity for the development of a pneumococcal conjugate vaccine
with extended coverage (Committee on Infectious, Diseases 2010).

3.3.3  Haemophilus influenzae

H. influenzae is one of the significant gram-negative bacteria associated

with respiratory infections, ranging from acute otitis media to chronic
obstructive pulmonary disease (COPD) and also includes invasive diseases
such as s­ epsis and meningitis (Murphy et al. 2009). The serotype variation
among the H.  influenzae can be differentiated by the encapsulated and
 Prevalence of Bacterial Infections in Respiratory Tract     89

nonencapsulated forms. Encapsulated H. influenzae is categorized into

six diverse serotypes such as a to f, whereas the nonencapsulated H. influen-
zae is labeled as no-typeable H. influenzae (NTHI) (Van Wessel et al. 2011).
Generally, H. influenzae exist in the mucosa, and NTHI is predominantly
associated with RTIs, while encapsulated H. influenzae serotypes including
H. influenzae type b (Hib) cause invasive diseases such as meningitis and
septicemia. Until the 1990s, Hib was considered to be the most common
serotype, but a dramatic decrease was observed in Hib infections was per-
ceived after the development of a conjugate vaccine. However, an accumula-
tive occurrence of invasive diseases caused by nontype b H. influenzae has
been recently reported throughout the world (Resman et al. 2011; Rubach
et al. 2011).

3.3.3.1  Pathogenesis – Once the human airway epithelium is immuno-

compromised, H. influenzae can create a respiratory infection. The first step
of pathogenesis is nasopharyngeal colonization (Figure 3.5). The pathogen
starts adhering to the respiratory tract epithelial cells and escapes from a
number of host defenses, including complement fixation, secretory IgA,
mucociliary clearance, lactoferrin, lysozyme, and a­ntimicrobial peptides.
Surface adherence of the H. influenzae to mucin can assist clearance, and
impairment of mucociliary clearance is a crucial stage in the virulence. When
mucociliary is damaged, the pathogen is able to interact with nonciliated
cells through one or few of its several bacterial adhesins. The p ­ athogen
either invades the epithelial cell or enters to the subepithelial layer by means
of transcytosis or aggregates to form biofilm. Still, it remains unclear how
H. influenzae forms carriage to form biofilm. Whereas, H. influenzae is not
habitually known as an intracellular pathogen, feasible bacteria have been
frequently found within host cells (Schaechter 2009). A report by Singh
et  al. ­suggest that H. influenzae interacts with the host epithelium and
H.  influenzae protein E  (PE) intermediates the binding to the epithelial
­surface, using an unknown receptor (Singh et al. 2013). The bacterial adhe-
sion leads to the induction of a pro-inflammatory response by the epithelial
cells. During the p­ atho­genesis, PE binds to laminin (Ln), which contributes
H. influenzae adhesion to the basement membrane and the host extracellu-
lar matrix (ECM). Moreover, H. influenzae binds with plasminogen by using
PE. Once, the plasminogen is bound to PE of H. influenzae, it is transformed
into active from called plasmin by host urokinase plasminogen activator
(uPA) or tissue plasminogen activator (tPA). Active plasmin may possibly
help in thebacterial invasion and degradation of the ECM (Singh et al. 2013).

3.3.3.2  Prophylaxis – Vaccines are available that can prevent H. ­influenzae

type b (Hib) disease, which is the most common serotype (“strain”) of
H. influenzae bacteria. But Hib vaccine does not prevent the diseases caused
90    Pocket Guide to Bacterial Infections

Figure 3.5  Microbial pathogenesis of Haemophilus influenzae in the lung epithelial

cells. The H. influenzae protein E (PE) molecule facilitates binding to the surface of
epithelial cell by using an unknown receptor. This interaction induces the bacterial
adhesion and elevates a proinflammatory response in the epithelial cells. (Extracted
and Modified from Singh, B. et al., Infect. Immun., 81, 801–814, 2013.)

by any other types of H. influenzae. Generally, Hib vaccine is endorsed for

children younger than 5  years of age in the United States and was also
prescribed to 2-month-old infants. In certain circumstances, individuals
at increased threat for getting invasive Hib diseases (when H. influenzae
invades other internal parts of the body and produces septicemia) and who
are completely vaccinated may possibly need additional doses of the Hib
 Prevalence of Bacterial Infections in Respiratory Tract     91

vaccine. It is also recommended that unimmunized children and adults with

certain medical circumstances should also be given the Hib vaccine. A child
with H. influenzae (including Hib) infection may not develop defending lev-
els of antibodies, providing chances for the individual to be affected with
H.  influenzae disease again. Children whose age is younger than 2  years
who have recovered from invasive Hib disease are not be considered safe
and must be given Hib vaccine immediately. In certain circumstances, a per-
son in close contact with someone who is infected with Hib should also
receive antibiotics to prevent them from receiving the disease (CDC 2016).

3.3.4  Mycobacterium tuberculosis

M. tuberculosis  arises globally and is reported to kill 2–3  million people

per year (Gagneux et al. 2006). Tuberculosis (TB) is considered one of the
ancient documented human diseases and still stands as one of the preva-
lent killers among the other well-known infectious diseases, even with the
worldwide usage of a live attenuated vaccine and a number of antibiotics.
The discovery of mutilated bones in various neolithic locations in Denmark,
Italy, and other countries in the Middle East Europe suggests that TB was
found all over the world 4,000 years ago. The origin of M. tuberculosis has
been the subject of recent investigations, and it is now thought that the
bacteria in the genus Mycobacterium, like Actinomycetes, were primarily
found in the soil and that some species of Mycobacterium evolved to live in
mammals (Smith 2003).

3.3.4.1  Pathogenesis – Generally, M. tuberculosis infection was initiated

by the nasopharyngeal inhalation of aerosol droplets that comprises the
infectious bacteria. The preliminary stages of TB infection are usually char-
acterized by the innate immune responses that involve in the r­ecruitment
of inflammatory immune cells to the alveoli and bronchioles of lung
(Figure  3.6). Following bacterial diffusion to the exhausting lymph node,
dendritic cell presentation of bacterial cells (including virulence factors) leads
to T cell priming and activates the development of antigen-specific T cells,
which are gathered to the lung. At this time, the recruitment of B  cells,
T cells, ­activated macrophages, and other leukocytes leads to the f­ ormation
of granulomas which can comprise the M. tuberculosis cells (Nunes-Alves
et al. 2014). Most of the time, the infected person will persist in a “latent”
state, which has no clinical symptoms. A small number of ­ colonies of
M. ­tuberculosis is enough to develop an active disease c­ ondition, which can
lead to the release of the bacteria into the airways. When individuals with
active TB cough, they can produce potentially infectious droplets that can
transmit the bacterial infection. The “central dogma” of defensive immunity
92    Pocket Guide to Bacterial Infections

Figure 3.6  Pathogenesis of Mycobacterium tuberculosis. Primary infection is com-

menced by the inhaled aerosol droplets that contain pathogenic bacteria. At the ini-
tial stages, infection is characterized by innate immune responses that comprise the
recruitment of inflammatory cells such as macrophages, antigen-specific T cells to the
lung. The recruitment of B cells, T cells, other leukocytes, and activated macrophages
leads to the formation of granulomas, which can have the Mycobacterium tuberculosis
bacteria. Once individuals acquire an active tuberculosis (TB) cough, they can produce
infectious aerosol droplets that transmit the TB infection. (Extracted and Modified
from Nunes-Alves, C. et al., Nat. Rev. Microbiol., 12, 289–299, 2014.)

to TB is that CD4+ T cells that produce interferon-γ (IFN-γ), T helper 1 (TH1)

cells, which associates with tumor necrosis factor (TNF; produced by the
macrophage of T cell), and also these activate antimicrobial activity that is
capable of controlling the development of M. tuberculosis further. Finally,
a number of components of the innate immunity, including vitamins and
interleukin-1 (IL-1) can collaborate with cytokines that are produced by
T cells to fight against the TB (Nunes-Alves et al. 2014).
 Prevalence of Bacterial Infections in Respiratory Tract     93

3.3.4.2 Prophylaxis – TB is a curable disease and general drug-­

susceptible TB disease can be treated with a standard 6-month passage
of antimicrobial drugs that are prescribed under the supervision of a phy-
sician and support to the patient by a trained volunteer. It is estimated
that 53  million people were saved through TB treatment and diagnosis
between 2000 and 2016. It was found that drug-resistant strains emerge
when anti-TB medicines are used improperly, through inappropriate pre-
scriptions of poor-quality medications by physicians, and patients stop-
ping antibiotics prematurely. Multidrug-resistant tuberculosis (MDR-TB) is
caused by the M. tuberculosis bacteria that do not respond to rifampicin
and isoniazid, the two most powerful first-line anti-TB drugs. Second-line
drugs can cure the MDR-TB, but second-line treatments are restricted and
require an extensive chemotherapy (up to 2  years of treatment). In sev-
eral cases, extreme drug resistance can develop and is called extensively
drug-resistant TB (XDR-TB); it is a more serious form of MDR-TB caused
by the M. tuberculosis bacteria that do not counter to the second-line
anti-TB drugs. WHO estimated that there were 600,000 new cases during
2016 with drug resistance to rifampicin (the most effective first-line drug)
of which 490,000  people had MDR-TB. The MDR-TB problem is mainly
estimated in three countries (India, China, and the Russian Federation),
which collectively account for close to half of the total cases throughout
the world. Further, it is estimated that around 6.2% of MDR-TB cases had
XDR-TB in 2016. However, WHO approved the use of a standardized short
treatment for MDR-TB in patients who were resistant to second-line TB
medicines. Individuals with XDR-TB cannot use this regimen; however,
one  of the new drugs (delamanid and bedquiline) may be used for the
regimen to control the prevalence for long time. Reports by WHO suggest
that as of June 2017, 54 countries had hosted delamanid and 89 nations
had hosted bedaquiline to develop the efficiency of MDR-TB treatments
(WHO 2017).

3.3.5  Klebsiella pneumoniae

K. pneumoniae was first isolated during the late nineteenth century and
was initially recognized as Friedlander’s bacterium owing the discoverer’s
name (Merino et al. 1992). It is a gram-negative, nonmotile, encapsulated
bacterium that is found in the environments including water and soil and
on medical devices such as catheters (Bagley 1985; Rock et  al. 2014).
Prominently, reports suggest that K. pneumoniae freely colonizes surfaces
of human mucosa, including the important organs such as oropharynx
and gastrointestinal (GI) tract and where the properties of its establish-
ment seems to be benign (Bagley 1985; Rock et al. 2014; Dao et al. 2014).
94    Pocket Guide to Bacterial Infections

From  those colonized sites, K. pneumoniae may enter into other nearby
tissues and cause severe infections. This can be represented by the abil-
ity of these bacteria to escape from the immune response and survive at
many sites in the hosts, rather than aggressively suppress various mecha-
nisms of the immune system (Paczosa and Mecsas 2016). As a common
route of infection, K. pneumoniae are typically seen in individuals with
an immune-suppressed condition. The individuals with K. pneumonia
infection is assumed to have weakened respiratory host defenses, includ-
ing a­ lcoholism, diabetes, liver disease, malignancy, glucocorticoid therapy,
COPD, and renal failure. Most of these infections are acquired while a per-
son is in the hospital for some other reasons and is known as a nosocomial
infection.

3.3.5.1  Pathogenesis – Klebsiella infections usually spread through the

pathogenic bacteria via respiratory tract, which initially causes pneumo-
nia or infection in the bloodstream (septicemia). Klebsiella can spread
promptly and easily but not through the air. Healthcare locations are the
most vulnerable places for Klebsiella infections, owing to the nature of
­trials that allow tranquil access of the pathogen into the body. Patients
who are on ventilators or have catheters or surgery lesions are highly
disposed to spreading this deadly nosocomial infection. Infection of
K. ­pneumoniae occurs in the lungs, where they cause necrosis, inflamma-
tion, and hemorrhage within the lung tissue. This is caused by aspirating
oropharyngeal microorganisms into the LRT. About 70% of hospitalized
pneumonia cases can be detected as aspiration pneumonia (ASP) based
on the description determined by Japanese hospital acquired pneumo-
nia (HAP) and nursing and healthcare-associated pneumonia (NHCAP)
guidelines (Teramoto et al. 2008; Kohno et al. 2013). Reports suggest that
the ratio of ASP cases to the incidence of hospital-acquired pneumonia
increases with age. ASP contains two pathological conditions: dysphagia-­
associated miss-­swallowing and airspace infiltration with b­ acterial patho-
gens. Microaspiration of oropharyngeal matters is particularly communal
in the elderly, including those who are affected after a stroke and may
cause small penetrations to the lungs, which then develop into ASP
(Kikuchi 1994; Teramoto 2009; Shimada et al. 2014). Occurrence of pneu-
monia among outpatients in communication with the healthcare system
such as hospitals is characterized as healthcare-associated p ­ neumonia.
The incidence of ASP is high in very elderly patients and those with
­healthcare-associated pneumonia (Teramoto et al. 2009).

3.3.5.2 Prophylaxis – Various reports clearly reveal that K. pneu-

moniae is resistant to a number of well-known antibiotics. Based on
the area of infection that has been affected by K. pneumoniae, the
 Prevalence of Bacterial Infections in Respiratory Tract     95

antibiotic treatment varies. The choice of antibiotic treatment is spe-

cifically improved for people affected with confirmed bacteremia.
The available antibiotics with high intrinsic activities against K. pneu-
moniae consist of carbapenems, cephalosporin, quinolones, and ami-
noglycosides. These treatments against K. pneumoniae are primarily
used as mono-antibiotic therapy or a combination with one or more
antibiotic therapy. An initial course of treatment, typically between 48
and 72 hours of combination aminoglycoside therapy, is suggested for
the patients who are severely ill with pneumonia and also bacteremia.
This would then be followed by other extended-spectrum cephalospo-
rin antibiotic. Carbapenem resistance is an emerging problem and is
considered to be remarkable due to the production of K. pneumoniae
carbapenemase beta-lactamase (KPC) (Arnold et  al. 2011). Due to the
widespread of KPC-producing bacteria, clinicians rely on tigecycline
and polymyxins for the K. pneumoniae treatment. It is found that poly-
myxins have been the only agent among other antibiotics that is active
against KPC-producing K. pneumonia. However, they were occasionally
used due to their association with neurotoxicity and nephrotoxicity. Very
few data support the polymyxin as a promising drug for KPC treatment.
Swallowing function assessment is significant for the management and
diagnosis of pneumonia. When performed in the elderly who require
a high level of attention, bedside simple swallowing provocation tests
and swallowing function assessments may be desirable (Teramoto et al.
1999, 2004; Osawa et al. 2013).

3.4 Conclusion

The human respiratory system contains a series of organs accountable for

the intake of oxygen and dismissing the waste product of cellular function
(i.e., carbon dioxide). Whenever something goes wrong with any of the
parts of respiratory tract, it makes it harder to intake the oxygen required
and to get rid of the excess carbon dioxide. Common respiratory symptoms
comprise difficulty in breathing, chest pain, and cough. Numerous patho-
gens have been found to endure the disease progression from lungs to
other organs by a mutual invasion through receptors. There are various anti-
biotic therapies available to treat RTIs, but the lack of adequate therapeutic
options for RTIs caused by emerging bacterial pathogens signifies a main
hazard to human health throughout the world. There are only few reports
collectively reviewed about the RTIs and bacterial pathogens starting from
etiology, treatment, and pathogenesis. This chapter summarizes the outline
of RTIs and the pathogenesis followed by prophylaxis of potential RTI bacte-
rial pathogens.
96    Pocket Guide to Bacterial Infections

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4
Oral Health
A Delicate Balance between
Colonization and Infection
Ana Moura Teles and José Manuel Cabeda

Contents

4.1 Shaping the oral microbiome 103

4.2 Oral anatomy influences the oral microbiome 105
4.3 Composition of the healthy oral microbiome 107
4.3.1 Nonbacterial oral microbiome 107
4.3.2 Bacterial oral microbiome 108
4.4 Shaping oral immunity 109
4.4.1 Saliva flow 111
4.4.2 Crevicular flow 112
4.5 From colonization to infection and to pathology 113
4.5.1 Dysbiosis—Disease trigger 113
4.5.2 From the early colonizers to oral biofilms: A holistic
perspective of the microbiome behavior 114
4.5.3 From a healthy microbiome to a disease-associated one 115
4.5.4 Caries, periodontal disorders, and endodontic lesions:
Manifestations of microbiome imbalances 116
4.5.4.1 Caries 116
4.5.4.2 Periodontitis 118
4.5.4.3 Endodontic lesions 119
4.6 Oral microbiome as health biomarker 120
References 121

4.1  Shaping the oral microbiome

The immunological reactions, although essential for microbial control, self-

maintenance, and vigilance are a real and severe potential problem. In fact,
the destructive nature of most immunological reactions leads the immune
system to be the causative agent of disease in chronic inflammation, hyper-
sensitivity, and autoimmunity (Devendra and Eisenbarth 2003; Hennino

103
104    Pocket Guide to Bacterial Infections

et al. 2006; Balfour Sartor 2007; Harre and Schett 2017). With this in mind,
it is not strange to consider the fact that the primary goal of the immune sys-
tem is not always to eliminate pathogens, but rather to keep them in check.
In fact, in most cases, the microbe itself may be both harmful or symbiotic,
depending on the body anatomical location and even the local microbiota
(Ramsey et al. 2016). Actually, it is now clear that the human microbiota is
indeed a regular provider of genes and biochemical pathways absent from
the human genome (Dethlefsen et al. 2007; Turnbaugh et al. 2007), render-
ing human subjects with a blend of their genome plus the metagenome of
microorganisms in the body (He et al. 2015). Thus, a microbe may need to
be tolerated in a “safe” anatomical location but must be eradicated from a
dangerous one. The obvious “safe” organs seem to be the skin, gut, and
aerial and oral cavities because these are continuously subjected to environ-
mental contamination and prone to damaging chronic ineffective immune
reactions. Thus, these surfaces are likely to harbor a complex set of interact-
ing microorganisms that influence the host health state in a variable way,
depending on age, robustness, nutrition, and other factors.

During the last few years, with the advent of next-generation sequencing
(NGS) techniques (Cabeda and Moreno 2014) and the Human Microbiome
Project (Turnbaugh et  al. 2007; Huttenhower et  al. 2012), scientists have
been able to better characterize complex environments where many dif-
ferent bacterial species coexist in complex pathogen-pathogen and patho-
gen-host interactions. These include commensal, mutualistic, agonistic, and
pathogenic interactions, leading to a delicate balance of pathogens not
only relative to species diversity, but also to their relative numbers (Ebersole
et al. 2017). These studies on the human microbiome have led to interest-
ing new concepts and have shed new light on some diseases previously
not expected to be related to bacteria such as tumors (Farrell et al. 2012),
preterm birth, and low birth weight in infants (Bobetsis et al. 2006; Sacco
et al. 2008; Huck et al. 2011; Walia and Saini 2015), diabetes (Mokkala et al.
2017; Knip and Honkanen 2017), hypertension (Raizada et al. 2017), obesity
(Roland et al. 2017), cystic fibrosis (de Dios Caballero et al. 2017; Frayman
et al. 2017; Boutin et al. 2017), and Alzheimer disease (Itzhaki et al. 2016;
Pistollato et  al. 2016; Tremlett et  al. 2017). The same was true in more
expected suspects such as bacteraemia (Bahrani-Mougeot et  al. 2008),
rheumatic diseases (Zhong et al. 2018), chronic bowel inflammatory disease
(Aleksandrova et al. 2017), and asthma (van Meel et al. 2017), among oth-
ers. These studies have even lead to some strange innovative treatments
such as fecal transplant therapy (Pai and Popov 2017; Brandt 2017; Khajah
2017) as well as provided evidence supporting more conventional therapies
such as probiotics (Bagarolli et al. 2017).
 Oral Health     105

Microbiome studies have not only been showing that imbalances of the
local microbiome contribute to in situ pathology, but has also shown sys-
temic and distant organ-related effects (Looft and Allen 2012; Arimatsu
et al. 2014; Hur and Lee 2015; Isolauri 2017). These seem to arise from both
metabolic consequences of the microbiome shift and disturbances in the
immune cell populations that propagate to other body sites. Thus, health-
related consequences can result from both microorganism-derived metabo-
lites and from soluble and cell surface mediators that communicate and
shape the immunological repertoire and responses (Looft and Allen 2012;
Arimatsu et al. 2014; Hur and Lee 2015; Isolauri 2017).

The NGS studies of the oral microbiome have revealed a complex commu-
nity with some 700  different species potentially colonizing the mouth, and
at least 200 different species present at any time in each individual human
mouth (Paster et al. 2006a; Chen et al. 2010; Wade 2013). The complexity is
even greater as different regions of the mouth tend to have different bacterial,
viral, and fungal communities, in elaborate three-dimensional biofilms that are
dynamically influenced by, at least, pH, saliva, immune cell reactions, immune-
derived soluble agents, food remains, and oxygen availability. Furthermore,
person-to-person contacts contribute to shaping the microbiome with infants
showing microbiomes closely related to the ones of their respective mothers
(Takahashi et al. 2017). Even sexual behavior may contribute to this diversity
with oral sex practitioners, showing sexual transmissible pathogens such as
human papillomavirus (HPV) (Tristao et al. 2012; Owczarek et al. 2015).

4.2  Oral anatomy influences the oral microbiome

The oral cavity shows a remarkably diverse array of anatomical niches with
different characteristics and susceptibilities. From the highly lubricated and
keratin protected tongue, to the thin junctional epithelium in the tooth-
gingiva interaction, a diverse set of opportunities and challenges presents
to both microorganisms and the immune system. Additionally, availability of
food remains is also variable according to the anatomical site (there are sig-
nificant differences in the proper tooth’ anatomy, with smooth or grooved
surfaces and if there is no correct alignment of teeth), as is the likelihood
of incomplete or inefficient hygiene procedures, rendering microbe popula-
tions with different microenvironments. Finally, the likelihood of mechanical
tissue damage as a result of not only mastication but also from hygiene
procedures or from the occurrence of parafunctional habits, like bruxism,
is also singular according to the anatomical site, rendering microbes in dif-
ferent anatomical locations with differential access to inner tissues and dif-
ferentially subjected to immunological surveillance.
106    Pocket Guide to Bacterial Infections

The highly vascularized base of the tongue and mouth is generally consid-
ered an intense interaction point between oral antigens and the immune
system. Its importance may be highlighted by the presence of lymphoid
tissues directly interacting with the oral cavity (the amygdales and the ade-
noids) and its intense dynamics particularly during early childhood when
adaptive immunity is intensively developing. It is also interesting to note
that this is the exit point of some of the salivary glands, which are impor-
tant delivery systems of immunity mediators. Thus, this may be an impor-
tant anatomical place for both immunological learning and immunological
response to the oral microbiota.

Oral tissues, like the tongue mucosa and the palate, that are subjected to
frequent mechanical stress from mastication, are protected by a thin layer
of keratin (Moutsopoulos and Konkel 2017). This coating provides a higher
mechanical protection from tissue damage, thus efficiently preventing patho-
gens from entering the body. On the other hand, lower pathogen adherence
may result from the physical stress of these surfaces constantly removing bac-
teria by mechanical forces. These surfaces are, at the same time, extensively
subjected to a constant saliva flow, which prevents bacterial adhesion and
promotes its mechanical wash into the digestive tract, while also promot-
ing some chemical elimination by enzyme mediated bacterial lysis. Similar
mechanisms are in action at the surface of teeth. They are subject to extreme
mechanical stress but are protected by saliva and specially enamel, the hard-
est substance in the human body. However, the teeth area closest to the
gingiva shows a different picture. This region is generally less involved in
mastication and more prone to low efficiency of hygiene procedures, leav-
ing it relatively more likely to bacterial adhesion and biofilm formation. For
the most part, the hardness of the teeth surface leaves little room for teeth
damage from this microbiota, at least while acid metabolic secretions do not
dissolve this protective layer. However, another danger is near the epithe-
lium of the gingival crevice, which lines inside the gingiva, surrounding the
teeth. In fact, the gingival epithelium, directed toward the gingival sulcus is
a nonkeratinized epithelium that progressively thins toward the base of the
sulcus where it meets the teeth and is only three to five cell layers thick. At
this point, the epithelium (named junctional epithelium) is linked to teeth by
hemi-desmosomes and is particularly vulnerable because it is a passageway
of host factors, including cells to the gingival crevice, and thus, a potential
gateway for pathogen entrance into the body. It is, however, also a major
oral immune system checkpoint because the crevice is filled with the gin-
gival crevicular fluid, constantly renewed through the junctional epithelium
and containing a continuous flow of immune system humoral factors and
immune system cells such as neutrophils (Moutsopoulos and Konkel 2017).
 Oral Health     107

4.3  Composition of the healthy oral microbiome

Recent estimates indicate that only about 10%–50% of the human body cells
are derived from a eukaryotic host (Savage 1977; Wilson 2008; Sender et al.
2016), with the rest composed of prokaryotic unicellular microorganisms,
residing in ecological niches of our body. Most of these are gut-resident
microbes, followed by the microorganisms present in the oral cavity of the
human body (Savage 1977; Wilson 2008; Sender et al. 2016).

The oral microbiome is composed of a large array of viruses, fungi, archaea,

and bacteria. Knowledge of its composition has been greatly increased in
recent years because of the use of NGS techniques that directly sequence
nucleic acid sequences of the genomes present, thus circumventing the dif-
ficulty of being able to culture these diverse set of microorganisms (Hasan
et al. 2014).

The human microbiome can be cataloged into a “core” microbiome and

a “variable” microbiome (Turnbaugh et  al. 2007). The essential microbi-
ome comprises the predominant species existing in healthy conditions and
is shared among most individuals (Turnbaugh et al. 2007; Zaura et al. 2009;
Sonnenburg and Fischbach 2011). The variable one is the product of indi-
vidual lifestyle, phenotypic, and genotypic determinants, thereby making
it exclusive, and it can be as inimitable to the individual as his fingerprint
(Dethlefsen et  al. 2007). It must however be stressed that microbiomes
from the same location on the body are more similar among different indi-
viduals than microbiomes from different locations on the same individual
(Sonnenburg and Fischbach 2011).

4.3.1  Nonbacterial oral microbiome

Virus: Viruses present in the oral cavity are mostly related to chronic or
transient infections. Examples are HIV, HPV, mumps, rabies virus, hepati-
tis virus, rhinovirus, influenza, and herpes viruses. Additionally, a number
of different bacteriophage viruses have been consistently found (Wade
2013), which is not surprising given the large array of bacteria present,
but may indicate that these viruses also have a role in shaping the micro-
biome composition.
Protozoan: Two protozoan species (Entamoerbagingivalis and
Trichomonastenax) were also present in the normal oral microbiome that
seem to be associated with poor hygiene and have little or no associa-
tion with pathology (Wade 2013).
Fungi: As many as 85 different species of fungi have been found in the normal
oral microbiota (Ghannoum et al. 2010). The most predominant genera
108    Pocket Guide to Bacterial Infections

were Candida, Cladosporidium, Aureobasidium, Saccharomycetales,

Aspergillus, Fusarium, and Cryptococcus (Ghannoum et al. 2010). It is,
however, uncertain if all detected species represented active coloniza-
tion, or if airborne spores were also detected.
Archae: The Archaea component of the oral microbiome seems to be small
with only a few methanogens detected in healthy volunteers. However,
its presence seems to increase with periodontitis (Wade 2013).

4.3.2  Bacterial oral microbiome

The complexity of the bacterial composition of the oral microbiome has

only recently started to be fully appreciated since, at least, 50% of its con-
stituents are noncultivable microorganisms revealed only by recent NGS
approaches (Paster et  al. 2006a; Wade 2013). The fact that most spe-
cies identified in the oral cavity have been described at a molecular level
(16S rRNA sequencing) has led to the development of an online database
describing the characteristics of the bacterial oral microbiome, the Human
Oral Microbiome Database (homd.org) (Chen et al. 2010).

NGS studies of different anatomical sites in the mouth and in different

individuals have revealed a core oral microbiome where at least 47% of
species-specific operational taxonomic units (OTU) are shared between all
individuals. This, however, also reveals a considerable heterogeneity because
53% OTUs are divergent (Wade 2013). Furthermore, there are significant
differences both between individuals and between anatomical sites. In gen-
eral terms, we can distinguish three different microbial oral communities:
the first one is present in the buccal mucosa, gingivae, and hard palate; the
second one is characteristic of the saliva, tongue, tonsils, and throat; and
the third one is present in supra and subgingival plaque (Paster et al. 2006a;
Wade 2013; Eren et al. 2014).

Bacteria at the Bucal Mucosa: The buccal mucosa has relatively a poor bacte-
rial flora compared to the other buccal anatomical sites. The tongue dorsum
of healthy populations is colonized mainly by Streptococcus salivaris, Rothia
mucilaginosa, and uncultivable species of Eubacterium. Despite this relatively
low density bacterial population, the presence of bacteria in the tongue is
clinically significant because it changes with pathology, and even seems to
be associated to a traditional tongue diagnosis in traditional Chinese medi-
cine (Jiang et al. 2012). Also halitosis is associated with an altered bacterial
composition in the tongue (Kazor et al. 2003).
Bacteria in the Saliva: At least seven different phyla are present in
the saliva (i.e., Actinobacteria, Bacteroides, Firmicutes, Fusobacteria,
 Oral Health     109

Proteobacteria, Spirochetes, and TM7) (Zaura et al. 2009; Huttenhower

et  al. 2012), making a community of a minimum of 100,000  cells per
microliter (He et al. 2015). This microbial community seems stable for a
limited time frame in each individual, is not geographically dependent,
and is variable between individuals (Lazarevic et  al. 2010); it has even
been suggested to be used as a disease marker (Relvas et al. 2015).
Bacteria in the Dental Plaque: The dental plaque is a complex three-
dimensional array of interacting bacteria, forming a biofilm on the sur-
face of teeth. It contains mainly Firmicutes and Actinobacteria bacterial
groups, but may vary with the plaque type (supragingival or subgingival
plaque) and the dental integrity because supragingival plaque shows
progressively lower bacterial diversity with the progression of caries; the
dominant bacteria may change with disease stage. A similar situation
was also found on root surfaces and the subgingival plate in association
with periodontal disease. Recently, a host genetic influence has been
described for the bacterial composition of healthy teeth plaque, whereas
the cariogenic bacteria seemed to be controlled by nongenetic factors
(Gomez et al. 2017).
Geographic Influences on Oral Bacteria: Although the geographical
diversity of the oral microbiome does not seem to be relevant at the
phyla level, significant differences can still be found between individuals
at the species level. This seems to indicate that diet and the environment
have a much lower influence than the individual per se in shaping the
oral microbiome (Wade 2013).

Generally, the oral microbiome includes bacterial representatives from the

phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Spirochetes,
Fusobacteria, Synergitetes, and Tenericutes as well as the uncultured GN02,
SR1, and TM7 (Wade 2013). Together, the first six phyla represent 96%
of the species present in the normal oral microbiota, although, as men-
tioned before, different anatomical locations may harbor different bacterial
populations.

4.4  Shaping oral immunity

The oral immune system may best be described separating the lumen of the
oral cavity from its surrounding tissues. In fact, as previously described, the
immune system, to some extent, tolerates microorganisms in the lumen of
the oral cavity but needs to eradicate them as soon as they gain access into
the interior of tissue. As different needs require different actions, differ-
ent immunological responses arise in these two different anatomical sites.
110    Pocket Guide to Bacterial Infections

Nevertheless, it must be noted that there is a continuum and a dependency

between these two reactions.

In the lumen of the oral cavity, most immunological components are con-
tained in the saliva, reaching it directly during its production in the vari-
ous salivary glands, or via the gingival crevicular fluid. Accordingly, saliva
may contain immunoglobulins (mostly dimeric IgA), complement’ factors,
neutrophils, macrophages, lysozyme, histatins, mucins, and even leucocytes
(Schenkels et al. 1995).

All these components reach saliva as a response to activation of the sup-

porting oral immunological system that includes the amygdales (palatines,
lingual, and pharyngeal) and a network of submucosal lymphoid aggre-
gates resembling the gut Peyer’s patches, although not so structurally orga-
nized. These lymphoid aggregates are part of a network of lymph capillaries
and drain lymph from the mucosa, gingiva, and the dental pulp, thus being
capable of detecting antigens present in all these structures.

Now, let’s consider the physiology of an immune response to microorganisms

that penetrate the mucosal barrier. First of all, we should not forget that, in
itself, this may be already considered a major accomplishment on the part of
the microorganism. To do that, it had to resist immunoglobulin opsonization
and consequent neutrophil or macrophage phagocytosis; it had to survive
crevicular complement activation and consequent microorganism lysis; it had
to circumvent neutrophil and macrophage direct phagocytosis via pathogen
pattern receptors (PPR); if in the masticatory mucosa, it had also to gain access
not only through the epithelial cells of the mucosa but also through the exter-
nal keratin layer; and after breaking through the epithelial layer, it also had to
penetrate the basal membrane of the mucosa. However, after accomplishing
all this, the microorganism still has to face the mucosal immunity. This starts
by the recognition of the microorganism by mucosal resident monocytes and
dendritic cells, and as they detect the invader, not only phagocyte it but also
migrate to the lymphoid aggregates where they stimulate B and T cells to
start responding by presenting to them the antigens of the phagocytized
bacteria. Even if resident macrophages fail to phagocyte the microorganism,
it will find itself sooner or later dragged by the lymph into the lymphoid
aggregates where it will be recognized by B-lymphocytes and start the lym-
phoid reaction. Similarly, the amygdales can intervene in the process. As fully
developed and structured secondary lymphoid organs, their function can be
seen to be similar to the lymphoid aggregates, although with a bigger chance
of harboring the specific T and B lymphocytes required to respond to the anti-
genic stimuli. All these stimulated lymphocytes will then divide, producing
large numbers of clones of themselves, differentiate from naive into memory
 Oral Health     111

or effector cells, and migrate to the tissue where they are most needed. This
may be done through the circulatory system or directly into the oral cavity.
In the circulatory system, the lymphoid cells can use chemical clues left by
the phagocytes that change the circulatory epithelium near the site of infec-
tion, making it express the adhesion molecules needed for the lymphocyte
to enter the tissue. In turn, the generated memory cells will migrate to other
secondary lymphoid organs, leaving with them the memory of the previous
encounter and rendering other sites of the body capable of recognizing and
responding to the microorganism first found in the oral microenvironment.

All these mechanisms, in turn, influence the set and diversity of immune
system cells and soluble mediators continuously being sent to the oral cavity
via the saliva and the crevicular flows.

4.4.1  Saliva flow

Saliva (produced at a rate of approximately 500mL per day) has a complex

mixture of components, accounting for a diverse set of functions rang-
ing from lubrication to digestion and immunological control of the oral
microbiota. Some functions may even be shared, as the lubrication and
the mechanical washing of bacteria can be seen as indirect immunologi-
cal actions as they are efficient ways of preventing oral bacterial adhesion
and of directing oral bacteria to the gut where they may help food diges-
tion and absorption. Saliva composition is not only derived from salivary
glands, as the crevicular flow (see next section) is also an important con-
tributor of immunological mediators that end up in this physiological fluid.
Nevertheless, the salivary glands (primary and accessory) are the major
source of saliva components production, including its immunological medi-
ators. In particular, it has been demonstrated that the dimeric IgA compo-
nent of saliva (including its dimerizing J chain) is mostly derived from plasma
B cells of salivary glands. Also, the salivary duct has shown to be responsible
not only for the release of saliva, but they also serve as a gateway for anti-
gens to stimulate salivary B lymphocytes to produce the immunoglobulins
depending on oral antigen composition (Nair and Schroeder 1983).

Saliva is also rich in lysozyme, peroxidase, lactoferrin, and immune-derived

glycoproteins that bind to bacterial adhesins and complement cascade pro-
teins (Schenkels et al. 1995; Fabian et al. 2012), all of which contribute to
oral native immunological reactions.

Saliva also harbors a large number of polymorphonuclear cells, mostly

reaching saliva via the crevicular flow. Despite the fact that its importance
out of the crevicular sulcus is not clear, these cells may react locally to oral
112    Pocket Guide to Bacterial Infections

antigens by phagocytosis and by releasing active antimicrobial agents such

as lysozyme, acid hydrolases, and reactive oxygen species (Moutsopoulos
and Konkel 2017).

4.4.2  Crevicular flow

As mentioned previously, the gingival crevice is a particularly sensitive

anatomical site, potentially allowing oral bacterial entry into the adjacent
tissues. This is even emphasized by the 10-fold increase in the gingival cre-
vicular area in cases of active inflammation, potentially increasing the leak-
age potential to the inner tissues. However, this is counterbalanced by the
fact that this is, in parallel, a major oral immunological active site. Actually,
the junctional epithelium of the gingival crevicular is the major source of
humoral and cellular immunological components that fill the crevicular sul-
cus and ends up in the saliva. Contrary to the described composition of the
saliva, the crevicular fluid has major influences from the systemic immuno-
logical system, as it receives its components not only from local secondary
lymphoid tissues, but also from the blood. Thus, the crevicular fluid is not
only rich in IgA, but also in IgG and IgM, complement components and
its labile fragments produced during complement activation. The presence
of these complement activation by-products not only shows that active
complement reactions are taking place in the crevicular sulcus, but also
indicate that immune cell chemotactic calling and capillary vasodilation with
inherent increased permeability is also a result of complement activation in
the crevicular sulcus, further potentiating the immune response to bacterial
presence in this anatomical area. Native response immune mediators such
as neutrophil-derived mediators, among others, have also been described
in the crevicular flow and may contribute to the immunological reactions
and to differences between deciduous and permanent teeth immunology
(Moriya et al. 2017).

Of particular importance to crevicular immunology is the flow of immu-

nological cells. In fact, the leaky nature of the junctional epithelium in this
area allows an important flow of immunological cells. Most of these cells
are neutrophils (more than 95% of the total leukocytes) migrating from the
circulation into the gingival tissue and to the crevicular sulcus at a rate of
over 30,000 neutrophils per minute in humans (Schiott and Loe 1970). This
is the most important source of immunological cells and renders the oral
cavity with a plethora of cells with varying levels of activation and func-
tional states and a potential repertoire of cells for phagocytosis, degranula-
tion, and secretions of immune mediators such as cytokines, anti-microbial
peptides, and neutrophil extracellular traps. The importance of these cells
has been demonstrated by studies of patients with genetic defects of
 Oral Health     113

neutrophils that show severe periodontal immunopathology. Interestingly,

it has been shown that both too few or too many neutrophils unbalance
the immune reaction toward periodontal immunopathology (Kantarci et al.
2003; Matthews et al. 2007; Dutzan et al. 2016), making the regulation of
neutrophils number in the crevice an unknown mechanism of rather critical
importance.

4.5  From colonization to infection and to pathology

4.5.1  Dysbiosis—Disease trigger

Once established, the oral microbiome is maintained by host and microbe-

derived factors, in a not fully understood process involving pro- and anti-
inflammatory bacterial signals (Devine et  al. 2015). However, despite the
establishment of this flora, infections on the immune-competent oral
mucosa are a rather rare event (Zaura et al. 2014). In contrast, patients who
are immunosuppressed can often experience life-threatening viral and fun-
gal infections of the mucous membranes (Soga et al. 2011; Petti et al. 2013;
Diaz et al. 2013), demonstrating that the pathological potential of this flora
exists but is controlled by the host immune system.

The concept of an “exposome” encompassing all nongenetic influences

from birth onward promote increased susceptibility or increased protection
to a disease has been proposed. The exposome includes the nutritional
status, antioxidant status, free radicals generated, and the microbiome
(Bogdanos et al. 2013). Inherent to the exposome concept is the notion that
maintenance of an oral microbiome healthy state (symbiosis) or a disease
state (dysbiosis) results from a complex and dynamic equilibrium between
all the resident species in the oral cavity. So, a dysbiotic microbiome is one
in which the diversity and relative proportions of taxa within the finely
tuned ecosystem is disturbed (Cho and Blaser 2012). Consequently, dys-
biosis can be defined as a compositional shift in the normal population of
a particular microbial community that promotes development of an inflam-
matory or disease state. Often it arises through external triggers like poor
oral hygiene, dietary habits, and intake of medications which, for instance,
change saliva flow or composition, induce gingival inflammation (Marsh
et al. 2014; Wu et al. 2016), reduce oxygen tension due to increase in bio-
film thickness, altered host defenses, or nutritional, metabolic, or structural
stresses within the ecosystem (Cho and Blaser 2012). Dysbiosis can also
result from microbes-host relationship shifts, turning from a mutualistic to
a parasitic connection, acquisition of virulence factors, or a shift to opportu-
nistic behavior instead of a commensal one (Avila et al. 2009; Parahitiyawa
et al. 2010). As a result of the ecological shifts, beneficial bacterial become
114    Pocket Guide to Bacterial Infections

less capable of inhibiting the growth of the usually well-controlled biofilm

pathogenic bacteria (Marsh et  al. 2015), resulting in infection (Ruby and
Goldner 2007) and disease (Amerongen and Veerman 2002).

4.5.2 From the early colonizers to oral biofilms: A holistic

perspective of the microbiome behavior

The oral cavity is a dynamic microbial ecosystem to several allochthonous

species (transient visitors) in addition to autochthonous members (stable
colonizers) (Krishnan, Chen, and Paster 2017). Furthermore, the ecosys-
tem varies dramatically throughout life from bacteria acquisition at birth
(Berkowitz and Jones 1985; Asikainen and Chen 1999) to elderly colo-
nization (Preza et  al. 2008). Some of these changes are also associated
with modifications in the oral surfaces and microenvironments such as the
crevice sulcus with teeth eruption and the availability of synthetic surfaces
with the use of prosthesis, for instance. All these changes provide different
opportunities for different bacteria depending on the availability of specific
adhesion-receptors for bacteria-to-bacteria and bacteria-to-surface con-
tacts (Kolenbrander 2000; Nobbs et al. 2011).

During the first months of life, species of Streptococcus are usually the first
pioneering microorganisms to colonize the oral cavity with Streptococcus
salivarius found mostly on the tongue dorsum and in saliva, Streptococcus
mitis on the buccal mucosa, and Streptococcus sanguinis on the teeth
(Socransky and Manganiello 1971; Gibbons and Houte 1975; Smith et al.
1993). The establishment of these herald microorganisms implies local eco-
logical transformations, namely, local redox potential, pH, co-aggregation,
and availability of nutrients, thereby enabling more fastidious organisms to
colonize after them (Marsh 2000). As a result, we can see the appearance of
Prevotellam elaninogenica, Fusobacterium nucleatum, Veillonella, Neisseria,
and nonpigmented Prevotella (Kononen et al. 1992). Latter, the appearance
of teeth surfaces (and with it, the gingival crevice) leads to increases of gen-
era such as Leptotrichia, Campylobacter, Prevotelladenticola, and members
of the Fusobacterium and Selenomonas genera (Kononen et al. 1994).

As individuals age, biofilm maturation leads to increases in the ­numbers

of Streptococcus, Veillonella, Granulicatella, Gamella, Actinomyces,
Corynebacterium, Rothia, Fusobacterium, Porphyromonas, Prevotella,
Capnocytophaga, Nisseria, Haemophilis, Treponema, Lactobacterium,
Eikenella, Leptotrichia, Peptostreptococcus, Staphylococcus, Eubacteria,
and Propionibacterium (Aas et  al. 2005; Jenkinson and Lamont 2005;
Zaura et al. 2009; Bik et al. 2010).
 Oral Health     115

Currently, it is generally accepted that bacteria are present in the oral cavity in
the form of biofilms. These are structured and dynamic communities attached
to a surface and enwrapped in an extracellular matrix. In this intricate com-
munity, different bacteria interact with each other to establish a complex
ecosystem where each member contributes in some form to the remaining
members of the community (Marsh 2000). These elaborate associations
create special microenvironments, modifying the virulence of the result-
ing biofilm and contributing to the associated pathogenesis (Marsh 2000;
Flemming et  al. 2016; Koo and Yamada 2016). Thus, microfilm dynamics
allows specific low-abundance pathogens to influence disease by altering the
“healthy” microflora to a disease state (Hajishengallis et al. 2012), thus chang-
ing a healthy community to a disease-inducing one (Krishnan et al. 2017). This
has been formally proposed as the “keystone pathogen hypothesis.”

4.5.3  From a healthy microbiome to a disease-associated one

The oral microbiome is so deeply linked to the host healthy or diseased state
that over the years, several convincing studies have uncovered correlations
between its qualitative compositions (Kilian et al. 2016). However, under-
standing the complexity of microbiome dynamics and its effects on hosts
requires a holistic view, including interactions among different residents
within the community (bacterial interspecies and interkingdom interactions)
as well as their interaction with the host (Baker et al. 2017). In other words,
the microbiome must be seen producing commensalism within microorgan-
isms and mutualism with the host (Ruby and Goldner 2007; Zaura et  al.
2009; Filoche et  al. 2010; Ebersole et  al. 2017). Commensal relationships
among microbes allow them to flourish at no expense to their cohabitants
and, in turn, maintain biodiversity within the oral cavity (Zarco et al. 2012).
Research has demonstrated such biodiversity to be crucial to health; chil-
dren suffering from severe dental caries have less diverse oral microbiome
than those who are healthy (Kanasi et  al. 2010). Similarly, symptomatic
lesions of endodontic origin have a lower level of diversity than asymptom-
atic ones. It could be suggested that health-associated biodiversity results
from the need for specific functions from each species to maintain oral
cavity equilibrium and homeostasis (Zarco et al. 2012).

Commensal bacteria not only protect the host simply by niche occupation
but also interact with host tissue, promoting the development of proper tis-
sue structure and function; it is now consensual that host-associated polymi-
crobial communities, such as those found in the oral cavity, co-evolved with
us and have become an integral part of who we are (Roberts and Darveau
2015). Also, the residual and commensal bacteria have a critical function
116    Pocket Guide to Bacterial Infections

called colonization resistance, as a result of their proficiency to adapt to a

variety of niches, preventing colonization by pathogenic bacteria. In fact, the
detrimental effects of commensal bacteria depletion are well demonstrated
by the use of broad-spectrum antibiotics (Brook 1999; He et al. 2014). This
is also highlighted by the normal commensal microbiota’s ability to rapidly
adapting to hostile changes in microenvironment, preventing pathogenic
bacteria from taking the opportunity. In fact, the normal microbiota rapidly
recovers after changes in pH, redox potential, atmospheric conditions, salin-
ity, and water activity from saliva (Badger et al. 2011), resulting from eating,
communicating, and oral hygiene (Avila et al. 2009)

Thus, diseases such as caries, gingivitis, and periodontitis may be seen as

the result of a failure of the preexisting healthy microbiome to adapt to
some trigger that has produced the right conditions for some preexisting
controlled bacteria to take over and change the biofilm dynamics into a
disease-promoting one. It can thus be expected that further elucidation of
these changes will allow us to greatly improve our understanding of oral
microbial physiology, pathogenesis, and ecology, as well as our ability to
diagnose and treat microbial infections (Baker et al. 2017).

4.5.4 Caries, periodontal disorders, and endodontic lesions:

Manifestations of microbiome imbalances

It is well established that the most frequent oral diseases (i.e., caries, peri-
odontal diseases, and endodontic lesions) are caused by microorganisms
that, nevertheless, are also present in the healthy human oral microbiota
(Socransky and Haffajee 2005; Paster et al. 2006b; Aas et al. 2008; Dewhirst
et al. 2010; Johansson et al. 2016). These species are likely living harmlessly
in low numbers (often below the limit of detection of conventional micro-
biology) and, hence, regularly receive the designation of “putative” patho-
gens or biomarkers of disease (Krishnan et al. 2017). Thus, the presence of
the bacteria is not, per se, sufficient to induce the disease.

A remarkable note is that these oral diseases have as a main etiological

cause a polymicrobial infection. Another important point to highlight is that
those disorders result from an opportunistic infection as a consequence of
diet, host immune response, complicating systemic or genetic disorders, pH
change, poor oral hygiene, and lifestyle fluctuations (Krishnan et al. 2017).
In a simple sentence: when dysbiosis happens.

4.5.4.1  Caries – The most prevalent oral disease, responsible for tooth pain
and lost, begins with minor lesions on the enamel and can evolve, without
treatment, into the dentin and into the pulp space. This destruction of the
 Oral Health     117

mineralized tooth surface results from metabolic actions of opportunistic

pathogens. The microorganisms are capable of dietary carbohydrates fermen-
tation with production of acidic by-products, eventually decreasing the micro-
environment’s pH and destroying the tooth’s mineralized surface.

In the early nineteenth century, the nonspecific plaque hypothesis (Rosier

et  al. 2014) stated that dental infections were the outcome of an over-
growth of plaque nonspecific bacteria, leveraged by its own accumulation
and ecological changes in consequence of either ineffective hygiene or diet
modification. Also, there was the concept that anyhow, every plaque had
the potential to cause disease and as preventive measure, every effort to
plaque removal was advised (Rosier et al. 2014).

Later, with classic culture development, isolation and characterization of spe-

cies were possible, which allowed the observation that kanamycin (Loesche
et al. 1977) was particularly effective against caries-associated species, such
as streptococci, leading to the emergence of the specific plaque hypothesis.
At this point, it was conceivable that only a few species participated in the
illness process; therefore, cure or prevention of sickness went through the
prescription of antibiotics (Loesche 1979; Rosier et  al. 2014). Restrictions
of this microbiological identification methods became evident when trans-
lation from day-to-day practice with poor long-term clinical benefits was
observed (Rosier et al. 2014).

The interaction between the resident oral microbiota and the host environ-
ment proposed, in the 1980s, by the ecological plaque hypothesis explained
the manifestation of caries and stills. Briefly, an increased frequency of sugar
intake, or a reduction in saliva flow, results in plaque biofilms in close prox-
imity to the tooth surface that are exposed for longer and more regular
periods to lower pH levels. As a result, there is an increase in the putative
pathogens, as they are better adapted to the new acidic conditions, either by
producing acids themselves or by being more tolerant to an acidic environ-
ment. This occurs at the expense of bacteria that thrive in neutral conditions
or contribute to pH neutralization (Liu et al. 2012; Marsh et al. 2015). That is,
dysbiosis occurs by the selection of efficient acid-producing and -tolerating
bacteria in contrast with daily health, where the biofilm undergoes multiple
pH cycles during one day, resulting in enamel de- and re-mineralization.
Accordingly, if there is insufficient time at neutral pH during frequent snack-
ing, then demineralization outweighs remineralization which results in min-
eral loss, and eventually, in enamel lesions (Kilian et al. 2016).

Although a specific microbiome that signals dental caries is yet to be

found (Ling et al. 2010), the most common bacteria responsible for dental
118    Pocket Guide to Bacterial Infections

caries are Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus

a­ cidophilus (Selwitz et al. 2007). NGS experiments have demonstrated that
in carious lesions with Streptococcus mutans, additional species belong-
ing to the genera Atopobium, Propionibacterium, and Lactobacillus were
also present at significantly higher levels. In those subjects with no detect-
able levels of S. mutans, Lactobacillus species, Bifidobacterium dentium,
and low-pH non-S. mutans streptococci were predominant (Aas et  al.
2008). Based on these findings, it was advocated that species of the genera
Veillonella, Lactobacillus, Scardovia, and Propionibacterium, low-pH non-
S. mutans streptococci, Actinomyces species, and Atopobium species may
play an important role in caries progression.

Other researchers supported by NGS methodologies on the microbiome of

populations with a low and high prevalence of caries found that adolescents in
Romania, who had limited access to dental care, were colonized with S. mutans
and S. sobrinus. In contrast, adolescents in Sweden, who benefit from good
dental care, were colonized only rarely with those two closely related spe-
cies of streptococci, but were colonized by more species of Actinomyces,
Selenomonas, Prevotella, and Capnocytophaga (Johansson et al. 2016).

Among primary and secondary dentitions as well as in root surface caries, there
are significant differences in the oral microbiome’s composition. In the first
one, not surprisingly, S. mutans was typically detected at high levels (Becker
et al. 2002). Other species like Actinomycesgerencseriae, Scardoviawiggsiae,
Veillonella, Streptococcus salivarius, Streptococcus constellatus, Streptococcus
parasanguinis, and Lactobacillus fermentum were found in the secondary
dentition. The root surface, unlike the dental crown, is not covered by enamel.
It was found that the predominant taxa included Actinomyces species,
Lactobacillus, Enterococcus faecalis, Mitsuokella sp. HOT131, Atopobium and
Olsenella species, Prevotella multisaccharivorax, Pseudoramibacter alactolyticus,
and Propionibacterium acidifaciens, although S. mutans was also present with
these kind of caries (Preza et al. 2008).

4.5.4.2  Periodontitis – Periodontal disease, a polymicrobial inflammatory

disorder of the periodontium (Pihlstrom et al. 2005), also results from sub-
gingival plaque accumulation that causes remodulation in the microbiota
(Horz and Conrads 2007). Its less severe form is gingivitis, a gingiva inflam-
mation caused by pathogenic biofilms that is often reversible (Pihlstrom
et al. 2005), if dealt with good oral hygiene in a timely manner (Horz and
Conrads 2007).

Periodontitis is an evolution of gingivitis that can be described as an infec-

tion that involves all soft tissue and bone that support the periodontium
 Oral Health     119

and teeth structures (Horz and Conrads 2007). The pathologic mechanism
is similar to the one described for caries: multiple opportunistic pathogens
overgrow in dental plaque, become pathogenic (Horz and Conrads 2007),
release proteolytic enzymes that break down host tissue, and may result in
gingival inflammation, loss of gingival attachment, periodontal pocket for-
mation, alveolar bone ,and even, root resorption. The predominant patho-
gens involved in periodontitis are Aggregatibacter actinomycetemcomitans,
Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum,
Tannerella forsythia, Eikenellacorredens, and Treponema denticola (Filoche
et al. 2010; Dashiff and Kadouri 2011).

If not treated, periodontal pockets occur, and periodontitis becomes irre-

versible (Pihlstrom et al. 2005) because the periodontium, once separated,
is unable to reattach to the bone. Furthermore, periodontal pathogens
develop virulent factors, such as encapsulation factors, that together with
the fact that they are deep within the periodontal pockets render them
resistant to antibiotics (Horz and Conrads 2007; Van Essche et  al. 2011).
Also, recolonization is also possible, as the nearby mucous membranes
­harbor bacterial reserves (Horz and Conrads 2007).

Periodontitis treatment is thus a hard challenge. Mechanical treatment is

often insufficient, making antibiotics a much-needed option, despite its
aforementioned limitations. Accordingly, the most appropriate and least-
invasive form to manage periodontitis consists of regular appointments,
acquisition of efficient ambulatory hygiene procedures, and on-time sup-
ported use of antibiotics. It is also important that the oral cavity maintains
certain gram-positive bacteria that shield pathogens from damaging hard
and soft tissues (Van Essche et al. 2011).

4.5.4.3  Endodontic lesions – Infection of the root canal system has been
established as the primary cause of apical periodontitis, a condition charac-
terized by the inflammation and destruction of periradicular tissues caused
by etiological agents of endodontic origin. The classic pathways between
the pulpal space and the outer tissues, in a two-way direction, include the
apical foramen, accessory root canals, and the dentinal tubules. Due to
ecological factors of the endodontic environment, such as availability of
nutrients (namely fermentable carbohydrates), oxygen and pH levels, the
surviving microorganisms, belong to restricted group of species which are
predominantly anaerobic, with the facultative anaerobes residing in the
more coronal part of the root canal system and the obligate anaerobes
apically (Alves et al. 2009; Aw 2016). Those species outlast from the break-
down of pulpal tissues and serum proteins (Figdor and Sundqvist 2007),
120    Pocket Guide to Bacterial Infections

which leads to rise of environment pH (Sundqvist 1992; Figdor and

Gulabivala 2008), favoring the proliferation of late-stage bacteria. The dif-
ferent species of the bacterial population also appear to be interdependent
for nutrition (Sundqvist and Figdor 2003) because the metabolic products
of one species may act as a source of nutrition for others.

In primary infections, predominant taxa detected include species of

Peptostreptococcus, Parvimonasmicra, Filifactoralocis, and P. alactolyticus,
and species of Dialister, F. nucleatum, T. denticola, P. endodontalis, P. gingiva-
lis, T. forsythia, Prevotella baroniae, P. intermedia, Prevotella nigrescens, and
Bacteroidaceae [G-1] HOT272 (Siqueira and Rocas 2009). Enterococcus faeca-
lis was detected, but in lower levels. However, in retreatment cases advocated
for secondary or persistent endodontic infections, the predominant taxa
include Enterococcus species such as E. faecalis, Parvimonas micra, Filifactor
alocis, P. alactolyticus, Streptococcus constellatus, Streptococcus anginosus,
and Propionibacterium propionicum (Aw 2016; Krishnan et al. 2017).

The microbiomes of endodontic-periodontal lesions, which evolve simulta-

neous infection of pulpal space and of the periodontum, possessed similar
profiles including E. faecalis, P. micra, Mogibacterium timidum, Filifactor
alocis, and Fretibacterium fastidiosum (Gomes et al. 2015).

4.6  Oral microbiome as health biomarker

The oral cavity is the primary gateway to the human body; therefore, micro-
organisms that inhabit that area are capable of spreading to different body
sites (Dewhirst et al. 2010).

Pathogens originated in the oral cavity can be often detected in blood cul-
tures as they destroy and pass through oral mucous membranes, periapi-
cal lesions, and periodontal pockets (Horz and Conrads 2007). As they get
access to the bloodstream, they induce immune responses or produce exces-
sive and deregulated amounts of inflammatory mediators and ultimately
can cause disease at different body sites (Williams 2008). Oral bacteria may
be used as biomarkers for certain systemic diseases, such as endocarditis
(Berbari et al. 1997), ischemic stroke (Joshipura et al. 2003), cardiovascular
disease (Beck and Offenbacher 2005; Teles and Wang 2011), pneumonia
(Awano et al. 2008), pancreatic cancer (Farrell et al. 2012), diabetes type II
(Demmer et al. 2015), pediatric Crohn disease (Docktor et al. 2012), and low
weight and preterm birth (Shira Davenport 2010).

Periodontal disease has been shown to predispose individuals to cardiovascu-

lar disease through its ability to induce chronic inflammation (Syrjanen 1990).
 Oral Health     121

Also, individuals who are diabetic can experience more often periodontal
disease. Similarly, the presence of several anaerobic oral bacterial species,
like A. actinomycetemcomitans and S. constellatus (Shinzato and Saito 1994)
has been shown to predispose to bacterial pneumonia. Other renal and car-
diovascular pathologies are nowadays a focus of investigation in relation to
changes in nitric oxide homeostasis and its effect on vasodilation of vascular
smooth muscle. An important role in those disorders maybe played by oral
bacteria that reduce dietary nitrates into nitrite (Hezel and Weitzberg 2015),
which, in turn, is absorbed by the blood and further reduced to nitric oxide
by a variety of mechanisms. In Alzheimer disease, inflammation, a key fea-
ture of the disease could be caused in part by peripheral infections, such
as periodontal disease (Olsen and Singhrao 2015). Periodontal pathogens
such as A. actinomycetemcomitans and P. intermedia are capable of elicit-
ing systemic inflammation, which results in the release of pro-­inflammatory
cytokines that traverse the blood–brain barrier. However, it is yet to be
established if there is a direct causal relationship between the oral microbi-
ome and these systemic disorders (Krishnan et al. 2017).

All this renders the oral microbiome a complex system capable of influenc-
ing health not only locally at the oral cavity, but also influencing the sys-
temic immunological status and capable of influencing distant anatomical
status health. Thus, it should be possible to manipulate the microbiome’s
potential to optimize personal health and identify microbial profiles with
potential use to assess disease risk, which is the next phase of the Human
Microbiome Project.

Thus, continuous understanding of the oral microbiome dynamics and its

influence in both health and disease is a complex endeavor that promises to
keep us occupied for many years to come. It may be anticipated that results
will change the way we diagnose and also the way we treat many diseases,
and not only oral cavity ones.

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Zaura, E., E. A. Nicu, B. P. Krom, and B. J. F. Keijser. 2014. Acquiring
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5
Bacterial Infections in
Atherosclerosis
Atherosclerosis Microbiome
Emil Kozarov and Ann Progulske-Fox

Contents

5.1 Introduction 135

5.2 Inflammation as the core process in atherogenesis: Could
infection of arterial wall be the culprit? 136
5.3 Epidemiology of AVD 138
5.4 Seroepidemiology 139
5.5 DNA evidence 139
5.6 Clinical isolates from atheromas. Atherosclerosis microbiome 140
5.7 Clinical trials with antibiotics 140
5.8 Atherosclerosis microbiome—The most critical segment of
human microbiome 141
5.9 Conclusion. Infectious component of vascular inflammations 141
5.10 Next steps 142
References 143

5.1 Introduction

Atherothrombotic vascular disease (AVD), to which atherosclerosis is the

main underlying pathology, is the leading cause of morbidity and mortality
in adults and the most critical area of medical sciences.

Significant advances in AVD area are crucial, specifically due to increased

prevalence with age. Myocardial infarction (MI) and stroke continue to be
major causes of morbidity and mortality.

Atherosclerosis is a chronic inflammatory disease. Despite meaningful

progress in the identification of risk factors and development of clinical
tools, such as statins and PCSK9 inhibitors, AVD-related deaths continue to
increase worldwide, due in part to the moderate efficacy of the available

135
136    Pocket Guide to Bacterial Infections

drugs. For example, after 104 weeks of maximal-dose therapy, atorvastatin

and rosuvastatin led to reductions of only 0.99% (95% confidence inter-
val [CI], –1.19  to –0.63) and 1.22% (95% CI, –1.52  to –0.90), respectively
(P  =  0.17), in percentage of atheroma volume, the primary efficacy end-
point (Nicholls et al., 2011).

At the same time, the current treatment modalities targeting hyperten-

sion, hyperlipidemia, and controlling hemostasis do not directly address the
inflammatory origin of atherosclerosis (Weber and Noels, 2011). Genetic
research has also not led to major breakthroughs. A large genome-wide
association study (GWAS) of 63,746  coronary artery disease (CAD) cases
and 130,681 controls brought the number of identified human genetic vari-
ants that are associated with increased coronary disease risk to 46, which
still accounts only for about 10% of CAD heritability (Deloukas et al., 2013).
The prospect of addressing hundreds of single nucleotide polymorphism
(SNP) loci immensely complicates genetic targeting of the predisposition to
vascular inflammation.

A large network analysis identified lipid metabolism and inflammation as

the two key biological pathways involved in the genetic predisposition
to CAD (Deloukas et  al., 2013). Nevertheless, many of the individuals
with multiple classic risk factors for AVD do not go on to experience
such events. Moreover, MI and stroke continue to occur in up to two-
thirds of all patients (Libby, 2005). Because many cardiovascular events
have not been explained by genetics or other risk factors, and multiple
epidemiologic studies have consistently suggested an infectious compo-
nent, entirely novel approaches for diagnostics and treatment are acutely
needed.

These novel approaches should be based on the concept of personalized

medicine and addressing additional manageable risk factors to control AVD
to achieve longevity, while also increasing the quality of life. There are a
variety of avenues that could enable a novel approach to AVD. These are
based on the discovery, characterization, and focusing on its infectious
component.

5.2 Inflammation as the core process in atherogenesis:

Could infection of arterial wall be the culprit?

The inflammatory nature of the atherosclerotic lesion from initiation (fatty

streak) to culmination into an acute ischemic event (plaque rupture) has been
established. The degree of inflammation correlates with the severity and
 Bacterial Infections in Atherosclerosis     137

clinical outcome. Inflammation influences plaque progression and plaque’s

vulnerability to rupture (Libby, 2002, 2012).

Markers of inflammation may predict development of disease in asymptom-

atic individuals. C-reactive protein (CRP), an acute-phase reactant elevated
in all inflammatory diseases, has been the most widely studied biomarker of
inflammation. Indeed, the acceptance of the high-sensitivity C-reactive pro-
tein (hsCRP) test as a biomarker for cardiovascular disease (CVD) risk assess-
ment in primary prevention (Ridker, 2016) naturally suggests considering
infections as an underlying cause of the inflammation. The increased risk
for heart attack associated with elevated CRP has been reported to have
prognostic value even among patients with negative cardiac troponin and
no evidence of myocyte necrosis (Heeschen et al., 2000), again suggesting
an alternative origin of the inflammation.

Defective resolution of inflammation is the basis of most prevalent chronic

inflammations, including AVD. Specialized pro-resolving mediators include
arachidonic acid-derived lipoxins, omega-3  fatty acid, eicosapentaenoic
acid-derived resolvins, docosahexaenoic acid-derived resolvins, protectins,
and maresins (Fredman and Tabas, 2017). For in-depth reviews on inflam-
mation in AVD, see also Pant et al. (2014).

Importantly, the abundant evidence accumulated so far points at infections

(often of periodontium) as a contributing factor for CVD. Periodontitis pro-
vides an “open gate” to the circulation for entry of oral bacteria into the
bloodstream, allowing them to activate the host inflammatory response in
the direction of atherogenesis (i.e., atheroma formation, maturation, and
exacerbation) (Reyes et al., 2013). Similarly, the “leaky gut” syndrome can
deliver intestinal species to a new, systemic location, inducing inflamma-
tion (Figure 5.1 shows the multiple way the infections contribute to chronic
inflammation, including atherogenesis).

Atherosclerosis is a disease of the vasculature characterized by inflamma-

tory lesions in the arterial bifurcations, which, when destabilized, can lead
to MI and stroke. AVD does have many characteristics of a chronic infec-
tious disease. For example, clinical studies have indicated that infections
with multiple pathogens result in chronic persistent inflammation (Epstein
et al., 2009; Campbell and Rosenfeld, 2015). In addition, internalization of
many types of bacteria can produce a “privileged niche,” where they per-
sist in a dormant, nonreplicating state, sheltered from humoral and cellular
immune responses (Tufariello et al., 2003). Lastly, DNA data suggest that
various and specific pathogens are associated with atherosclerotic tissue
(Kozarov et al., 2006; Ott et al., 2006).
138    Pocket Guide to Bacterial Infections

Figure 5.1  Contribution of bacterial pathogens to the “response to injury” model

of atherogenesis. Mentioned in red font is the bacterial component of vascular
inflammations as suggested in recent publications.

5.3  Epidemiology of AVD

The presently accumulated epidemiological data support the hypothesis

that infections contribute to AVD (Amar et al., 2009; Epstein et al., 2009).
In particular, periodontal inflammatory components were recognized as
contributors to or triggers for systemic inflammatory responses and the risk
of developing heart disease was shown to increase by 168% in patients with
periodontitis (Genco et al., 2002).

As another example, in the Northern Manhattan Study (NOMAS), a pro-

spective cohort study of stroke incidence and prognosis, it was shown
that infectious burden is associated with risk of stroke, a carotid plaque
thickness (Elkind, 2010), and also with cognition (Katan et al., 2013). The
Atherosclerosis Risk In Communities (ARIC) study found systemic antibody
response to periodontal bacteria associated with coronary heart disease in
ever and never smokers (Beck et al., 2005). Similarly, the data from the Oral
Infections and Vascular Disease Epidemiology Study (INVEST) demonstrated
a direct relationship between tooth loss and carotid plaque prevalence
(Desvarieux et al., 2003), colonization with pathogenic periodontal patho-
gens to be associated with subclinical vascular disease (i.e., carotid artery
 Bacterial Infections in Atherosclerosis     139

intima-media thickness) (Desvarieux et al., 2005), and showed that severe

periodontal bone loss is associated independently with carotid atheroscle-
rosis (odds ratio 3.64, P < 0.05) (Engebretson et al., 2005).

5.4 Seroepidemiology

Supporting these data, seroepidemiological investigations showed that

infections caused by major periodontal pathogens, Porphyromonas gingiva-
lis and Aggregatibacter actinomycetemcomitans (in seropositive subjects) is
associated with future stroke (Pussinen et al., 2004). Seroepidemiology also
demonstrated that anti-P. gingivalis antibody is associated with atrial fibril-
lation as well as carotid artery atherosclerosis. In addition, anti-Prevotella
intermedia antibody may be associated with stroke through its association
with carotid atherosclerosis (Hosomi et al., 2012). Finally, murine and larger
animal models of infection corroborated the results from in vitro and studies,
demonstrating the exacerbation of the vascular inflammation upon adminis-
tration of infectious agents (Jain et al., 2003; Brodala et al., 2005).

Most often, direct systemic exposure to infectious agents, such as bacte-

remia with an oral origin, is communicated. In one study, 80.9% of the
patients presented positive cultures after dental cleaning procedure, and
19% of the patients still had microorganisms in the bloodstream after
30 minutes (Lafaurie et al., 2007).

5.5  DNA evidence

Microbial pathogens associated with atheromatous tissues have been identi-

fied at DNA level (Haraszthy et al., 2000; Kozarov et al., 2006).Using genomic
(16S rDNA signatures) and bioinformatic (molecular phylogenies) tools, the
diversity of bacteria in atherosclerotic lesions in patients with AVD was system-
atically explored. Polymerase chain reaction (PCR), clone libraries, gradient gel
analyses, and fluorescence in-situ hybridization (FISH) were used to demon-
strate the presence of bacterial DNA in patient tissues. Mean bacterial diversity
in atheromas was high, with a score of 12.33 ± 3.81 (range, 5–22), suggest-
ing that diverse bacterial colonization may be important (Ott et  al., 2006).
Further, another survey of bacterial DNA signatures in atherosclerotic plaques
suggested that both oral and intestinal bacteria may correlate with disease
markers of atherosclerosis (Koren et  al., 2011). We have also shown, using
molecular, genetic, and immunological approaches, the association of variety
of bacterial species with atheromatous tissues (Kozarov, 2005, 2012), creating
for the first time the concept of atherosclerosis microbiome. Interestingly, in a
recent study, P. gingivalis was the most abundant species detected in coronary
and femoral arteries (Mougeot et al., 2017).
140    Pocket Guide to Bacterial Infections

5.6 Clinical isolates from atheromas.

Atherosclerosis microbiome

However, DNA detection does not fulfill Koch’s postulates for association
of infectious agents with disease. In a significant advancement, we were
able to demonstrate, at several levels, that microbial infection of arterial
plaques may be the missing link, a major contributing factor to acute isch-
emic events. We designed and implemented a breakthrough technology,
immune-mediated resuscitation (IMR), for cultivation and identification of
a variety of live bacteria in plaques from patients (Rafferty et  al., 2011a,
2011b), yielding clinical isolates, which are the basis for establishment of
the atherosclerosis microbiome and for development of new diagnostics.
Using immunology, cellular microbiology, genomics, and fluorescence
microscopy, we also demonstrated vascular cell transmission of bacterial
pathogens (Li et al., 2008), association of dormant invasive bacteria with
atheromatous tissue (Kozarov, 2005), and most importantly, developed a
technology to isolate and identify previously uncultivable bacterial patho-
gens from such tissue from patients (Rafferty et al., 2011a, 2011b). Patient-
specific drug targets were essentially identified, opening the possibility to
introduce personalized medicine in the critical area of AVDs.

It is known that periodontal infection accelerates both lipid deposition in

arteries and atherosclerosis in animal models (Jain et  al., 2003; Li et  al.,
2008), suggesting that identification of the infectious agents in the athero-
matous tissue, followed by a treatment regimen specific for the infecting
bacteria, would open an additional avenue to address not only infections,
but also hyperlipidemias as a causative factor in atherosclerosis.

The reemergence of bacterial pathogens as potential initiators or exacer-

bators of AVD poses new challenges to medical research and to an over-
whelmed healthcare budget; however, there is a silver lining to it. The
possibility of a new approach to CVD creates a perfect opportunity to
develop entirely novel theranostics and to introduce personalized medicine
in the most critical area of human health.

5.7  Clinical trials with antibiotics

Importantly, the antibiotic clinical trials for the secondary prevention of late-
stage coronary heart disease a decade ago failed because all patients were
treated with the same antibiotic (O’Connor et al., 2003; Cannon et al., 2005;
Grayston et al., 2005). We now know that the negative results were actually
expected due to (i) the fact that the trials were not designed to demonstrate
 Bacterial Infections in Atherosclerosis     141

causation and (ii) to the fact that we isolated (years later) different pathogens
from the plaques from different patients. However, we realized this had not
evaded the attention of the clinicians in the field. When presented with our
investigational data at meetings and seminars, clinicians asked for further
advancements. Identifying the patient-specific pathogens in the vasculature
would for the first time provide clinicians with a tool to adequately inform
patient-specific treatment, which is the basic tenet of personalized medicine.

5.8 Atherosclerosis microbiome—The most

critical segment of human microbiome

The discovery of the atherosclerosis microbiome highlights a rare possibility

for chronic inflammation-related biomarker discovery and for individualized
diagnosis and treatment of vascular inflammations that lead to MI, stroke,
and peripheral vascular disease. From the perspective of public health, cur-
rently there is no more critical inflammatory disease than AVD (Figure  5.2
illustrates the variety of avenues exploited by bacterial pathogens for sys-
temic dissemination and internalization in vascular walls, contributing or trig-
gering chronic inflammation). Besides being the number-one killer globally,
AVD is estimated to cost the European Union (EU) €169 billion and causes
nearly half of all deaths in Europe (48%) and in the EU (42%), making it the
main source of morbidity and mortality in the EU (Leal et al., 2006). Similarly,
AVD accounted for 32.8% (811,940) of all 2,471,984 deaths in the United
States (Roger and O’Donnell 2012), costing the United States $448.5 billion
in 2008, greater than any other medical condition (www.americanheart.org).
Thus, immediate development of new approaches to treating AVD is needed.
Accomplishing this would have tremendous outcomes, in addition, to the
economic aspects. For example, successful treatment of MI is estimated to
increase the human life span by 16.6 years (Go et al., 2013).

5.9 Conclusion. Infectious component

of vascular inflammations

Atherogenesis involves both the innate and adaptive arms of the immune
system, similar to bacterial infections. The “response to injury” hypothesis
of atherogenesis is presented here as a pathogen-accelerated inflammatory
process, leading to endothelial activation, growth factors release, monocytes’
adhesion and transmigration (extravasation), while maturing into macro-
phages, followed by foam cell formation and smooth muscle cell proliferation.
This progressive process leads to arterial thickening and eventually to grad-
ual occlusion of the vascular lumen and inflammatory atheromatous lesion
formation (Figure 5.2 depicts the bacterial component of atherogenesis).
142    Pocket Guide to Bacterial Infections

Figure 5.2  Model representing what is now known about the microbial component of
atherogenesis. Both bacteremic- and phagocyte-mediated avenues of bacterial delivery
to the site of inflammation are proposed. Bacteremia-related bacteria (left) are shown
invading the endothelial layer and further spreading into deeper tissue. Activation of
infected endothelia are represented with the release of pro-inflammatory chemokines
(such as MCP-1) in the lumen, activating blood monocytes (MN) and macrophages
(MΦ) and promoting their adhesion and diapedesis. Transmigrating leucocytes (in the
center) can harbor internalized viable bacteria, which represents the second avenue
for systemic bacterial dissemination to distant sites. The internalization of bacteria
can switch them into uncultivable state (red into green), while their internalization by
phagocytes can reactivate them (from green to red). Atheromas can grow because
of macrophage-secreted growth factors-mediated smooth muscle cell proliferation.
Bacteria are also released upon host cell death (depicted at right). Apoptotic EC, apop-
totic endothelial cell releasing intracellular bacteria; EC, endothelial cell; MN, monocyte;
MΦ, macrophage with internalized bacteria; SMC, smooth muscle cell.

Thus, the thrombosis and the consecutive acute ischemic events that often
follow, possibly leading to a fatal outcome, can also be pathogen related.

5.10  Next steps

We strongly suggest closer interaction between interdisciplinary teams of bio-

medical investigators and better-trained clinicians, including those with both
medical and dental clinicians to efficiently address this plague on today’s soci-
ety. To translate the data from the often complicated laboratory and clinical
investigations into tangible results, clinicians are needed who will be able to:
 Bacterial Infections in Atherosclerosis     143

1. Address the most critical conditions of AVD in an innovative, efficient

manner;
2. Adequately update their core competencies during continuous medical
education courses;
3. Place specific emphasis on infections as a key risk factor, thus introducing
personalized medicine;
4. Pay critical attention to infections as modifiable risk factors for AVD, inde-
pendent of lifestyle; and
5. Communicate the importance of control of infections, such as periodon-
titis, to patients.

Creating synergy of research and education, clinicians are those individuals

who are empowered to lead the shift in healthcare expenditures from the
costly tertiary care toward early diagnostics and prevention, thus reducing
the healthcare budgets and their burden on governments, and ushering in
the era of personalized (i.e., precision) medicine.

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Tufariello, J. M., J. Chan, and J. L. Flynn. 2003. Latent tuberculosis:
Mechanisms of host and bacillus that contribute to persistent infec-
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Weber, C. and H. Noels. 2011. Atherosclerosis: Current pathogenesis and
therapeutic options. Nature Medicine 17:1410–1422.
6
Neonatal Bacterial Infection
Insights into Pathogenic Strategy
and Onset of Meningitis and Sepsis
Koilmani Emmanuvel Rajan and Christopher Karen

Contents

6.1 Introduction 148

6.2 Causative organisms of meningitis and sepsis 149
6.3 Etiology 149
6.4 Pathophysiology of bacterial meningitis 151
6.4.1 Infection 151
6.4.1.1 Mode of transmission 151
6.4.1.2 Colonization and invasion 152
6.4.2 Bacteremia 152
6.4.3 CNS invasion 152
6.4.3.1 Bacterial entry into CNS 152
6.4.3.2 Survival of pathogens in CNS 153
6.4.3.3 Bacterial multiplication in CSF 154
6.4.3.4 Host response to invasion 154
6.5 Pathophysiology of neonatal sepsis 155
6.5.1 Infection 156
6.5.1.1 Onset of sepsis 156
6.5.1.2 Immune system of neonatal sepsis 156
6.5.1.3 Factors that cause sepsis 156
6.5.1.4 Microbiology of sepsis and septic shock 157
6.5.2 Molecular and cellular events 157
6.5.2.1 Signaling of PRRs, PAMPs, and DAMPs 157
6.5.2.2 Proinflammatory response 158
6.5.2.3 Interplay between anti-inflammatory and
proinflammatory responses 158
6.5.2.4 Complement system 158
6.6 Concluding remarks 159
References 160

147
148    Pocket Guide to Bacterial Infections

6.1 Introduction

Bacterial invasion in neonates can cause life-threatening diseases like men-

ingitis and sepsis. Accordingly, it accounts a major cause of morbidity and
mortality in global scenario (Park et al. 2001; Neher and Brown 2007; Tzialla
2015). Despite several preventive measures, including the improvement of
socioeconomic status, appropriate antimicrobial therapy, and modern vac-
cination strategies, the episodes of meningitis and sepsis remains alarming
(Harvey 1999; Park et al. 2001; Agrawal 2011; Van Sorge 2012; Miller et al.
2013; Camacho-Gonzalez 2013).

Meningitis is a neurologic emergency characterized by inflammation of the

meninges in response to microbial infection (Heckenberg 2014). The hall-
mark feature of meningitis is the inflammation in pia matter, arachnoids,
and subarachnoid space. Moreover, some forms of neurological sequelae in
neonatal meningitis have been associated with deficit in learning and mem-
ory due to prominent neuronal injury or damage in the two brain structures,
namely the cortex and the hippocampus (Bifrare et  al. 2003; Leib et  al.
2003; Blaser 2011; Barichello et al. 2013a, 2013b). Sepsis is defined as the
systemic response to infection. The infection manifests two or more clini-
cal symptoms, including changes in body temperature (>38˚C or <36˚C),
heart rate (>90 beats/min), respiratory rate (>20 breaths/min) or partial CO2
(<32 mm Hg), and white blood cell (>12,000/cu  mm or <4,000/cu  mm).
Severe sepsis condition causes hypotension and multiple organ dysfunc-
tions. Septic shock is the sepsis-induced hypotension, which is unresponsive
to adequate fluid resuscitation, associated with abnormalities (Bone et al.
1992; Markiewski 2008). As soon as the infection occurs, it deteriorates the
neonatal health, and the neonate develops septic shock even before the
identification of the cause (Segura-Cervantes 2016).

Generally, the immune system (i.e., humoral and cellular immunity and
phagocytic function) of neonates are immature; thus, they are prone to
bacterial infections. Due to immature immune system functioning, neo-
nates are particularly susceptible to infection and can lead to chronic
sequelae such as deafness, blindness, cerebral palsy, and other neuro-
development disorders. Newborn babies, especially who are premature
and have very-low birth weight are vulnerable for any infection. The rapid
change in epidemiology of meningitis and sepsis due to immunization prac-
tices makes it more challenging to develop new antibiotics. Also preterm
infants will be lacking enough immunoglobulins transplacentally derived
from their mother (Markiewski et al. 2008; Agrawal and Nadel 2011). The
introduction of conjugate vaccines has virtually controlled the epidemiology
 Neonatal Bacterial Infection     149

of meningitis among neonates. Yet, average rates of 0.1–0.4 incidence have

been reported per 1000 live births. The prevalence of the disease is higher
in preterm with very-low birth weight and chronically hospitalized infants.
In diseased victims, it causes severe injury and even causes death in one-
fifth of infected population (Bogaert et al. 2004; Gordan 2017). Globally,
one million deaths have been occurring every year due to neonatal sepsis;
this accounts for 26% of all neonatal deaths (Miller et al. 2013; Tzialla et al.
2015). The possible routes of infection of sepsis are respiratory, abdominal,
and urogenital tracks (Markiewski et al. 2008).

In this chapter, we focus on the causative bacteria and its pathophysiology

in neonatal meningitis and sepsis.

6.2  Causative organisms of meningitis and sepsis

The common bacterial pathogens that cause meningitis are Group

B Streptococcus (GBS), which is also called Streptococcus agalac-
tiae (50%), Escherichia coli (20%), Listeria monocytogenes (5%–10%),
Streptococcus pneumonia (6%), Staphylococcus aureus, Neisseria men-
ingitidis, Haemophilus influenza type b (Hib), other Enterobacteriaceae
(e.g., Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp.,
and Proteus mirabilis (Harvey et al. 1999; Bonacorsi 2005; Agrawal and
Nadel 2011; Van Sorge and Doran 2012; Barichello et al. 2013a, 2013b;
Ramakrishnan 2013) (Table 6.1).

Bacteria involved in sepsis infection are GBS (43%), E. coli (15.5%–29%),

coagulase-negative Staphylococcus (CoNS; 42%–45%), S. aureus (10%–13%),
S. pneumonia, Klebsiella spp., Pseudomonas aeruginosa, L. monocytogenes
and other Enterobacteriaceae like Cirtobacter, Serratia, and Enterobacter
(Markiewski 2008; Heath and Okike 2010; Miller 2013; Camacho-Gonzalez
et al. 2013; Shah and Padbury 2014; Tzialla et al. 2015) (Table 6.1).

6.3 Etiology

The etiology of meningitis and sepsis includes several factors like infec-
tious pathogens, pathophysiology, and virulence factors used by pathogen,
host-pathogen interaction, host immune response, and evading strategies
adapted by pathogen/factors and age of the host. Not to mention that
exposure to invasive procedures during delivery, neonates are often han-
dled with medical facilities (e.g., iatrogenic devices, tracheal cuff balloons in
ventilation); contaminated neonatal intensive care environment, parenteral
150    Pocket Guide to Bacterial Infections

Table 6.1  Organisms Associated with Early and Late Onset of Meningitis
and Sepsis
Disease
Condition Type Organism Occurrence
Group B Streptococcus/ Both Early- & Late-onset
Streptococcus agalactiae meningitis
Gram Listeria monocytogenes Both Early- & Late-onset
positive meningitis
Streptococcus pneumoniae Late-onset meningitis
Staphylococcus aureus Late-onset meningitis
Escherichia coli K1 Both Early- & Late-onset
meningitis
Meningitis
Neisseria meningitidis Late-onset meningitis
Haemophilus influenza Late-onset meningitis
Gram type b
negative Other enterobacteriaceae Late-onset meningitis
e.g., Klebsiella spp,
Enterobacter spp, Citrobacter
spp, Serratia spp, Proteus
mirabilis
Group B Streptococcus/ Early-onset sepsis
Streptococcus agalactiae
Coagulase-negative Late-onset sepsis
Gram
Staphylococcus
positive
Staphylococcus aureus Late-onset sepsis
Streptococcus pneumoniae Late-onset sepsis
Listeria monocytogenes Late-onset sepsis
Sepsis
Escherichia coli K1 Both Early- & Late-onset
sepsis
Pseudomonas aeruginosa Late-onset sepsis
Gram
Klebsiella spp Late-onset sepsis
negative
Other enterobacteriaceae Late-onset sepsis
Cirtobacter spp, Serratia spp
and Enterobacter spp
Source: Tzialla, C. et al., Clin. Chim. Acta, 451, 71–77, 2015; Barichello, T. et al., J.
Med. Microbiol., 62, 1781–1789, 2013a; Bonacorsi, S. and Bingen, E., Int. J. Med.
Microbiol., 295, 373–381, 2005.

nutrition, and powdered infant formula are the major causes. The sequential
steps of bacterial infection to the progress of meningitis are invasion, pro-
liferation, and colonization in central nervous system (CNS), inflammation,
and acute brain damage (Cahill 2008; Grandgirard 2010; Ramakrishnan
2013; Pai et al. 2015).
 Neonatal Bacterial Infection     151

6.4  Pathophysiology of bacterial meningitis

Generally, the bacterial pathogens colonize in the mucosal epithelium take

hematogenous route from nasopharynx (especially in neonates and chil-
dren) and infect the subarachnoid cavity. Later in further stages, the infec-
tion reaches the meninges. Mostly, neonatal meningitis is associated with
sepsis (Koedel et al. 2010).

6.4.1 Infection

6.4.1.1 Mode of transmission – Neonates are thought to acquire

meningitis in two possible ways, such as vertical and horizontal mode
of transmission. Vertical transmission that occurs during the first week
of life is the early onset of the disease, and it gets transmitted from
mother to her newborn either in utero or during the passage through
birth canal. Some studies show that the disease presents itself within
24 hours to first 7 days of life. On the other hand, horizontal transmis-
sion is also called the late onset of disease that occurs after the first
week of life; it can be acquired by nosocomial infections. It can manifest
from 7th day of life to the 89th day of life (Grandgirard and Leib 2010;
Puopolo and Baker 2012; Barichello et al. 2013a). Frequently, the former
are more prone to sepsis, pneumonia, respiratory disease, and menin-
gitis and the latter to fever and meningitis (Zaidi et al. 2009; Heath and
Okike 2010; Camacho-Gonzalez 2013; Shah and Padbury 2014; Tzialla
et al. 2015) (Table 6.2).

Table 6.2  Risk Factors for Neonatal Meningitis/Sepsis

Risk Factors for Neonatal Meningitis/Sepsis
Early-Onset Late-Onset
Maternal colonization Damage in primary defense (skin and
mucosa)
Chrorioamnionitis Invasive procedures
Premature rupture of membranes Contaminated catheters
Maternal urinary tract infection Infant powder formula
Preterm delivery <37 weeks Very low birth weight </= 2500 g
High gravidity Prolonged hospital stay
Lack of prenatal care
Urinary tract infection
Intrapartum fever
Source: Camacho-Gonzalez, A. et al., Pediatr. Clin. North Am., 60, 367–389, 2013;
Wynn, J.L. and Wong, H.R. Clin. Perinatol., 37, 439–479, 2010.
152    Pocket Guide to Bacterial Infections

6.4.1.2  Colonization and invasion – The colonization of nasopharynx,

oropharynx, or sinuses is the critical prerequisite for the development of
bacterial meningitis. The virulent type of the pathogen determines the
mechanisms behind invasion and colonization. Adhesion to the host tissue
is the essential and initial step in bacterial infection. Bacterial adhesions
extend to attach, colonize, and evade most of the defensive mechanisms
of the host. The appendage or cell surface components that facilitate
bacterial adhesion are called adhesins. Among gram-negative pathogens,
pili or fimbriae are the major bacterial adhesive surface structures. They
are mostly filamentous and can pass through or between capsular layers.
Type 1, P-pili, type IV, and curli are the well-characterized pilus structures
in gram-negative bacteria. On the other hand, in gram-positive bacteria,
surface proteins, polysaccharides, or lipids displayed the adhesive func-
tions (Kline 2009; Grandgirard and Leib 2010). Adhesins helps menin-
geal pathogens to successfully colonize the mucosal epithelial surfaces
(i.e., respiratory, intestinal, and genitourinary tract) of the host (Kc et al.
2017). E. coli K1, GBS, and N. meningitidis use pili or fibrils to initiate
binding in brain microvasculature endothelial cells (BMEC). Occasionally,
the toxins released by the bacteria can lower the blood–brain barrier and
facilitate penetration. Penetration of the mucosal barrier by the bacteria
through or between the epithelial cells is very specific to most of the bac-
teria (Van Sorge and Doran 2012).

6.4.2 Bacteremia

Bacteremia is defined as the presence of bacteria in the bloodstream. It may

be transient, intermittent, or continuous. When bacteria cross the muco-
sal barrier, it gains entry into the systemic circulation. The polysaccharide
capsule helps in the survival of the pathogen in bloodstream of host by
rendering resistance against host responses like complement-mediated lysis
and phagocytosis by polymorphonuclear leukocytes and macrophages.
Moreover, the survival of the internalized bacteria can also be enhanced
by the inactivation of secretory IgA by the specific bacteria endopeptidase
(Grandgirard and Leib 2010; Pai et al. 2015).

6.4.3  CNS invasion

6.4.3.1  Bacterial entry into CNS – Bacteria enters the CNS either tran-
scellularly or paracellularly, through cerebral vasculature (i.e., blood–brain
or blood–choroid barrier). The blood–brain barrier acts as a structural and
functional barrier of brain. It helps to maintain the homeostasis inside
the brain. It is composed of specialized BMEC composed of pericytes,
astrocytes, and a basal membrane. It helps to prevent and protect the
 Neonatal Bacterial Infection     153

brain from harmful substances found in the bloodstream (i.e., these cells
keeps a checkpoint and infilters the blood proteins and cells). The blood–
brain barrier can also provide necessary nutrients to the brain and make
it an immune-privileged organ (Dejana et al. 1995; Pulzova et al. 2009).
Additionally, it also possesses an adherens junction (AJ) and tight junction
(TJ). AJs are dependent on the interaction between the cytoplasmic tail of
the components and TJs are composed of membrane proteins (Schulze and
Firth 1993; Wolburg and Lippoldt 2002). The critical function of AJs and
TJs are to regulate the permeability of blood–brain barrier. Breakdown or
any disruption in the blood–barrier is the hallmark characteristic in patho-
physiology of bacterial meningitis. Interestingly, GBS and S. pneumoniae
disturb the blood–brain barrier by producing a pore-forming toxin (Van
Sorge and Doran 2012).

Bacteria that cross the blood–brain barrier in live condition called neuro-
tropic pathogens where they form complex interplay with host receptors
on the surface endothelial cells of the brain. For the traversal, they require
the use receptors like endoplasmin, CD46, the 37  kDa laminin, and the
platelet-activating factor. It has been shown that L. monocytogenes cross
the blood–brain barrier by transmigration of L. monocytogenes-infected
monocytes, which is called a Trojan horse mechanism (Kim 2010). When
the pathogen enters CNS, it leads to the activation of endothelium, inflam-
matory, and proinflammatory cytokine. Rather than the pathogen itself,
the immune response of the host can cause major neurological damages.
For instance, during an infection, the blood–brain barrier becomes more
permeable and allows the influx of leukocytes. This influx of a large number
of leukocytes leads to edema within the cerebrum and swelling of meningi-
tis. Though, the mechanism of how the pathogen crosses the blood–brain
barrier is poorly understood, several studies have reported that the first
step could be the destruction of the endothelial cell layers with the help of
pneumolysin (PLY), which subsequently disrupts the tight junctions in the
blood–brain barrier during traversal and makes a way for pathogen to cross
the blood–brain barrier by transcytosis (Kim 2008; Grandgirard and Leib
2010; Barichello et al. 2013a; Lovino 2013).

6.4.3.2  Survival of pathogens in CNS – The CNS compartment serves

as favorable environment to pathogens when compared to the blood-
stream because they get benefitted and can rapidly multiply inside the
CNS. Additionally, they also penetrate brain, spinal cord, and the Virchow-
Robin space along the vessels. In the CNS, particularly in the subarachnoid
space, there is limited host defense because of the anatomical design of the
blood–brain barrier and less access to the cerebrospinal fluid (CSF).
154    Pocket Guide to Bacterial Infections

• The subarachnoid space does not have a completely organized drainage

by the lymphatic system (Johnston et al. 2004).
• Absence of the complement factors of soluble pattern recognition recep-
tors (PRRs) help in recognition of bacteria and in phagocytosis (Dujardin
et al. 1985; Stahel et al. 1997).
• The systemic circulation contains more blood components that are pre-
vented from crossing the blood–brain barrier to enter the CSF (Pachter
et al. 2003).
• Generally CSF contains anti-inflammatory and immunosuppressors that
actively suppresses immune reactivity (Niederkorn 2006).

6.4.3.3 Bacterial multiplication in CSF – However, the macrophages

and dendritic cells are present in the tissues lining the CSF, namely, lepto-
meninges, choroid plexus, and perivascular spaces that function as senti-
nel cells. Sentinel cells through PRRs help to recognize the bacteria in CSF.
Some PRRs expressed can act as a macrophage scavenger receptor, the
mannose receptor, and complement receptors. They bind to the bacteria,
thus mediating their internalization by phagocytes. While in brain, toll-like
receptors (TLRs) are present in the immunocompetent cells. These make the
CSF more susceptible and suppress the immune reactivity. When bacteria
invade the CSF, they can easily proliferate and spread throughout the brain
without any hindrance (Grandgirard and Leib 2010; Koedel et al. 2010). The
bacterial concentration in infected CSF can reach a similar value to the bac-
terial concentration of the broth-culture in vitro (Small et al. 1986).

6.4.3.4  Host response to invasion – During inflammation, both microg-

lia and astrocytes get activated, and their morphology changes in a process
called astrogliosis. Microglia interacts with neurons and astrocytes to neu-
tralize infections as quickly as possible and acts as a first immune defense
against infections. Additionally, macrophages can phagocytose the patho-
gens present in the brain and spinal cord and present them to T cells. It can
also travel to the site of infection and release proinflammatory cytokines.
Astrocytes (astroglia) are star-shaped abundant cells found in the brain
(Lovino et al. 2013).

Neurological damage is not only caused by viable bacteria but also by sub-
capsular components. Moreover, the unsheathed pathogens that are at
stationary phase or damaged by the antibiotic treatment undergo autoly-
sis. It concomitantly results in the release of the bacterial components like
peptidoglycan, teichoic acid, lipoteichoic acid (LTA), bacterial DNA, PLY, and
proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α),
interleukin 1 beta (IL-1β) and interleukin 8 (IL-8), free radicals, and excitatory
amino acids (Schmidt et al. 1999; Koedel et al. 2010).
 Neonatal Bacterial Infection     155

Table 6.3  TLR Dependant Activation and Bacterial Components

Bacterial
TLR Components Neurodegeneration Reference
2 Peptodoglycan Hirschfeld et al. (1999),
Han et al. (2003),
Schröeder et al. (2003)
2 Lipoteichoic acid Hirschfeld et al. (1999),
Han et al. (2003),
Neuronal loss and Schröeder et al. (2003)
4 Pneumolysin damage, Apoptosis Poltorak et al. (1998),
Malley et al. (2003)
1/2 Lipoprotein Barichello et al. (2013a)
4 Lipopolysaccharide Poltorak et al. (1998),
Malley et al. (2003)
5 Flagellin Hayashi et al. (2001)

During meningitis, TLRs play the key inflammatory mediators that trigger
immune response and promote proinflammatory cytokines production,
which finally causes neuronal damage (Amor et al. 2010). On the cell surface,
TLRs that have been identified are TLR1, 2, 4, 5, 6, and 10, which facilitate to
identify the subcapsular bacterial components; additionally, TLR3, 4, 7, 8, and
9 trigger signaling cascades. It has been reported that TLR2 along with TLR1
and CD14  recognize LTA, lipoproteins, and peptidoglycan (Hirschfeld et  al.
1999; Han et al. 2003; Schröeder et al. 2003), wherein lipopolysaccharides
(LPS) and PLY can be engaged by TLR4 (Poltorak et  al. 1998; Malley et  al.
2003) and bacterial flagellin can be recognized by TLR5 (Hayashi et al. 2001)
(Table 6.3). TLRs activate mitogen activated protein kinase (MAPK) and
nuclear factor κB (NF-κB). These findings suggest that TLRs have to combine
to activate inflammatory response. The individuals with meningitis show gen-
eral characteristics of high levels of proinflammatory cytokines like IL-1β, IL-6,
and TNF-α in CSF (Dunne and O’Neill 2003; Mogensen et al. 2006).

6.5  Pathophysiology of neonatal sepsis

Neonatal sepsis is the growth of microorganism in the blood of a newborn

with systemic signs of infection and hemodynamic compromise (Ganatra
et  al. 2010; Tosson and Speer 2011). Neonates with sepsis may progress
to septic shock where the progressions of the infection have not been
stopped; multiple organ damage can occur and results in death (Wynn and
Wong 2010).
156    Pocket Guide to Bacterial Infections

6.5.1 Infection

6.5.1.1  Onset of sepsis – Neonatal sepsis is a blood infection that contin-

ues to be the common cause of mortality in neonates. Infection during early
stages is termed early-onset sepsis (EOS), which is transmitted prenatally
or during time of birth from mother (i.e., occurs within 72 hours to first
week of life), whereas the infection that occurs after 1 week is termed late-
onset sepsis (LOS), which is considered to be acquired from the environ-
ment sources (i.e., occurs after 1 week of life) (Miller et al. 2013; Shah and
Padbury 2014; Tzialla et al. 2015).

6.5.1.2  Immune system of neonatal sepsis – Among all age groups, neo-
nates are susceptible to infectious pathogens because of the developmental
immunodeficiencies, and suboptimally functioning of innate immune sys-
tem, hyporesponsive mononuclear phagocytes to physiologic and patho-
logic signaling mechanisms. In addition, preterm neonates have very low
IgG levels because maternal IgG passively transfers across the placenta dur-
ing pregnancy. Low serum levels will hinder opsonization. Deficiency in cel-
lular immunity decreases the production of proinflammatory cytokines like
interferons IL-12/IL-23 and IL-18, and this weakens immune system, which
in turn fails to fight against the infection (Zaidi 2009).

6.5.1.3  Factors that cause sepsis – During the antenatal, intrapartum,

and neonatal period, intrinsic and extrinsic factors include the risk of sep-
sis faced in developing countries. Intrinsic factors in the developing world
are premature births, intrauterine growth restriction, and prolonged rup-
ture of membranes during pregnancy, maternal peripartum or intrapar-
tum infections, rectovaginal colonization with GBS, and chorioamnionitis.
Extrinsic factors include no care taken during pregnancy and insanitary
delivery practices. Based on the World Health Organization (WHO) sta-
tistical report on birth practices in developing countries, only 35% of the
births are handled by professionals. This results in the unhygienic practices
like using unsterile instruments and lack of sterile environment during
delivery and postnatal period. Early-onset sepsis also includes factors like
socioeconomic and cultural practices and the burden of poverty (Ganatra
et al. 2010).

Infections are also transmitted by the multiuse syringes, surgical tools,

and failure to follow aseptic techniques, especially during invasive pro-
cedures. Further it has been reported that the use of intravenous ali-
mentation and prolonged use of mechanical ventilation/central venous
catheters for the neonates has increased the risk of sepsis and septic
shock (Wynn and Wong 2010) (Table 6.2).
 Neonatal Bacterial Infection     157

6.5.1.4  Microbiology of sepsis and septic shock – Sepsis is caused pre-

dominantly by bacteria, but viruses also can cause it; in both cases, there is
a high mortality. Among neonates who are septic, gram-positive bacteria
are prevalent, whereas gram-negative infection accounts for 38% of septic
shock and 62.5% sepsis mortality. Sepsis is dominated by GBS and coag-
ulase-negative Staphylococcus (Hyde et  al. 2002; Kermorvant-Duchemin
et al. 2008; Chen et al. 2015).

6.5.2  Molecular and cellular events

6.5.2.1  Signaling of PRRs, PAMPs, and DAMPs – Once the local bar-
rier has been compromised, the pathogen is recognized by body’s first
line of defense (i.e., local immune sentinel cells). This recognition has been
done with the help of activated PRRs and TLRs (Kawai and Akira 2009).
TLRs are capable of recognizing extracellular and intracellular pathogens,
and they are present in multiple cell types. LPS is one of the products that
is termed a pathogen-associated molecular patterns (PAMPs). This is the
mediator for systemic inflammation, septic shock, and multiple organ fail-
ure (Trinchieri and Sher 2007; Kumagai et al. 2008; Rittirsch et al. 2008).
LPS signals through TLR4 in conjugation with cell surface adaptor proteins
CD14 and myeloid differentiation MD265, and another PAMP (i.e., LTA) sig-
nals through TLR2. Generally, more than one TLR are activated at the same
time for the specific host-pathogen interaction. In neonates, during both
gram-positive and -negative infection, there is upregulation of TLR2 and
TLR4 mRNA in leukocytes (Zhang et al. 2007).

Along with PAMPs, TLRs is also activated by damage or danger associ-

ated molecular patterns (DAMPs). High-mobility group box-1 (HMGB-1) is
a DAMP produced by endothelial cells and macrophages. It gets activated
with LPS/TNF-α and signals through TLR2/TLR4/receptor for advanced
glycation end products (RAGE). HMGB-1 helps in cytokine production,
coagulation activation and recruitment of neutrophil, and involves the dis-
ruption of epithelial junctions in the gut. This HMGB-1 advances the dis-
ease progression of sepsis to septic shock (Mullins et al. 2004; Lotze et al.
2005; Zhang et  al. 2007; Rittirsch et  al. 2008; Van Zoelen et  al. 2009);
in addition, heat shock proteins (Hsps) and uric acid are DAMPs that are
also involved in the physiological process of septic shock. In pediatric septic
shock, there were elevated levels of Hsp60 and Hsp70 and are associated
to death. Although uric acid can increase cytokine production and dendritic
cell stimulation, it acts as an anti-oxidant. It is found in lower levels in septic
neonates (Batra et al. 2000; Pack et al. 2005; Wheeler et al. 2005, 2007;
Kapoor et al. 2006; Kono and Rock 2008).
158    Pocket Guide to Bacterial Infections

Nucleotide binding oligomerization domain (NOD), a NLR (i.e., NOD-like

receptor) is an intercellular non-TLR PRRs. Peptidoglycan of gram-positive
bacteria in the cytosol is detected by NLR. After the pathogens are engaged,
immune response is initiated by PRRs and through MAPKNF-κB, proin-
flammatory cytokines are produced (Trinchieri and Sher 2007; Wynn and
Wong 2010).

6.5.2.2 Proinflammatory response – In response to infection, innate

inflammatory response is amplified following the stimulation of PRR and
cytokine production. It has been reported that during sepsis and septic shock,
proinflammatory cytokines such as IL-1β, IL-6, IL-8, IL-2, IL-18, INF-γ, and TNF-
α are elevated (Ng 2004). Septic neonates have decreased cytokine produc-
tion compared to adults who are septic, which is another reason why the
neonates are at risk (less IL-1β, IL-12, INF-γ, and TNF-α). These proinflamma-
tory cytokines activate endothelial cells, which result in increased expression
of cell adhesion molecules (CAMs) and chemokines. CAMs include soluble
ICAM, VCAM, L-selectin, P-selectin, and E-selectin, which helps in leukocyte
recruitment and diapedesis (Dollneer et al. 2001; Kourtis et al. 2003; Turunen
et al. 2005; Figueras-Aloy 2007). Leukocyte recruitment is an important key
inflammatory response step because it prevents the propagation of systemic
infection (Sullivan et  al. 2002). Generally, in sepsis condition, chemokines
­(IL-18  and interferon gamma-induced protein 10  [IP-10] can be used as a
sensitive marker during infection), and other chemo-attractive molecules like
complement proteins (C3a and C5a), host defense proteins (i.e., cathelicidins
and defensins) and invaded bacterial components are increased (Ng 2004;
Kingsmore 2008; Rittirsch 2008; Zaidi 2009; Wynn and Wong 2010).

6.5.2.3 Interplay between anti-inflammatory and proinflammatory

responses – Sometimes in the immune mechanism, there is inappropriate
response that results in systemic inflammatory response syndrome (SIRS).
When there is no homeostasis in the inflammatory system, it ends up either
in excessive immunity or meager immunity. The stage when former immunity
occurs is termed as cytokine storm, and latter immunity is called immune
paralysis. Apoptosis occur in both of these stages. To prevent this during infec-
tion, anti-inflammatory products (i.e., IL-4, IL-10, IL-11, IL-13, and TGF-β) are
produced simultaneously to counter the activity of proinflammatory cytokines.
Using receptor antagonists like TNFR2, sIL-6R, sIL2, and IL-1ra, the activity of
proinflammatory mediators can be monitored along with anti-inflammatory
cytokines (Sriskandan and Altmann 2008; Wynn and Wong 2010).

6.5.2.4  Complement system – The complement system is like an alarm

system, which gets activated when pathogens get invaded or also during
tissue damage. Complement activation pathway is the one that is the central
 Neonatal Bacterial Infection     159

constituent of immune system. It orchestrates and connects the inflamma-

tory responses of innate and adaptive immunity. They help in coagulation,
cytokine production, and activation of lymphocytes. Three properties of
complement are opsonization, phagocytosis, and lysis of pathogens, which
are events associated with immune response to infection. Three pathways
are involved: classical, lectin, and alternative (Notarangelo et  al. 1984;
Wolach et al. 1997).

Generally, neonates have lower complement proteins and poor response

in opsonization mediated by complement. The contribution of the comple-
ment system to the invasion of pathogens appears to be contradictory in
some cases. The C3 component helps in the dysfunctioning of the effector
which leads to an increase in septic infection (Quezado et al. 1994). It has
been studied that C3a and C5a are up-regulated during sepsis infection;
therefore, inhibition of C5a signaling can prevent from further infection
(Ward 2008). Apparently, during early stages, the proinflammatory sub-
stance of complement pathway helps in the widespread of sepsis, but in
the later stages, C5a along with sepsis leads to systemic infection and finally
multiple organ failure.

During sepsis, there is up-regulation of complement-mediated activation of

leukocytes along with cell surface receptors (i.e., CR1 and CR3). The receptor
for C3b is CR1 (i.e., CR1–C3b) and helps in opsonization. C5a-C5aR facilitates
the redistribution of blood flow, inflammation, aggregation of platelets, and
release of reactive oxygen intermediates (ROI) (Snyderman and Goetzl 1981;
Vogt 1986). CR3 is involved in adhesion of leukocytes, phagocytosis, and
the recognition of pathogen and its products. Unfortunately, in neonates,
the activation of CR3 on neutrophils is less compared to adults, which is the
another reason infants get infected. Similarly, in neonates, C5aR is deficient,
which eliminates the possibility to respond to C5a. Thus, it leads to increase
in infection (Markiewski et al. 2008; Wynn and Wong 2010).

6.6  Concluding remarks

Despite advancements in treatment, sepsis and meningitis remain the most

infectious diseases. Neonatal systemic bacterial infection (i.e., meningitis
and sepsis) causes burden of mortality in some cases, and in survivors, it
lasts as long-term morbidity in newborns. The pathophysiology of these
diseases is complex and multifactorial. A key rhetorical question to neona-
tologists, neurobiologists, and obstetricians is how much the fetus or a neo-
nate can withstand the inflammatory stress during infection and whether
there is time until the infection has been identified to start medication.
160    Pocket Guide to Bacterial Infections

To address these queries, there is a need to recognize authentic biomarkers

for meningitis and sepsis. This may contribute to control several neonatal
incidences of meningitis and sepsis.

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7
Bacterial Infections of
the Oral Cavity
Bacterial Profile, Diagnostic
Characteristics, and
Treatment Strategies
P. S. Manoharan and Praveen Rajesh

Contents

7.1 Introduction 168

7.2 Morphological attributes of the structures in the oral cavity 169
7.2.1 Tooth or the dental apparatus 169
7.2.2 Periodontal apparatus 170
7.2.3 Junctional epithelium 170
7.2.4 The oral mucosa 171
7.3 Bacteria of dental caries and periodontal diseases 172
7.4 Mechanism of colonization and adherence of bacteria 173
7.5 Microorganisms in dental caries 176
7.5.1 Streptococcus mutans 176
7.5.2 Lactobacilli 177
7.5.3 Actinomyces 177
7.6 Microorganisms of periodontal infections 177
7.7 Pathogenesis of dental caries and periodontal diseases 180
7.7.1 Dental caries 180
7.7.1.1 Remineralization–demineralization cycle 182
7.7.1.2 Zones of infection 182
7.7.1.3 Root caries 183
7.7.1.4 Intraradicular infections 184
7.7.1.5 Extraradicular infection 184
7.7.2 Periodontal disease 185
7.8 Diagnostic characteristics 186
7.8.1 Dental caries 186
7.8.2 Periodontitis 187

167
168    Pocket Guide to Bacterial Infections

7.9 Treatment strategies 189

7.9.1 Dental caries 189
7.9.2 Endodontic treatment 189
7.9.3 Periodontal disease management 190
7.10 Oral health and systemic diseases and conditions 192
7.10.1 Reversal of paradigm 192
7.11 Miscellaneous: Diseases and their possible associations 194
7.12 Future perspectives 195
References 197

7.1 Introduction

The oral hygiene status, pathogenicity of a microorganisms, and host

response influence the general oral health and overall well-being of an
individual. There are various commensals present in the mouth and throat.
An understanding of the normal commensals in the oral environment, its
symbiosis, and interaction is needed for the healthcare provider. It helps to
appreciate pathogenesis of a microorganism and understand the nature of
a disease (Gendron et al., 2000).

Bacterial infections contribute to a major deal in the dental, oral, and gen-
eral health of the individual. Dental caries and periodontal disease are the
most common diseases of the oral cavity. The disease-causing microorgan-
isms seem to exhibit a definite site specific pathogenicity. Streptococcus
mutans—a caries-producing microorganism causes lesion only when on the
tooth structure. Lactobacillus acidophilus is commonly seen in deep carious
lesions. Aggregatibacter actinomycetemcomitans and Porphyromonas gin-
givalis, which are associated with periodontal infections when present on
enamel structure, was not found to be cariogenic. But species particular to
caries or periodontal disease are not isolated, although associations were
present (Aas et al., 2005). The complexity of the oral environment demands
the study of pathogens to be carried out as a consortium of microorgan-
isms. Culture-independent molecular techniques, site, and subject specific-
ity of the microorganisms seem to play a major role in isolating the etiology
and understanding the behavior of the bacterial infections (Loesche et al.,
1992). Periodontal disease is not a universal phenomenon. It is surprising
that severe forms of this disease affect a group of population who are
abnormally susceptible (Genco and Borgnakke, 2013).

The oral cavity being the abode to diverse microbiota was found to influ-
ence the systemic health of the individual. Lifestyle, diet, habitat, and
personal habits have been found to influence the oral microbiome. These
 Bacterial Infections of the Oral Cavity     169

complexities have opened new avenues for translatory research. The course
of systemic diseases and conditions were also found to be altered by oral
infections. This reversal of paradigm has also directed the healthcare pro-
vider to consider the oral health status in handling other medical conditions.

This chapter provides an overview on the present trend in understanding

the nature and behavior of various pathogens of common dental and oral
diseases. Moreover, an insight on the applied aspects for the healthcare pro-
vider in handling patients who present with such infections is also discussed.

7.2 Morphological attributes of the structures

in the oral cavity

7.2.1  Tooth or the dental apparatus

The uniqueness of the tooth begins with the description of it being partly
embedded in the hard connective tissue, the bone, and part of it open
to the oral environment (Figure 7.1). The surface is covered by the hard-
est substance in the human body—the enamel. The surface of the coronal
tooth structure is not smooth and well rounded, but present with eleva-
tions called cusps and depressions called pits and fissures (Figure 7.2). The
second layer from the enamel is the dentin, which is composed of tubular
structures that travel down into the living pulpal tissue layer. These also pro-
vide channels for microorganisms to percolate if the dentin is exposed. The
enamel does not extend to the root structure. The root dentin is covered
with cementum all over the surface. The nature of disease and progression

ENAMEL
DENTIN PIT AND
PULP FISSURE
ENAMEL
CEMENTUM
PERIODONTAL
LIGAMENT
ALVEOLAR BONE

APICAL
FORAMEN
(a) (b)

Figure 7.1  Longitudinal section of the tooth and supporting apparatus. (a)
Longitudinal section of the tooth showing various layers and the periodontal appa-
ratus; (b) caries in the pits and fissures.
170    Pocket Guide to Bacterial Infections

Figure 7.2  Tooth structure. Pits and fissures of a molar tooth—susceptibility to

­caries. Arrow indicates hypocalificed area on a smooth surface which may break-
down further.

of disease is different in a smooth surface compared to that of pit and fis-

sure of the enamel. The type of bacteria that harbor the tooth also depends
on the various locations mentioned previously.

7.2.2  Periodontal apparatus

The root of the tooth is embedded in the alveolar bone of the maxilla and
mandible. The cementum is a hard tenacious layer that covers the root sur-
face, which is 15–150 microns thick. The tooth is anchored to the alveolar
bone through periodontal ligament, a fibro-epithelial tissue layer that is sen-
sitive to pressure stimuli, but at the same time protective for the tooth by
acting as a cushion against microtrauma. The periodontal ligament is seen
extending from the cementum of the root to the cortical plate of the alveolar
bone (supporting bone surrounding the tooth structure of maxilla and man-
dible). The thin cortical plate aids in attachment to the periodontal ligaments,
which is described as a lamina dura and is a radiopaque line in radiographs.

7.2.3  Junctional epithelium

The attachment of the soft tissue, gingiva, to the tooth is through junctional
epithelium, which can be considered a weak zone and is prone for infection
(Figure 7.3). The harbored microorganisms and its products proximal to this
area can ingress to the supporting periodontal apparatus. Another point of
entry is through the apical foramen present at the end of the tooth where the
pulpal nerves and blood supply enters. The course of the disease and nature
of treatment is completely different, depending on the pathway of infection.
 Bacterial Infections of the Oral Cavity     171

(a) (b) (c) (d)

Figure 7.3  Junctional epithelium in health and disease. (a) In health. The instrument
probed shows normal depth of sulcus—yellow marking; (b) plaque deposits harbor-
ing microorganisms; (c) bacteria and its toxins infliterating the connective tissue,
breakdown of junctional epithelium; (d) deepening of sulcus. The instrument probed
shows periodontal pocket—red marking.

The epithelial component, which is described as the attachment appara-

tus, has been extensively studied (Schroeder and Listgarten, 2003). It is the
most active yet sensitive area of tooth structure. A high turnover of cells are
noticed in this junction and under continuous shedding process as new
cells are formed. The junctional epithelium is seen at the base of the sulcus,
15 to 30-cell-layer thickness and is found attached partly to the enamel at
the neck region of the tooth and partly over the cementum. Plaque deposits
that harbor bacteria at this junction can be potentially harmful. The subgin-
gival aggregation of plaque forms a niche from which the disease process is
aggravated by further accumulation of plaque and other debris.

7.2.4  The oral mucosa

The mucosa covering the oral cavity is described as stratified squamous
epithelium. Some areas like the gingiva palate are keratinized. The cheek
part of the lip and floor of the mouth are covered by nonkeratinized epi-
thelium. The tongue has epithelium modified to specialized mucosa and
papilla, which lodge taste buds. Candida infections are more common on
the tongue than bacterial infections. Reports of bacterial infections are seen
with tongue piercing. Autoimmune desquamative diseases, such as lichen
planus and pemphigus vulgaris, can occur in relation to mucosa tongue
and cheek. Viral infections, like herpetic gingivostomatitis and ulcerative
gingivitis, are more common than bacterial in the gingival area. However,
bacterial superinfection was found to be associated in most of the cases.
Superinfection can also be observed in simple traumatic to recurrent
172    Pocket Guide to Bacterial Infections

aphthous ulcers. Bacterial colonies are also seen in salivary films adhering
to mucosa of lip cheek and tongue. However, their active role in producing
illness as an entity by itself is not recognized.

7.3  Bacteria of dental caries and periodontal diseases

The oral environment is flooded with more than 108 bacteria, which amount
to 400 of the 700 species or phenotypes in the oral cavity. There is a chance to
harbor at least 150 of the bacterial species at any point in a lifetime. Many spe-
cies have not been isolated, but their role in producing oral infections is obscure.
This microbiota can be seen adherent to the surface of the tooth mucosa and
tongue surface as biofilms. In the context to the oral cavity, the bacterial depos-
its can be termed dental plaque or bacterial plaque. The pathogenesis can be
attributed to the irritants that form as a result of bacterial colonization, and
it follows a carefully choreographed pathway (Socransky et al., 1998). Acids,
endotoxins, antigens from the bacteria, and the chemical mediators released
in response to these products decide the course of the disease. The outcome is
based on the balance between pathogenicity of the bacteria and the response
of the host to the irritants. The degeneration of the tooth, epithelium, peri-
odontal ligament, and bone happens, and it becomes progressively severe, if
the disease process is not controlled in the initial stage.

Bacterial plaque are colorless and transparent when they form. With time, the
presence of chromogenic bacteria imparts color to the plaque. Long-standing
plaque gets mineralized to a hard tenacious deposit called the calculus, which
can be creamish white, dark yellow. or even brown. Heavy deposits are seen
adjacent to salivary gland excretory ducts (i.e., lingual aspect of lower anterior
teeth and buccal aspect of maxillary molars). Plaque, when calcified from the
minerals in the saliva and blood, is called calculus. The calculus present above
the marginal gingiva on the tooth is called supragingival calculus and below
the marginal gingival is called subgingival calculus. Subgingival calculus are
not visible unlike supragingival deposits. They can be found only with tactile
exploration with a diagnostic instrument like an explorer and might get noted
in radiographs. The time required for a supragingival calculus to form is 2
weeks with 80% of mineralization. Mature deposit formation may take sev-
eral months to years. The subgingival calculus should be regarded a secondary
product following infection and should not be treated as if it was the primary
etiology. Calculus deposits can occur in any rough surface at a faster rate.
The demineralized rough surface enamel by the acid products of plaque is
prone to calculus deposition. Rough surfaces of restorations, fixed or remov-
able dentures, implants, and improper contours may also lead to deposition
of plaque and calculus. The tooth or replacement structures lose their surface
 Bacterial Infections of the Oral Cavity     173

smoothness. The crystals of calculus penetrate the surface and are virtually
locked to the tooth. In the root surfaces the covering layer, cementum, gets
an irregular surface, and deposits may be seen that are extremely difficult to
remove. The nature of such a plaque was found to be similar to that of the
deposit that was noticed on the teeth.

The relationship of microorganism to the disease is not clearly understood

because there are number of other criteria related such as association,
elimination, response of the host, virulence factors, animal model stud-
ies, and assessment of risk. All these criteria put together contribute to
the “weight of evidence” and would lead to specific diagnosis. The con-
sensus report from World Workshop in Periodontology have concluded
that A. actinomycetemcomitans and Tannerella forsythia as periodontal
pathogens. Classification of all the types of periodontal diseases was also
put forth (Armitage, 1999). Table 7.1 shows the weight of evidence for
the three common periodontal pathogens mentioned above (Lindhe et al.,
2008).

Spirochetes are seen in some wet mount preparations with stains specific to
Spirochetes and dark field microscopy in destructive periodontitis. Fusiform
bacteria was also found frequently isolated with destructive periodontitis
and also in a kind of diphtheria like pseudomembranous infection—acute
necrotizing ulcerative gingivitis [Vincent in 1889]. Streptococci was also
found to be commonly isolated from supragingival plaque.

7.4  Mechanism of colonization and adherence of bacteria

Plaque biofilm is seen as a firmly adherent mass that develops soon after
immersion into a fluid media. The mouth is constantly bathed in saliva, and
the adsorption of the contents of the saliva to form a matrix takes place in
a sequential manner.

The first step is the formation of a conditioning film called acquired pellicle
(Phase I). The film contains glycoproteins (i.e., mucins) and antibodies from the
saliva. This acts as a matrix to harbor the microorganisms. Primary colonization
is mainly by facultative anaerobic gram-positive cocci. In the first 24  hours,
the plaque consists mainly of Streptococcus sanguis. The bacteria along with
polymorphonuclear leucocytes and epithelial cells adhere to the tooth surface.
Early deposits can be easily removed. The surface energy is altered, which leads
to the adhesion of bacteria on to the tooth. Loose bacteria (i.e., planktonic)
slowly transform to a consolidated adherence (Gibbons and Houte, 1975). A
typical corncob appearance of plaque is because of adherence of cocci on a
filamentous bacteria (Listgarten et al., 1973).
Table 7.1  Weight of Evidence Table
Bacteria Association Elimination Host Response Virulence Factors Animal studies
A. Localised aggressive or Therapy Antibody in Endotoxin, Leucotoxin, epitheliotoxin, Gnotobiotic rats
Actinomycet­ prepubertal periodontitis successful serum or saliva of collagenase, fibroblast inhibiting factor, showed positive
emcomitans adult periodontal lesions Recurrent Localised aggressive bone resorption inducing factor, cytokines disease presentation
Healthy, edentulous lesions periodontitits, from macrophages, cytolethal distending Caused subcutaneous
sites, gingivitis species seen Chronic toxin[apoptosis], neutrophil function abscess in mice
periodontitis modified, immunoglobulin degradation
P. gingivalis Periodontitis aggressive Therapy Antibody in Collagenase, endotoxin, proteolytic trypsin Gnotobiotic rats,
forms, attachment loss successful serum or saliva of like activity, fibnrinolysin, hemolysin, sheep, monkeys and
and bone loss Reccurrent various forms of proteases including gingipain, dog experiments
lesions periodontitis phospholipase A, Fibroblast inhibiting showed positive
species seen factor, hydrogen sulphide, ammonia, fatty disease presentation
174    Pocket Guide to Bacterial Infections

acids, neutrophil modifying factors, Negative impact of

cytokine production from host cells, immunization on
migration of Polymorophonuclear disease in
lympocytes, immunoglobulin degradation experimental animals
T. forsythia Periodontitis Therapy Antibody in Endotoxin, fatty acids, methylglyoxal Increased ligature
Actively progressing lesion, successful serum production, cytokine production, induced periodontitis
Abscess, refractory Reccurrent More antibody apoptosis and invasion of epithelial cells in dogs
periodontitis, periodontal lesions in refractory Gnotobiotic rats
pockets, attachment and species seen periodontitis showed positive
bone loss disease presentation
health, gingivitis

Source: Lindhe, J. et al., Clinical Periodontology and Implant Dentistry, Blackwell Munksgaard, Oxford, UK, 2008.
 Bacterial Infections of the Oral Cavity     175

Bacteria adheres by means of fimbria and extracellular polymeric substances.

Slowly gram-positive rods dominate, especially the Actinomyces species. The
gram-negative bacteria (i.e., Veillonella and Fusobacterium) find difficulty in
attaching to the pellicle. The receptors found on the surface of the gram-
positive rods and streptococci allow adhesion of the gram-negative bacteria
(Phase II). Once attached, they rapidly grow and multiply (Phase III) and syn-
thesize cellular membrane component (Phase IV). As the thickness increases,
they present with deeper layers under an anaerobic environment. The bacte-
ria are deprived of the nutrition from the dietary products, so they produce
enzymes that can breakdown host macromolecules of the periodontium into
peptides and amino acids. The destructive disease onset thus occurs and can
cause irreversible damage to the periodontal apparatus. The matrix material
seen between the microorganisms amounts to almost 25% of the plaque
volume. This intermicrobial matrix can be either filamentous or granular in
appearance. The polysaccharides are mainly levans (i.e., fructans from the
diet) and glucans (i.e., dextran and mutans). The stability of intercellular
matrix is offered by mutans. Uncharacterized lipids also are seen in the matrix,
which are mainly the lipopolysaccharide toxins from gram-negative bacteria.

This adherent biofilm becomes complex and resistant as it gains more shear
strength against removal. The bacterial deposits become site specific, and
a distinct composition are noted on various surfaces (e.g., pit and fissure/
smooth surface plaque, shallow/deep crevices of sulcus). The time period
of the lesions also determines the type of bacteria. Established lesions are
presented with more gram-negative bacteria and anaerobes. Around 109
bacteria can be found on a supragingival plaque in a tooth. Millions to
billions of bacteria may be found to colonize the subgingival crevice in dis-
eased and even in healthy oral cavity. The inverse relationship between the
development of disease and the presence of microorganisms has opened
up avenues of research in this field.

Some of the commonly isolated pathogens in relation to periodontal disease

are A. (Actinobacillus)actinomycetemcomitans, T. forsythia, Campylobacter
rectus, Eubacterium nodatum, Fusobacterium nucleatum, Peptostreptococcus
micros, P. gingivalis, Prevotella intermedia, Prevotella nigrescens, Streptococcus
intermedius, and Treponema spp. (Socransky et al., 1998).

The subgingival plaque presents with all the aforementioned characteristics

along with a special entity—the cuticle, which is presumed to be secretory
product of the epithelial cells (Schroeder and Listgarten, 2003). Apart from
gram-positive and -negative anaerobes, Spirochetes and other flagellated
bacteria are also seen.
176    Pocket Guide to Bacterial Infections

7.5  Microorganisms in dental caries

No single microorganism or group of microorganisms may be held solely

responsible for the initiation and progression of caries. Thus, it is less
important to remember the specific names of the bacteria than to under-
stand their potential role in the biofilm community in health and disease.
However, few bacteria, which have a very strong association with caries
pathogenesis, need to be discussed.

Several studies have shown Streptococcus mutans to have strong associ-

ation with initiation of dental caries. (Loesche et  al., 1975; Loesche and
Straffon, 1979). They are mainly isolated from the surface of teeth. They
synthesize a variety of extracellular polysaccharides, including water-soluble
and nonwater-soluble glucan and fructan from sucrose. These polysaccha-
rides promote bacterial colonization and are key virulence factors in the
formation of dental caries

Lactobacilli were more common from sites with soft and necrotic dentin
(Schüpbach et al., 1996). Their role is still important in contributing to the
proteolytic and collagenolytic activities associated with breakdown of the
tissue. Recently, molecular analyses have been performed on carious dentin
to describe more fully the microbial diversity of lesions. A range of lacto-
bacilli, comprising 50% of the species detected, were identified (Nadkarni
et al., 2004; Chhour et al., 2005).

Root surface caries have a different environment, and hence, a differ-

ent flora can be expected. Gram-positive filamentous bacteria, especially
Actinomyces spp., play a key role in root-surface caries. A close relationship
between A. odontolyticus and the earliest stages of enamel demineraliza-
tion and the progression of small caries lesions have been reported. The
most important human pathogen is A. israelii.

7.5.1  Streptococcus mutans

S. mutans is a general term for several closely related species of Streptococcus

originally described as different serotypes of S. mutans. The name mutans
results from its frequent transition from coccal phase to coccobacillary
phase. They are facultative anaerobes. The specific name, S. mutans, is now
limited to human isolates previously belonging to serotypes c, e, and f. This
is the most common species isolated from human dental biofilms. The next
most prevalent species is S. sobrinus (previously S. mutans serotypes d and
g), which has the potential to produce more acids from sucrose compared
to S. mutans.
 Bacterial Infections of the Oral Cavity     177

Mutans streptococci are catalase-negative, gram-positive spherical or oval

cocci occurring in pairs and chains; 0.7–0.9  µm in diameter. Colonies of
S.  mutans grown on blood agar after 48-hour anaerobic incubation are
either regular and smooth or irregular, hard, and sticky. The diameter of
colonies is 0.5–1.0 mm. Zones of α- or γ-hemolysis can be observed around
colonies (Xuedong and Yuqing, 2015).

7.5.2 Lactobacilli

Lactobacilli are gram-positive, with cocco-bacillary forms (mostly bacillary),

alpha or nonhemolytic, facultative anaerobes. These organisms ferment
carbohydrates to form acids (i.e., they are acidogenic) and can survive well
in acidic milieu (i.e., they are aciduric); they may be homofermentative or
heterofermentative. A special selective medium, tomato juice agar (pH 5.0),
promotes the growth of lactobacilli while suppressing other bacteria. The
question as to whether they are present in carious lesions because they
prefer the acidic environment or whether they generate an acidic milieu and
destroy the tooth enamel, has been debated for years—the classic “chicken
and egg” argument (Samaranayake, 2006).

7.5.3  Actinomyces

Most Actinomyces are soil organisms, the potentially pathogenic species are
commensals of the mouth in humans and animals. A number of Actinomyces
species are isolated from the oral cavity. These include A. israelii, A. gerenc-
seriae, A. odontolyticus, A. naeslundii (genospecies I and 2), A. myeri, and
A. georgiae. They are gram-positive filamentous branching rods that are
non-motile, non-sporing, and non-acid-fast. Clumps of the organisms can
be seen as yellowish “sulfur granules” in pus discharging from sinus tracts,
or the granules can be squeezed out of the lesions. The colonies resemble
breadcrumbs or the surface of “molar” teeth. Sulfur granules in lesions are
a clue to their presence. When possible, these granules should be crushed,
gram-stained, observed for gram-positive and branchin filaments, and also
cultured in preference to pus (Samaranayake, 2006).

7.6  Microorganisms of periodontal infections

Some common bacteria and their associations with diseases are mentioned
as discussed in various studies and reports given in the literature. In 1968,
Sigmund Socransky classified bacterial species into colored complexes.
Bleeding-associated bacteria were named red complex. P. gingivalis, T. for-
sythiam, and T. denticola are the bacteria in this group, and they are also
associated in deep periodontal pockets (Haffajee et al., 2006). The second is
178    Pocket Guide to Bacterial Infections

the orange complex which constitutes F. nucelatum, P. intermedia, P. nigres-

cens, P. micros, S. constellatus, E. nodatum, C. showae, C. gracilus, and C.
rectus. P. intermedia along with P. gingivalis was found to occur in deep
pockets. The third yellow complex include bacteria such as S. sanguis,
S. oralis, S. mitis, S. gordonii, and S. intermedius. Capnocytophaga spp.,
Eikkenella corrodens, Campylobacter concisus. and Actinobacillus actino-
mycetemcomitans [serotype a] form the fourth green complex. Veillonella
parvula and Actinomyces odontolyticus form the fifth purple complex.

A. actinomycetemcomitans was found to adhere the oral epithelium

through a protein adhesion Aae, which binds to a carbohydrate receptors
on buccal epithelial cells. It was also found that it migrates to gingival area
and to the tooth surface. The bacterial fimbriae with extracellular carbohy-
drate polymer attaches on hard tooth surface. Sometimes coaggregation to
other bacteria can be seen as a means of colonization. Among the six sero-
types of A. actinomycetemcomitans a, b, c, d, e, and f and sero types a, b,
and c are globally dominant (Brigido et al., 2014). Serotype b was found to
be associated with localized aggressive periodontitis in American subjects
(Zambon, 1985) and serotype a with chronic periodontitis. Sero type c was
more commonly associated with periodontally healthy subjects. The isola-
tion of such stereotypes is not similar in other countries (i.e., Korea, Finland,
Japan, Taiwan, and Brazil) (Brigido et al., 2014). There is an association of this
bacteria in localized aggressive periodontitis; progressive lesions are proved
by many researchers in human and animal models. Increased leukotoxin and
cytolethal distending toxin was also reported. Previous reports stated that a
certain subset of species was not seen in samples of subgingival plaque and
did not show enhanced antibody response (Loesche et al., 1992; Moore and
Moore, 1994).

P. gingivalis are observed as black colonies in culture on blood agar plates.

Burdon in 1928 has grouped such black/brown colony-forming organisms
as Bacteroides melanogenicum. Deeper pockets have shown presence of
serotype I, whereas II and III are isolated commonly from shallow pock-
ets and gingivitis (Dahlen, 1993) In the Bacteroides group, the P. gingiva-
lis group are saccharolytic (i.e., breaking down sugar products to produce
energy), whereas P. intermedia and P. melanogenica are sacharolytic.
P.  gingivalis group of microorganisms are found to produce collagenase,
hydrogen sulfide, indole, proteases (including those which breakdown
immunoglobulins), gingipain, hemolysins, endotoxin, ammonia, and fatty
acids. P. gingivalis inhibits polymorphonuclear leucocytes to diapedise
through epithelium, and it has been demonstrated to influence production
and destruction of cytokines by mammalian cells. Its absence in healthy
 Bacterial Infections of the Oral Cavity     179

gum or gingivitis and its presence in an advanced and destructive form

of periodontal disease where there is increased pocket depth indicate its
aggressiveness, behavior, and nutritional source. Immunization using hem-
agglutinin B, capsular polysaccharide, heat shock protein, gingipain R, and
the active sites of RgpA and Kgp proteinases have found to reduce alveolar
bone loss in mouse models. And their action was found to raise the level of
specific antibodies to P. gingivalis antigens.

In 1979, Tanner in 1979 described T. forsythia as “fusiform” Bacteroides. It

requires two weeks to form minute colonies. It is a gram-negative anaero-
bic, spindle-shaped, highly pleomorphic rod. It is seen commonly in subgin-
gival sites as reported by Socransky et al. (1998). In electron microscopy, this
bacteria is found to exhibit a serrated S layer, which was found to mediate
hemagglutination, adhesion of epithelial cells, and demonstrate subcutane-
ous abscess formation in rats. This layer was found to possess two types of
glycoproteins of different molecular mass. Along with macrophages and
epithelial cells, if these bacteria are cultured, it was found to express proin-
flammatory cytokines, chemokines, PGE2, and MMP9 as reported by Bodet
et al. (2007).

Higher counts and prevalence of this microorganism was found in individ-

uals with different forms of periodontitis than in healthy individuals (Yang
et al., 2016; Haffagee et al., 2009; van Winkelhoff et al., 2016). Presence
of this microorganism was also demonstrated in progressing active peri-
odontal lesions and dormant lesions (Dzink et  al., 1989). Machtei et  al.
(1992) stated that the subjects with bone loss, attachment loss, and tooth
loss have harbored T. forsythia compared to subjects who are free of dis-
ease. Elimination of this microorganism was found to reduce in counts
after scaling and root planning. T. forsythia was also found to be the most
commonly isolated from periodontal pocket epithelial cells (Dibart et al.,
1998).

Spirochetes are gram-negative, highly motile microorganisms commonly seen

in destructive periodontal diseases. The etiology of acute necrotizing ulcerative
gingivitis was strongly related to spirochetes because large numbers were seen
in tissue specimens after biopsy of the infected sites (Listgarten and Socransky,
1964; Listgarten, 1965). Their role is not clear in other forms of periodontitis.
However, their occurrence in subgingival plaque, deeper pockets, and severe
disease states were demonstrated (Moore and Moore, 1994; Haffagee et al.,
2006) Among the virulence factors, dentilisin, coded by prtP gene, affects
many protein substrates like fibronectin, fibrinogen, and laminin (Ishihara,
2010).
180    Pocket Guide to Bacterial Infections

P. intermedia and P. nigrescens are part of the black pigmented Bacteroides

group of microorganisms that are gram-negative, anaerobic rods. Elevated
levels are seen in specimens isolated in acute necrotizing ulcerative gingivitis
(Loesche et al., 1982). Some authors have reported elevated levels in some
forms of periodontitis (Moore and Moore, 1994; Papapanou 1996).

7.7  Pathogenesis of dental caries and periodontal diseases

7.7.1  Dental caries

Dental caries is the localized destruction of susceptible dental hard tissues by

acidic by-products from bacterial fermentation of dietary carbohydrates. Its
pathogenesis is best explained by the Keyes tetrad, which states that dental
caries can occur on a susceptible host only when both acidogenic bacteria and
a carbohydrate-rich diet are present for a definite period of time (Figure 7.4).

The carious process begins with the tenaciously adhered biofilm on the
tooth surface expressing acidic metabolites that dissolve the hydroxyapa-
tite of enamel. S. mutans are largely responsible for initiating caries due
to their exceptional ability to produce extracellular polysaccharides, which
are the building blocks for the biofilm. They can also metabolize a number
of sugars and glycosides such as glucose fructose sucrose, lactose, galac-
tose, mannose, cellobiose, glucosides, trehalose, maltose group of sugar
alcohol. S. mutans synthesize intracellular glucose and sucrose polysaccha-
rides and also produce mutacins (bacteriocins) what is considered to be

Figure 7.4  Keyes Tetrad—Interplay of four factors for caries pathogenesis.

 Bacterial Infections of the Oral Cavity     181

important factor in the colonization and establishment in the biofilm; these

biofilms produce acids from carbohydrates that result in caries (Karpinski
and Szkaradkiewicz, 2013).

Gradual demineralization of involved dental hard tissues will be active

because of the disturbance in physiologic equilibrium in the biofilm of den-
tal plaque covering the affected site. The bacteria of the genus lactobacillus
are the important factor for further progression of dental caries, especially
in dentin (Figure 7.5). The caries process consists of three reversible stages.
The microflora ions on sound enamel surface contains mainly nonmutants
streptococci and actinomyces in which acidification is mild and infrequent;
this is compatible with the equilibrium of the demineralization and remin-
eralization balance or shifts the mineral balance toward net mineral gain
(dynamic stability stage) when sugar is supplied frequently; acidification
becomes moderate and frequent, which may enhance the acidogenicity
and acidurance of the non-mutans adaptively. These microbial acids induce
the adaptation and selection process and may over time shift the deminer-
alization and remineralization balance toward net mineral loss, leading to
progression of dental caries (i.e., acidogenic stage). Under severe and pro-
longed conditions, more aciduric bacteria becomes dominant through aci-
duce selection by temporary acid impairment and acid inhibition of growth
(i.e., aciduric stage).

The organic components of dentin do not allow acids to cause caries like
lesions in dentin, rather the appearance of such lesion requires activation
of enzymatic proteolysis by metallo-proteinase (MMPs) in mildly acidic

Figure 7.5  Caries progression showing breakdown of tooth structure with

discoloration.
182    Pocket Guide to Bacterial Infections

SUSCEPTIBLE TOOTH SURFACE

FORMATION OF BIOFILM AND MOLECULAR DEPOSITS

ACID PRODUCTION AND CHANGES IN PH

SHIFT IN DYNAMIC EQUILIBRIUM OF MINERALS

DISSOLUTION OF MINERALS

INITIATION OF DENTAL CARIES

Figure 7.6  Flow chart showing development of dental caries.

condition that leads to cavity formation. It constitutes a large family

of calcium-zinc dependent endo-peptidase that contributes extracellular
matrix degradation (Femiano et al., 2016) (Figure 7.6).

7.7.1.1 Remineralization–demineralization cycle – The etiological

agents of dental caries are S. mutans and lactobacillus. These bacteria can
generate acids from fermentable carbohydrates. The level of infection that
causes damage to the teeth depends on multiple factors. Acids generated
by these bacteria diffuse into the subsurface of the enamel and can dissolve
calcium and phosphate, which then diffuse out of the tooth; this process is
called demineralization.

The presence of healthy saliva will provide buffers so that calcium and
phosphate will reverse the early damage caused by demineralization.
­
Due  to  this repair, the salivary buffers must first neutralize the acid and
stop the demineralization. When the calcium and phosphate concentration
becomes higher outside the tooth than inside, they will diffuse back into
the tooth. This reversal process is called remineralization.

7.7.1.2  Zones of infection – In 1935, Fish attempted to explain the patho-

genesis of periradicular inflammation using an animal model (Figure 7.7).
He inoculated bacteria into the pulp chamber of virgin teeth and observed
the following items.
 Bacterial Infections of the Oral Cavity     183

Zone of infection

Zone of contamination

Zone of irritation

Zone of stimulation

Figure 7.7  Zones of periapical lesion.

A central zone containing pus, polymorphonuclear leukocytes (PMNL) and

microorganism are known as a zones of infection.

Zone of contamination are seen around the central zone characterized by

the presences of PMNLs and macrophages. The toxins from the bacteria are
diluted, and the inflammatory fluid contains antibacterial action. There was
an empty lacunae created by autolysis of dead bone cells.

Zones of irritation contain macrophages, lymphocytes, and plasma cells,

mediators of inflammation and immune system collagen matrix degraded
by the macrophages; the bone is resorbed, leaving a small space. This space
is filled with granulomatous tissue, and this prevents the spread of necrosis
and initiates repair because it contains new capillaries and fibroblast russell
bodies.

Zone of stimulation is the peripheral zone characterized by fibroblast

and osteoblast. The fibroblasts lay down collage fibers, creating a wall of
defense around the zone of irritation on which osteoblasts reside to deposit
new bone in irregular fashion.

7.7.1.3  Root caries – Root caries, which is seen apical to the cemento-
enamel junction, is generally characterized by a soft active progressive
lesion. Etiology is gingival recession as a result of periodontitis, age,
radiation therapy, xero-stomia, abrasion, erosion, abfraction, primary
root caries, recurrent caries, or diabetes. Microorganism responsible for
root caries are S. mutants, Lactobacillus, and Actinobacillus. Root car-
ies is mostly seen when there is periodontal ligament attachment loss.
184    Pocket Guide to Bacterial Infections

This exposes the root surface to the oral environment, which leads to
infiltration of caries. They appear as a white or discolored, irregular, and
progressive lesion (Zaremba et al., 2006).

Histologically, dental caries have been characterized as having two distinct

layers: the outer zone (stains with caries detector dyes) where the dentin
is highly demineralized and the collagen is denatured and heavily infected
with bacteria (often referred to as the infected zone), and the inner zone
(does not stain with a caries detector dye) where the dentin is demineralized
but the collagen is intact and minimally infected (often referred to as the
caries affected zone).

7.7.1.4 Intraradicular infections – Endodontic infections have a poly-

microbial nature and obligate anaerobic bacteria conspicuously dominate
the microbacterial factors in primary infections. The endodontic micro-
biota presents a high interindividual variation that can significantly vary
in species diversity and abundance from individual to individual, indi-
cating that apical periodontitis has a heterogeneous etiology, and mul-
tiple bacterial combinations can play role in diverse causation. As the
breadth of bacterial diversity in endodontic infection has been unraveled
by the molecular biology methods, the list of endodontic pathogens has
expanded to include several cultivable and as-yet uncultivated species that
had been underrated by culture-dependent methods. Endodontic bac-
teria are now recognized to belong to 8 of the 12  phyla that have oral
representatives namely firmicutes, bacteroides, spirochetes, fusobacte-
rial, actinobacteria, and proteobacteria. Studies have demonstrated that
predictable disinfection of the root canal system is only achieved after
proper antimicrobial medications are placed in the canals and left therein
between appointments.

7.7.1.5  Extraradicular infection – An apical periodontitis lesion is formed

in response to intraradicular infection and comprises an effective barrier
against the spread of the infection to the alveolar bone and the other body
sites. The most common form of extraradicular infection is the acute apical
abscess characterized by the purulent inflammation in the periapical tissue
in the root canal. Acute abscesses are usually characterized by absence of
over symptoms. Extracellular infection can be dependent on or independent
of the intraradicular infections. Once the intraradicular infection is properly
controlled by root canal treatment or tooth extraction and drainage of pus
is achieved, the extraradicular infection is handled by the host defense and
 Bacterial Infections of the Oral Cavity     185

usually subsides. There are some situations that permit intraradicular bac-
teria to reach the periapical tissue and establish an extraradicular infection.

1. The infection may be a result of direct advance of some bacterial species

that overcome host defenses concentrated near the apical foramen or
that manage to penetrate into the lumen of pocket cyst, which is in direct
communication with the apical foramen.
2. The infection may be due to bacterial persistence in the apical periodon-
titis lesion after remission of acute apical abscess.
3. The infection may be a sequel to apical extrusion of debris during root
canal instrumentation after overinstrumentation. The virulence and the
quantity of the involved bacteria as well as the host ability to deal with
the infection (Figure 7.8).

7.7.2  Periodontal disease

Periodontitis, commonly known as pyorrhea, is a complex disease entity of

the oral cavity, affecting the supporting tissues of the tooth. The disease
process is likely to initiate at the junction where the tooth emerges out into
the oral cavity.

The number of species of pathogenic bacteria present may not be directly

related to the periodontal infections. Even microorganism and site specific-
ity is not directly related to the pathology or course of infection (Socransky
et al., 1987). It was demonstrated that even an otherwise healthy gingiva or

Figure 7.8  Flow chart showing development of pulpal and periapical lesions.
186    Pocket Guide to Bacterial Infections

periodontium could harbor pathogens but still could not establish any kind
of disease pattern. However, the reduction of periodontal pathogens has
been used as a parameter in assessing the positive outcome of periodon-
tal therapy. Fastidious, strict anaerobes may cause initiation of periodontal
disease in a shallow periodontal pocket or progression of disease in a deep
pocket to some extent. The plaque that is accumulated may be supragingi-
val on the crown surface or subgingival in the sulcus or on the tooth surface
root. The nature of microbiota would be different in the aforementioned
two situations, which would be based on the partial pressure of oxygen in
the environment, depth of the periodontal pocket, redox potential, avail-
ability of excretory products from sulcular fluid, and blood products.

7.8  Diagnostic characteristics

7.8.1  Dental caries

Dental caries present as black to black brown discoloration primarily

in pit and fissure. It can be either a cavitated or a noncavitated lesion.
Noncavitated lesions are not so easy to identify because a physical exami-
nation tool like an explorer can push the microorganisms deeper when it is
used. Advanced diagnostic tests are made available to disclose the presence
of lesions. Smooth surface caries occur on cusp tips and tooth surfaces that
may be hypocalcified white spots, which are often marred by the presence
of salivary film.

Diagnosis is made possible by drying the tooth surface and examination

under good light source. Caries can also be diagnosed by radiographs like
intraoral periapical or bitewing radiographs. They present as radiolucent
lesion extending from the deepest part of the fissure in a triangular fashion
with their base at the dentinoenamel junction. Smooth surface caries break
down is also triangular, but their base is at the surface and apex toward
the pulp at the dentinoenamel junction. Diagnosis of caries is important for
planning the treatment.

Cervical caries and root caries can also be seen in individuals who are geri-
atric. Nursing bottle caries are common in bottle-fed infants where the
milk bottle is left in the mouth. Caries are seen in maxillary central incisors.
Rampant caries, yet another entity, presents as caries in the proximal areas
of the teeth, and they seem to spread from one tooth to the other, which is
common in individuals who neglect their oral hygiene or who have reduced
salivary secretion. Reduced salivary secretion is yet another factor for caries.
This may be due to any salivary gland pathology, autoimmune conditions of
 Bacterial Infections of the Oral Cavity     187

salivary glands like Sjogren syndrome, after radiation, use of antihyperten-

sive, antipsychotic, antineurotic drugs, diabetes, salivary duct obstruction,
increased antibiotic usage, and many others.

Caries risk-assessment analysis can be performed using a systematic chart-

ing that includes dietary history, genetic factors, and other local presenta-
tions (Hillman, 2002). Salivary buffer capacity by analyzing the pH of saliva
can tell the risk range from high to low. Such analysis can help the clini-
cian understand the nature of the disease and its severity and to develop
preventive measures to preserve, prevent, and restore the tooth. Dental
caries can progress to pulp and result in pulpal inflammation, which can
be either acute or chronic and is based on the symptoms. It can be classi-
fied as reversible or irreversible pulpitis based on the nature of symptoms.
Irreversible pulpitis needs a root canal. Irreversible pulpitis can progress to
spread the infection beyond the tooth to the surrounding bone where it
results in periapical abscess. The abscess can consolidate to a granuloma or
spread to the surrounding areas and into potential spaces of the face and
oral cavity, resulting in serious infection. Sometimes, the abscess can divert
its course to the path of least resistance and drain to the oral cavity in the
form of a sinus opening. Granuloma can be a chronic presentation or can
progress to a cyst with breakdown of the cells and development of lining of
the cavity filled with fluid. The cyst related to the tooth is termed a radicular
cyst and can either progressively increase in size, expanding to the bone, or
can present as a symptomatic painful swelling if it is infected.

All the chronic destructive lesions like granulomas, cysts, or chronic

abscesses can be diagnosed by only radiographs. Cysts are characterized by
a radiopaque border surrounding a radiolucent lesion, whereas granulomas
and abscesses do not have a radiolucent border. The tooth becomes non
vital in irreversible pulpitis, abscess, granuloma, and cystic lesions.

7.8.2 Periodontitis

Gingivitis, the inflammation of the gingiva, presents with redness, swelling,

bleeding on probing, and altered contour. The inflammation can be either
acute or chronic based on the presentation of symptoms.

Periodontal disease presents as bleeding on probing when it affects the

gingiva and the superficial soft tissues in an acute phase. Several Indices
are used to detect the health of gingiva (i.e., Ramjford’s, Sillness and Loe,
and Loe and Sillness). Noninflammatory enlargement of gingiva may be
as a result of chronic use of certain drugs like phenytoin sodium, nife-
dipine, and certain immunosuppressants. Severe bleeding may be due
188    Pocket Guide to Bacterial Infections

to vitamin  C deficiency (i.e., scurvy) or acute myeloid leukemia. Linear

­erythema of marginal gingiva with bleeding is noted in HIV. Gingival
inflammation can also be present in acute necrotizing ulcerative gingi-
vitis and herpetic gingivostomatitis. Gingival enlargement may be due to
benign or malignant lesions.

Bleeding upon slightest provocation along with clinical signs and symptoms of
surface color and texture absence of stippling will denote reactive inflamma-
tion. Breakdown of junctional epithelium and deepening of sulcus is known as
a periodontal pocket, which is measured using a probing instrument such as
a William’s periodontal probe or Community Periodontal Index for Treatment
Needs (CPITN) probe, which has got a blunt end. Bone loss can be appreciated
with radiographs (i.e., orthopantomography or intra-oral periapical).

Bone-loss patterns commonly observed are craters, both horizontal

and vertical in nature. Bone sounding with deep probing can be done
to perceive the configuration of the defect. Confirmation is done with
radiographs. Treatment varies depending on the type of bone defect.
Periodontal inflammation can progress to destruction of periodontal cells
and accumulation of inflammatory exudate. Periodontal abscess can result
in the destruction of the apical area of the tooth or the lateral surface
of the tooth. Clear diagnosis can be made if the abscess is in the lateral
surface of the tooth and is sensitive to lateral percussion with handle of a
mouth mirror.

Apical periodontitis is often confused with periapical abscess. Clinical

correlation is of utmost importance to diagnose whether it is a periodon-
tal or an endodontic infection. The periodontal abscess can also take
the course similar to periapical abscess. Multiple periodontal abscesses
and weak periodontium are characteristic of diabetes mellitus. Other
diseases like neutrophil dysfunction (Chedak Higashi Syndrome), palmo-
plantar keratosis (Papillon Levfre Syndrome), hypophosphatasia can be
associated with a typical form of periodontitis called as aggressive peri-
odontitis. It can also occur in silos as a separate entity. This periodontitis
is formerly known as juvenile periodontitis and presents as early loss
of teeth and characteristic bone-loss patterns in a young patient in the
molar and incisor areas. Refractory periodontitis is another type of peri-
odontitis where the lesions do not respond to conventional modality of
treatments. Periodontal diseases are also found to be aggravated by the
presence of systemic diseases and vice-versa where it is understood as a
reversal of paradigm in understanding lesions accompanied by systemic
diseases. The clinician’s role is to correlate the findings and treat the
patient appropriately.
 Bacterial Infections of the Oral Cavity     189

7.9  Treatment strategies

7.9.1  Dental caries

The treatment is based on the type of lesion and presenting condition.

The use of nonoperative prevention-oriented strategy for caries manage-

ment is often put aside in favor of restoration placement. One approach is
caries management by risk assessment (CAMBRA). The goal is to determine
if caries is reduced in high-risk patients receiving treatment with a nonop-
erative anti-caries agent. The general approach to active caries should be
preventive treatment like advice to take less sugar, brushing twice daily with
effective fluoride toothpaste, using dental floss, and stimulating saliva by
use of sugar-free gums such as xylitol. When active fissure caries have been
diagnosed, and fissures are with susceptible morphologic characteristics,
sealant with a low-filled resin is indicated.

The operative management of dental caries has traditionally involved

removal of all soft demineralized dentin before filling is placed. This tissue is
heavily infected with bacteria is removed slowly using a slow speed bur or
hand-excavating instruments. There are five basic reasons to place restora-
tions when cavitation occurs as a result of caries:

• To remove infected dentin,

• To protect the pulp and avoid pain,
• To remove the habitat for cariogenic bacteria, and
• To facilitate plaque control.

The primary goal is to regain the lost form, function, and esthetics. In
deep carious lesions, direct or indirect pulp capping procedures are recom-
mended to preserve vitality of pulp.

Deep carious lesions can penetrate the root canal system and cause an array
of problems for the patient. The complex anatomy of the root canal system
forms the ideal playground for microorganisms. They can lodge in the nar-
row crevices and cause periradicular inflammatory responses. This in turn
can cause a sequelae of events

7.9.2  Endodontic treatment

Treatment of a vast majority of endodontic infections include nonsurgical

or surgical root canal. The infected root canals are shaped and cleaned until
the microorganism load is brought down, and they are filled three dimen-
sionally to achieve a hermetic seal.
190    Pocket Guide to Bacterial Infections

7.9.3  Periodontal disease management

Many interesting anecdotes were used previously in the management of

periodontal disease. Pierre Fauchard, a French physician known as the
father of modern dentistry, advocated techniques to remove deposits using
his specially developed instruments. As early as 1929, Rasmussen reported
that ultraviolet light was found to reduce the overall bacterial load, thus
reducing the oral microbiota. Caustic chemicals have been used to control
infections. Some felt that the microorganisms played only a secondary role
in the progression of the disease. They attributed the presence and prog-
ress of infections to the individual’s constitutional defect or a traumatic
occlusion. Early researchers also proposed the role of a mixed group of
microorganisms causing infections. They found patterns that could cause
infections like fuso-spirochetal combination in Vincent disease or Trench
mouth. After the mid-1950s, the role of biofilm or plaque and not just a
single bacteria was found to be causative for periodontal infections. The
treatment strategy also changed accordingly. Plaque was mentioned as a
biomass (i.e., nonspecific plaque hypothesis) irrespective of the pathogenic-
ity of some potential microorganisms causing infections (Theilade, 1986).
Specific plaque hypothesis stated to tackle only the specific bacteria related
to the infections (Loesche and Straffon, 1979). The ability of certain bac-
teria like Actinomycosis viscoses to produce infection in rats free of dis-
ease, and experiments such as those strengthened the theory of specificity
(Figure  7.9). Drug therapy can be delivered in the form of oral antibiot-
ics or local drug delivery in the form of a miniature wafer or chip using
metronidazole and tetracycline. They can be used as adjuvants apart from
active mechanical debridement, removal of granulation tissue, and robust
maintenance.

The cause of infection due to endogenous or exogenous organisms can also

alter the treatment strategy. An opportunistic infection caused by an endog-
enous organism, which may be a normal commensal in oral cavity, may resolve
if the ecologic and immune response is under control, which regulates the
bacterial growth and multiplication (Takahashi, 2011). On the other hand,
the mere presence of some uncommon microorganisms (i.e., exogenous) can

Association of specific pathogen Non specific plaque hypothesis Specific plaque hypothesis
Attempt to identify a pathogen Disease associated with host defects Treatment for causative agent

1880 1900 1930 1960 1990 2000

Golden Age of Microbiology Plaque Control Biofilm

Figure 7.9  Diagramatic representation of various hypothesis.

 Bacterial Infections of the Oral Cavity     191

trigger and complicate the course of infection. In such cases, therapy should
be aimed to prevent exposure and elimination of the microorganisms.

It is also vital to understand the risk factors associated with periodontal

disease. Some are modifiable such as lifestyle factors (e.g., smoking and
alcohol consumption) and some are diseases or conditions like diabetes,
obesity, osteoporosis, metabolic syndrome, low calcium in diet, and vita-
min D. Managing periodontitis includes control of these modifiable risk
factors. Genetics plays a major role in aggressive periodontitis. However,
association of a genetic factor with chronic adult periodontitis remains
unclear.

The most important phase in periodontal therapy is the maintenance phase

where the relapse of the disease can happen after treatment. Motivation
to practice and maintain oral hygiene measures are crucial in the progno-
sis of the disease after treatment. Goal setting, self-monitoring, and plan-
ning are found to be effective in improving oral hygiene-related behavior
in such patients (Newton and Asimakopoulou, 2015). Various psychological
models were proposed to handle periodontal infections after therapy with
behavior modification. In a systematic review, all the models were found
to play some role in the reduction of plaque and the increased motivation
for the initial time period. But no difference was noted at 6 or 12 months
follow-up.

The mechanical removal of biofilm is mandatory rather than trying to fight

against the microorganisms with antimicrobials and antibiotics. Mechanical
debridement can be carried out using hand instruments like scalers. These
instruments are modified to various shapes to access the inaccessible areas.
Machine-driven instrumentation like the use of an ultrasonic vibration in
scaler tips can also effectively remove plaque and tenacious deposits of cal-
culus. The surface of the root is covered by a thin cementum layer. Deposits
on this surface along with the granulation tissue should be removed by
root planing. The diseased cementum should be removed and the soft tis-
sue approximated back, expecting for healing with long junctional epithe-
lium formation. This procedure was also found to reduce the pocket that
resulted because of the disease process.

The instruments mentioned can remove the deposits on the tooth and root
surfaces, thus restoring the periodontium to health. The loss of bone and
the attached epithelium may not be possible to recover to its original state.
With advanced periodontal surgical therapy, guided tissue regeneration
and use of graft and other scaffolds can restore the attachment loss to
some extent.
192    Pocket Guide to Bacterial Infections

Attachment of deposits to the prosthesis intraorally can be mechanically

debrided using the mentioned instrumentation. Fabrication of highly glossy
surfaces and precision in restorations and replacements can prevent bacte-
rial adhesion. As the adage goes, “Prevention is always better than cure.”
Early recognition of disease and timely visits to the dentist can prevent com-
plications associated with the disease progression.

7.10  Oral health and systemic diseases and conditions

Several systemic diseases or conditions can increase the severity of gingivi-

tis and periodontitis. Diabetes, HIV, and some drugs like phenytoin, cyclo-
sporine, immunosuppressants, and nifedipine were found to influence the
prognosis of bacterial infections and gingival health in individuals. Smoking
and mouth breathing may also trigger gingival and periodontal disease and
complicate bacterial infections. Prognosis of patients with periodontitis is
poor in patients with poorly controlled diabetes.

7.10.1  Reversal of paradigm

Specific pathologic conditions of the oral cavity like caries or periodontal

disease can act as foci of infection to trigger systemic conditions elsewhere
in the body. It can affect vital systems like the cardiovascular, renal, and
endocrine systems. Certain bacteria like P. gingivalis is found to increase
the formation of atheroma in blood vessels, causing atherosclerosis and
thereby increasing the risk of myocardial infarction (Humphrey et al., 2008).
It is also shown that increase in bacterial foci of infection can reduce the
insulin uptake by the cells and cause poor glycemic control. Its effect was
also shown to precipitate obesity, preterm low birth-weight infants, glo-
merulonephritis, and bacterial endocarditis.

Dental procedures may carry the bacterial load through the bloodstream
elsewhere and trigger an inflammatory response in certain targeted tissues.
The bacterial endocarditis is an acute condition that can be triggered by an
extraction or a simple invasive procedure in patients with artificial valves.
The bacterial colonies may aggregate in the artificial valves, causing a local
reaction via an inflammatory response and a systemic response (Sen et al.,
2017).

Oral periodontopathic bacteria were found to be aspirated by some

patients, resulting in aspiration pneumonia. An association between poor
oral hygiene and chronic obstructive pulmonary disease was also noted.
Periodontal disease-associated enzymes in saliva were found to modify the
 Bacterial Infections of the Oral Cavity     193

mucosal surface to promote adhesion and microbial colonization by patho-

gens lining the respiratory tract (Scannapieco, 1999).

A report of three unrelated patients with interleukin receptor-associated

kinase (IRAK) deficiency are resistant to pyogenic infections as opposed to
healthy children who developed pyogenic infections (Picard et  al., 2008).
Toll interleukin receptor (TIR) mediates the recruitment of IRAK complex.
This TIR-IRAK signal pathway is found to be important for immunity against
some bacteria, but their role is negligible against other microorganisms. In
a case control study of 150 patients who were pregnant or who were post-
partum revealed that periodontal disease can be significant risk factor of
preterm low birth-weight infants (Messinis et al., 2010).

In a meta-analysis, it was found that with active intervention of periodon-

tal therapy one cannot reduce the incidence of preterm low birth-weight
infants (Polyzos, 2010). However, pregnant women should undergo peri-
odontal screening and avoid any oral foci of infection. They have also men-
tioned that quality trials provide strong evidence of no correlation to exist,
whereas randomized clinical trials have overestimated the effect of treat-
ment (Offenbacher et al., 1996; Khader and Ta'ani, 2005).

A positive association was seen between bacterial infections and respira-

tory infection. In a review, there was a mention of possible mechanism of
pathogenesis like bacterial aspiration (P. gingivalis and A. actinomycetem-
comitans), modification of respiratory mucosa by enzymes of breakdown
products, and cytokines released from periodontal diseases (Scannapieco,
1999). Periodontal and gingival bacterial super infections of individuals
affected with HIV was found to present a similar picture to those of indi-
viduals who were immune suppressed. In a study done on 31 intact teeth
affected by pulp and marginal infections, polymerase chain reaction was
used to isolate pathogens like A. actinomycetemcomitans, B. forsythus,
Eikenella corrodens, F. nucleatum, P. gingivalis, P. intermedia, and T. den-
ticola (Reichart, 2003). The organisms were present in the chronic apical
periodontitis and chronic periodontitis. This was suggestive that any refrac-
tory course of endo-perio lesions may be attributed to the endodontic and
periodontic pathway of infections (Rupf et al., 2000).

Presence of Helicobacter pylori in the oral cavity was noted in patients with
gastric infection. They have also commented that the oral cavity is the first
extragastric reservoir.

Some studies have proven association between bacterial infection and ischemic
cerebrovascular disease (Syrjanen et al., 1989). An extremely rare case of brain
194    Pocket Guide to Bacterial Infections

abscess was found to occur with the causative being P. gingivalis, which was
identified with culture methods. The bacterial source was found to be from the
oral cavity as the patient suffered from recurrent periodontitis (Yoo et al., 2016).

Association of periodontal disease with aortic atheromas was also found.

A systematic review and meta-analysis showed that periodontal disease is
a strong risk factor for ischemic heart disease, which was found to be inde-
pendent of other risk factors (Sen et al., 2017). In a systematic review, an
association was noted between obesity and periodontal status (Haffajee and
Socransky 2009). Although, periodontitis as a risk factor for obesity could
not be established because there were other events that could be associ-
ated. In clinical practice, a high prevalence of periodontal disease can be
noted with individuals who are obese (Chaffee and Weston, 2010).

In another meta-analysis, it was found that Type II diabetes was found to

be associated more with destructive periodontitis, than Type I (Chavvary
et al., 2009).

In a meta-analysis with 456  patients, a positive association was found in

patients with reduction of glycemic index with active periodontal treatment
in patients with diabetes. However, none of the reduction was statistically
significant (Janket et al., 2005).

7.11  Miscellaneous: Diseases and their possible associations

Tonsillitis and pharyngitis are caused by beta hemolytic streptococci. Severe

purulent forms are associated with Staphylococcus. Scarlet fever caused by
streptococci is characterized by reddish diffuse skin rashes of face and neck.
Hemolytic property of streptococci can present as petechiae (small hemor-
rhagic spots) on the palate or strawberry tongue. F. necrophorum was also
isolated in one study. More studies were recommended to confirm their
association (Pallon et al., 2018).

Syphilis caused by Treponema pallidum can present itself as various presen-

tations based on the stages of syphilis. Transmitted through the genito-oral
route commonly, the primary lesion occurs at the site of inoculation (usually
the lip as an ulcer, which is called the chancre). Intraoral presentation of the
ulcer is painful and covered by greyish white membrane. It is a highly infectious
stage and heals usually in 3–4  months. Secondary stage appears 6  weeks
after primary lesion if left untreated. Diffuse eruptions of skin and mucous
membrane are noted as macules. A thick papule may be noted as a greyish
plaque over an ulcerated surface. They are seen commonly in tongue, gin-
giva, and buccal mucosa. Snail track ulcers are characteristic of the secondary
 Bacterial Infections of the Oral Cavity     195

stage. Tertiary stage appears after 6  months. They are not infectious and
may present as perforations of mucosa of oral cavity as a punched-out ulcer
called a gumma. Sloughing of tissues are seen at the base of the ulcer. Active
antibiotic therapy is recommended to treat the infection. Gonorrhea, caused
by Neisseria gonorrhea, is a genitourinary bacterial infection. Transmitted
through the genito-oral route, the oral lesions may be seen in mucosa of the
tongue as red, dry lesions, which are shiny and painful erosive ulcers.

Tuberculosis caused by Mycobacterium tuberculae is characterized by

granulomatous inflammation presenting as a painful nonhealing ulcer in
the intraoral presentation along with other general characteristics. Chronic
active treatment is recommended with antibiotics for a longer period of
time (e.g., up to 12–24  months). Systemic spread is dangerous and gen-
eralized lymphadenopathy with presentation on the skin as “scrofula” is
characteristic of tuberculosis.

Rhinoscleroma, caused by Klebsiella rhinomatosis is a rare bacterial infec-

tion characterized by reactive proliferative nodular presentation of skin and
mucosa of nose lacrimal gland and sinuses. Oral lesions present as ulcer of
palate and attached gingiva. It may also present with lymphadenopathy.

Actinomycosis israelii is found to be associated with cervicofacial skin and

bone lesions or tongue infections with pus collection. Sulfur granules in the
exudate seem to be characteristic of this infection. Long-term high doses of
antibiotics are recommended to handle this infection.

Clostridium tetani can cause tetanus, which presents symptoms like lockjaw
due to a spasm of the masseter muscle. Risus sardonicus—a condition due
to spasm of rhizorius—the grinning muscle is characteristic of this disease.

7.12  Future perspectives

The oral microflora exists as commensal in the oral cavity, which benefits the
host as well. There is a symbiotic relationship between the host and the flora
that may be identified in the disease process as dysbiosis (Marsh et al., 2014).
Identification of factors that can affect the symbiosis, reduction, and elimi-
nation of such factors can be the modality of treatment. Oral antimicro-
bial therapy should be aimed to modulate the activity and growth of oral
pathogens. Silico models on biofilms have shown how sublethal concentra-
tions of agents that mildly fluctuate the pH of the oral environment can
alter the competitiveness of the bacterial colonies. Buffering capacity of the
plaque film can modify to large extent the composition of microorganisms
in the biofilm. Elimination of microorganisms to render a disease-free state
196    Pocket Guide to Bacterial Infections

is almost next to impossible. Maintaining the normal commensals in the

patient’s mouth, deriving their benefits, maintaining the pH, and buffering
capacity of the oral cavity through a holistic approach may prevent dysbiosis.
A holistic approach and systemic thinking have also been proposed by many
others in handling oral infection (He et  al., 2009) Interspecies interaction,
microbial community, and microbial interactions in polymicrobial diseases
may need to be further researched to obtain insight into the etiology of end-
odontic and periodontal infections. The role of bacteria was also questioned
by many authors, which has been reviewed systematically and have shown
that Streptococcus angiosus, Capnocytophaga gingivalis, Prevotella melano-
genica, Bacteroides fragilis, and Streptococcus mitis may have an association
with carcinogenesis. Possible mechanisms like inhibiting apoptosis, activating
cell proliferation, promoting cellular invasion, and inducting chronic inflam-
mation may set a predisposition for carcinogenesis. However, the author
states that in all the studies mentioned, the methodology standardization is
questionable because there is a wide variation in tissue sampling, microbial
profiling, and DNA extraction (Perera et al., 2016).

The human body has coevolved with the microorganisms and has con-
stantly been modified by lifestyle, diet, habits, and psychological factors. At
some point in the state when the balance is lost, the disease process sets in
as the immune mechanisms set in an inflammatory pathway, which leads to
destruction of the tissues. The human body was mentioned by halobiont or
the superorganism where the human being coexist with the commensals by
adaptation and functional integration.

With evolution, S. mutans has developed a mechanism to compete against

other oral species. It has developed defenses against increased oxida-
tive stress and remained resistant to by-products of its own metabolism.
Worldwide promotion of toothbrush and flossing have also made consid-
erable modification in oral environment, making resistant biofilms loaded
with microorganisms which turn pathogenic. Virulence and pathogenic
potential in the recent decades are understood by quorum sensing, which
has opened a new avenue for research. The host substrate are the mucosa
and the teeth. The mucosa sheds continuously and does not provide a sta-
ble home for the microorganisms, whereas the tooth and artificial crown
or replacements and restorations can serve as a stable substrate to harbor
biofilms, which become more complicated with aforementioned factors.

In the recent age, after the launch of Human Microbiome Database and
16s ribosomal RNA gene community profiling, identification of a bacteria
in its original or a variant can be done by simple matching with the avail-
able database. Next-generation sequence (NGS) coding tools can allow a
 Bacterial Infections of the Oral Cavity     197

high-volume study of genetic material in samples to provide better and

specific understanding of oral microbiome.

Treatment should be focused in future in prevention strategies, awareness

of the public in the role of diet, lifestyle, and habits on oral health (Kilian
et al., 2016). Awareness of effective plaque control measures on tooth sur-
faces, restorations, replacements, and implants can also minimize the bac-
terial biofilm from nonshedding surfaces of the oral cavity to an acceptable
level, which is compatible to oral health.

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8
Prognosis and Impact
of Recurrent Uveitis, the
Ophthalmic Infection Caused
by Leptospira spp.
Charles Solomon Akino Mercy and
Kalimuthusamy Natarajaseenivasan

Contents

8.1 Introduction 204

8.2 History 204
8.3 Bacteriology 205
8.3.1 Morphological characteristics 205
8.3.2 Growth condition 206
8.3.3 Cultural characteristics 206
8.3.4 Molecular characteristics 207
8.3.5 Classification 207
8.3.5.1 Serological classification 208
8.3.5.2 Genotypic classification 209
8.4 Etiology 209
8.5 Epidemiology 210
8.6 Infectious cycle 210
8.6.1 Pathogenic mechanism 211
8.6.2 Risk factors 213
8.7 Ocular manifestations 213
8.8 Morbidity and mortality 215
8.9 Complications 215
8.10 Diagnosis 215
8.11 Treatment 217
8.12 Prevention 218
Acknowledgments 219
Financial support 219
References 219

203
204    Pocket Guide to Bacterial Infections

8.1 Introduction

The eye, a functionally and structurally complex organ, can be infected by

the varieties of bacterial, fungal, viral, and parasitic infection. Among this
most often ocular infections occur through bacterium. Ophthalmologists
are still facing difficulties in managing bacterial eye infections. External
and intraocular infections can lead to visual impairments, which is a major
public health problem. Many microorganisms have been implicated in
the pathogenesis of uveitis in humans, including certain bacteria species
of Salmonella, Campylobacter, Shigella, Klebsiella, Yersinia, Chlamydia,
Mycoplasma, Leptospira, Treponema, or Borrelia or viruses such as rubella or
HIV. In equine ophthalmology, numerous microorganisms, such as Brucella
abortus, Borrelia burgdorferi, Streptococcus equi, Rhodococcus equi,
Onchocer cacervicalis, and Toxoplasma gondii, have also been associated
with equine recurrent uveitis (ERU), but the most important pathogen impli-
cated in the pathogenesis of ERU is Leptospira (Verma 2012). Leptospirosis,
which is caused by Leptospira, a spirochete bacterium, is transmitted directly
or indirectly from animals to humans and are reported to frequently infect
the ocular structure, which occurs worldwide but is most common entity
in tropical and subtropical areas. The spectrum of the disease remains
extremely wide, ranging from subclinical infection to a severe multiorgan
failure with high mortality. Ocular manifestation develops in both acute bac-
teremia phases and the second immunologic phases but is mostly noted in
the second phase of illness, and the severity of the infection ranges from
mild self-limiting conditions to those that could be extremely serious and
visually threatening. Leptospiral infected persons frequently develop uveitis,
with ocular manifestations reported in between 3% and 92% and this often
occurring after a year from the initial acute form of leptospirosis. Detection
of ocular manifestations remains underdiagnosed because of the prolonged
symptom-free period and is frequently misdiagnosed due to its nonspecific
symptoms that mimic better-known diseases. Management of patients with
ocular infection may involve nothing more than supportive and palliative
therapy along with aggressive intervention with antimicrobial and anti-
inflammatory agents. This chapter deals with leptospirosis-­associated uveitis
and its prognosis, impact, diagnosis, and prevention strategies.

8.2 History

Initially leptospires were not cultured but have been observed from an autopsy
specimen who died of yellow fever and was named Spiro­cheta inter­
rogans by Stimson (1907). Its contagious nature and microbial origin were
 Leptospiral Recurrent Uveitis      205

proved independently, first in Japan by Inada et al. (1915) (Spiroch aeta ictero-

haemorrhagiae), and soon after in Germany (Spirochaeta icterogenes) by
Uhlenhuth and Fromme (1916). Following detailed microscopical and cultural
observations Noguchi (1918) proposed the name Leptospira, which means
thin spirals.

Leptospiral uveitis was first reported by Adolf Weil (1886) in his original
article and subsequently, several authors found its varying presentation.
The importance of occupation as a risk factor was recognized early during
1917, and rat as a source of human leptospiral infection was discovered sub-
sequently. DukeElder (Duke-Elder and Perkins 1966) reported that among
all uveitis cases, 10% contribute to leptospiral uveitis. European authors
reported an incidence of 10% to 44%, whereas Brand (Brand and Benmoshe
1963) and Heath (Heath et al. 1965) reported figures of 13% and 2% from
Israel and the United States, respectively. In India, leptospirosis was known
to occur for many decades in Andaman Islands.

8.3 Bacteriology

8.3.1  Morphological characteristics

Leptospires belong to the order Spirochaetales, family Leptospiraceae,

genus Leptospira. These bacteria are long thin, about 0.1  µm in diam-
eter by 6–20  µm in length. Electron microscopy shows a cylindrical cell
body (i.e.,  protoplasmic cylinder) wound helically around an axistyle
­(0.01­– 0.02 µm in diameter), which comprises two axial filaments (a spiro-
chetal form of a modified flagellum) inserted subterminally at the extremi-
ties of the cell body, with their free ends directed toward the middle of the
cell (Bharti et al. 2003). They are helical bacteria with tight coils and have
a typical double-membrane structure in which the cytoplasmic membrane
and peptidoglycan cell wall are closely associated and are overlaid by an
outer membrane that contains porins that allow solute exchange between
the periplasmic space and the environment. Within the outer membrane,
the leptospiral lipopolysaccharide (LPS) constitutes the main antigen and
has a composition similar to that of other Gram-negative bacteria with
lower endotoxic activity. It is characterized by active motility due to the
presence of two periplasmic flagella with polar insertions in the periplasmic
space and exhibits two distinct forms of movement, translational and non-
translational. Either or both the ends of the single organism are blended or
hooked. The free living (L. biflexa) and parasitic leptospires (L. interrogans)
are morphologically indistinguishable, although the morphology of individ-
ual isolates varies with subculture in vitro and can be restored by passage
206    Pocket Guide to Bacterial Infections

in hamsters. Both saprophytic and pathogenic leptospires are present in

nature. However, pathogenic leptospires mostly present in renal tubules of
animals, whereas saprophytic leptospires were found to be present in many
types of wet or humid environments.

8.3.2  Growth condition

Leptospires can able to survive in alkaline soil, mud, swamps, streams, riv-
ers, organs, and tissues of live or dead animals and diluted milk. Survival
of pathogenic leptospires in the environment is dependent on several fac-
tors including pH, temperature, and the presence of inhibitory compound.
In general, they are sensitive to dryness, heat, acids, and basic disinfectants
(Mohammed et al. 2011). Leptospires are obligative aerobic with an optimal
growth temperature of 28°C to 30°C, and an optimal pH 7.2 to 7.6 is required
for their growth. Their generation time varies from 7 to 12 hours. They grow
in simple media enriched with vitamins B1, B12, and long-chain fatty acids
are the only organic compounds required for their growth. Long-chain fatty
acids are used as the sole carbon source and are metabolized by β-oxidation.
Free fatty acids cause inherent toxicity and hence must be supplied either
bound to albumin or in a nontoxic esterified form because Leptospira can-
not synthesize fatty acids de novo (Faine et al. 1999). Ammonium salts are
an effective source of cellular nitrogen. Several liquid media enriched with
rabbit serum were described in the past by Noguchi (1919), Fletcher (1928),
Korthof (1932), and Stuart (1946). Currently, the most widely used medium
is based on the oleic acid, bovine serum albumin, and polysorbate (Tween)
medium EMJH. Some strains require the addition of pyruvate or rabbit serum
for initial isolation. In nucleic acid of leptospires purine bases were incor-
porated but not pyrimidine bases. Hence, it is resistant to the antibacterial
activity of the pyrimidine analogue. Therefore, 5-fluorouracil can be used
in the leptospiral growth media to eliminate other contamination sources.
Other antibiotics have been added to media for culture of veterinary speci-
mens, such as gentamicin, nalidixic acid, or rifampicin, in which contamina-
tion is more likely to occur (Adler and De La Pena Moctezuma 2010).

8.3.3  Cultural characteristics

Growth of the Leptospira with a pure culture takes 10 to 14 days, whereas

primary isolates often grow slow and the cultures are retained for up to
13 weeks. Leptopsira can be cultivated in liquid, semi-solid, and solid media.
Liquid media are the most commonly used medium for the maintenance
of leptospires by periodical subculture for the serological diagnosis of infec-
tion and for typing. Semi-solid media can be prepared by incorporating
agar at lower concentration (0.1% to 0.2%), and it is used for the long-term
 Leptospiral Recurrent Uveitis      207

maintenance of the strains. Growth in the semi-solid media is easily visual-

ized as one or more rings form from mm to cm below the surface of the
medium, which is related to the optimum oxygen tension and is known as
a Dinger’s ring or disk (Levett 2001; Natarajaseenivasan et  al., 1996). It is
mainly useful for storing the culture and for long-term storage lyophilization
was preferred, and also it can be stored at −70°C for several months. In solid
medium, colonial morphology is dependent on agar concentration (1%–
1.5%) and serovar; it can be used for cloning the strains, isolating leptospires
from contaminated sources, and for the detection of hemolysin production.

8.3.4  Molecular characteristics

The genome of Leptospira is composed of two circular chromosomes of the

larger replicon, with the sizes various between 3.6 and 4.3 Mb in length; a
smaller chromosome, 277- to 350-kb in size contains a plasmid-like origin
of replication (Ren et al., 2003). However, the genomes size of L. interro-
gans serovar Copenhageni is approximately 4.6 Mb, whereas the genome
of L.  ­borgpetersenii serovar Hardjo is only 3.9 Mb in size. The saprophyte
L. biflexa possesses a third circular replicon of 74 kb, designated p74, not
present in the pathogens. The presence of housekeeping genes on p74,
which have orthologs located on the large chromosome in pathogenic
Leptospira, suggests that p74 is essential for the survival of L. biflexa (Adler
and De La Pena Moctezuma 2010). The guanine plus cytosine (GC) con-
tent is between 35% and 41%. Leptospira contains two sets of 16S and
23SrRNA genes and one set of 5S rRNA. Several insertion sequence (IS)
coding for transposases were found among that IS1533; IS1500 are found in
many serovars; IS1533 has a single open reading frame; and IS1500 has four.
Copy number of IS varies between different serovars and also among same
serovars isolated in different resources. Comparative genomics of the two
pathogenic and one saprophytic species has identified 2052 genes com-
mon to all; the core leptospiral genome is consistent with a common origin
for leptospiral saprophytes and pathogens. Genome comparisons allow the
identification of pathogen-specific genes (Adler and De La Pena Moctezuma
2010). The relationships between the different Leptospira strains provide for
horizontal gene transfer and create in the bacterial population the pool of
genes important for adaptivity to various conditions (Voronina et al. 2014).
The reported genes and their functions are given in Table 8.1.

8.3.5 Classification

The Spirochaetales are an order of bacteria dividing itself into two families:
Spirochaetaceae and Leptospiraceae. The Spirochaetaceae family includes
Treponema types, Serpulina and Borrelia, whereas the Leptospiraceae
208    Pocket Guide to Bacterial Infections

Table 8.1  A Number of Leptospiral Genes Reported with Various Function

Sl. No. Function Corresponding Genes
1. DNA repair rec A
2. Encoding RNA rpoB
polymerase
3. Encoding rRNA Rrs
4. Encoding ribosomal fus, rplC, rplB, rplE, rplF, rplD, rplS, rplP, rplP,
proteins rplN, rplR, rplO, rplQ, rplW, rplV, rplX, rpsC,
rpsQ, rpsM, rpsS, rpsS, rpsN, rpsK, rpsJ, rpsH,
rpsE, rpsD, rpmD, rpmJ, rpmC, adk, rpoA, infA,
secY, tuf
5. Amino acid synthesis proA, proB, proC, leuA, leuB, leuC, leuD
6. Encoding heat shock hsp10, hsp58
proteins
7. Encodes outer lipL21, loa22, ligB, ligA, fecA, cirA, ostA, gspD,
membrane proteins bamA, cirA, ompA, ompL1,omp85, ompL37,
ompL47, ompL54, mcE, fecA, tolC, fadL, lenA,
lenF, lp29, lp30, lp49, lipL32, lipL41, lipL36,
lipL46, tlyC, hbpA, lepA, lruC, lsA21,
8. Encodes flagellar flaA, flaB, flg B–D, flgF, flgG, flgE, flgK, flgL, fliE,
protein fliD, fliK, flgH, fliF, flgI, flhA, flhB,  flhF, fliL, fli
Q-S, motB, motA, fliG, fliM, fliN, fliW and fliH,
bolA, ftsA, ftsH, ftsI, ftsK, ftsW, ftsZ, gldF, gldG
9. Lipopolysaccharide rfb loci
(LPS) synthesis

family includes Leptospira, which is further classified into Leptospira spp.

are of importance because different serovars can exhibit different host
specificities and may not be associated with a particular clinical form of
infection. Classification of Leptospira is based on the expression of the sur-
face exposed epitopes in a mosaic of the lipopolysaccharide (LPS) antigens,
whereas the specificity of epitopes depends on their sugar composition and
orientation (Dikken and Kmety 1978).

8.3.5.1  Serological classification – Basically, Leptospira has been divided

into pathogenic and nonpathogenic (saprophytic) species. Of that patho-
genic species have the potential to cause disease in both animals and
humans whereas saprophytic, the free living, are generally considered as non-
pathogenic. All pathogenic strains classified under L. interrogans (Dikken
and Kmety 1978). Saprophytic strains include L. biflexa. Serovars that are
antigenically related have traditionally been grouped into serogroups; within
the species L. interrogans more than 300 serovars and with L. biflexa more
 Leptospiral Recurrent Uveitis      209

Table 8.2  Classification of Leptospira

Sl. No. Classification Leptospira Genus
1. Pathogenic L. interrogans, L. borgpetersenii, L. kirschneri,
L. noguchii, L. santarosai, L. weilii,
L. alexanderi, L. alstonii, L. kmetyi
2. Intermediated L. broomii, L. fainei, L. inadai, L. licerasiae,
pathogenic L. wolffii
3. Non pathogenic L. biflexa, L. meyeri, L. terpstrae, L. vanthielii,
L. wolbachii, L. yanagawae, L. idonii

than 60 serovars have been recognized. They have no taxonomic standing

but is useful for epidemiological understanding.

8.3.5.2  Genotypic classification – The genus is classified into 12 patho-

genic and 4 saprophytic species, with more than 300 pathogenic serovars
reported. The genomospecies of Leptospira do not correspond to the pre-
vious two species (L. interrogans and L. biflexa), and indeed, pathogenic
and nonpathogenic serovars occur within the same species (Ramadass et al.
1992; Levett 2001). The new genomic classification system has revealed
a pathogenic species, which can contain both pathogenic and nonpatho-
genic serovars as well as intermediate species such as L. meyeri, L. inadai,
and L. fainei. Taxonomy, which is still in progress, classifies Leptospira into
22 species. All recognized species have been classified as pathogenic, inter-
mediate, and non-pathogenic (Fouts et al. 2016) (Table 8.2).

8.4 Etiology

It is endemic in tropical countries because of their geoclimatic and social

conditions, which influence the epidemiological and geographical distribu-
tion of specific entities. The highest prevalence rates remain in tropical,
developing countries where leptospirosis cases are on the rise. This rise
is associated with urban population growth, urban decay, and flooding.
Outbreaks have been related to heavy rainfall in various parts of the world,
including Tamil Nadu, India, (Natarajaseenivasan et  al. 2011; Prabhakaran
et al. 2014) and Salvador, Brazil (Planka and Dean 2000). The incidence of
ocular complications were variable, but this probably reflects the long time
over which they may occur (Rathinam et al. 1996). In the United States, the
incidence was estimated at 3%, whereas in Romania an incidence of 2%
was estimated between 1979 and 1985. However, in abattoir workers with
evidence of recent leptospirosis, the latter authors reported an incidence
of 40%. For example, ERU has various etiologies, with Leptospira infection
210    Pocket Guide to Bacterial Infections

and genetic predisposition being the leading risk factors. In Europe and the
United States, Leptospira serovars Grippotyphosa, Pomona, and Bratislava
have been implicated in pathophysiology of ERU, whereas in the United
Kingdom, Leptospira infection is not a major factor in the etiology of ERU.

8.5 Epidemiology

Leptospirosis has become an endemic disease in the geographical regions

with a mild climate and a high precipitation of flood. It is endemic to areas
of the Caribbean, Central America, South America, Southeast Asia, and
Oceania (Dunay et al. 2016). The number of severe human cases worldwide
is estimated above 500,000. Incidences range from 0.1 to 1/100,000 per
year in temperate climates, 10 to 100/100,000 per year in the humid tropics
to more than 100/100,000 per year during outbreaks and in high-exposure
risk groups (Musso and La Scola 2013). Epidemic outbreaks are common
after rainfall or flooding. Endemic transmission occurs because of factors
such as tropical humid environments and poor sanitation. Urban epidem-
ics are reported in cities throughout the developing world and will likely
intensify as the world’s slum population doubles to two billion by 2030
(Costa et al. 2015). Leptospira persist in entire continent except Antarctica.
Specifically, leptospiral ERU in North America is commonly associated with
the species L. interrogans serovar Pomona type kennewicki, whereas in
Europe, ERU case studies show the implication of species L. kirschneri serovar
Grippotyphosa (Malalana et al. 2015). European investigators reported that
vitreous samples from 78% of ERU clinical cases were positive for Leptospira
spp. Interestingly, 81% of the horses positive with Leptospira showed no
further recurrences after vitrectomy, whereas in 83% of horses negative
with Leptospira further recurrences occurred (Witkowski et  al. 2016). In
the United Kingdom, where both the L. interrogans serovar Pomona and
L. kirschneri serovar Grippotyphosa are found to be rare, but the species
L. interrogans serovar Sejroe are common. L. interrogans serovars Australis,
Canicola, Hardjo, and Icterohaemorrhagiae are less commonly associated
with ERU (Malalana et al. 2015).

8.6  Infectious cycle

Autoimmunity plays an important role in the ocular pathogenesis.

Leptospirosis occurs biphasic in which an acute or septicemic phase lasts
about a week and is characterized by high fevers (39°C–41°C) for 7–9 days
after the initial exposure; a second immune phase occurs during the sec-
ond week of illness, in which the disappearance of the organism from the
 Leptospiral Recurrent Uveitis      211

bloodstream coincides with the appearance of antibodies. After the initial

bacteremia the leptospires are eliminated by the immune system from all
host tissues except from immunologically privileged places like the brain or
eyes, resulting in immunological pathology in the eyes like uveitis (2 days to
4 weeks). Leptospiral antibodies are first detectable in serum 4–8 days after
exposure and may be maintained for at least 7 years. Conjunctival suffusion
is seen in most patients in some series. Uveitis may present weeks, months,
or occasionally years after the acute stage. Chronic visual disturbance can
persist 20  years or more after the acute illness. Chronic uveitis develops
after a few days of unrelenting severe inflammation or following multiple
recurrent episodes of uveitis (Gilger 2016).

8.6.1  Pathogenic mechanism

All pathogenic serovars associated with animal leptospirosis can also be

pathogenic to humans. Transmission to humans occurs through penetra-
tion of the organism into the bloodstream via cuts, skin abrasions, or mucus
membranes. Mechanisms behind the leptospiral infection that trigger the
ocular infections are not clearly understood. Leptospires penetrate mucus
membranes and abraded skin and rapidly gain access to the vascular space,
such as in the ocular structure that causes inflammation and damage to
the small blood vessels and results in vasculitis with leakage and extrava-
sation of cells, hemorrhages which directs to cytotoxicity and cell death,
and exposing the immune system to an immune privileged site (Figure 8.1).
Most of the complications of leptospirosis are associated with localization
of leptospires within these immune privileged sites such as the placenta,
renal tubules, and anterior and posterior chambers of the eyes during the
immune phase (Frellstedt 2009). In early ERU, congestion of uveal vessels
and inflammatory cellular infiltrates are observed. Neutrophils are the first
cells infiltrating the uvea and can result in hypopyon when accumulated in
the anterior chamber. They are soon replaced by lymphocytes, plasma cells,
and macrophages. With time and further recurrence, organization of the
lymphocyte infiltrate is evident. Nodules in the ciliary body and iris are com-
posed of B lymphocytes in the center and T lymphocytes in the periphery
(Deeg 2002). Humoral and cellular immune reaction occur when interphot-
oreceptor-retinoid binding protein (IRBP) and predominance of CD4 + T-cell
infiltrates the affected area. An immune response to cellular retinaldehyde-
binding protein (CRALBP) was detected in a large percentage of ERU cases
(Gilger 2016). Generally, leptospiral LPS stimulated adherence of neutrophils
to endothelial cells and platelets, causing aggregation and suggesting a
role in the development of thrombocytopenia. The other reason may be
that some of the proteins LruA, LruB, and LruC have been reported to cross
 212    Pocket Guide to Bacterial Infections

Figure 8.1  Schematic representation of (a) a normal healthy eye and (b) clinical complications due to infection with Leptospira spp.
 Leptospiral Recurrent Uveitis      213

react between the antibodies present in equine cornea, lens, and retina,
which serve as an initiative factor for pathogenesis of leptospiral-induced
uveitis. Due to the delayed development of uveitis, it is difficult to confirm
a diagnosis of leptospiral uveitis (Malalana et al. 2015).

8.6.2  Risk factors

Though leptospirosis is a zoonotic disease, the carriage of Leptospira

has been found in virtually all mammalian species human often infected
accidentally. Transmission occurs in both industrialized and developing
countries. Leptospirosis in humans is always acquired from an animal
source; human-to-human transmission is for practical purposes nonexis-
tent. Pathogenic Leptospira have been found in the proximal renal tubules
of the kidneys of carriers, although other tissues and organs may also
serve as a source of infection. From the kidneys, leptospires are excreted
in urine and may then contaminate soil, surface water, streams, and riv-
ers. The carriers may be wild or domestic animals, especially rodents and
small marsupials, cattle, pigs, and dogs (Adler and De La Pena Moctezuma
2010). Leptospirosis is an occupational disease for veterinarians, farmers,
abattoir workers, butchers, hunters, rodent control workers, and other
occupations requiring contact with animals. Indirect contact with con-
taminated wet soil or water is responsible for the great majority of cases
in the tropics, either through occupational exposure as in rice or taro
farming, flooding after heavy rains, or exposure to damp soil and water
during avocational activities (Musso and La Scola 2013). The expansion of
urban slums worldwide has increased the chance of rat-borne transmis-
sion (Dunay et al. 2016). Genetic factors are strongly implicated in ERU.
Uveitis occurs in all horse breeds, but the strong predilection of some
breeds for ERU suggests a genetic link. ERU is most commonly seen in
Appaloosas, European Warm-bloods, draught breeds, and Standard bred
trotters (Witkowski et al. 2016).

8.7  Ocular manifestations

The prolonged symptom-free period between the systemic and ocular

manifestations makes it difficult for the ophthalmologist to link uveitis
to leptospirosis. The uveal tract is highly vascular, usually pigmented, and
provides most of the blood supply to the eye. Because of the direct prox-
imity to the peripheral vasculature, any disease of the systemic circulation
may also affect the uveal tract (Malalana et  al. 2015). Table 8.3 gives
the broad spectrum of clinical manifestations involved during leptospiral
­ocular infection.
214    Pocket Guide to Bacterial Infections

Table 8.3  Ocular Manifestations during Leptospiral Infection

Period of
Sl. No Infection Infection Signs and Symptoms
1. Leptospiral Anterior Ocular pain, blepharospasm,
uveitis uveitis lacrimation, chemosis, corneal
changes (i.e., edema, vascularization,
cellular infiltrate, keratic precipitates),
aqueous flare, hypopyon, hyphema,
miosis, iris color changes, and low
intraocular pressure
Posterior Vitritis with liquefaction of the
uveitis vitreous, the presence of vitreal
floaters and retinal changes
2. Leptospiral Classic ERU Active intraocular inflammation,
ERU cataract, intraocular adhesion, and
phthisis
Insidious ERU Low-grade inflammation with very
few external signs
Posterior ERU Vitreal opacities, retinal
inflammation and degeneration
with no anterior sign of uveitis
3. Common Immunological Conjunctival congestion without
symptom of phase (After discharge, conjunctival chemosis,
leptospiral 4 to 7 days of aqueous flare, fibrin, hyphema,
ocular infection) miosis, cloudy-yellow-green
infection vitreous, subconjunctival
hemorrhages, panuveitis often
accompanied with retinal
periphlebitis and hypopyon
Posterior Vitritis, pars planitis, periphlebitis,
segment choroiditis, papillitis, macular edema,
manifestations retinal hemorrhages, retinal
exudates, arteritis, retinal
detachment resulting in blindness,
corneal stromal opacities,
inflammatory cells in the anterior
chamber, hyperemic disc, optic
neuritis, neuroretinitis,
nongranulomatous uveitis hypopyon,
cataract, vitreous inflammatory
reaction, retinal vasculitis, retinal
hemorrhages, phthisis bulbi or
glaucoma and papillitis
ERU, equine recurrent uveitis.
 Leptospiral Recurrent Uveitis      215

8.8  Morbidity and mortality

Leptospirosis is a leading zoonosis cause of morbidity and mortality. Overall,

leptospirosis was estimated to cause 1.03 million cases and 58,900 deaths
each year. Although leptospirosis is a life-threatening disease and recog-
nized as an important cause of pulmonary hemorrhage syndrome, the
lack of global estimates for morbidity and mortality has contributed to its
neglected disease status. Leptospiral ERU usually leads in to moon blindness
among horses (Verma et al. 2013).

8.9 Complications

Cataracts tend to be the most common complication of ocular eye infec-

tion and occurs during steroid treatment of leptospiral uveitis. Steroids
are the mainstay of treatment for leptospiral uveitis. Ocular involvement
is seen both in the systemic bacteremic phase as well as in the immuno-
logical phase. The incidence of ocular signs during acute systemic phase
varies from 2% to 90%. However, in some instances, the ocular manifesta-
tions may be subclinical or of such low order as to be overlooked; they
are usually found by those who search for it (Rathinam 2005). One of the
studies shows that 14% of sero-positive cases of leptospiral uveitis develop
cataracts and of them, 76% develop significant cataract before the ste-
roid treatment. Leptospiral uveitis usually responds promptly to treatment
and cataract removal and intraocular lens implantation results in complete
recovery of vision among human cases.

8.10 Diagnosis

Slit lamp bio-microscopic examination of the anterior segment of the eye

revealed the presence of inflammatory cell collection or nongranulomatous
keratic precipitates (KPs) at the back of the cornea. In case of severe inflam-
mation, the cells gravitate down to form hypopyon, which has been noted
to occur in 12% of patients with leptospiral uveitis. Leptospiral DNA can
be detected in ocular fluids of affected horses even when they are sero-
negative. This means that lack of detectable serum antibodies does not rule
out leptospirosis as a potential contributing factor (Witkowski et al. 2016).
Other various laboratory procedures involved in the diagnosis has been rep-
resented in Table 8.4.

Misdiagnosis of cases leads in to unilateral uveitis with hypopyon and

arthralgia as Behcet’s disease, but the severe vitreous reaction, vasculitis,
216    Pocket Guide to Bacterial Infections

and hyperemic disc can differentiate leptospiral uveitis from other uveitis.
The onset of leptospiral uveitis is acute and of shorter duration, whereas
Behcet’s uveitis is chronic, recurrent, and insidious.

Table 8.4  Various Diagnosis and Identification Methods for Leptospirosis

Diagnostic
Sl. No. Methods Technique Used References
1. Bacteriological Isolation, animal Inada et al. (1915); Noguchi
methods inoculation and Kligler (1920)
2. Microscopic Silver staining Warthin and Starry (1992);
methods Direct dark-field Koshina et al. (1925)
microscopy,
3. Serological CF Sturdza et al. (1960); Torten
diagnosis Immunofluorescence et al. (1966); Terpstra et al.
Counter (1979); Terpstra et al.
immunoelectrophoresis (1980); Tu et al. (1982)
Microscopic
agglutination test
RIA
ELISA
Patoc slide
agglutination.
IgM dot-ELISA dipstick
test
4. Molecular In-situ hybridization Terpstra et al. (1980); Tu
diagnosis PCR et al. (1982); Terpstra et al.
Dot-blotting (1987); Lilenbaum et al.
Real-Time PCR (2009)
LAMP
5. Molecular PFGE Ciceroni et al. (2002);
typing Ribotyping Perolat et al. (1994); Brown
REA and Levett (1997);
RFLP Kawabata et al. (2001);
Detection of VNTR Majed et al. (2005); Ahmed
MLST et al. (2006); Moreno et al.
AFLP (2016)
Arbitrarily primed
multiple locus
sequence typing,
insertion sequences-
based typing

(Continued)
 Leptospiral Recurrent Uveitis      217

Table 8.4 (Continued)  Various Diagnosis and Identification Methods for

Leptospirosis
Diagnostic
Sl. No. Methods Technique Used References
6. Kit method Leptospira-MC test Arimitsu et al (1982); Smits
(microcapsule et al. (2001); Levett et al.
agglutination assay) (2006)
LeptoTek LFA (lateral
flow assay)
IHA
Test-it (lateral flow
assay)
Leptocheck-WB (lateral
flow assay)
SD Leptospira LF
(lateral flow assay)
IgM LFA (lateral flow
assay)
LeptoTek DriDot
(latex-card
agglutination test)
Leptorapide (latex-card
agglutination test)
AFLP, amplified fragment length polymorphism; CF, complement fixation; ELISA,
enzyme-linked immunosorbent assay; IHA, indirect hemagglutination; MLST, multiple
locus sequence typing; PCR, polymerase chain reaction; PFGE, pulsed field gel elec-
trophoresis; REA, restriction endonucleases analysis; RIA, radioimmunoassay; RFLP,
restriction fragment length polymorphism; VNTR, variable number of tandem repeats.

8.11 Treatment

It depends on the severity and duration of symptoms at the time of infec-

tion. Antibiotic treatment with doxycycline, vibramycin, oracea, adoxa,
penicillin, oxytetracycline, streptomycin, cefotaxime, erythromycin, and flu-
oroquinolones is more effective when initiated early in the course of illness.
Topical 1% atropine alone or combination with 10% phenylephrine is used
to relieve ciliary muscle spasm and achieve mydriasis. Topical or systemic
nonsteroidal anti-inflammatory drugs (NSAIDs) such as corticosteroids are
used to decrease the inflammation, and dexamethasone and prednisolone
are the two most commonly used topical corticosteroids (Jabs et al. 2005;
Taylor et al. 2010). Topical steroids do not penetrate the posterior segment
of the eye; it may be adequate for disease affecting the anterior uvea, but
218    Pocket Guide to Bacterial Infections

other strategies, such a systemic corticosteroid administration may be nec-

essary in case of posterior uveitis. However, systemic administration of cor-
ticosteroids for ERU is less commonly performed because it can sometimes
be associated with significant adverse effects in horses such as laminitis
and flunixin meglumine has better ocular penetration than other NSAIDs.
Subconjunctival and intraocular injections of triamcinolone acetonide have
been reported in horses with no overt adverse effects observed (Yi et al.
2008 and Gilger 2016). Studies in horses have shown that intravitreal injec-
tion of rapamycin appears to be safe, although its efficacy in ERU has not
yet been established. Injection of the anterior chamber with tissue plas-
minogen activator (TPA) can be performed to accelerate fibrinolysis in cases
with severe fibrin accumulation in the anterior chamber. Surgical treatment
includes the implantation of cyclosporine-releasing devices and vitrectomy
to remove fibrin, inflammatory cells, and debris trapped in vitreous fluids
to improve vision and delay the progression of the clinical signs (Witkowski
et al. 2016). Another surgical option in the treatment of ERU is pars plana
vitrectomy (PPV). The main goals of this procedure are the clearance of the
ocular media and removal of cells and inflammatory mediators from the pos-
terior segment. Recurrence of ERU was prevented in 73%–92% of horses in
which PPV were performed. Long-term antibiotic therapy is not well estab-
lished to work in a high transmission endemic setting. Phacoemulsification
for the treatment of cataract secondary to ERU combined with cyclosporine
implantation or PPV can be performed in an attempt to restore vision. When
vision is lost, and the eye is still painful, enucleation might be necessary for
the animal’s comfort. Enucleation can be performed using a trans-palpebral
or trans-conjunctival approach; a prosthesis can be placed within the orbit
to improve the cosmetic outcome. Alternatively, 15  intrascleral prosthesis
placement can be performed in cases in which the fibrous tunic of the eye
is healthy enough to support the prosthesis (Malok et al. 1999; Deeg et al.
2002; Gilger 2016; Yi et al. 2008).

8.12 Prevention

Human vaccines have not been applied widely in Western countries.

Immunization with polyvalent vaccines has been practiced in China
and Japan, where large numbers of cases occur in rice field workers.
In France, a monovalent vaccine containing serovar Icterohaemorrhagiae
is licensed for human use. A vaccine containing serovars Canicola,
Icterohaemorrhagiae, and Pomona has been developed recently in Cuba.
Vaccines to prevent human leptospirosis are available in some countries,
and large-scale clinical trials have been reported from Russia, China,
Japan, and Vietnam, but they are serovar specific and require annual
 Leptospiral Recurrent Uveitis      219

boosters. A vaccine against multiple serovars has been developed in Cuba

and shown to be 78.1% effective, but it is currently in the early stages
of clinic trials and safety testing (Bharti et al. 2003; Dunay et al. 2016).
General measures such as rodent and infection control, self-sanitation
approach, and avoiding of contaminated water reservoirs can prevent
from leptospirosis. Prophylactic antibiotics beneficial for short-term, well-
defined exposures such as those involved in military training or recre-
ational sports like swimming, although long-term measure is difficult to
practice in tropical countries.

Acknowledgments

Authors like to thank Dr. John F. Timoney, Keeneland Chair of Infectious

Diseases, Gluck Equine Research Center, University of Kentucky, Lexington,
USA, for the critical scientific suggestions for this work.

Financial support

This study was supported by the Indian Council of Medical Research

(Sanction No:Leptos/34/2013-ECD-l), Ministry of Health (Sanction No:
Leptos/33/2013-ECD-I), Department of Biotechnology (DBT) (No. BT/
PR12133/ AAQ/3/707/2014), DBT-NER (No. BT/PR16685/NE/95/249/2015),
Ministry of Science and Technology, Government of India, New Delhi.

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9
Beneficial Lactic Acid Bacteria
Use of Lactic Acid Bacteria in
Production of Probiotics
Galina Novik and Victoria Savich

Contents

9.1 Introduction 225

9.2 Morphology, cytology and physiology of LAB 226
9.3 Growth. Optimization of medium composition and
physicochemical conditions 231
9.4 Production of bacteriocins. Genetic regulation of bacteriocin
production 233
9.5 Biosafety assessment of lactic acid bacteria 235
9.6 Biochemistry and genetics of antibiotic resistance 236
9.7 Taxonomy of LAB. Significance of LAB systematics 237
9.8 Beneficial lactic acid bacteria 242
9.9 Medical application of lactic acid bacteria 250
9.10 Functional food and nutritional supplements 252
9.11 Conclusions and future prospects 257
References 258

9.1 Introduction

Lactic acid bacteria (LAB) are one of commercially valuable groups of microor-
ganisms. LAB have a long application record starting from the ancient times.
The bacteria have been employed in dairying, baking, fish, and meat process-
ing. However, the first pure culture of LAB (Bacterium lactis, now known as
Lactococcus lactis) was obtained by Lister in 1873 (Santer 2010). The devel-
opment of microbiological methods allowing investigations of morphologi-
cal, physiological, biochemical, and genetic properties of LAB led to deeper
insight into their biology and originated new applications for this bacterial
group. LAB have been shown as sources of bioactive compounds. These sub-
stances as well as LAB themselves provide for health benefits. Mechanisms
of favorable action are diverse and play important roles in the modulation

225
226    Pocket Guide to Bacterial Infections

of immunological and gastrointestinal functions and control of pathogens.

Bacteria do not necessarily possess similar features, so that the development
of selection criteria for probiotics is vital for their further use. Besides medicine
and food processing, LAB are applied in agricultural and industrial sectors.

9.2  Morphology, cytology and physiology of LAB

Metabolic pathways of lactic acid bacteria. Biosynthesis of bioactive second-

ary metabolites. Gene clusters for secondary metabolic pathways. LAB rep-
resentatives are gram-positive, nonsporulating, catalase-negative, anaerobic
or microaerophilic, acid-tolerant, organotrophic, and strictly fermentative
rods or cocci producing lactic acid as a major end product (König and Fröhlich
2009). Cell wall of LAB has the typical gram-positive structure formed by
a thick, multilayered peptidoglycan envelope decorated with proteins, tei-
choic acids, and polysaccharides and surrounded in genus Lactobacillus by
an outer shell of proteins packed in a paracrystalline layer (S-layer) (Delcour
et al. 1999). Lactobacillus S-layer proteins differ from those of other bacteria
in their smaller size and highly predicted pI. The positive charge in S-layer
proteins is concentrated in the more conserved cell wall binding domain,
which can be either N- or C-terminal depending on the species. The more
variable domain is responsible for the self-assembly of the monomers into a
periodic structure (Hynönen and Palva 2013). Peptidoglycan is the main con-
stituent of the gram-positive cell wall consisting of glycan chains composed
of alternating N-acetylglucosamine and N-acetylmuramic acid and linked by
β-1,4 bonds. In most bacterial species, peptidoglycan basic structure is par-
tially altered; either glycan chains undergo N-deacetylation or O-acetylation
or free carboxyl groups of the amino acids in the peptide chains are ami-
dated. Teichoic acids, anionic polymers made up of alditol-phosphate
repeating units, also can be modified by replacing free hydroxyl groups of
the alditol-phosphate chains with various sugars or D-alanin. Bacterial poly-
saccharides of cell wall exhibit great diversity in sugar composition, linkage,
branching, and substitution. The structural divergence shown by these cell
wall components may underlie differences in processes such as autolysis
and characteristics such as stress resistance, probiotic properties, or phage
sensitivity (Chapot-Chartier and Kulakauskas 2014). The extracellular and
surface-associated proteins also can be involved in cell wall metabolism,
degradation, and uptake of nutrients, communication, and binding to sub-
strates or hosts (Zhou et al. 2010).

The genomes of LAB and bifidobacteria have low and high guanine-
cytosine (GC) contents and vary in size from 1.3 to 3.3 and 1.9 to 2.9 Mb,
respectively (Klaenhammer et al. 2005; Wu et al. 2017). In bifidobacterial
 Beneficial Lactic Acid Bacteria     227

genomes adapted for colonization of the gastrointestinal tract, 1,604–

2,588  genes have been identified (Pokusaeva 2011). The number of pre-
dicted protein-encoding genes in LAB ranges from about 1,700 to about
2,800. The distinctive feature of LAB genomes is gene loss due to adaptation
to nutritionally rich environments (Makarova et al. 2006). Lactobacillales is
presumed to have lost 600–1200 genes inherited from its Bacilli ancestor,
including genes encoding biosynthetic enzymes and sporulation (Makarova
and Koonin 2007). Some key genes for LAB survival in a new environment
could be acquired via horizontal gene transfer (Wu et al. 2017). The pres-
ence of multiple pseudogenes when compared with other groups of bac-
teria is also evidence of genome reduction. Loss of carbohydrate transport
and metabolism genes and amino acid biosynthesis genes accompanies
adaptation to the environment rich in lactose and protein (Schroeter and
Klaenhammer 2009). Plasmids found in many LAB vary in size and gene con-
tent. Some plasmids carry genes that potentially contribute to adaptation
of the host cell and code for bacteriocins, amino acid or sugar transport-
ers, and restriction-modification systems (Wu et al. 2017). With progress of
genomic research, key features of LAB genomes continue to be discovered
leading to better understanding of the physiology and metabolism of this
microbial group.

LAB do not possess a functional respiratory system, so they derive the

energy required for their metabolism from the oxidation of chemical com-
pounds, mainly sugars. Sugars are fermented by LAB via homofermenta-
tive or heterofermentative pathways. Homofermentative bacteria produce
lactic acid as the only product of glucose fermentation through glycoly-
sis or Embden–Meyerhof–Parnas pathway. Heterofermentative bacteria
use the pentose phosphate pathway generating carbon dioxide (CO2) and
ethanol or acetate, besides lactic acid. Other hexoses are also fermented
by LAB after preliminary isomerization or phosphorylation. In addition,
LAB with heterofermentative type of fermentation successfully metabo-
lize pentoses. Disaccharides are split enzymatically into monosaccharides
entering the appropriate pathways (Von Wright and Axelsson 2011). Genus
Bifidobacterium degrades hexose sugars through a particular metabolic
pathway or bifid shunt allowing to produce more energy in the form of
ATP. Bifidobacterial pathway yields 2.5 mol of ATP, 1.5 mol of acetate, and
1  mol of lactate from 1  mol of fermented glucose, while the homofer-
mentative LAB produce 2 mol of ATP and 2 mol of lactic acid and hetero-
fermentative LAB produce 1 mol each of lactic acid, ethanol, and ATP per
1 mol of fermented glucose. Fructose-6-phosphoketolase enzyme plays a
key role in this pathway and is considered to be a taxonomic marker for the
family of Bifidobacteriaceae (Pokusaeva 2011). Recently a novel metabolic
228    Pocket Guide to Bacterial Infections

pathway (galacto-N-biose (GNB)/lacto-N-biose (LNB) I pathway) that utilizes

both human milk oligosaccharides and host glycoconjugates and is essen-
tial for colonization of the infant gastrointestinal tract was found in genus
Bifidobacterium (Fushinobu 2010). This route was suggested to be specific
for Bifidobacterium; however, further studies showed ability of some LAB
to metabolize LNB and GNB. Nevertheless, metabolic pathways responsible
for catabolism of these compounds in LAB are completely different from
those described for Bifidobacterium species (Bidart et al. 2014).

The proteolytic system of LAB provides amino acids essential for bacterial
growth by protein conversion. It is also engaged in generation of flavor
compounds, accounting for the development of organoleptic properties
of fermented food (Liu et  al. 2010). Two major pathways convert amino
acids to flavor compounds: elimination reactions catalyzed by lyases and
pathways initiated by aminotransferases. Lyases take part in the production
of methanethiol from methionine, while aminotransferases convert amino
acids to corresponding α-keto acids. The α-keto acids are key intermedi-
ates in aroma generation and can be further transformed into other com-
pounds: α-hydroxyacids, acetyl-CoA derivatives, and aldehydes while the
latter turn into alcohols and carboxylic acids (Steele et al. 2013). The pro-
teolytic system of LAB contains cell wall–bound proteinase degrading milk
proteins into oligopeptides, peptide transporters transferring peptides into
the cell, and various intracellular peptidases breaking down the peptides
into shorter peptides and amino acids (Liu et al. 2010).

Lipid metabolism is the enzymatic break down of lipids into fatty acids and
glycerol by lipases with either intracellular or extracellular localization in
LAB strains. The latter are able to perform unique fatty acid transformation
reactions, including isomerization, hydration, dehydration, and saturation
(Hayek and Ibrahim 2013). Such products of lipid metabolism as conju-
gated fatty acids have beneficial effects on health, making them a target
of intensive study. LAB were found to successfully produce conjugated lin-
oleic acid (CLA) through two consecutive reactions: hydration of linoleic
acid to 10-hydroxy-12-octadecenoic acid and dehydrating isomerization
of the hydroxy fatty acid to CLA. Ricinoleic acid also can be transformed
into CLA. On the other hand, linoleic acid can be used in production of
conjugated trienoic acid through alkali-isomerization (Ogawa et al. 2005).
Bifidobacterium species show ability to conduct isomerization of linoleic
acid to CLA (Raimondi et al. 2016).

Besides the aforementioned compounds, LAB are sources of many other

substances: bacteriocins, vitamins, enzymes, exopolysaccharides, and sweet-
eners. A wide range of LAB are able to produce capsular or extracellular
 Beneficial Lactic Acid Bacteria     229

polysaccharides with various chemical composition and properties. The

term exopolysaccharide (EPS) often denotes all forms of polysaccharides
located outside of the microbial cell wall. Polysaccharides are divided into
two groups: homopolysaccharides composed of only one type of monosac-
charide and heteropolysaccharides containing two or more types of sugars.
Two pathways for biosynthesis of exocellular polysaccharides have been
described for LAB: the Wzy-dependent pathway and the extracellular glyco-
syltransferase pathway for synthesis of glucans and fructans. Genes encod-
ing Wzy-dependent proteins in LAB are typically organized in a cluster with
an operon structure and can be chromosomal as well as plasmid-borne.
These gene clusters are extremely diverse, and their nucleotide sequences
are among the most variable sequences in LAB genomes. Genes in the
operon can be categorized into several groups: modulatory genes, poly-
saccharide assembly machinery genes, genes encoding glycosyltransferase
involved in the assembly of the repeating units, and genes required for the
synthesis of activated sugar precursors and modification of the sugar resi-
dues. Clusters are usually 15–20 Kb in size and comprise less than 30 genes.
Genes of LAB have the same orientation and are transcribed as a single
mRNA. Extracellular glycosyltransferase pathway is a simple biochemical
route employing a specific glucansucrase or fructansucrase and an extracel-
lular sugar donor for the synthesis of glucans or fructans (Zeidan et al. 2017).
All EPS-producing bifidobacteria appear to synthesize EPS through internal
heteropolysaccharide pathways. Their EPS biosynthesis is concentrated in
the 25.6 Kb region composed of 20 genes, 18 of which are positioned in
oppositely directed but adjacent transcriptional loci. Regulatory genes have
not been identified in Bifidobacterium spp. to date (Ryan et al. 2015).

LAB are known as producers of vitamins, mainly K, and some vitamins of the
B group (Patel et al. 2013). Riboflavin (vitamin B2) plays a significant role in
cellular metabolism, acting as the precursor of electron carriers in oxidation–
reduction reactions. Biosynthesis of riboflavin dependent on the precursors
guanosine triphosphate and D-ribulose 5-phosphate occurs through seven
enzymatic steps. B2 synthesis genes in LAB form a single operon, including
riboflavin-specific deaminase and reductase (ribG), riboflavin synthase alpha
subunit (ribB), a bifunctional enzyme catalyzing the formation of 3,4-dihy-
droxy-2-butanone 4-phosphate from ribulose 5-phosphate (ribA), and ribo-
flavin synthase beta subunit (ribH). Riboflavin biosynthesis and transport are
controlled by conserved regulatory region located upstream of the operon
(LeBlanc et al. 2011; Capozzi et al. 2012). Folate (vitamin B11) is involved in
essential functions, like DNA replication, repair, methylation, and synthesis
of nucleotides, vitamins, and some amino acids. The biosynthetic pathway
in LAB includes several consecutive steps, wherein the precursor guanosine
230    Pocket Guide to Bacterial Infections

triphosphate is converted into tetrahydrofolate (Capozzi et  al. 2012). The

folate biosynthetic genes of L. lactis MG1363 are organized in a folate
gene cluster, consisting of six genes (folA, folB, folKE, folP, ylgG, and folC)
(Sybesma et al. 2003). The similar structure of the cluster was revealed for
Lactobacillus species (Santos et al. 2008). Cobalamin (vitamin B12) is one of
the vital vitamins whose deficiency affects hematopoietic, neurological, and
cardiovascular systems. Cobalamin is synthesized only by prokaryotes via
aerobic and anaerobic pathways. The biosynthetic route of the vitamin from
5-aminolaevulinic acid is divided into three sections: biosynthesis of uropor-
phyrinogen III from 5-aminolaevulinic acid; conversion of uroporphyrinogen
III into the ring-contracted, deacylated intermediate precorrin 6 or cobalt-
precorrin 6; transformation of the intermediate to form adenosyl cobalamin
(Scott and Roessner 2002). Production of B12 was observed mainly in other
groups of bacteria, although the analyses revealed the presence of 32 open
reading frames related to coenzyme B12 production (cbi, cob, hem, and cbl
gene cluster) in Lactobacillus coryniformis (Torres et al. 2016). Lactobacillus
reuteri JCM 1112T has a unique cluster of 58 genes encoding biosynthesis
of reuterin and cobalamin. At least two independent insertion events can be
engaged in formation of the cluster (Morita et al. 2008). Vitamin K plays a
significant role in blood clotting, bone, and kidney function, tissue calcifica-
tion, and atherosclerotic plaque prevention. The vitamin is usually available
in two forms: phylloquinone (K1) and menaquinone (K2). The latter is pro-
duced by some intestinal bacteria, like LAB (Patel et al. 2013). Biosynthesis
of menaquinone was studied in other groups of bacteria, starting from cho-
rismite converted from shikimate (Meganathan and Kwon 2009). However,
operon men encoding menaquinone synthesis was also found in some LAB
(Bolotin et al. 2001; Wegmann et al. 2007).

The other substances produced by LAB are mannitol, sorbitol, tagatose,

and xylitol used as sweeteners in food industry. Mannitol is a six-carbon
sugar alcohol synthesized by bacteria from fructose using mannitol dehy-
drogenase (Patra et al. 2009; Papagianni 2012). The research revealed that
one-third of fructose could be replaced with glucose, maltose, galactose,
mannose, raffinose, or starch with glucoamylase, and two-thirds of fruc-
tose could be replaced with sucrose for mannitol production (Saha and
Nakamura 2003). Tagatose is an isomer of fructose showing prebiotic effect
and antioxidant activity, and it can be used for control of diabetes and obe-
sity. D-tagatose can be produced from D-galactose by L-arabinose isom-
erase (araA) (Chouayekh et al. 2007; Patra et al. 2009). Sorbitol is another
six-carbon sugar alcohol produced by catalytic hydrogenation of glucose,
with applications in the food and pharmaceutical industries. Only a few
organisms are able to synthesize sorbitol. LAB strains are often subjected
 Beneficial Lactic Acid Bacteria     231

to metabolic engineering to  achieve sorbitol hyperexpression (Patra et  al.

2009; Papagianni 2012). Xylitol is a five-carbon sugar alcohol produced by
reduction of xylose. LAB have not been reported to produce xylitol natu-
rally, but recombinant strains with xylose reductase were able to generate
this compound (Papagianni 2012). Bacteriocins are considered in the sepa-
rate chapter.

9.3 Growth. Optimization of medium composition

and physicochemical conditions

Substrate pretreatment. Selection of supplements, autoregulators and

autoinducers. LAB are industrially attractive microorganisms that are able
to produce a number of valuable compounds. Bacteria require appropriate
medium and physicochemical conditions to grow and express normal meta-
bolic activities. LAB are known as fastidious microorganisms that demand
specific cultural conditions. It urges optimization of medium composition
and cultivation factors. Optimal physicochemical parameters (e.g., tempera-
ture, pH, water activity, and redox potential) are diverse among LAB strains.
Bacteria of this group can grow in pH range 3.5–9.6 and temperatures
5°C–45°C (Abdel-Rahman et al. 2013). Nevertheless, temperature rise led
to the decrease of growth yield based on ATP production as well as specific
growth rate of bacteria, whereas specific lactate production rate remained
constant or even increased. pH fall resulted in inhibition of both growth rate
and lactate production (Adamberg et  al. 2003). Autolysis of Lactococcus
lactis strains is necessary for cheese ripening and is highly strain dependent.
However, autolysis in most of the strains is favored by low NaCl concen-
trations (0.17  M) and acidic pH (5.4) (Ramírez-Nuñez et  al. 2011). Some
Lactobacillus strains successfully ferment glucose and produce lactic acid
even at 8% salt level (Rao et al. 2004).

LAB cannot grow on simple mineral media supplemented only with a car-
bon source. These microorganisms often demand various free amino acids,
peptides, nucleic acid derivatives, fatty acid esters, minerals, vitamins, and
buffering agents in the medium. As a carbon source glucose is commonly
preferred by the majority of LAB cultures, but some strains have opted for
alternative sugars (Hayek and Ibrahim 2013).

To cheapen production of various compounds generated by LAB, especially

lactic acid, new substrates have been tested because of high cost of the raw
materials, such as starch and refined sugars. Lignocellulosics due to their
abundance, low price, high polysaccharide content, and renewability have
232    Pocket Guide to Bacterial Infections

been chosen as potential carbohydrate feedstock; however, LAB were not

able to use these substrates without pretreatment. Various physical, chemi-
cal and biological methods were used to remove lignin, separate cellulose
and hemicellulose, increase the accessible surface area, partially depolymer-
ize cellulose, and enhance porosity of the materials to promote the subse-
quent action of the hydrolytic enzymes. Enzymatic hydrolysis converts the
polysaccharides remaining after pretreatment in the water-insoluble solid
fraction into soluble sugars further utilized by LAB (Abdel-Rahman et  al.
2011). Compounds toxic to fermentative organisms such as furfural, phe-
nolic derivatives, and inorganic acids are also released during pretreatment,
urging to seek resistant bacteria or carry out detoxification (Guo et al. 2010).
Other wastes can be used as substrates for LAB; however, they are often
subjected to either pretreatment or supplementation of missing compounds
as carbon sources or minerals (Dumbrepatil et al. 2008; Pacheco et al. 2009;
Panesar et al. 2010; Özyurt et al. 2017).

Besides external factors, bacteria of the same or different species are able
to conduct regulation via quorum sensing. Quorum-sensing bacteria pro-
duce and release chemical signal molecules termed autoinducers altering
gene expression and behavior in detecting bacterium (Waters and Bassler
2005). Three-component regulatory operon involving quorum-sensing
mechanism was found in Lactobacillus plantarum NC8. The presence
of specific bacteria could act as an environmental signal able to switch
on bacteriocin production in L. plantarum NC8 mediated by the induc-
tion factor PLNC8IF. Supernatants of other species (Lactococcus lactis
MG1363) expressing this gene also promoted bacteriocin production in
NC8 (Maldonado et al. 2004). Lactobacillus plantarum DC400 synthesized
pheromone PlnA both under mono- or co-culture conditions. PlnA repre-
sents an induction factor for gene regulation (pheromone behavior), and it
acts as an antimicrobial peptide. Its biosynthesis was induced to different
extent depending on microbial partnership with Lactobacillus sanfrancis-
censis DPPMA174 influencing the highest yield of PlnA. The mixed culture
carried out biosynthesis and the concentrations of specific volatile organic
compounds (e.g., furanone B and decanoic acid) were suggested to act as
signal molecules (Di Cagno et al. 2010). Autoinducer-2 (AI-2) was used as
signaling molecule in one of the primary bacterial interspecies communica-
tion mechanisms known as the luxS-mediated universal signaling system.
The Lactobacillus sakei NR28 produced a significant reducing effect on the
expression of virulence factors in enterohaemorrhagic Escherichia coli by
AI-2 signaling inhibition (Park et al. 2014). On the other hand, luxS-medi-
ated system of bifidobacteria takes part in gut colonization and protection
from pathogens (Christiaen et al. 2014). Production of some compounds
 Beneficial Lactic Acid Bacteria     233

can be regulated by product itself. External nisin has been shown to induce
its own synthesis, functioning as signaling molecule (Kuipers et al. 1995;
Qiao et al. 1996). PepR1 of L. bulgaricus was involved in regulation of the
prolidase PepQ biosynthesis. In the absence of glucose PepR1  does not
stimulate pepQ transcription and is constantly synthesized. In the presence
of glucose PepR1 blocks transcription of its own gene and induces pepQ
transcription (Morel et al. 2001).

Co-cultivation may exert positive effect on growth of the both bacteria.

Streptococcus thermophilus in combination with L. bulgaricus (basonym
Lactobacillus delbrueckii ssp. bulgaricus) showed up-regulation of peptides
and amino acid transporters and of specific amino acid biosynthetic path-
ways, notably for sulfur amino acids as well as genes and proteins involved
in the metabolism of various sugars (Herve-Jimenez et  al. 2008). Further
research of interactions between these bacteria showed that formic acid,
folic acid, and fatty acids were provided by S. thermophilus. The cleavage of
casein into peptides by membrane-resident protease of L. bulgaricus and the
enhanced expression of peptidases in S. thermophilus supported increased
growth rates of both species in mixed culture. Genes involved in iron uptake
by S. thermophilus were affected and genes coding for exopolysaccharide
production in both organisms were up-regulated in mixed culture as com-
pared to monocultures (Sieuwerts et al. 2010).

9.4 Production of bacteriocins. Genetic regulation

of bacteriocin production

Bacteriocins are ribosomally synthesized peptides possessing antimicrobial

activity. They often demonstrate activity toward a specific group of bacteria
over a wide pH range in contrast to antibiotics. Bacteriocins are also readily
degraded by proteolytic enzymes due to their proteinaceous nature, which
makes them harmless to the human body and the surrounding environment
and useful for food and clinical applications (Perez et al. 2014). Bacteriocins
are generally divided into several classes.

1. Class I (lantibiotics) unites small thermostable peptides (<5  kDa) pos-

sessing unusual post-translationally modified residues such as lanthio-
nine or 3-methyllanthionine. Such atypical residues form covalent bonds
between amino acids, resulting in internal “rings” and giving lantibiotics
their characteristic structural features.
2. Class II is represented by unmodified heat stable bacteriocins (<10 kDa)
lacking unusual modifications.
234    Pocket Guide to Bacterial Infections

3. Class III includes unmodified heat unstable bacteriocins sized over 10 kDa
with bacteriolytic or non-lytic mechanism of action (Perez et  al. 2014;
Alvarez-Sieiro et al. 2016).
4. Class IV embraces complex bacteriocins containing lipid or carbohydrate
moieties (Ahmad et al. 2017). Recently the members were re-classified as
bacteriolysins (i.e., hydrolytic polypeptides), leaving only three classes of
bacteriocins (Mokoena 2017).

In turn, the classes are divided into subclasses based on the biosynthesis
mechanism and biological activity (Alvarez-Sieiro et al. 2016).

Mode of action of these compounds depends on group of bacteriocins.

Nevertheless, they often affect cell membrane. Different charges of mem-
brane and bacteriocins lead to electrostatic interaction between them, facil-
itating attraction of the molecules to the membranes. Bacteriocins often
influence gram-positive bacteria, whereas gram-negative cells contain extra
lipopolysaccharide outer membrane, so that additional agents are required
to compromise its integrity. Bacteriocins are able to form pores leading to
the dissipation of the membrane potential, take part in the efflux of cell
metabolites, or induce membrane permeabilization (Perez et al. 2015).

Majority of the genes encoding bacteriocins are clustered in operons lying

in the bacterial chromosome, plasmids, or transposons. The expression
needs at least two genes: gene directly encoding bacteriocin and gene of
immunity protein providing protection from the compound (some operons
of class II bacteriocins). In most cases, the production is also dependent on
specific export machinery and regulation factors. The operons of lantibiotics
are more complex because they require additional enzymes for posttrans-
lational modifications. Bacteriocins are mainly synthesized as propeptides
with leader sequences (Dimov et al. 2005). Some bacteriocins possess more
complex structure consisting of several peptides (Stephens et al. 1998).

Nisin is the best-studied compound among lantibiotics as well as bacte-

riocins. The nisin gene cluster contains 11 genes (nisABTCIPRKFEG). nisA
encodes the precursor peptide of 57  amino acid residues; nisB and nisC
encode putative enzymes involved in the posttranslational modification
reactions; nisT encodes a putative transport protein of the ABC transloca-
tor family, which is probably engaged in the extrusion of modified nisin
precursor; nisP encodes extracellular protease responsible for precursor pro-
cessing; nisI encodes lipoprotein involved in the producer self-protection
against nisin; nisFEG encodes putative transporter proteins which are also
implied in immunity mechanism. nisR and nisK take part in the regulation of
nisin biosynthesis. NisR is a response regulator, and NisK is a sensor histidine
 Beneficial Lactic Acid Bacteria     235

kinase which belongs to the class of two-component regulatory systems

(Kuipers et  al. 1995). Biosynthesis of bacteriocin goes the following way.
nisA is translated to pre-nisin A followed by transformation to precursor
nisin A with formation of several disulfide bridges and modification of some
amino-acids. Then the precursor nisin A is exported out of the cell, while the
leader peptide is cleaved and the final product nisin A is obtained (Dimov
et al. 2005). Added to regulatory genes of the operon, transcription of nisin
can be stimulated by nisin itself, nisin mutants, or analogs, but not by the
unmodified precursor peptide or by other antimicrobial peptides (Kuipers
et al. 1995).

Among class II bacteriocins pediocin PA-1 was most thoroughly studied bio-
chemically and genetically and, unlike nisin, pediocin PA-1 is heterologously
expressed in other genera (Moon et  al. 2006). The bacteriocin operon
is represented by only four genes pedA, pedB, pedC, and pedD. pedA
encodes the precursor of pediocin PA-1; pedB encodes the immunity pro-
tein; products of pedC and pedD take part in bacteriocin transport. PedC is
suggested to be involved in the channel formation. PedD shows homology
with other bacteriocin ABC-transporters and also is capable of processing
pediocin by cleavage of the leader sequence (Venema et al. 1995).

Besides direct genetic regulation, various factors influence bacteriocin pro-

duction. The optimal conditions for producing bacterial strains are also
favorable for generation of antimicrobial compounds. Temperature, pH,
the presence or lack of certain substances were able to affect bacteriocin
production (Diep et al. 2000; Li et al. 2002; Leroy and De Vuyst 2005; Van
den Berghe et al. 2006). Class I and II bacteriocin regulation relies on signal
transduction systems mostly differentiated with regard to peptide inducer.
The bacteriocin of class I has a two-component regulatory system activating
auto expression. Class II regulation is almost identical to class I regulation
pathways, but it is generally associated with peptide pheromone induction
as opposed to autoregulation (Snyder and Worobo 2014).

9.5  Biosafety assessment of lactic acid bacteria

LAB have a long application history. They find use in production and pres-
ervation of fermented food and probiotics and have “qualified presump-
tion of safety” (QPS) status and are “generally recognized as safe” (GRAS)
microorganisms by the European Food Safety Authority (EFSA) and Food
and Agriculture Organization of the United Nations (FAO), respectively.
Nevertheless, there are several criteria to ensure safety of LAB used as pro-
biotics for the consumers.
236    Pocket Guide to Bacterial Infections

1. Correct assessment of taxonomic identity should be defined in the

screening process for new probiotic strain. It allows to affiliate the stud-
ied strain with the identified variants and obtain scientific and techno-
logical information, including data on growth conditions, metabolic and
genomic characteristics.
2. The strain should adhere to colonization site and multiply with beneficial
impact on the host organism.
3. The strain should not carry transmissible antibiotic resistance genes and
virulence factors and should not induce adverse effects in the host body.
4. The strain should be viable and genetically stable to ensure that specific
health-promoting characteristics and functionalities are not affected dur-
ing long-term preservation and production.
5. Administration of D(−)-lactate-producing probiotics should be carefully

traced in patients with risk of developing D-lactic acidosis, in cases of bowel
surgery and subsequent short gut syndrome, in the newborn category.

In addition to probiotic strain characteristics, evaluation of biosafety should

take into account purity of the culture from contaminating microbes or
other substances, including allergenic materials, physiological status of the
consumers, supplied dose, and method of administration.

Assessment of LAB safety is carried out by various methods, but general

health status of the animal models and the specific parameters are mainly
studied (Sanders et al. 2010).

9.6  Biochemistry and genetics of antibiotic resistance

Antibiotics are antimicrobial agents killing or inhibiting growth of bacte-

ria. Intensive pursuit of substances targeting only disease-causing microbes
began in the twentieth century, although antibacterial properties of molds
have been known since ancient times. Penicillin discovery by Fleming and
the following description of its purification process led to mass production
of antibiotics and search of new ones. In the course of time resistant bacte-
ria have been revealed. Abuse of antibiotics increased antibiotic resistance,
especially since the sixties. Moreover, bacteria not susceptible to several
antimicrobials (multidrug-resistant bacteria) have emerged (Aminov 2010;
Davies and Davies 2010; Ventola 2015).

Antibiotics demonstrate their antibacterial activity via different ways. They

may inhibit synthesis of proteins, nucleic acids, cell wall components, disor-
ganize membrane, and so on. Biological mechanisms of antibiotic resistance
are diverse, but they all can be summarized:
 Beneficial Lactic Acid Bacteria     237

1. Antibiotic degradation or transformation: Bacteria can produce one

or more enzymes decomposing or chemically modifying antimicrobial
agents.
2. Active efflux that pumps out the antibiotic molecules penetrating into
the cell until they reach concentration subminimal for antibacterial
activity.
3. Receptor modification interfering with binding function and leading to
loss of antibacterial effect.
4. Changes in permeability of cell wall restricting antimicrobial access to
target sites.
5. Acquisition of metabolic pathways alternative to those inhibited by

the drug.
6. Hyperproduction of the target enzyme (Alanis 2005; Van Hoek et al. 2011).

Antibiotic resistance can occur in two ways: via mutation of regulatory

or structural genes or acquisition of a resistance gene from an exoge-
nous source (horizontal gene transfer). LAB can be carriers of antibiotic
resistance genes to pathogenic species, although LAB are not generally
targeted by antibiotic treatments since they are considered to be non-
pathogenic. It makes them objects of studies to secure safety of probiotics.

Antibiotic resistance of LAB can be related to the absence of the target.

Most species are resistant to metronidazole because they do not possess
hydrogenase activity. Insensitivity to sulfonamides and trimethoprim is
determined by LAB limited biosynthetic capabilities and lack of the folic
acid synthesis. Antibiotic profiles of different representatives of this group
are quite various. Lactobacillus species are generally resistant to glycopep-
tides, such as vancomycin, whereas Lactococcus is usually susceptible to this
antibiotic (Ammor et al. 2007). To detect transmissible antibiotic resistance
genes, screening by PCR technique and nucleotide sequencing were carried
out. However, antibiotic resistance can be encoded by several genes, as in
tetracycline case. At least 40 different tetracycline resistance genes (tet)
have been characterized (Roberts 2005). Different bacterial groups carried
diverse sets of tet determinants (Roberts 1996).

9.7  Taxonomy of LAB. Significance of LAB systematics

The first pure culture of LAB was isolated late in the nineteenth century
(Santer 2010). After publication of the monograph by Orla-Jensen in 1919,
the principles of modern LAB classification were formulated. Taxonomic
affiliation of the bacteria was based on cellular morphology, mode of glu-
cose fermentation, growth temperatures, and range of sugar utilization
238    Pocket Guide to Bacterial Infections

(Orla-Jensen 1919). Taxonomic classification has long been focused solely

on phenotypic characteristics, with genetic data analysis introduced only
in the 1960s. Since the 1980s, the development of PCR technique and
sequencing of the 16S rRNA gene led to major changes in prokaryotic sys-
tematics (Sentausa and Fournier 2013).

At present most of LAB belong to phylum Firmicutes, order Lactobacillales,

including genera Aerococcus, Alloiococcus, Carnobacterium, Enterococcus,
Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus,
Streptococcus, Symbiobacterium, Tetragenococcus, Vagococcus, and Weissella.
Species of Bifidobacterium genus from phylum Actinobacteria are referred to
LAB in some cases because of their ability to produce lactic acid, but these bac-
terial groups are phylogenetically distinct (Biavati 2001; Liu et al. 2014).

Due to industrial and medical implications, correct identification and tax-

onomic affiliation are significant for further LAB application. Systematic
approach allows to classify bacteria into groups or taxa on the basis of their
mutual similarity or evolutionary relatedness, distinguish the known strains
and recognize novel ones. A proper identification of the probiotic strain
defines safety risks associated with the specific microorganism and rules
out the inclusion of potential pathogenic species in commercial product
formulas (Gueimonde et al. 2006).

Identification of lactic acid bacteria. Instrumental Technique.

Identification on generic and species level. Gene sequencing: 16S rRNA
and constitutive genes. Identification to species, subspecies, strain
level. Molecular typing: REP-PCR, ERIC-, BOX-, (GTG)5-PCR, RAPD-PCR.

Due to their extensive use, especially in food fermentations, LAB have been
thoroughly characterized. This bacterial group is one of the well-studied
microbial objects. The correct identification of LAB is essential to improve
technological aspects and provide safety and quality. Currently various identi-
fication techniques are available. Phenotypic methods are traditionally used in
bacterial description and classification. These methods include morphological
and physiological characterization, carbohydrate fermentation patterns, and
protein profiling. They usually show a relatively poor reproducibility and low
taxonomic resolution, often allowing differentiation only at the genus level.
Phenotypic methods do not afford unique descriptions for each bacterium
and strains of various genera may possess similar features that not necessarily
represent evolutionary relationships between species. However, these meth-
ods do not require special equipment, and the experiments can be carried out
at any laboratory. Moreover, multiple phenotypic techniques can be combined
to correct identification (Temmerman et al. 2004; Moore et al. 2010).
 Beneficial Lactic Acid Bacteria     239

Molecular-genetic techniques exhibit various levels of discriminatory power,

from species level to differentiation of individual strains (typing). Many
methods are based on PCR, conducting the selective amplification of tar-
geted DNA fragments using specific oligonucleotide primers. DNA-DNA
hybridization (DDH) is one of the first genomic methods used for the com-
parison of bacteria. A DDH similarity of approximately 70% serves as the
recommended demarcation value for bacterial species. Nevertheless, the
method is tedious and complicated for wide application (Moore et al. 2010).
DDH should be performed in cases where the new taxon contains more
than a single strain or when strains share more than 97% of 16S rRNA gene
sequence similarity (Mattarelli et al. 2014).

Nowadays 16S rRNA gene sequence analysis is the most practiced molec-
ular-genetic technique. Popularity of the method is supported by ubiqui-
tous distribution of the gene, its functional stability and large size. Besides,
16S rRNA exhibits both evolutionarily conserved regions and highly variable
structural elements (Ludwig and Klenk 2005). The gene structure allows to
design universal primers to identify different species as well as primers to dis-
tinguish separate species and strains, including LAB (Chagnaud et al. 2001;
Caro et al. 2015). However, because of the conserved nature of the gene, in
some cases 16S rRNA analysis is not sufficient for differentiation between
LAB species, like Enterococcus spp. (Moraes et al. 2013). The 16S rRNA gene
can be present in multiple copies, which might cause identification prob-
lems. Lactobacillus strains have been shown to possess usually from four to
seven copies of the gene, while copy number of rRNA per bifidobacterial
genome can vary from 1 to 5 (Candela et al. 2004; Lee et al. 2008).

Internal transcribed spacer (ITS) separating 16S and 23S rRNA genes is also
used in bacterial identification. 16S-23S ITS displays considerable variation
in both the length and the nucleotide sequence suitable for application in
molecular-genetic techniques (Gürtler and Stanisich 1996). 16S-23S region
exhibits larger variation than rRNA genes, which is appropriate to discrimi-
nate taxonomically proximal species. 16S rRNA analysis cannot always cope
with distinguishing closely related Lactobacillus species as compared to DDH
procedure or Southern type hybridization. PCR with the primers tuned to
16S-23S region allows the same accuracy of species identification as the lat-
ter techniques (Berthier and Ehrlich 1998). Moreover, spacer sequence iden-
tification showed the advantage of distinguishing between Lactobacillus
rhamnosus and Lactobacillus casei strains, which could not be accomplished
by comparison of 16S V2-V3 region sequences (Tannock et al. 1999).

Housekeeping genes encode products essential for cell survival, and there-
fore, they undergo changes rarely. However, these genes have been reported
240    Pocket Guide to Bacterial Infections

to evolve much faster than rRNAs; hence, they can be engaged in bacterial
identification (Ochman and Wilson 1987). The phenylalanyl-tRNA synthase
alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial
gene sequences can be used as genomic markers alternative to 16S rRNA
gene sequences, and they have a higher discriminatory power for LAB. Strains
of the same enterococcal species have at least 99% rpoA and 97% pheS gene
sequence similarity, whereas different enterococcal species have at maximum
97% rpoA and 86% pheS gene sequence similarity (Naser et al. 2005). The
pheS gene sequence analysis provided the highest discrimination for the iden-
tification of different species of lactobacilli. pheS provided an interspecies gap,
which normally exceeded 10% divergence and an intraspecies variation up
to 3%, while rpoA revealed a lower resolution with an interspecies gap nor-
mally exceeding 5% and an intraspecies variation up to 2% (Naser et al. 2007).
Additionally, the pheS and rpoA genes were successfully used in identification
of novel species (Yi et al. 2013; Chang et al. 2015; Kadri et al. 2015).

To discriminate different bacterial strains of the same species, methods of

molecular typing are preferred. Pulsed field gel electrophoresis (PFGE) is the
technique used for separating larger pieces of DNA by applying electrical
current that periodically changes direction (three directions) in a gel matrix,
unlike the conventional gel electrophoresis where the current flows only in
one direction (Adzitey et al. 2013). PFGE allows to study genotypic diversity
of species, select and optimize cultures with desired properties for industry
(Psoni et al. 2007; Kahala et al. 2008; Bouchard et al. 2015).

Restriction fragment length polymorphism (RFLP) is characterized by the

use of restriction enzymes to digest DNA and following separation of the
restriction fragments according to their length by agarose gel electrophoresis
(Adzitey et  al. 2013). Single digestion with AciI of rpoB gene (coding for
RNA polymerase β-subunit) in Leuconostoc, Oenococcus, Pediococcus, and
two or three digestions (AciI, HinfI and MseI) in Lactobacillus spp. allowed
to identify LAB species commonly isolated from wine (Claisse et al. 2007).
Restriction patterns of the tuf gene, encoding the elongation factor Tu and
universally distributed in gram-positive bacteria, derived by enzymes AluI and
HaeIII could effectively differentiate closely related Lactobacillus species (Park
et al. 2012). However, sometimes strain variations could not be demonstrated
by the RFLP analysis. Morphologic differences (colony shape and size) were
evident between Lactobacillus kefir strains ATCC 35411 and ATCC 8007, but
genotypic results failed to differentiate them (Mainville et al. 2006).

Random amplified polymorphism DNA (RAPD) is the method in which

arbitrary primers (typically 10-mer primers) are used to randomly amplify
 Beneficial Lactic Acid Bacteria     241

segments of target DNA under low-stringency PCR conditions with genera-

tion of the set of finger printing patterns of different sizes specific to each
strain (Adzitey et al. 2013). The technique was successfully tested in identifi-
cation of Lactobacillus, Lactococcus, Enterococcus, and Streptococcus strains
(Cocconcelli et al. 1995; Samarzija et al. 2002; Rossetti and Giraffa 2005).
Additionally, RAPD can follow and study the progression of starter cultures
in food fermentations (Plengvidhya et al. 2004; Siragusa et al. 2009).

Amplified fragment length polymorphism (AFLP) involves the use of restric-

tion enzymes to digest total genome DNA followed by amplification of
a subset of selected restriction fragments (Adzitey et al. 2013). The AFLP
method enables to delineate closely related strains, like Lactococcus lactis.
Advanced analysis of the AFLP profiles in combination with genome analy-
sis can facilitate the recognition of genetic markers responsible for specific
phenotypic traits, contributing further to development of rapid and predic-
tive screening procedure for culture collections (Kütahya et al. 2011).

Some methods of molecular typing are based on use of oligonucleotide

primers complementary to repetitive sequences. Diverse regions of DNA
flanked by the rep sequences are amplified, leading to amplicon patterns.
The conserved repetitive sequences are divided into four types: the repeti-
tive extragenic palindromic (REP), the enterobacterial repetitive intergenic
consensus (ERIC), the BOX, and the polytrinucleotide (GTG)5 sequences
(Mohapatra et  al. 2007). Repetitive extragenic palindromic (REP) primers,
the enterobacterial repetitive intergenic consensus (ERIC) primers, and the
(GTG)5 primer can be used in the typing of Lactobacillus strains. DNA con-
centration and quality did not affect the ERIC-PCR profiles, indicating that
this method, unlike other high-resolution methods, can be adapted to high-
throughput analysis of isolates. Also, ERIC-PCR simultaneously types isolates
to the strain and species levels, compared to PFGE that can type only to the
strain level (Stephenson et al. 2009). PCR using the BOXAIR primers provides
differentiation at species, subspecies and strain level, acting as the tool con-
firming phenotypic identification (Mohammed et al. 2009).

Thus, there are a lot of techniques available for identification of LAB.

Methods differ in taxonomic resolution, labor expense, and cost. Phenotypic
techniques are simple and affordable, but they do not possess high dis-
criminatory power. Molecular-genetic methods provide more accurate iden-
tification, allowing to distinguish even separate strains. However, in most
situations identification to the species level is required, where 16S rRNA
sequencing or DDH suffice. Typing methods delineate strains and permit to
study and select LAB with set characteristics for industrial processes.
242    Pocket Guide to Bacterial Infections

9.8  Beneficial lactic acid bacteria

Selection criteria for probiotic strains. Industrial strain development.

Technological properties. Beneficial properties of LAB are diverse. They are
able to improve nutritional value of foodstuffs, stimulate lactose digestion,
control infections, allergic reactions and some types of cancer, and enhance
immune system functions (Gilliland 1990; Wedajo 2015). Valuable proper-
ties of products largely depend on selected LAB strains. There are several
important selection criteria allowing to choose strains, which can be further
used in manufacturing of probiotic products with desired characteristics.

1. Origin of strain: Probiotic bacterium should originate in microflora of host

organism. It will guarantee safety and better attachment to intestinal wall
as compared with LAB from other groups of organisms.
2. Accurate identification to genus, species, and strain level allows to

screen variants possessing favorable properties and to avoid pathogenic
species.
3. Probiotic should be safe for consumers.
4. The strain should be resistant to bile acids and the gut environment to
survive in the gastrointestinal tract and exercise its beneficial properties
on the organism.
5. Probiotic culture should be able to adhere to and colonize intestinal epi-
thelium. It prevents cells from wash-out and ensures immune modula-
tion, competitive expulsion of pathogens, production of enzymes, lactic
acid, and vitamins by LAB.
6. The probiotic strain should be capable of producing antimicrobial substances
such as organic acids, hydrogen peroxide, bacteriocins, and so on. Synthesis
of these compounds is one of the mechanisms of LAB beneficial action.
7. LAB should stimulate immune response and provide protection from vari-
ous types of diseases (Shewale et al. 2014).

Compliance with these criteria will allow variants with beneficial properties
to be picked up. Nevertheless, coupled to health-promoting effects, the
LAB strains should possess certain technological properties ensuring their
use in industrial processes.

1. The cultures should be stable and viable. LAB are chosen for the ability
to survive in fermentation process, during manipulations and storage, to
achieve maximum biomass concentration in a simple, and cheap nutrient
medium. Poor endurance of bacteria restrains scope of LAB application,
even strains with good selected characteristics.
 Beneficial Lactic Acid Bacteria     243

2. Scale-up of probiotic technology is indispensable for mass fabrication of

the product.
3. The strain should have good organoleptic properties contributing to

attractive product taste, flavor, appearance, and texture.
4. The strain should be resistant to phages. Sensitivity to bacteriophages
is one of the most grave challenges in food industry. LAB viruses cause
great economic losses due to fermentation failure; 0.1% to 10% of all milk
fermentations are negatively affected by virulent phages. Their presence
results in deterioration of product quality parameters, like taste, flavor,
texture, and development of contaminating microbiota (Szczepankowska
et al. 2013; Shewale et al. 2014; Wedajo 2015).

Applications of probiotics face many problems. The proper selection cri-

teria and technological properties allow to choose strains with beneficial
properties sustained during fermentation process. The probiotic product
favorably influences host health. Mechanisms of LAB-positive action are
diverse and some compounds produced by LAB are especially valuable to
the organism.

Lactic acid bacteria as sources of bioactive compounds. Biologically

active polar lipids and polysaccharides. Chemical composition and
structure. Health benefits of bioactive compounds.

LAB employed in the food industry usually produce biologically active

compounds, giving the final product an additional nutritional and health-
promoting value. LAB are sources of vitamins, bacteriocins, exopolysac-
charides, enzymes, conjugated linoleic acid, and so on. These compounds
differ in chemical structure and beneficial properties. Biosynthesis and com-
position of some substances (e.g., bacteriocins, vitamins, and sweeteners)
were discussed in previous chapters.

γ-aminobutyric acid (GABA) is the amino acid acting as the major inhibitory
neurotransmitter in the mammalian central nervous system. GABA displays
hypotensive, tranquilizing, diuretic, and antidiabetic effects and additionally
upgrades plasma concentration, growth hormones and protein synthesis in
the brain. The biosynthesis of GABA is catalyzed by glutamate decarboxyl-
ase transforming glutamate to the bioactive compound (Dhakal et al. 2012).
The ability to produce GABA was found in some Lactobacillus, Lactococcus,
Leuconostoc, and Weissella strains (Kim and Kim 2012; Lacroix et al. 2013;
Kook and Cho 2013). In LAB, GABA takes part in acid resistance mechanism
(Sanders et al. 1998).
244    Pocket Guide to Bacterial Infections

Conjugated fatty acids, products of lipid metabolism, are important bio-

active compounds, especially CLA. CLA belongs to the family of isomers
of octadecadienoic acid (18:2) carrying a pair of conjugated double bonds
along the alkyl chain. As mentioned previously, it is synthesized through
two consecutive reactions from linoleic acid. Many LAB demonstrate the
ability to produce CLA isomers; hence meat and milk from ruminants and
the derived products are the natural sources of these bioactive compounds.
Properties of CLA administration depend on isomer, doses administered, and
the period of study. Biological activities of CLA are expressed as inhibition of
various types of cancer, immunoregulatory, antioxidant, anti-osteoporotic,
and anti-atherosclerotic effects, and decrease in body fat mass. However,
not all isomers are absorbed to the same extent, and there are some reports
of possible adverse impact. Pro-carcinogenic effects and increased produc-
tion of prostaglandins attributed to CLA 10-trans and 12-cis isomers have
been reported. A negative alteration in the serum lipid profile and probability
of developing insulin resistance have been demonstrated (Van Nieuwenhove
et al. 2012; Lehnen et al. 2015; Kuhl and De Dea Lindner 2016).

EPS form a diverse group of compounds divided into homo- and heteropoly-
saccharides, depending on the number of monosaccharide types in their
structure. Homopolysaccharides are made from sucrose using glucansucrase
or levansucrase, while the synthesis of heteropolysaccharides involves sugar
transportation, sugar nucleotide synthesis, repeating unit synthesis, and
polymerization of the repeating units. In bacterial cells, EPS take part in protec-
tion from various adverse factors such as phage attack, toxic metal ions, and
desiccation (Harutoshi 2013). Extensive EPS applications are determined by the
specific compound. They can be used as adjuvants, emulsifiers, carriers, stabi-
lizers, humectants, bio-thickeners, prebiotics, sweeteners, plasma substitutes,
matrices of chromatography columns, anticoagulants, and so on. EPS may be
applied not only in food processing and pharmaceutics, but in paper industry,
metal-plating, and oil recovery. However, in some cases, EPS cause food spoil-
age. Mass production of EPS requires correct knowledge of EPS biosynthesis
mechanisms and optimized bioprocess technology (Patel et al. 2012).

Bioactive peptides are produced from proteins during LAB fermentation

and have positive influence on the organism. These peptides exert a wide
range of effects. They control arterial blood pressure through the contrac-
tion of smooth muscles of blood vessels; express free radical-scavenging
activity and promote synthesis of antioxidants, retarding lipid peroxidation;
reduce or inhibit the formation of blood clots; repress the reabsorption of
 Beneficial Lactic Acid Bacteria     245

bile acid in the ileum, decreasing blood cholesterol level; show agonistic
or antagonistic action toward opiate receptor; suppress the appetite, pre-
venting weight build-up; display bacterial membrane-lytic activities; exert
immunomodulatory and cytomodulatory effects; transfer different minerals
by forming soluble organophosphate salts; and play a growth-promoting
role. Milk and colostrum of dairy species are considered as the most impor-
tant sources of bioactive peptides (Park and Nam 2015).

LAB are producers of many ferments that can be used in various fields.
Enzymes degrade caseins yielding key flavor components, which contrib-
ute to the sensory perception of dairy products (Smit et al. 2005). These
bacteria also possess a broad array of enzymatic activities influencing wine
composition and quality of wine (Matthews et al. 2004). Some representa-
tives of genera Lactobacillus, Lactococcus, and Streptococcus demonstrated
amylolytic activity. It provides opportunity to produce lactic acid directly
from starch as a carbon source (Petrova et  al. 2013). β-galactosidase, or
lactase, is extensively used in food and pharmaceutical industries due to
its capability to hydrolyze lactose to monosaccharide and eliminate lactose
intolerance problem (de Vrese et al. 2001).

The main distinctive feature of LAB is the ability to produce lactic acid,
conferring the group name. Since its discovery in 1780 by Scheele, the
compound has become the important chemical used in food, cosmetic,
pharmaceutical, and chemical industries. Lactic acid is regarded as the
raw material in manufacturing of a number of products such as lactate
ester, propylene glycol, 2,3-pentanedione, propanoic acid, acrylic acid,
acetaldehyde, dilactide, and even biodegradable polymer polylactic acid
mainly applied in packaging. Lactic acid functions as a descaling agent,
pH regulator, neutralizer, chiral intermediate, solvent, humectant, clean-
ing aid, skin-lightening and rejuvenating substance, moisturizer, slow
acid-releaser, metal complexing and antimicrobial agent. It is also applied
in tableting, prostheses, surgical sutures, controlled drug delivery sys-
tems, as electrolyte in solutions (Wee et  al. 2006). The food industry is
the main consumer of lactic acid accounting for approximately 85% of its
total demand (John et al. 2007). It is widely used in almost every segment
of the food industry for flavoring, pH regulation, mineral fortification,
increasing shelf life, and better control of foodborne pathogens (Wee
et al. 2006). The estimated global lactic acid demand of 714.2 kilo tons in
2013 is expected to reach 1,960.1 kilo tons by 2020 (SpecialChem 2014).
The compound can be produced by two ways: via chemical synthesis or
246    Pocket Guide to Bacterial Infections

microbial fermentation. LAB produce lactic acid by homofermentative or

heterofermentative pathways, whereas Bifidobacterium cultures use spe-
cial metabolic route.

Mechanisms of beneficial action of lactic acid bacteria.

Gastroenterological effects. Regulation of lipid metabolism. Immunity
enhancement. Cancer prevention.

LAB play a fundamental role in health maintenance. LAB and the derived
products can be used in treatment of various infections and cancer, immu-
nity enhancement, and regulation of diverse functions of the organism.
Positive action of the bacteria can be achieved by several mechanisms.

Pathogenic microorganisms can cause adverse effects on the body, lead-

ing to death ultimately. LAB helps to prevent proliferation and growth of
pathogens. One of the mechanisms of antimicrobial activity is produced
by secretion of bioactive compounds. Some chemicals, like bacteriocins
demonstrate activity toward specific group, species, or strain of bacteria.
Eliminating pathogenic bacteria, the broad-spectrum bacteriocins may
change microbiota diversity (Rea et al. 2011). Short-chain fatty acids (SCFA),
like lactic acid, affect bacterial fitness via acid stress, additionally modulat-
ing host immune functions and serving as metabolic substrates (Sun and
O’Riordan 2013). Lactic acid, besides pH reduction, also functions as a per-
meabilizer of the gram-negative bacterial outer membrane and may act
as a potentiator of the effects of other antimicrobial substances (Alakomi
et al. 2000). Hydrogen peroxide shows microbicidal properties by damag-
ing cell structure. Some studies demonstrated that hydrogen peroxide dis-
played enhanced killing activity in the presence of lactic acid, while in other
cases pathogens could be suppressed with acid but not peroxide (Atassi
and Servin 2010; O’Hanlon et al. 2011).

Other LAB strategies to prevent spread of pathogens are displacement,

exclusion, and competition. The studies show that LAB were able to
attach to mucosa of intestinal epithelial cells, blocking pathogen adhe-
sion, reducing colonization and invasion and preventing infection.
Bacteria act as a barrier to avoid direct contact between pathogens and
epithelial cells, protecting thereby cells from the damage inflicted by
pathogenic species (Jankowska et al. 2008; Abdel-Daim et al. 2013). LAB
may not elicit bactericidal effect on pathogens, but decrease their toxin
production. Shiga-toxin-producing Escherichia coli inhibits protein syn-
thesis in eukaryotic cells and plays a role in hemorrhagic colitis and hemo-
lytic uremic syndrome. Bifidobacterium, Lactobacillus, and Pediococcus
strains have been shown to down-regulate shiga toxin expression, but the
 Beneficial Lactic Acid Bacteria     247

probiotic effect was strain-specific and might be related to pH effect as a

result of organic acid production by LAB (Carey et al. 2008).

Nutritional requirements may be shared by pathogens and microflora, so

that they may potentially compete for growth-limiting resources. In case
pathogen competes with host microbiota for only one limiting nutrient, the
pathogen will be eliminated if its R* (steady-state resource concentration
balancing the pathogen’s birth rate versus mortality loss) exceeds the cor-
responding values of the host competing microflora. Where key indigenous
microbes show low abundance, pathogen control by indigenous microor-
ganisms can be enhanced by the introduction of novel populations of non-
pathogenic competitors (Smith and Holt 1996).

Positive effect of LAB can be expressed via direct action on the organism,
like enhancement of barrier function. The intestinal epithelium acts as a
selectively permeable barrier regulating the absorption of nutrients, elec-
trolytes and water, and providing effective defense against toxins, antigens,
and enteric flora (Groschwitz and Hogan 2009). Breakdown of intestinal
barrier function plays a crucial role in development of such pathologies as
infectious enteritis and inflammatory bowel diseases (Halpern and Denning
2015). Mucins, large complex glycoproteins, protect intestinal mucosal sur-
faces by limiting access of environmental matter to their epithelial cells.
It was shown that Lactobacillus strains increased extracellular secretion
of mucin, leading to reduced adherence of enteropathogen E. coli during
coincubation experiments (Mack et al. 2003). The increase in intestinal per-
meability induced translocation of antigens across the epithelium, provok-
ing inflammation. Bifidobacterium strains possess the capacity to prevent
disruption of intestinal epithelial barrier and to promote its integrity. The
up-regulation of the production of SCFA (acetate and formate) restores the
barrier (Hsieh et al. 2015).

Additionally, LAB can exert an immunomodulatory effect on the organism.

The intestine contains 70%–80% of all immunoglobulin A (IgA) producing
cells. Macrophages, regulatory T cells, and effector B and T lymphocytes
induce the protective IgA function-associated with the mucosal surfaces.
The inductive sites are represented by the Peyer’s patches (PP), the appendix
and the small lymphoid nodules in the large intestine (Perdigón et al. 2001).
It was shown that concentrations of IgA+ cells and IL-6-producing cells
increased after 7  days of Lactobacillus casei administration. IL-6 released
by epithelial cells or macrophages takes part in the enhancement of IgA
secretion by inducing the terminal development of B cells in plasmatic cells,
which express the corresponding immunoglobulin (Galdeano and Perdigón
2006). The use of high doses of antibiotic kanamycin resulted in increased
248    Pocket Guide to Bacterial Infections

IgE levels and decrease in IgA with the number of PP cells, but LAB treat-
ment led to the reverse situation (Kim and Jeung 2016). The administration
of L. bulgaricus raised both interferon γ and interleukin 17 production by
CD4 + T cells from PP. The population of CD4 + T cells is about 10% in PP,
and they are in charge of responding to exogenous antigens by building
up anti-inflammatory and anti-infectious functions (Kamiya et  al. 2017).
Dendritic cells are crucial immune cells linking innate immune response and
acquired immunity by distinct capacity to recognize pathogenic and endog-
enous inflammatory signals. These cells vary in tissue distribution, pattern of
cytokine/chemokine production, and interactions with other immune cells.
Plasmacytoid dendritic cells (pDC) take part in various processes ranging
from the enhancement of anti-viral immunity to augmentation of differ-
entiation of CD4 + induced regulatory T cells. Lactococcus lactis stimulated
pDC capacity to induce CD4 +CD25+ regulatory T cell generation (Jounai
et al. 2012). LAB were even shown to promote the production of cytokines
in macrophage cells (Hong et al. 2009).

LAB are able to influence lipid metabolism and synthesis. Visceral fat
accumulation may spur up progress of several diseases, including diabe-
tes, hyperlipidemia, hypertension, and arteriosclerosis. Sterol regulatory
element-binding protein (SREBP) expression leads to the transcriptional
activation of lipogenic genes in the liver and the development of beta-cell
dysfunction in the pancreas caused by elevated levels of free fatty acids.
Administration of Lactobacillus gasseri exerted anti-lipogenic effects mani-
fested as decrease in expression of the mRNA SREBP and fatty acid synthase
gene in the liver and reduction of free fatty acids in the blood (Yonejima
et  al. 2013). Lactobacillus plantarum displayed multiple effects on lipid
metabolism. It significantly lowed intracellular triglyceride deposits and
glycerol-3-phosphate dehydrogenase (GPDH) activity, mRNA expression of
transcription factors, like peroxisome proliferator-activated receptor γ and
CCAAT/enhancer-binding protein α involved in adipogenesis, the expres-
sion level of adipogenic markers, like adipocyte fatty acid binding protein,
leptin, GPDH, and fatty acid translocase (CD36). Thus, the bacterium inhib-
ited lipid accumulation in the differentiated adipocyte by down-regulating
the expression of adipogenic transcription factors and other specific genes
responsible for lipid metabolism (Park et  al. 2013). On the other hand,
10-oxo-12(Z)-octadecenoic acid produced by LAB induced adipocyte dif-
ferentiation via stimulation of peroxisome proliferator activated receptors γ
predominantly expressed in white adipose tissue, and increased adiponectin
production regulating glucose levels as well as fatty acid breakdown and
insulin-stimulated glucose uptake. As a consequence, LAB fatty acids could
 Beneficial Lactic Acid Bacteria     249

be involved in the regulation of host energy metabolism (Goto et al. 2015).

Application of mixed culture of LAB in the other experiment resulted in inhi-
bition of fat absorption. It also inhibited 3-hydroxy-3-methyl glutaryl-CoA
reductase activity, a major regulatory enzyme in cholesterol biosynthesis.
It was also presumed that bacteria facilitated conversion of cholesterol to
bile acids (Banjoko et al. 2012). Lactobacillus fermentum demonstrated abil-
ity to remove cholesterol from the cultural medium by assimilation (Pereira
and Gibson 2002).

Another remarkable feature of LAB is cancer treatment potential. Cancer is

one of the major causes of morbidity and mortality worldwide, with approxi-
mately 14 million new cases in 2012 and 8.8 million deaths in 2015. Nearly one
in six deaths is due to cancer. Disease incidence is expected to rise by about
70% over the next two decades, making it an acute global challenge (WHO
2017). Some LAB strains have been shown to activate antitumor mechanisms
regulating the host immune response. Probiotic Lactobacillus acidophilus was
able to promote apoptosis, genetically programmed cell death, in murine
colon adenocarcinoma cells (Chen et al. 2012). Lactobacillus kefiri selectively
induced apoptosis in gastric cancer cells in a dose-dependent manner, show-
ing no effects in breast cancer cells and human peripheral blood mononu-
clear cells (Ghoneum and Felo 2015). Administration of Lactobacillus casei
and its extracts revealed anti-proliferative and pro-apoptotic effects in regard
to colon carcinoma cells (Tiptiri-Kourpeti et al. 2016). Reactive oxygen species
(ROS) can provoke carcinogenesis. LAB possess antioxidant properties and
may prevent neoplasm development. However, studies indicate that impact
of LAB on DNA damage is ambivalent. Majority of strains showed protective
action against oxidative stress, while some of them induced DNA damage
in untreated cells probably triggered by release of hydrogen peroxide from
bacterial cells (Koller et  al. 2008). Some compounds initiating cancer (car-
cinogens) may get into food consumed by humans. LAB can bind or degrade
these substances preventing cancer development. The binding is a physical
phenomenon, mostly expressed via cation-exchange mechanism. Intact cell
wall and peptidoglycan show higher binding activity than the bacterial cells.
However, binding process does not entail drastic changes in absorption and
distribution of carcinogens. It is rapidly reversed in the gut by unfavorable
conditions or factors inhibiting binding (Bolognani et  al. 1997; Rajendran
and Ohta 1998). Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus
salivarius ssp. thermophiles prevented DNA damage induced by N-methyl-
N′-nitro-N-nitroso guanidine in isolated primary rat colon cells. Possible
mechanism of this protection is associated with thiol-containing products of
protein breakdown catalyzed by bacterial proteases (Wollowski et al. 1999).
250    Pocket Guide to Bacterial Infections

The immune system takes part in control of cancer promotion and progres-

sion. Some LAB strains display immunomodulatory effect. and therefore they
may contribute to development of anti-tumor effect (Takagi et al. 2001).

Mechanisms of LAB beneficial action are diverse. They are manifested as

antimicrobial activity, enhancement of barrier function, immunomodula-
tory, and anti-cancer effects, influence on lipid metabolism of host organ-
ism. Health-promoting action of LAB facilitates its potential use in medicine
and formulation of functional food.

9.9  Medical application of lactic acid bacteria

Beneficial features of LAB make them an attractive target in diverse spheres,

including medicine. Many studies deal with treatment of various diseases by
LAB application. LAB can be used to control a wide range of diseases: diar-
rhea of various etiology, allergy, inflammatory bowel diseases, cancer, and so
on. Inflammatory bowel diseases represent a group of inflammatory dysfunc-
tions of the colon and small intestine, with Crohn disease (CD) and ulcerative
colitis (UC) as the principal types. Increase in the ratio of harmful bacteria
and reduction in the levels of beneficial bacteria is commonly associated
with inflammatory bowel diseases. Apart from it, abnormal host response to
luminal antigens, including the resident microflora, and enhanced mucosal
permeability characterize this condition. As mentioned previously, LAB dem-
onstrate antimicrobial activity, change intestinal permeability and modulate
immune response, so that LAB and bifidobacterial probiotics can provide a
remedy by improving clinical symptoms. The use of probiotics produced a
favorable effect on treatment and maintenance of UC, while effectiveness for
CD control was less significant (Bai and Ouyang 2006; Saez-Lara et al. 2015).

Diarrhea results from disequilibrium in the water flows in the gut mainly
generated by Na-solutes cotransport systems (Na-glucose) or chloride
secretion through the apical membrane of enterocytes. The first system is
related to water absorption, while the latter determines water secretion in
the intestinal lumen. These transporters or channels are highly regulated
structures and various factors may affect their performance leading to
diarrhea development. LAB are used in treatment of the disease caused
by viruses, pathogenic bacteria, antibiotics, and radiation. The same LAB
properties that improve condition of patients suffering from inflammatory
bowel diseases are able to mitigate effects of diarrhea of various etiolo-
gies. Lactose intolerance also may lead to this disorder. In the latter case
positive action of probiotics is related to the presence of β-galactosidase
hydrolyzing lactose to monosaccharide and eliminating intolerance problem
(Heyman 2000; Samaržija et al. 2009).
 Beneficial Lactic Acid Bacteria     251

Allergies are widespread health problems in the world. It is estimated that as

much as 30%–40% of global population is susceptible to allergenic agents
(Żukiewicz-Sobczak et al. 2014). Allergic state emerges as the result of an
inappropriate reaction to usually innocuous substances. Immune hypersen-
sitivity reactions are mediated predominantly by IgE antibodies or T cells
(Schnyder and Pichler 2009). As LAB are able to modulate immune system,
they can diminish effects of allergic reactions. Type I allergy is characterized
by shift in the T helper cell type 1 (Th1) and 2 (Th2) balance towards Th2-
dominated response, with increase in the levels of Th2 cytokines IL-4 and
IL-5. Lactobacillus plantarum strain inhibits allergic response through modu-
lation of Th1/Th2 balance and promotion of regulatory T cells (Ai et al. 2016).
The use of recombinant LAB producing the major birch pollen allergen Bet
v 1 leads to reduced allergen-specific IgE level concomitantly with increased
allergen-specific IgA concentration and offers a promising approach to pre-
vent systemic and local allergic immune responses (Daniel et al. 2006). Even
heat-killed strains show stimulating effect on IL-12p70  production, which
in turn shifts the balance between the T helper type 1 and 2 cell response
(Sashihara et al. 2006). LAB inducers of IL-12p70 and IL-10 in dendritic cells,
supporting IFN-and IL-10 production in CD4 + T cells reduce hyperrespon-
siveness, bronchial inflammation, and proliferation of specific T cells in cer-
vical lymph nodes (Van Overtvelt et al. 2010).

Hepatic encephalopathy is a common and usually reversible neurocognitive

syndrome occurring in patients with cirrhosis. It manifests itself as a spec-
trum of changes from the state of low-level cognitive dysfunction detect-
able in up to 70% of the patients leading to the plausible risk of cerebral
edema and death. Treatment of hepatic encephalopathy with Streptococcus
thermophilus and strains of Lactobacillus and Bifidobacterium exerted long-
term positive effects (Shavakhi et  al. 2014). Strain Enterococcus faecium
SF68 demonstrated the same efficiency as lactulose in treatment of chronic
hepatic encephalopathy, with no adverse symptoms and a 2-week remis-
sion (Loguercio et al. 1995).

As mentioned previously, LAB are able to regulate lipid metabolism in the

body and prevent cancer development. Various mechanisms of action pro-
vide opportunity to apply these bacteria in treatment of obesity, hypercho-
lesterolemia, colon cancer, and so on. Owing to production of a number of
beneficial compounds and immunomodulatory action, LAB probiotics may
be used as bioactive food supplements.

Production and application of probiotics and prebiotics. Management

and research. Market demand of probiotics and prebiotics. Current
market situation. Strategies.
252    Pocket Guide to Bacterial Infections

Probiotics are health-promoting microbial agents. The global probiotics

market totaled $31.8  billion and $34.0  billion in 2014 and 2015, respec-
tively. The market capacity should reach $50.0 billion by 2020, growing at
compound annual growth rate of 8.0% from 2015 to 2020 (Kumar 2016).
Expansion of probiotics market is driven by rising application in the animal
feed sector and consumer demand for natural products with health ben-
efits represented by beverages, probiotics, and supplemented foods (Global
Industry Analysts Inc. 2016). Functional foodstuffs and beverages segment
accounted for more than 80% of the probiotics market share in 2015. Dairy
products, cereals, baked food, fermented meat, and dry food were the
major commodities. The experts expect significant growth in the functional
food segment owing to rising demand instigated by a range of diseases
(Global Market Insights Inc. 2016). Prebiotics are usually nondigestible food
materials for supporting probiotic growth. Prebiotics consumption favors
growth of probiotic cultures, helping to fight chronic pathologies. Prebiotics
market size was above USD 3.5 billion in 2016 and may exceed USD 7 billion
in 2024, with consumption rate exceeding 1.4 million tons (Global Market
Insights Inc. 2017).

Production of probiotics is carried out in the following way. Using selec-

tion and technological criteria, a probiotic strain with defined features is
chosen. Composition of the medium and cultural conditions for bacterial
growth also pass through the optimization stage. A pretreatment phase is
essential to adjust substrate characteristics and eliminate microorganisms
able to interfere with fermentation process. After the pretreatment stage,
bacterial cultures are inoculated into the medium and incubated for the
definite period. After fermentation process, the product may be subjected
to processing and upgrading to acquire additional characteristics, like flavor,
taste, and so on. Finally the product is packed for further storage and mar-
ket supply (Novik et al. 2017).

9.10  Functional food and nutritional supplements

Functional food includes products imparting some health benefits to the

consumers. The presence of functional microorganisms such as LAB and
Bifidobacterium provides valuable properties of food. Fermented products
are used from ancient times; however, the first proposal of deliberate bacterial
use in food as probiotic was formulated by Metchnikoff (Mackowiak 2013).
Now functional foodstuffs are manufactured worldwide with the global mar-
ket worth about USD 129.39 billion in 2015. This market is expected to reach
USD 255.10 billion by 2024 (Grand View Research Inc. 2016).
 Beneficial Lactic Acid Bacteria     253

Bacteria in food demonstrate a whole spectrum of properties. Microorgan­isms

transform chemical constituents during food fermentation, enhancing acces-
sibility of nutrients, enriching food flavor and taste, rendering bio-preservative
quality, upgrading food safety, and degrading toxic components and anti-
nutritive factors (Tamang et al. 2016). They may take part in prevention and
treatment of diarrhea, inflammatory bowel disease, allergies, cholesterolemia
and lactose intolerance, immunomodulation and cause anticancer effects via
diverse mechanisms of action. Moreover, probiotic bacteria produce health-
promoting bioactive compounds. Summing up, LAB and bifidobacteria used
in food industry should meet certain selection criteria and possess technologi-
cal properties.

Biotechnological products. Agricultural and agro-industrial applica-

tions of lactic acid bacteria.

LAB find wide use in food processing as sources of probiotics and bioactive
compounds. As mentioned previously, the main part of probiotics is applied
in functional food and beverages, but some of them can be used in main-
tenance of health and meat quality as well as treatment and prevention of
various animal diseases. Lactobacillus pentosus strain LB-31 distinguished by
the best characteristics concerning growth parameters, lactic acid produc-
tion, acidic pH and bile salts tolerance, cell surface hydrophobicity, antimi-
crobial susceptibility, and antagonistic activity proved beneficial to broilers
due to ability of modulating the immune response and upgrading morpho-
physiological, productive, and health parameters of fowl (García-Hernández
et al. 2016). Commercial product FM-B11 containing eleven LAB poultry gut
isolates significantly reduced viability of Salmonella in day-of-hatch broilers.
Possible mechanisms of action involve competitive exclusion or stimulation
of a host innate immune response (Higgins et al. 2007). Wheat germ agglu-
tinin (WGA) lectins are components that protect wheat from insects, yeast,
and bacteria. WGA has toxic effects on intestinal epithelial cells obtained
from 14-d-old broilers depending on time of exposure and lectin concen-
tration. Adherent and nonadherent strains of LAB could avoid eukaryotic
cells-WGA interactions by different mechanisms. Nonadherent bacteria
could capture WGA in the intestinal lumen, reducing the amount of free
lectin able to interact with epithelial cells. Adherent bacteria attached to
intestinal cells interfered with the interaction between WGA and eukaryotic
cells. Despite bacterial binding to epithelial cell surface, in some cases the
latter remains vulnerable to damage (Babot et al. 2017). Supplementation
of Lactobacillus strains into broiler rations could improve the body weight
gain and feed conversion rate from 1 to 42 days of age and was effective
in cutting abdominal fat deposition but only after 28 days of age. Such diet
254    Pocket Guide to Bacterial Infections

additionally reduced serum total cholesterol, low-density lipoprotein cho-

lesterol and triglycerides in broilers from 21 to 42 days of age, but there
was no significant difference in serum high-density lipoprotein cholesterol
and in the weight of organs between control and Lactobacillus-fed broil-
ers (Kalavathy et al. 2003). Other experiments showed that the addition of
probiotic Lactobacillus spp. to the feed did not significantly improve weight
gain, feed intake, and feed conversion rate of broiler chickens, but tended
to increase the total number of anaerobic bacteria in the ileum and caeca,
the number of LAB in the caeca and to significantly raise the weight of small
intestine (jejunum and ileum), reducing the number of Enterobacteria in the
ileum, when compared with the control (Olnood et al. 2015).

Probiotics fed to ruminant livestock have been shown to decrease scours in

neonatal calves, to promote milk yields in dairy cows, decrease morbidity
in newly weaned calves and new calves at the feedlot, and increase daily
gains and carcass weight in feedlot cattle. Moreover, strains of Lactobacillus
acidophilus were shown to reduce fecal shedding of E. coli by feedlot cat-
tle at harvest time (Krehbiel et  al. 2003). Feeding microbial inoculum of
Lactobacillus casei, Lactobacillus salivarius, and Pediococcus acidilactici with
milk replacer, when young calves consumed a large quantity of spray-dried
whey powder generating intestinal imbalance, promoted earlier consump-
tion of starter, and indirectly, may have stimulated earlier development of
the rumen, omasum, and reticulum, favoring early weaning. Inoculated
calves showed better growth performance, which could be related to
improved digestion of lactose and spray-dried whey proteins (Frizzo et al.
2010). LAB are able to inhibit E. coli, preventing metritis development in
dairy postpartum cows mainly by acid production (Otero et al. 2006). Steers
fed with Enterococcus faecium EF212 had numerically lower concentrations
of blood CO2 than control steers, which is consistent with a reduced risk of
metabolic acidosis (Ghorbani et al. 2002).

LAB display a lot of beneficial effects on newborn and weaned piglets,

growing pigs, and sows. Studies demonstrated probiotic ability to increase
average dairy and weight gains, feed conversion, to modulate immune sys-
tem, to show antibacterial effects, to promote apparent ileal digestibility of
crude protein, crude fiber, and organic matter to alleviate diarrhea, to raise
the ratio of monounsaturated and polyunsaturated fatty acids to upgrade
meat quality (Yang et al. 2015).

The growth of aquaculture has accelerated over the past decades.

Aquaculture allows a selective increase in the production of species used for
human consumption, industry, or sport fishing. Viral, bacterial, and fungal
infections cause devastating economic losses worldwide. Use of chemical
 Beneficial Lactic Acid Bacteria     255

additives and veterinary medicines, especially antibiotics, generates signifi-

cant risks to public health by promoting the selection, propagation, and
persistence of bacterial-resistant strains. Therefore, probiotics are consid-
ered as the means to prevent and treat various diseases as well as to stimu-
late growth of aquatic organisms and feed conversion efficiency (Martínez
Cruz et al. 2012). Lactobacillus rhamnosus reduced the mortality of rainbow
trout (Oncorhynchus mykiss) significantly from 52.6% to 18.9% from furun-
culosis caused by Aeromonas salmonicida ssp. salmonicida (Nikoskelainen
et  al. 2001). Blue shrimps Litopenaeus stylirostrisfed treated with probiotic
Pediococcus acidilactici displayed lower infection (20% instead of 45% in the
control group) and mortality (25% instead of 41.7% in the control group) rates
under exposure to Vibrio nigripulchritudo. Compared to the infected control
group, probiotic-fed shrimp exhibited higher antioxidant status and lower oxi-
dative stress level (Castex et al. 2010). The rations supplemented with 0.01%
Lactobacillus acidophilus powder caused positive influence on growth, feed
utilization, and survival of snakehead (Channa striata) fingerlings (Munir et al.
2016). Administration of Lactobacillus plantarum for 60 days exerted favor-
able effects on the specific growth rate and feed utilization efficiency of Labeo
rohita juveniles, additionally increasing the serum lysozyme and alternative
complement pathway activities, phagocytosis, and respiratory burst activity
against Aeromonas hydrophila infection. The serum IgM levels were consider-
ably higher in the experimental groups as compared to the control group after
30 days of feeding. The treated fish displayed enhanced survival rate (77.7%)
(Giri et  al. 2013). Specific and relative growth rate, protein efficiency, and
feed-conversion ratio, survival, blood parameters, and total immunoglobulin
concentrations were much better in African catfish (Clarias gariepinus) fed
the ration supplemented with L, acidophilus when compared with the control
(Al-Dohail et al. 2009). Dicentrarchus labrax (European sea bass) juveniles sup-
plied with LAB showed 81% increment of body weight in a long-treated group
(59 days) and 28% rise in short-term experiment (25 days) with respect to the
control. Probiotics decreased cortisol levels in treated ­animals and affected
the transcription of two antagonistic genes involved in the regulation of body
growth—IGF-I and myostatin (MSTN). IGF-I transcription was increased, while
MSTN was inhibited (Carnevali et al. 2006).

Some experiments concerned the use of LAB in treatment of plant diseases.

Plant pathogen Ralstonia solanacearum causes bacterial wilt. Lactobacillus
sp. strain KLF01 isolated from rhizosphere of tomato reduced disease sever-
ity of tomato and red pepper as compared to nontreated plants (Shrestha
et  al. 2009a). Lactobacillus KLF01  and Lactococcus KLC02  strains showed
55% and 60% bio-control efficacy, respectively, in regard to Pectobacterium
carotovorum subsp. carotovorum, soft rot pathogen, on Chinese cabbage
256    Pocket Guide to Bacterial Infections

(Shrestha et  al. 2009b). These LAB significantly reduced bacterial spot
caused by Xanthomonas campestris pv. vesicatoria on pepper plants in com-
parison with untreated plants in both greenhouse and field experiments.
Additionally, LAB are able to colonize roots, produce indole-3-acetic acid,
siderophores, and solubilize phosphates (Shrestha et al. 2014). LAB are effec-
tive in the removal of the root-knot nematodes. The decreased pH levels in
agricultural soil due to lactic acid produced by bacteria are correlated with
reduced population of nematodes (Takei et al. 2008). Microalgae are used
as feed for live prey (rotifers, Artemia), larvae and adult fish, mollusks, and
crustaceans. The growth of microalgae Isochrysis galbana was enhanced by
LAB, both in the absence and in the presence of nutrients in the culture. The
highest final biomass concentration was achieved by adding Pediococcus
acidilactici, whereas Leuconostoc mesenteroides spp. mesenteroides and
Carnobacterium piscicola provided for maximal growth rates. However, the
latter species also showed inhibitory effect on Moraxella (Planas et al. 2015).

Agriculture, medicine, and food industry are not the only application fields for
LAB and their products. Many cosmetic ingredients have been developed using
LAB and bifidobacteria. Supernatants of these bacteria contain lactate and
amino acids, which contribute to the hydration of the skin. Studies revealed
that skim milk fermented by Streptococcus thermophilus had skin hydrating,
antioxidative, cytoprotective, and pH control effects. Aloe vera fermented by
Lactobacillus plantarum possessed four times higher skin moistening effect
than nonfermented A. vera juice. Soybean milk fermented by Bifidobacterium
breve demonstrated the potential to enhance hyaluronic acid production in
human cell culture. Streptococcus thermophilus YIT 2084 proved capable to
produce hyaluronic acid used as conventional cosmetic ingredient (Izawa and
Sone 2014). Lactic acid itself is primarily used as moisturizer and pH regulator,
additionally possessing multiple other properties such as antimicrobial activity,
skin lightening, and hydration (Vijayakumar et al. 2008).

In the chemical industry, lactic acid undergoes a variety of chemical con-

versions into potentially useful chemicals and takes part in diverse techno-
logical processes. The compound is used in dyeing of silks and other textile
fabrics, as a mordant in printing of woolens, in bating and plumping of
leathers, in deliming of hides, in vegetable tanning, and as a flux for soft
solders (Vijayakumar et  al. 2008). Recently lactic acid has drawn atten-
tion as substrate for the production of polylactic acid (PLA) or biodegrad-
able plastic. Lactic acid can be used to produce PLA of variable molecular
weight, but usually the polymer with high molecular weight has the supe-
rior commercial value. There are three main methods to synthesize PLA:
 Beneficial Lactic Acid Bacteria     257

direct condensation polymerization; direct polycondensation in an azeo-

tropic solution; polymerization through lactide formation. The major PLA
application sphere today is packaging (nearly 70%); however, the poly-
mer is used in other fields. It is applied in implants and medical devices.
Because PLA degrades with time, the removal of implanted detail is not
required. Nevertheless, in some cases, such implants may cause rejection
reaction from the human host. In medical devices, PLA is considered as an
alternative to metal implants responsible for possible corrosion and distor-
tion of magnetic resonance images. The polymer can be readily processed
into fibers due to its ability to absorb organic compounds and its wick-
ing properties. Some other potential applications of PLA include paints,
cigarette filters, three-dimensional printing, parts for space exploration,
and environmental remediation (Jamshidian et  al. 2010; Castro-Aguirre
et al. 2016).

9.11  Conclusions and future prospects

LAB are gram-positive rods or cocci mostly belonging to order

Lactobacillales. These bacteria are fastidious microorganisms requiring
definite cultivation conditions and various components in the medium.
In turn, LAB produce a number of valuable compounds such as bacte-
riocins, vitamins, enzymes, exopolysaccharides, sweeteners, lactic acid,
and others. Provided the defined cultural conditions and nutrient medium
composition, diverse strains display the ability to ferment substrates and
synthesize bioactive compounds. To demonstrate vital properties and gain
maximum benefits from them, LAB cultures are selected according to the
established criteria.

LAB have long history of application, and they are usually considered safe
microorganisms for use in medicine and food industry. LAB can contribute
as probiotics into maintenance of human and animal health, prevention, and
treatment of diarrhea of various etiology, allergy, inflammatory bowel diseases,
and cancer. They are widely used in production of fermented foodstuffs, espe-
cially dairy products, cereals, fermented meat, and baked and dry food. The
market of probiotics grows steadily and is expected to reach USD 50.0 billion
by 2020 due to rising demand in the animal feed sector and consumer interest
in health-promoting beneficial food. Some studies revealed favorable effect
of bacteria on plants. Moreover, LAB and the derived products demonstrate
attractive commercial prospects in other fields, like chemistry and the manu-
facture of plastics. New LAB applications are likely to emerge in the near future.
258    Pocket Guide to Bacterial Infections

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10
Role of Bacteria in
Dermatological Infections
Thirukannamangai Krishnan Swetha and
Shunmugiah Karutha Pandian

Contents

10.1 Introduction 280

10.2 Skin, an indispensable innate barrier 281
10.3 Skin microflora 282
10.4 Acute bacterial skin and skin structured infections 284
10.4.1 Impetigo 286
10.4.1.1 Epidemiology 286
10.4.1.2 Diagnosis 287
10.4.1.3 Prevailing treatment options 287
10.4.2 Folliculitis 287
10.4.2.1 Epidemiology 287
10.4.2.2 Diagnosis 287
10.4.2.3 Prevailing treatment options 288
10.4.3 Cellulitis 288
10.4.3.1 Epidemiology 288
10.4.3.2 Diagnosis 289
10.4.3.3 Prevailing treatment options 289
10.4.4 Scarlet fever 289
10.4.4.1 Epidemiology 290
10.4.4.2 Diagnosis 290
10.4.4.3 Prevailing treatment options 290
10.4.5 Staphylococcal scalded skin syndrome 290
10.4.5.1 Epidemiology 291
10.4.5.2 Diagnosis 291
10.4.5.3 Prevailing treatment options 291
10.4.6 Wound infections 291
10.4.6.1 Epidemiology 292
10.4.6.2 Diagnosis 292
10.4.6.3 Prevailing treatment options 292

279
280    Pocket Guide to Bacterial Infections

10.4.7 Cutaneous abscess 293

10.4.7.1 Epidemiology 293
10.4.7.2 Diagnosis 293
10.4.7.3 Prevailing treatment options 293
10.4.8 Necrotizing fasciitis 294
10.4.8.1 Epidemiology 294
10.4.8.2 Diagnosis 294
10.4.8.3 Prevailing treatment options 295
10.5 Predominant bacteria in skin infections 295
10.5.1 S. aureus 296
10.5.2 Streptococcus spp. 299
10.5.3 P. aeruginosa 302
10.5.4 Predominant skin commensals as opportunistic
skin pathogens 304
10.5.4.1 S. epidermidis 304
10.5.4.2 P. acnes 305
10.5.4.3 Corynebacterium spp. 306
10.6 Conclusion and future prospects 307
Acknowledgments 308
References 308

10.1 Introduction

Skin serves as an effective barrier that comes into immediate action as the
first line of defense by providing shelter to the inside of an organism from
environmental assaults such as ultraviolet (UV) irradiations, chemical toxins,
oxidative stress, and so on (Proksch et al., 2008). It also harbors a diversified
microbiota including bacteria, fungi, and viruses that render the host with a
protective shield against colonization of pathogenic microorganisms (Chiller
et al., 2001). Any breach in the dermal layer disgruntles the barrier action
of the skin and aids easy entry of pathogens, which in turn, lead to the
development of skin and skin structure infections (SSSIs; also known as skin
and soft tissue infections [SSTIs]) (Proksch et al., 2008; Ibrahim et al., 2015).
Dermatological dysfunctions, with special emphasis on those influenced by
bacterial attack, cover a wide spectrum of infections, from mild superficial
cutaneous infections such as folliculitis to life-threatening necrotizing fasciitis
(Palit and Inamadar, 2010). Based on the depth of the skin layer infected,
there are massive conditions that define the severity of bacterial skin infec-
tions as complicated SSSIs (cSSSIs) and uncomplicated SSSIs (Eisenstein,
2008). In 2008, new terminology was proclaimed by the U.S. Food and
Drug Administration (FDA), namely acute bacterial SSSI (ABSSSI), to describe
bacterial-aided SSSIs with a characteristic lesion size of at least 75  cm2
 Role of Bacteria in Dermatological Infections     281

(Pulido-Cejudo et al., 2017). ABSSSIs are a huge encumbrance in clinical set-

tings because the sensitivity pattern of infecting bacteria to current antibiotic
treatment is found to be altered (Eisenstein, 2008). Besides, bacteria form
biofilm on healthy and diseased epidermal surfaces (Vlassova et al., 2011).
Biofilm is a sessile microbial population encased in an extracellular polymeric
matrix and is a defensive action presented by microbes to sustain antibiotic
treatment and host immune attack (Subramenium et al., 2015a). Skin biofilm
formed in diseased state worsens the severity of infection by enduring the
antibiotic treatments (Vlassova et  al., 2011). The incessant increase in the
development of resistance to A recent generation of antibiotics by bacteria
impairs the management of ABSSSIs and demands alternate therapies to
tackle the situation (Shah and Shah, 2011). This standpoint describes the
requisite of deep insights to the clinical presentations, pathogen involved,
mode of pathogen entry, severity of infection, and proper choice of treat-
ment for effective management of ABSSSIs (Ki and Rotstein, 2008). This
chapter mainly reviews different ABSSSIs, their pathophysiology, clinical pre-
sentations, diagnosis, prevailing treatment strategies, and other emerging
alternative approaches for efficacious control of ABSSSIs outbreak.

10.2  Skin, an indispensable innate barrier

Skin furnishes the magnificent integumentary system and is the largest

organ of higher eukaryotes that makes up to approximately 16% of whole
body weight of an individual and covers a surface area of about 1.8 m2. It is
bequeathed with three structural layers, namely epidermis (outermost), der-
mis (middle), and hypodermis (innermost). Despite the fact that it is reliably
structured throughout the body, the age of an individual and the anatomi-
cal location greatly influences the thickness of the skin (Kanitakis, 2002).

Skin is an indispensable inborn barrier that constitutes the first layer of defense
and concretes protective armor to the inside of an individual against environ-
mental assaults. It plays numerous roles as a barrier (Figure 10.1) such as:

• Physical barrier to shield against mechanical disturbances,

• UV barrier to resist UV mutilations,
• Oxidant barrier to protect the cell membrane and lipids from oxidative
stresses,
• Thermal barrier to acclimatize the body from different climatic conditions,
• Permeability barrier to thwart water loss and resist entry of undesirable
allergens, and
• Microbial barrier to preclude the colonization of other pathogens on host
(Menon and Kilgman, 2009).
282    Pocket Guide to Bacterial Infections

Figure 10.1  The barrier functions of skin. Skin is an indispensable barrier that pro-
tects the inside of an individual from mechanical and chemical assaults, oxidative
stresses, ultraviolet mutilations, different climatic conditions, water loss, and entry
of pathogenic microbes. Disruption of skin integrity paves way for skin infections.

Despite the multiple barrier functions performed by the skin, there are mul-
titude cutaneous infections caused by the mutilation of epidermal integrity
and alterations in skin microbial community influenced by environmental
factors (Ibrahim et al., 2015).

10.3  Skin microflora

Ever since birth, the skin serves as the host for diversified collection of
microbes such as bacteria, fungi, and viruses and affords an apposite environ-
ment for microbial growth (Chiller et al., 2001). A handful of researches have
used amplicon sequencing and shotgun metagenomics sequencing tech-
niques, which have erected the primary pipelines for understanding the diver-
sity of skin microbial communities (Costello et al., 2009; Grice et al., 2009;
Findley et  al., 2013; Oh et  al., 2014, 2016). The exploration of skin micro-
bial community in healthy individuals has divulged the primary abundance
of bacterial kingdom at almost all sites of the skin and fungal species as the
least abundant kingdom (Oh et al., 2016). Commonly touted skin residential
 Role of Bacteria in Dermatological Infections     283

Figure 10.2  Skin microflora. Skin harbors a diversified collection of microbes such
as bacteria, fungi, and viruses. The abundant bacterial kingdom majorly constitutes
the microbial barrier, which includes S. epidermidis, P. acnes, and Corynebacterium
spp. These bacterial species produce lantibiotics and prevent the entry of other
pathogens. S. epidermidis in a competitive environment triggers toll-like receptor
(TLR) pathway, which in turn recognizes the pathogen-associated molecular pat-
terns and activates the host immune system accordingly. The production of free fatty
acids by P. acnes as a result of triglyceride metabolism prevents the colonization of
S. aureus, S. pyogenes, several gram-negative species, yeasts, molds, and so on.

bacterial species descend as Staphylococcus spp., Corynebacterium spp., and

Propionibacterium spp. (Cogen et  al., 2008). However, the skin microflora
greatly diverges in proportions and in structure among individuals as a conse-
quence of host genetics, sex, age, and regional and local environmental fac-
tors (Rosenthal et al., 2011). These commensals prominently play a beneficial
role of protecting the skin either directly or indirectly from the attack of other
pathogens (Figure 10.2). For instance, S. epidermidis armors the skin from
colonization of infectious S. aureus by binding the keratinocyte receptors on
skin. Similarly, P. acnes, an indigenous colonizer of sebaceous glands, catabo-
lizes the sebum lipids and releases fatty acids, which in turn, establishes an
inapt milieu for surviving virulent S. pyogenes (Chiller et al., 2001).
284    Pocket Guide to Bacterial Infections

In circumstances, environmental factors such as climatic conditions, individ-

ual’s lifestyle, choice of clothing, usage of antibiotics, usage of cosmeceuti-
cal products such as soaps, cream, lotion, and so on, altogether impacts
the normal microflora of skin. Alterations in skin microflora are now deliber-
ated to play essential role in skin infections. Additionally, dermal breaches
perturb the barrier function of the skin and permeate the invasion of com-
mensals as opportunistic pathogens and colonization of other pathogens,
which eventually culminate in skin infections with various degree of severity
(Grice and Serge, 2011).

Though understanding the interaction of skin microbiota with host and

with other pathogens remains unclear, it is an important prerequisite to
gain deep insights into the severity and underlying risk factors of skin infec-
tions, which will help to gain advancement in designing the appropriate
treatment strategies to preclude the difficulties in the management of skin
infections.

10.4  Acute bacterial skin and skin structured infections

Impairment in barrier activities of skin paves way for the infiltration of skin
commensals as pathogens and other invading microbes into the underly-
ing soft tissues and layers of skin, which results in the development of
SSSIs (Ki and Rotstein, 2008). Until 2008, SSSIs was stratified based on
the severity of the infection into two categories namely, uncomplicated
SSSI and cSSSI. Uncomplicated SSSIs generally include mild infections such
as impetigo, furunculosis, carbuncles, cellulitis, erysipelas, and other mild
abscesses. cSSSIs encompass spreading and deeper tissue infections such
as necrotizing cellulitis, infected wound burns, deep-tissue abscesses, and
infected ulcers, which require immediate attention (Ramana et al., 2013). A
decade ago, the FDA categorized cSSSIs and uncomplicated SSSIs as acute
bacterial skin and skin structured infections (ABSSSIs) (Figure 10.3) (Shah
and Shah, 2011). According to the guidelines of the FDA, the skin infections
with minimum lesion size of 75 cm2 and characteristic symptoms such as
redness, edema, and other local signs have been recognized as ABSSSIs
(Moran et al., 2013).

Bacterial skin infections are opportunistic and have a great predilection for
patients who are immunocompromised and individuals with other predis-
posed conditions. Identification of nature of infection such as mild or deep
and localized or spreading is quintessential, which lays foundation for deter-
mining the urgency and proper strategy of the treatment. It is generally
seen as almost curable if prompt diagnosis and immediate and appropriate
Figure 10.3  Acute bacterial skin and skin structured infections (ABSSSIs). ABSSSIs include a wide spectrum of infections ranging from mild super-
ficial impetigo, folliculitis to deep-seated cellulitis and life-threatening necrotizing fasciitis.
Role of Bacteria in Dermatological Infections     285
286    Pocket Guide to Bacterial Infections

treatment is provided to the patient. However, delayed or improper treat-

ment worsens the scenario, and the resulting secondary effects can be life
threatening (Hedrick, 2003). Thus, reviewing the etiologic agents, clinical
presentations, diagnosis, and treatment options of common bacterial skin
infections is essential.

10.4.1 Impetigo

Impetigo is a common superficial skin infection, which is greatly contagious

and usually affects children ages 2 to 5. It is classified into two types, non-
bullous impetigo (impetigo contagiosa) and bullous impetigo.

Impetigo contagiosa is slightly pruritic, which is mainly characterized by the

development of small pustules that exude at later stages and form honey-
colored crust. S. aureus is the predominant etiologic agent, whereas group
A Streptococcus (GAS) hits several cases either alone or in association with
S. aureus. The dissemination of nonbullous form to surrounding areas
occurs through autoinoculation. Secondary impetigo, a subtype of nonbul-
lous impetigo, increases the severity of systemic infections (such as diabetes
mellitus) and also prompts the individual with dermal breaches to develop
common impetigo. The recovery rate usually involves 2 to 3 weeks without
any treatment, whereas antibiotic treatment speeds up the rate of recovery
and lessens discomfort (Parker, 1955; Hedrick, 2003).

Bullous form of impetigo mainly develops due to the exfoliative toxin

A (ETA) produced by coagulase positive S. aureus. Neonates remain to be
the primary choice of bullous impetigo. However, it affects the elderly, too.
The clinical presentations of bullous form comprise development of small
vesicles that enlarge to become flaccid bullae and ooze out on exudation
(Amagai et al., 2000).

The severe and deeper form of impetigo is referred to as ecthyma, which

spreads to the dermis and is characterized by ulceration under crusted surface.
The management of ecthyma remains the same as that of impetigo because
the bacterial etiologic agents implicated in the infections are same (Pye, 2010).

10.4.1.1  Epidemiology – Impetigo accounts for 10% of overall skin prob-

lems and 30% of it are observed to be bullous form. Staphylococcal scalded
skin syndrome (SSSS) is the localized form of bullous impetigo, which causes
less than 3% of deaths in children and more than 60% in the elderly popula-
tion (Hanakawa et al., 2002). Moreover, it has been reported that the global
incidence of impetigo in neonates at any particular time point hits more than
162 million, which also heightens the incidence of life-threatening rheumatic
heart disease and arthritis due to rheumatic fever (Bowen et al., 2015).
 Role of Bacteria in Dermatological Infections     287

10.4.1.2  Diagnosis – Impetigo is generally identified clinically by cultur-

ing the lesion material acquired from infected area. Gram staining is rarely
performed to confirm the infection (Cole et al., 2007).

10.4.1.3  Prevailing treatment options – The current treatment options

of impetigo include topical and oral antibiotics. Mupirocin (commercially
known as Bactroban), a topical antibiotic, is used as the first-line of defense
for treating impetigo. However, patients who remain non-responsive to
topical treatment are treated with oral antibiotics such as penicillin V, ceph-
alexin, amoxicillin/clavulanate, and dicloxacillin. Antibiotic resistance among
bacterial species increases the treatment cost and incapacitates the man-
agement of impetigo (Bangert et al., 2012).

10.4.2 Folliculitis

Folliculitis is the inflammation of hair follicle caused due to virus, fungi,

or bacteria. Poor hygiene, exposure to certain chemicals, humid environ-
ment, maceration, hyperhydration, and occlusion are several predisposing
factors that conciliate the development of folliculitis. Folliculitis is charac-
terized by formation of small pustules with erythematous base that later
expand into clusters and crusts. Bacterial folliculitis may be superficial or
deep that occurs at different sites such as the scalp, buttocks, axillae, medial
thigh, and face of children and adults. S. aureus is the major cause of bac-
terial folliculitis; however, other bacterial species such as Pseudomonas,
Streptococcus, Proteus, and some Coliform bacteria are also involved in the
infection (Luelmo-Aguilar and Santandreu, 2004).

The symptoms usually include boils, scarring, furunculosis, and permanent

hair loss at the site of infection. The spread of infection into the tissues of
follicles culminates in the development of furuncles and carbuncles and the
untreated folliculitis may transpire into life-threatening necrotizing fasciitis
(Palit and Inamadar, 2009).

10.4.2.1  Epidemiology – A survey of bacterial skin infections in the north-

ern part of India among schoolchildren had reported that 64.4% cases of
skin infections were observed to be folliculitis together with impetigo (Palit
and Inamadar, 2009). Also, it accounts a total of 11% of all primary scarring
hair loss or cicatricial alopecia cases (Otberg et al., 2008).

10.4.2.2  Diagnosis – The physical examination of patient for clinical signs

and studying the patient history for analyzing other predisposing factors
helps in preliminary identification of folliculitis. And, the laboratory exami-
nation of pustular material collected from the site of folliculitis is usually
considered for identification of etiologic agent (i.e., bacteria, fungi, or virus)
288    Pocket Guide to Bacterial Infections

implicated in the infection. Histopathologic studies confirm and ease the

process of diagnosis and proper classification of infection (Luelmo-Aguilar
and Santandreu, 2004).

10.4.2.3 Prevailing treatment options – The use of topical antibiotics

such as mupirocin, neomycin, and fusidic acid for 7 to 10 days remains to be
the first-line of defense for generalized therapy. If treatment fails to work and
or the infection is spreading and severe, proper diagnosis of etiologic agent
usually soothes the process of identifying specified antibiotic treatment. In
the latter case, use of systemic antibiotics such as erythromycin and flucloxa-
cillin for 1 week are the prime choice of treatment. Heavy dose of trime-
thoprim prevails as the choice of treatment, if the etiologic agent implicated
in the infection is identified to be a gram-negative microorganism (Pye, 2010).

10.4.3 Cellulitis

Cellulitis is a common, deeper, and generally nonpurulent inflammatory

skin infection caused by bacteria. This disseminating skin infection is usu-
ally characterized by edema, pain, tenderness, swelling, warmth, local ery-
thema, lymphangitis and white blood cell infiltration (Hook et al., 1986). The
dermal and subcutaneous layers of skin remain inflamed in cellulitis, due
to infection caused by S. pyogenes, coagulase positive S. aureus, P. aeru-
ginosa, Haemophilus influenzae and other beta hemolytic Streptococcus
(BHS) that gain their access through impaired skin barrier, dermatitis, and
skin ulceration. However, S. pyogenes is the major etiologic agent of celluli-
tis, which generates inflammation of skin by enzymatic hydrolysis of cellular
components (Hedrick, 2003; Chira and Miller, 2010).

Frequently witnessed facial cellulitis is now a rarely observed type, which

is recently demarcated as erysipelas (superficial cellulitis) in some literature.
The vital complication associated with facial cellulitis comprises odonto-
genic or orbital infections, which require immediate attention and surgical
episodes (Stevens et al., 2014). Perianal cellulitis is witnessed in young chil-
dren and is characterized by purulent drainage, perianal pruritus, fissures,
and rectal bleeding. Periorbital cellulitis is observed in eyelid portion and
periorbital tissues. Buccal cellulitis is greatly observed in children before vac-
cination with conjugated H. influenzae type b vaccine and is responsible for
25% of the facial cellulitis cases (Swartz, 2004). Purulent cellulitis may also
be observed in several cases, which occur as an extension of initial abscess
and culminate in secondary cellulitis followed by purulent drainage and exu-
dation (Ibrahim et al., 2015).

10.4.3.1  Epidemiology – In the United States, 14.5 million cases of cellu-

litis were observed annually and hospitals specialized for cellulitis were also
 Role of Bacteria in Dermatological Infections     289

mounted (Arakaki et al., 2014). Moreover, cellulitis is an important compli-

cation associated with HIV, wherein, a retrospective study of epidemiol-
ogy of cellulitis in patients with HIV has shown that cellulitis accounted for
3.02% of overall admissions (Manfredi et al., 2002)

10.4.3.2 Diagnosis – Analysis of morphological characteristics of infec-

tion and patient history for identification of predisposing factors remains
as a standard method for diagnosing cellulitis. Identification of bacterial
species implicated in infection is quite difficult, unless pus or open wound
have developed on the skin (Hedrick, 2003). Elevated white cell count and
C-reactive protein (CRP) are several inflammatory markers employed for
diagnosis, which supports detection, but cannot be used solely to identify
the infection because of non-specificity. Skin biopsies and magnetic reso-
nance imaging (MRI) are engaged for distinguishing the fatal necrotizing
fasciitis from cellulitis because misdiagnosis occurs frequently and increases
the treatment cost (Schmid et al., 1998; Swartz, 2004).

10.4.3.3 Prevailing treatment options – Management of mild cel-

lulitis involves oral antibiotic treatment such as penicillin and flucloxacillin
(500 mg to 1 g for four times a day). The recovery rate usually takes about
1 to 2 weeks. Patients allergic to penicillin are treated with clarithromycin
as an alternate. Intravenous antibiotic therapy is suggested for severe cel-
lulitis cases and patients diagnosed with other systemic infections and other
complications (Pye, 2010).

10.4.4  Scarlet fever

Scarlet fever, or scarlatina, is the infection caused by GAS, typically as an

extension of strep throat and is characterized by fever, erythematous sore
throat, sand-paper like rash, and enlarged papillae on tongue (strawberry-
like tongue). The papillary eruptions generally initiate from groin, then dis-
seminate jointly to the trunk and axillae, and finally reach extremities in a
week’s time, followed by desquamation on palms and soles. Though, it
affects people of all ages with predominance among males over females,
the episodes of scarlet fever in children is still high (Basetti et al., 2017).

The erythrogenic toxins produced by GAS (now classified as pyrogenic

exotoxins) are the major etiologic agent of this infection (Wessels, 2016).
Recent researchers have divulged a collection of related streptococcal pyro-
genic exotins (SPE). Eleven such toxins are involved in infection and no single
toxin was found in all the cases because each Streptococcal strain was capa-
ble of generating four to six toxins (Spaulding et al., 2013). However, the
prime toxins SpeA, SpeC, and SSA are often found in combination in several
episodes (Davies et  al., 2015). Complications that are possibly associated
290    Pocket Guide to Bacterial Infections

with scarlet fever include rheumatic fever, more invasive streptococcal toxic
shock syndrome, suppurative arthritis, toxic myocarditis, osteomyelitis,
allergic glomerulonephritis, and meningitis (Hedrick, 2003).

10.4.4.1  Epidemiology – Previously demoted as less episodic, scarlet fever

has surged as a major health issue with massive episodes in recent decades.
The outbreaks of scarlet fever were stated to be surged in England, Hong
Kong, Vietnam, South Korea, and China. The epidemic of scarlet fever in
2009 was found to be very high, affecting nearly 23,000 individuals in
Vietnam and 100,000 individuals in mainland China (Basetti et al., 2017).

10.4.4.2  Diagnosis – Because the severities linked with scarlet fever are
extensive, early clinical diagnosis remains difficult. Clinical examination
and case history analysis are useful in diagnosis. Centor score is an aid-
ing technique used for the identification of scarlet fever across numerous
healthcare settings in various countries. This robust technique includes four
clinical signs and symptoms and is used to range the infection as “specific”
and “very specific” for GAS pharyngitis, when the clinical condition satis-
fies more than three or four symptoms, respectively. However, prolonged
pyrexia and tachycardia together with dissemination of rashes to trunk
ascertains the clinical condition of scarlatina (Aalbers et al., 2011).

10.4.4.3  Prevailing treatment options – Beta-lactam antibiotics remain

as the classic choice of treatment. Penicillin is prescribed frequently because
of its reduced costs and ability to outstrip cephalosporin and macrolides.
Macrolides are reported to favor antibiotic resistance, produce adverse
effects, and found to be toxic at effectual doses. For managing scarlet fever,
phenoxymethylpenicillin penicillin (V) is prescribed for 10 days (four times
a day) and the repercussions are usually lessened using over-the-counter
drugs such as paracetamol, ibuprofen, and so on (Basetti et al., 2017).

10.4.5  Staphylococcal scalded skin syndrome

SSSS is blistering skin disorder with preponderance for neonates and chil-
dren younger than ages 5 or 6 and not in adults. In 1878, Baron Gotfried
Ritter von Rittershain witnessed 297  cases of SSSS, and referred to it as
“dermatitis exfolitiva neonatorum,” which was later demarcated as Ritter’s
disease (Patel and Finlay, 2003).

SSSS is mediated by epidermolytic exfoliative toxins A and B (ETA and ETB)

produced by S. aureus. These ETA and ETB exaggerate the epidermal blis-
tering by proteolytic cleavage of desmoglein 1 (a cell-cell adhesion mole-
cule), which is expressed by keratinocytes in epidermal zona granulosa (Ross
and Shoff, 2017). SSSS initially commences with fever, pruritus, discomfort,
 Role of Bacteria in Dermatological Infections     291

redness of skin, and development of fluid-filled blisters on groin, armpits,

and body orifices such as the nose and ears, which disseminates to the
trunk and extremities followed by epidermal exudation within 72 hours that
leaves marks similar to that of burns. The clinical indications include chill-
ness, fever, and malaise with early signs of conjunctivitis or sore throat. The
potential complications comprise electrolytic imbalance, pneumonia, and
sepsis (Hedrick, 2003).

In neonates, SSSS is frequently observed in the diaper area and axillary and
naval regions. Adults are usually less prone to SSSS because of the presence
of antibodies specific to the toxins, which are developed during childhood
period. However, adults with a compromised immune systems and renal
failure are predisposed to the attack of SSSS (Lamanna et al., 2017).

10.4.5.1 Epidemiology – A retrospective study in France of the Czech

Republic estimated the outbreak of SSSS to be 2.53 cases per million per
year. In Europe, 0.56  cases per million per year was admitted with SSSS
(Lamanna et  al., 2017). The mortality rate associated with SSSS was
reported to be approximately 4% in children and about 50%–60% in adults
(Pye, 2010; Li et al., 2014).

10.4.5.2  Diagnosis – An assessment of case history along with evaluation

of clinical features of infection is usually done. Clinical diagnosis of SSSS is
performed by skin biopsies, analysis of frozen sections of blisters, and exfo-
liative cytology. Culturing of samples obtained from blisters in affected area
is helpful in identifying the etiologic agent of infection (Hedrick, 2003; Ross
and Schoff, 2017). Also distinguishing SSSS from toxic epidermal necrolysis
(TEN) is vital because the latter involves high mortality rate.

10.4.5.3  Prevailing treatment options – Intravenous antibiotics are usu-

ally administered for management of SSSS. Flucloxacillin remains as the pre-
ferred choice of treatment. Substitution of intravenous antibiotics with oral
antibiotics can be done after several days based on treatment response.
The risk of electrolytic imbalance is resolved by the administration of intra-
venous fluids. The recovery of skin health and heat loss reduction is carried
out using nonadherent dressings and emollients on skin (Pye, 2010; Ross
and Schoff, 2017).

10.4.6  Wound infections

The wound is generally abrasions, breaches, or breaks on anatomical struc-

tures such as skin that can extend to underlying tissues, dermal layers, and
even to the bone as a consequence of trauma or surgery (Boateng and
Catanzano, 2015). Wound infections are caused by the bacterial colonization
292    Pocket Guide to Bacterial Infections

on chronic wounds that prolongs the recovery rate and remains too unfa-
vorable for wound-healing process (Edwards and Harding, 2004). Burn
wounds predispose patients for high pathogenic attack and raise the prob-
ability of mortality and morbidity by increasing the risks of other complica-
tions such as multiple-organ dysfunction syndrome, systemic inflammatory
response syndrome, and severe sepsis (Schwacha et al., 2005).

In 2000, a retroactive study conducted by Giacometti et al. (2000) among

676 patients who underwent surgery for the analysis of possible bacte-
rial strains associated with wound infections showed the prominence of
aerobic bacteria in almost all cases with different percentage coverage by
different bacterial strains such as S. aureus (28.2%), P. aeruginosa (25.2%),
Escherichia coli (7.8%), S. epidermidis (7.1%), and Enterococcus faecalis
(5.6%). Polymicrobial infections were also witnessed with more predomi-
nance of P. aeruginosa and S. aureus combination. Another experimen-
tal study by Bessa et  al. (2015) using 312  wound samples collected from
213  patients at different wound sites have witnessed the prominence of
bacterial strains in the order S. aureus (37%), P. aeruginosa (17%), Proteus
mirabilis (10%), E. coli (6%), and Corynebacterium spp. (5%) with 59.2% inci-
dence of polymicrobial infections.

Bacterial biofilms are highly perceived on chronic wounds, including pres-

sure ulcers (PUs), diabetic foot ulcers (DFUs), and venous leg ulcers (VLUs).
Biofilms are a great menace to the management of wound infections
because most of the antibiotics administered remains ineffective against
bacterial biofilms (Malone et al., 2017).

10.4.6.1  Epidemiology – During mid of nineteenth century, surgical site

wound infections were predominantly observed that accounted for approx-
imately 70% to 80% of mortality. Surgical site infections ranks third place
of all recurrently reported infections in patients who are hospitalized, and
it responsible for 12% to 16% of all nosocomial infections (Cooper, 2013).

10.4.6.2  Diagnosis – The conventional methods for the identification of

causative agents of wound infections include tissue biopsies, wound swabs,
culturing of samples recovered from infected site, and polymerase chain
reactions (PCR) (Weinstein and Mayhall, 2003).

10.4.6.3 Prevailing treatment options – As identification of etiologic

agent of wound infections is quite time consuming, empirical antibiotic treat-
ments are often preferred, wherein generalized antibiotics that target a broad
spectrum of bacteria ire administered (Cooper, 2013). However, proper iden-
tification of causative agents eludes the risk of drug resistance and reduces
 Role of Bacteria in Dermatological Infections     293

the health expenditure. Also, wound dressings that deliver the actives such as
antiseptic agents or antibiotics at the wound sites are used often to reduce
the bacterial load and soothe the process of wound healing. Topical antimi-
crobials in the form of lotions, creams, and liquids are also used in the man-
agement of wound infection (Boateng and Catanzano, 2015).

10.4.7  Cutaneous abscess

Cutaneous and other soft skin abscesses are usually trailed by skin trauma
and are characterized by pus collection, redness, edema, and swelling of
soft tissues accompanied by erythema. Lesions may be encountered on
neck, face, extremities, trunk, axillae, and perianal regions. Based on the
severity, abscesses may be simple or uncomplicated skin abscesses (super-
ficial) or complicated skin abscesses (disseminating and deeper). The latter
type is likely to be associated with complications such as cellulitis, regional
lymphadenopathy, septic phlebitis, lymphangitis, and leukocytosis (Hedrick,
2003). It is predominantly observed among injection drug users (IDUs). The
use of heroin and cocaine mixtures (also called “speedballs”) containing
injections is reported to induce soft-tissue ischemia and predispose the
individual to develop abscessws (Murphy et al., 2001). The major etiologic
agents that spurred the development of infectious abscess include S. aureus
(especially community acquired methicillin-resistant S. aureus [CA-MRSA]),
alpha hemolytic and nonhemolytic Streptococcus, Propionibacterium spp.,
and Bacteriodes spp. (Hedrick, 2003; Lee et al., 2004).

10.4.7.1  Epidemiology – In urban medical centers, cutaneous abscesses

are reported to account for approximately 2% of all cases that visited emer-
gency facilities with predominance for IDUs (Talan et al., 2000). In the United
States, the annual epidemic of SSSI has been chronicled to be inclined from
1.2 to 3.4 million between 1993 and 2005, primarily with massive episodes
of cutaneous abscesses at emergency departments (Qualls et al., 2012).

10.4.7.2  Diagnosis – The medical history and drug practices of the patient
are studied initially. Then, the type of skin abscess such as postoperative
wound abscess, perirectal or perianal abscess, abscess developed from
infected breast cysts, or other kind of skin and soft tissue abscess, is ana-
lyzed (Talan et al., 2000). The pus or draining material collected from major
abscess site is cultured to identify the bacteriology of infection (Murphy
et al., 2001).

10.4.7.3 Prevailing treatment options – Empirical antibiotic therapy

is usually prescribed. For uncomplicated or simple skin abscess, a simple
incision followed by drainage of purulent material is the preferred treat-
ment. Doxycycline and minocycline administration together with incision
294    Pocket Guide to Bacterial Infections

and drainage remains successful in the management of uncomplicated

MRSA infections (Stevens et  al., 2014). Trimethoprim sulfamethoxazole
(TM-Sulpha) is also prescribed as an alternate for uncomplicated MRSA
infections (Talan et al., 2016). Oral or intravenous antibiotics are adminis-
tered to patients with complicated skin abscess to recover from the risk of
CA-MRSA infection (Lee et al., 2004).

10.4.8  Necrotizing fasciitis

Necrotizing fasciitis (NF) is a rare, deep, and aggressive soft-tissue infection

that involves the deeper layer of skin and spreads across dermal fascia and
remains detrimental for surrounding tissues. The physical manifestations
include skin lesions with redness, pain, swelling, erythematous, and edema
(Giuliano et  al., 1977). It usually occurs as an extension of impaired skin
integrity (Pye, 2010). NF is classified microbiologically into three types:

Type I NF: It is mostly polymicrobial infection with high incidence of combina-

tions of gram-positive and -negative microorganism along with anaerobes.
Type II NF: It is mainly a monomicrobial infection instigated by GAS, non-
GAS, S. aureus, and Clostridia spp.
Type III NF: It is rarely encountered type of NF wherein, Vibrio vulnificus
gain access through dermal breaches exposed to seawater and cause NF
(Elliott et al., 2000).

The aforesaid microorganisms instigate NF by producing endotoxins or exo-

toxins and invade subcutaneous layer leading to the ischemic necrosis of
tissue (Stevens and Bryant, 2017).

10.4.8.1  Epidemiology – Between 2001 and 2003, a population-based

surveillance conducted in Canada by Eneli and Davies (2007) revealed that
the epidemic of GAS associated NF was 2.12 cases per million children and
that of non-GAS NF was 0.81 cases per million children with mortality rate
of 5.4%. In New Zealand, a national study between 1990 and 2006 con-
ducted by Das et  al. (2011) showed that the annual outbreak of NF has
increased from 0.18 to 1.69 cases per 100,000 persons with mortality rate
of 0.3 cases per 100,000 persons. In the United States, a nationwide study
conducted by Psoinos et al. (1993) between 1990 and 2010 witnessed an
annual incidence of NF ranging from 3,800 to 5,800 cases.

10.4.8.2 Diagnosis – A Laboratory Risk Indicator for NF score (LRINEC)

is used for early diagnosis of NF from other soft-tissue infections. LRINEC
involves estimation of white cell count, CRP, sodium, creatinine, glucose,
and hemoglobin levels. Based on the level of variables, a score is assigned.
 Role of Bacteria in Dermatological Infections     295

Potential NF is feared, when the score is more than 6. Score ≤5 indicates

low risk of NF. Computed tomography (CT) and MRI are also performed
for the diagnosis of NF, when LRINEC scoring system remains ambiguous
(Wong et al., 2004).

10.4.8.3  Prevailing treatment options – Early diagnosis and proper dis-

tinction of NF from other soft-tissue infections is quintessential to elude
the risk of complications and for proper treatment. Surgical debridement
of the affected part is usually suggested in severe NF cases. However, the
management of early diagnosed NF can be achieved using administration
of broad-spectrum antibiotics (Pye, 2010).

10.5  Predominant bacteria in skin infections

A tremendous uplift of diagnosis of SSSIs in both community and health-

care settings has been observed lately. Over recent decades, visits at ambu-
latory care and emergency departments and hospitalization of patients are
reported to be enormously inclined as a ramification of substantial surge in
SSSIs (Esposito et al., 2016).

A bacterial role in dermatological diseases is a well-known fact. However, the

understanding of bacterial pathogenesis, the behavioral pattern of bacteria
to antibiotic treatments, and the likelihood of pathogenesis and antibiotic
resistance is still in its nascent period of development, which plays a potential
role in manipulating proper treatment strategies to manage infections.

Recent findings have divulged that bacterial ecology of skin is more mul-
tifaceted and harbors a wide spectrum of no-fastidious or noncultivable
bacteria in healthy and in diseased states, which posit the possible role of
commensals in skin infections (Vlassova et al., 2011). This viewpoint shows a
departure from previously established views of beneficial role of skin micro-
flora in maintaining skin health. Thus, reviewing the virulent traits of bac-
teria involved in pathogenesis, altered behavior of commensal microflora in
diseased condition, probable use of recent technological advancements as
alternate approaches for managing the global burden of emerging antimi-
crobial resistance is a dire need.

Frequently addressed gram-positive and -negative opportunistic human

pathogens in SSSIs include S. aureus, Streptococcus spp., and P. aeruginosa.
Beneath these formidable bacterial species are the profuse and underrated
skin commensals such as S. epidermidis, P. acnes, and Corynebacterium
spp., which have recently evolved as opportunistic pathogens in SSSIs
(Figure 10.4). These commensals are also molecular reservoirs of virulence
296    Pocket Guide to Bacterial Infections

Figure 10.4  The basic cell structure and virulent determinants of predominant
bacteria in skin infections. The left column includes predominant pathogens such
as S. aureus, S. pyogenes, and P. aeruginosa in skin infections, whereas the right
column includes predominant skin commensals as opportunistic pathogens such as
S. epidermidis, P. acnes, and C. jeikeium in skin infections. The virulence determinants
hosted by these bacterial species in addition to their ability to form biofilm on animate
and inanimate surfaces help to elude a multitude of antibiotic treatments and host
immune system and pose a great menace to the clinical settings.

and antimicrobial resistance traits for other pathogens, which elicit the
severity of infections (Levy and Marshall, 2004). Thus, the subsequent part
of this chapter details the essentials of virulence traits of the aforemen-
tioned pathogens and their role in SSSIs.

10.5.1  S. aureus

The gram-positive and coagulase positive S. aureus is a member of Firmicutes

and commensal in one-third of the human population (Sethupathy et al.,
2017). However, it is also an extensively isolated and leading human oppor-
tunistic pathogen (Alegre et al., 2016). The reportage of S. aureus contribu-
tion in SSSIs hit 80%, of which 63% was observed in cellulitis and cutaneous
abscesses (Malachowa et al., 2015). This remarkable escalation of infectious
 Role of Bacteria in Dermatological Infections     297

role is the result of the advent of MRSA in community and healthcare set-
tings. This notion affords a hostile situation for medical practitioners to
manage SSSIs, especially at emergency departments (Frazee et al., 2005).

Acquisition of type IV staphylococcal cassette chromosome mec (SCCmec,

a transposable element) comprising the mecA gene, which codes for
penicillin-binding protein (PBP) 2a is the actual cause of the emergence
of MRSA. The site-specific recombination of SCCmec and genomic DNA
makes MRSA to resist beta-lactam antibiotics, which basically targets cell
wall PBP to interrupt the synthesis of peptidoglycan layer and ultimately kills
the bacterium (Cogen et al., 2008).

An epidemiological population based study of S. aureus instigated SSSIs

at Georgia had unveiled the occurrence of 72% of MRSA in SSSIs. Among
MRSA isolates, CA-MRSA accounted for 87%, of which 99% CA-MRSA were
MRSA USA 300  clones. Moreover, notorious role of two types of clones
namely, USA 300 and USA 400 in CA-MRSA associated SSSIs in the United
States have been recognized by Centers for Disease Control and Prevention
(CDC). It has been stated that 65% of preliminary antibiotic treatments car-
ried out for CA-MRSA infections remains inadequate and ineffective than
infections associated with methicillin-susceptible S. aureus (MSSA). This dif-
ferential feedback of treatments used for CA-MRSA and MSSA infections
is primarily the consequence of presence of virulent factor named Panton–
Valentine leucocidin (PVL) in CA-MRSA strains (King et al., 2006).

The prolonged cohabitation of S. aureus with the host, together with its abil-
ity to express numerous secreted and cell-associated virulence factors has led
to its adaption to host immune attack at many stages of SSSIs (Malachowa
et al., 2015). The repertoire of virulence determinants of S. aureus include bio-
film formation, secretion of toxins (exfoliative toxins ETA and ETB), hemolysin
(α, β, δ, and γ), staphylococcal enterotoxins (A to E), leukocidin, PVL, toxic
shock syndrome toxin, etc.), secretion of enzymes (coagulases, proteases,
lipases, collagenases, hyaluronidases and nucleases), and surface expressed
virulent factors (collagen-binding protein, elastin-binding protein, fibro-
nectin-binding protein, protein A, and clumping factor) (Sethupathy et al.,
2017).

The virulent enzymes sturdily aid survival of S. aureus by host tissue dam-
age, whereas opsonization of S. aureus by surface expressed protein A and
clumping factor helps in the evasion of host phagocytic attack. The produc-
tion of hemolysins triggers the nuclear factor (NF-κB) inflammatory path-
way through pore formation of targeted host cell membranes (Cogen et al.,
2008). The exfoliative toxins ETA and ETB disrupt the cell-cell adhesion
298    Pocket Guide to Bacterial Infections

protein molecule desmoglein-1, which causes cutaneous blistering in SSSS

(Hedrick, 2003). PVL in CA-MRSA complicates SSSIs by polymorphonuclear
karyorrhexis, capillary dilation, and skin necrosis prompted by the pro-
duction of severe inflammatory lesions (Dufour et al., 2002). The surface-
expressed binding proteins elicit the severity of infections by promoting the
invasive lifestyle of S. aureus through initial adhesion of S. aureus to host
cell surface, followed by colonization and invasion (Shinji et al., 2011). Also,
a novel class of virulent determinant (observed initially in CA-MRSA USA
300 clone and later in other S. aureus lineages) named arginine catabolic
mobile element (ACMB) is involved in immune modulating functions such
as conferring tolerance to polyamines (a nonspecific immune response),
which in turn facilitate the successful survival of S. aureus by outnumbering
the competitors, host colonization, and invasion (Shore et al., 2011).

In addition, the novel lantibiotics synthesized by commensal coagulase nega-

tive Staphylococcus spp. were found to synergize with cathilicidin (a cationic
antimicrobial peptide), which curtails the growth of S. aureus (Nakatsuji
et al., 2017). However, S. aureus evade the attack of host cationic antimicro-
bial peptides (CAMP) by modulating its cell surface charge, which is actively
accomplished by Dlt protein and MprF enzyme that neutralize the negative
charge of cell wall surface by substituting d-alanine in cell wall teichoic and
lipoteichoic acids, and adding l-lysine to phosphatidyl glycerol, respectively
(Cogen et al., 2008). Besides, staphyloxanthin, a golden-colored carotenoid
pigment produced by S. aureus, protects it from oxidants and neutrophilic
attack by exhibiting antioxidant activity (Sethupathy et al., 2017).

Apart from the aforementioned virulence determinants that provide protec-

tion from host immune attack, biofilm formation of S. aureus is rated as one
of the important virulence factors that support antibiotic tolerance. The bio-
film formation of CA-MRSA strain, a leading notorious pathogen in SSSIs,
makes it to resist beta-lactam antibiotics and other non-beta-lactam antibi-
otics such as erythromycin, oxacillin, kanamycin, and tetracycline and con-
fers intermediate resistant to fusidic acid (Dufour et al., 2002; Vanhommerig
et al., 2014).

The current treatment options for MSSA associated skin infections include
first-generation cephalosporins, clindamycin, oxacillin, and nafcillin (Stevens
et al., 2014). On the other hand, the inefficiency of beta-lactam antibiotics
to treat MRSA-associated infections was remedied by vancomycin in 1980.
However, the emergence of vancomycin-intermediate MRSA (VIMRSA) in
late 1990s and subsequent vancomycin-resistant MRSA (VRMRSA) strains
momentously incapacitates the previous treatment strategies. Acquisition
of vanA gene complex from vancomycin-resistant enterococci (VRE) has led
 Role of Bacteria in Dermatological Infections     299

to the advent of VIMRSA, wherein vancomycin could still bind to thickened

cell wall of VIMRSA but efficiently curtails the diffusion of vancomycin. In
the current scenario, the inefficiency of vancomycin is balanced by tedizolid,
daptomycin, linezolid, oritavancin, dalbavancin telavancin, ceftobiprole, and
ceftaroline (Chambers and Leo, 2009). Lately, TM-sulfa is clinically used as
efficient antimicrobial agent to tackle MSSA and MRSA infections (Frazee
et al., 2005).

Alternative treatment strategies such as formulation of anti-biofilm and

anti-virulence regimen, which effectively targets only the virulence of the
pathogen rather than its growth, could be used in clinical settings for
evading the emergence of drug resistance because the use of antibiotics
impose Darwinian pressure on the pathogen (Sivasankar et al., 2016). Also,
an experimental study carried out in mouse model by Wang et al. (2016)
have posited that a nanoparticle-based anti-virulence vaccine apprehended
with staphylococcal α-hemolysin (Hla) could be used in clinical settings for
managing MRSA infections, which curtails pathogenesis and invasiveness
of MRSA through active elicitation of anti-Hla antibodies. Additionally, an
in vitro study by Ramsey et al. (2016) involving commensal Corynebacterium
striatum as a modulator of pathogenic and invasive lifestyle of S. aureus to
commensal without affecting the growth has unveiled the possibility of
new treatment options. Thus, nonbactericidal anti-virulent strategies are
the requirement of current age for combating S. aureus infections for elud-
ing antibiotic resistance.

10.5.2  Streptococcus spp.

The important bacterial species witnessed frequently after S. aureus in both

complicated and uncomplicated SSSIs is the gram-positive Streptococcus
spp., which causes numerous skin infections ranging from mild infections
such as impetigo, folliculitis, abscess, and scarlet fever to deep-seated cellulitis
and life-threatening invasive necrotizing fasciitis (Abrahamian et al., 2008).

Streptococcus, a member of Firmicutes is a chain-forming, catalase

negative, coagulase-negative coccus, and a non-spore-forming faculta-
tive anaerobe. Several streptococcal strains are indigenously colonized
on human skin and throat and become opportunistic pathogen at suit-
able predisposal conditions, whereas certain strains remain pathogenic
(Ralph and Carapetis, 2013). A backdated grouping of Streptococcus and
Enterococcus under the same genus Streptococcus was distinguished as
discrete genera after 1984. The grouping of Streptococcus was found
to be complicated during past. Initially, based on the pattern of hemo-
lytic activity observed on blood agar plates, Streptococcus was classified
300    Pocket Guide to Bacterial Infections

into three groups namely, α-hemolytic, β-hemolytic, and non-hemo-

lytic Streptococcus. In 1933, Lancefield performed serological typing of
β-hemolytic Streptococcus isolated from humans, other animals, cheese,
and milk based on anti-C precipitin test. The anti-C precipitin test majorly
relied on the carbohydrate content (such as polysaccharide and teichoic
acid) of antigens found on bacterial cell wall by classifying Streptococcus
into groups A, B, C, D, and E, wherein group D and E included Enterococcus
(Lancefield, 1933; Hardie et al., 1997).

Among three different hemolytic streptococci, β-hemolytic streptococci are

extensively associated with skin infections, whereas GAS (S. pyogenes) is
the predominant etiologic agent followed by group B streptococci (GBS,
Streptococcus agalactiae) (Bisno and Stevens, 1996; Schuchat, 1998). A ret-
roactive population-based study including adults with purulent SSSIs has
unveiled the frequency of streptococci isolates as 7% in abscesses, 9% in
infectious wounds, and 13% in purulent cellulitis (Abrahamian et al., 2008).
In addition, the advent of highly invasive GAS remains as the predominant
etiologic agent of series of severe and life-threatening SSSIs (Bisno and
Stevens, 1996).

Similar to S. aureus, GAS also presents an arsenal of virulent determinants

for eluding host immune attack and antibiotic treatments. These virulence
determinants include biofilm formation, surface-associated lipoteichoic
acids (LTA), M protein and pili, hyaluronic acid capsule and pyrogenic exo-
toxins such as leukocidin, streptolysins (O & S), streptokinase, superanti-
gens, hyaluronidase, hemolysins, and proteases. Biofilm formation of GAS
is a devastating and multifaceted process, which involves M-proteins, pili,
and LTA for initial attachments and includes numerous other virulent factors
to build a strong impediment against host immune attack and antibiotic
treatments and further capable of complicating the treatment by causing
severe invasive diseases using surface-associated invasins (Subramenium
et al., 2015a; Ibrahim et al., 2016).

The surface-associated pilus proteins and M-proteins of GAS strains

involved in skin and throat infections are found to be different, which are
discriminated in clinical settings using T-antigen and M-protein serotyp-
ing (Cogen et al., 2008). The T-antigen genes sheltered in FCT region (i.e.,
loci of Fibronectin, collagen-binding protein, and T-antigen) encode pilus
proteins and adhesins, which establish mechanical stabilization of covalent
bonds and supports endurance of shearing forces formed during initial
attachment of GAS to integrin found on host extracellular matrix (ECM)
(Bessen, 2016). Also, the interaction of host integrin α5β1  with bacterial
fibronectin-binding protein is reported to promote invasion by eliciting the
 Role of Bacteria in Dermatological Infections     301

cellular signaling pathways, which in turn, leads to conformational changes

of host cytoskeletal actin (Walker et al., 2014).

Furthermore, the surface associated M-proteins comprising M1, M3, and

M6  proteins are helpful in bacterial colonization, aggregation, and inva-
sion of host ECM, wherein the surface-exposed hypervariable N-terminus
of M-proteins facilitate attachment. M-proteins also bind and interfere with
immunoglobulins and complementary regulatory components, thereby elud-
ing host immune attack. The secreted proteases SpeB curtail the employment
of phagocytes at the infection site by cleaving chemokines such as comple-
ment component c5a and interleukin 8 (IL8). Similar to S. aureus, GAS recruits
Dlt proteins to neutralize the negative charge of cell wall LTA, thereby hinder-
ing the attachment of CAMP. Hyaluronic acid capsulation of GAS helps in
opsonophagocytosis and also to evade the attack of antimicrobial peptides
produced by commensals. The cytolysins such as streptolysin O and streptoly-
sin S help in the survival of host immune attack by facilitating the rupture of
neutrophils and macrophage (Walker et al., 2014).

Established with multitude of virulent determinants, this host-adapted

GAS exhibits an archetypal defense system to escape host immune attack
and antibiotic treatment. The antibiotic resistance profile of GAS has been
reported to include macrolides, tetracycline, and fluoroquinolone, which
pose a great threat to the management of streptococcal infections (Ibrahim
et al., 2016). The empiric antibiotic treatment administered in several cases
of SSSIs executes a greater risk of emergence of antibiotic resistance.

Despite of less resistance reported for penicillin, it is used currently as

the first-line antibiotic for streptococcal-associated SSSIs. For individuals
allergic to penicillin, macrolides are administered (Ibrahim et  al., 2016).
Phenoxymethyl penicillin (V) is used for managing scarlet fever (Basetti
et al., 2017). Daptomycin is reported to be safe and efficient in managing
Streptococcus-associated cSSSIs (Arbeit et al., 2004).

Numerous studies report anti-biofilm and anti-virulent agents from natural

sources such as 2, 4-Di-tertbutylphenol from Bacillus subtilis (Viszwapriya
et  al., 2016), limonene found in citrus fruits (Subramenium et  al., 2015),
cinnamaldehyde and its derivatives from cinnamon (Shafreen et al., 2014),
usnic acid from lichen (Nithyanand et al., 2015), and morin from orange and
guava (Green et al., 2012) against GAS, which reduces the virulence of the
pathogen and helps in natural clearing of bacterial load by host immune
system and aids effective penetration of antibiotics. These anti-virulence–
based treatment strategies could be established in clinical settings as alter-
nate for evading the risk of advent of antibiotic resistance.
302    Pocket Guide to Bacterial Infections

10.5.3  P. aeruginosa

P. aeruginosa, a ubiquitous gram-negative, rod-shaped and highly motile

aerobic bacterium is the part of normal skin microflora that innocuously
colonizes the human skin, mouth, and some nonsterile regions of healthy
individuals. But at suitable predisposing conditions, it could effectively
infect any region of the body it comes into contact, thus behaving as an
opportunistic pathogen (Cogen et  al., 2008). It is encountered predomi-
nantly in hot-tub folliculitis and acute and chronic wound and burn wound
infections, which are reported to be associated with higher risk of morbidity
and mortality (Percival et al., 2012; Serra et al., 2015).

P. aeruginosa greatly colonize the moist skin surfaces (such as burn wounds)
rather than dry skin and exhibits pathogenic mechanisms with great predi-
lection toward individuals who are immunocompromised and hospitalized
(Morrison and Richard, 1984). Moreover, it has been stated that 2.5% of
patients with acute thermal injuries or burns are highly prone to develop
Pseudomonas septicemia, wherein the rate of mortality has been observed
to be 76% (Stieritz and Holder, 1975).

Also, P. aeruginosa infects a broad area of wound site and complicates

the wound-healing process owing to its high rate of acquired antibiotic
resistance. The wound-healing process is reported to be still more complex,
when P. aeruginosa is found to be coinfected on wounds with S. aureus.
This bacterial interaction modulates the virulence rate, curtails the wound-
healing process, and exhibits altered feedback for antibiotic treatments,
which mystifies the identification of suitable treatment strategy (Serra et al.,
2015). These recalcitrant P. aeruginosa–infected chronic and acute wounds
and burn wounds stance a great economic as well as medical burden.

Furthermore, P. aeruginosa–colonized burn wounds rapidly worsen and

leads to complete dissemination followed by death within few weeks,
whereas, P. aeruginosa–colonized chronic wounds lasts for a long time
and shows less probability for mortality. Altogether, all the aforementioned
notions necessitate the unpinning of associated virulent determinants and
molecular mechanisms underlying the differential response of P. aeruginosa
infected burn and chronic wounds, which in turn, would facilitate the man-
agement of wound infections (Turner et al., 2014).

The secreted (i.e., alkaline protease, elastases LasA and LasB, phospholipases,
exoenzyme S and exotoxin A) and cell-associated (i.e., alginate, flagellum, pili
and adhesins) virulence determinants afford multiple benefits to P. aerugi-
nosa such as evasion from host immune attack, host invasion, and endur-
ance of antibiotic treatments. Additionally, quorum sensing systems las and
 Role of Bacteria in Dermatological Infections     303

rhl of P. aeruginosa are reported to be involved in chronic wound infections,

which are putatively known to control several virulence factors (Rumbaugh
et al., 1999). Quorum sensing (QS) is the cell-to-cell communication process
that occurs at the onset of increased signaling molecules called autoinducers
(AI) attained during high cell density (HCD) and monitors the level of signal-
ing molecules and modulates gene expression suitably to endure adverse
situations. The las QS system involves AI synthase LasI to secrete 3-oxo-
C12-homoserine lactone (3OC12HSL), which in turn, triggers the transcrip-
tion activator LasR at high AI level to regulate the expression of target genes
encoding exotoxin A, proteases, and elastases. The rhl QS system works in
series with las QS system, wherein the LasR-3OC12HSL complex formed at
high AI level targets the AI synthase RhlI and triggers it to produce butanoyl
homoserine lactone (C4HSL). This in turn, induces the activation of transcrip-
tion regulator RhlR at high AI level and regulates the expression of target
genes encoding proteases, rhamnolipid, biofilm formation, swarming, elas-
tases, siderophores and pyocyanin (Rutherford and Bassler, 2012).

The type IV pili and flagella are reported to be involved in burn wound
infections by mediating the twitching motility of P. aeruginosa through
liquid interface of wound, thereby helping the establishment of appro-
priate adhesion with skin surface. The flagellar components also aid dis-
semination of P. aeruginosa from infected wound site. Additionally, the
flagellar glycosylation supports the colonization of P. aeruginosa and
increases the virulence, which leads to death (Arora et  al., 2005). Cell-
associated alginate mediates attachment and biofilm viscoelasticity and
confers resistance toward host immune defenses and antibiotics during
chronic infections. Rhamnolipids are supportive in spreading of infection
by mediating the dispersal of P. aeruginosa from biofilm and helps in rees-
tablishment of biofilm in a new niche (Kostakioti et  al., 2013). Elastase,
a type of protease, plays and essential role in burn infections by exert-
ing tissue-damaging activity, including degradation of plasma proteins
such as complement factors, immunoglobulins, and so on (Wretlind and
Pavlovskis, 1983).

Altogether with robust defense strategies, P. aeruginosa remains as an

intractable multidrug resistant (MDR) pathogen and poses a great menace
to clinical settings for managing the infections. The antibiotic resistant pro-
file of P. aeruginosa includes a multitude of antibiotics such as gentamicin,
ceftizoxime, cephalothin, carbenicillin, ceftazidime, ciprofloxacin, tetracy-
cline, amikacin, and so on. The empiric antibiotic treatment is found to be
futile. Early diagnosis, appropriate identification of strain type of wound
colonizers, and their antibiotic sensitivity pattern is reported to be appropri-
ate for managing the burn infections (Pruitt et al., 1983).
304    Pocket Guide to Bacterial Infections

Alternative approaches, including development of quorum quenchers,

anti-biofilm, and anti-virulence agents, could act as efficient therapeutic
regimen. For instance, curcumin, a natural compound found in turmeric
plants, is reported to act as a potent quorum quencher of P. aeruginosa
(Sethupathy et al., 2016). Recently, ciprofloxacin-loaded keratin hydrogels
developed by Roy et al. (2015) were evidenced to progress the healing pro-
cess of P. aeruginosa–associated wound infections. Thus, these alternate
strategies could be adopted in clinical settings after appropriate clinical tri-
als, which are posited to help in eluding the advent of antibiotic resistance.

10.5.4 Predominant skin commensals as

opportunistic skin pathogens

10.5.4.1  S. epidermidis – S. epidermidis is a ubiquitous gram-positive skin

and mucosal membrane colonizer, which exerts a mutualistic relationship
with host. It forms the major part of microbial barrier that precludes the col-
onization of other pathogens. In a competitive environment, it secretes lan-
tibiotics (i.e., lanthionine-containing antibacterial peptides) often referred
as bacteriocins, which prevent the colonization of S. aureus and GAS (Sahl,
1994; Cogen et al., 2007). Also, accessory gene regulator (agr) locus found
in commensal S. epidermidis produces peptide pheromones that activate
the agr QS system of competing bacteria, which in turn, reduces coloniza-
tion and down-regulates the expression of virulence factors by increasing
the production of pheromones such as phenol soluble modulin (Otto, 2001).
In addition, S. epidermidis boosts the host immune defense by eliciting the
signaling of toll like receptor (TLR). The pattern recognition receptors TLRs,
in turn, specifically recognize different pathogen-associated molecular pat-
terns and activate the host immune system accordingly.

Despite the aforesaid beneficiary roles of S. epidermidis, it has been identified

as opportunistic pathogen during past two decades predominantly affecting
drug abusers and individuals who are immunocompromised. S­ . ­epidermidis
is reported to be encountered less frequently in abscesses, cellulitis, and
­several wound infections (Cogen et  al., 2008). It is also reported to elicit
the severity of miliaria (prickly heat), a skin ailment frequently witnessed
in profusely sweating individuals and neonates with undeveloped sweat
glands. Miliaria occurs as a result of occlusion of sweat glands by periodic
acid sciff (PAS) positive extracellular polysaccharide substance produced by
S. epidermidis (Mowad et al., 1995).

These detrimental infectious roles of S. epidermidis are well allied with

its ability to form biofilm and produce virulence factors such as autolysin
protein, proteases, lipases, polysaccharide intracellular adhesion (PIA), and
 Role of Bacteria in Dermatological Infections     305

surface-associated fibronectin and collagen-binding protein. The surface-

associated fibronectin and collagen-binding proteins mediate the attach-
ment of S. epidermidis (Williams et al., 2002). Autolysin proteins facilitate
autolysis process of competing bacteria and uses the extracellular DNA
(eDNA) attained through autolysis for establishment of robust biofilm.
Proteases and lipases are involved in the tissue-damage process, which
increases the severity of infection. Once the biofilm is established, PIA helps
intracellular adhesion and increases the virulence (McKenney et al., 2000).

These virulence factors equip a robust and recalcitrant biofilm and inca-
pacitate the antibiotic treatments at clinical settings. Thus, exploration of
novel treatment strategies to combat biofilm-assisted infections are still in
progress. Antibodies designed against S. epidermidis surface-associated
proteins and interferon therapy is reported to possess positive feedbacks
against recalcitrant biofilms (Boelens et  al., 2000). Alpha-mangostin, an
anti-biofilm agent found in mangosteen, has been reported to eradicate
S. epidermidis biofilms (Sivaranjani et  al., 2017). These treatment options
could be included in clinical trials for finding the suitability of these strate-
gies in clinical settings.

10.5.4.2  P. acnes – P. acnes is a gram-positive and anaerobic bacillus,

which is an indigenous colonizer of sebaceous glands. It also forms the part
of microbial and plays mutualistic role similar to S. epidermidis. It catabo-
lizes the sebum lipids of sebaceous glands and release fatty acids, which
precludes the colonization of sebaceous gland by other pathogens. P. acnes
are also known to produce antimicrobial peptides called bacteriocins, which
include jenseniin, acnecin, and propionicin. These bacteriocins prevent the
colonization of other Propionibacterium spp., several gram-negative bacte-
ria, yeast, molds, and so on.

P. acnes also exhibits infectious role by causing inflammatory acne vulgaris

majorly and synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO)
syndrome rarely. The free fatty acids produced by P. acnes as the result of
triglyceride metabolism trigger the inflammatory response. Acne vulgaris is
ailment of pilosebaceous follicles caused as result of hormonal imbalance,
immune hypersensitivity, P. acnes infection, and follicular keratinization. It
is frequently witnessed in 80% youngsters of the U.S. population. Several
putative predisposal factors such as genetic factors, stress, androgens, fol-
licular pattern of the individual, and use of steroids are known to influence
the onset of acne vulgaris.

Excessive sebum production attained as the result of high level andro-

gen mediates the colonization and biofilm formation of P. acnes
306    Pocket Guide to Bacterial Infections

(Coenye et al., 2008). In addition to biofilm formation, P. acnes secretes

several virulence determinants such as proteases, hyaluronidase, lipases,
and chemotactic factors, which play substantial role in acne vulgaris.
Furthermore, it has been reported that the aforementioned virulence fac-
tors of P. acnes triggers the nonspecific immune response by activating
the proinflammatory cytokines and supports differentiation and prolif-
eration of keratinocytes. The matrix metalloproteases (MMP) produced
by keratinocytes deteriorates the hair follicles and induces inflammatory
acne lesions (Dessinioti et al., 2010; Saising and Voravuthikunchai, 2012).

A number of treatment options are available to manage acne vulgaris,

which include topical retinoids, oral antibiotics, oral isotretinoin, and
administration of benzyl peroxide (Cogen et  al., 2008). However, alter-
native strategies such as anti-biofilm therapy would be useful in manag-
ing recalcitrant biofilm-assisted P. acne infections. Sivasankar et al. (2016)
investigated the in vitro and in vivo anti-biofilm potential of ellagic acid
and tetracycline against P. acnes biofilm and other associated virulence
factors, which remain a good revelation for managing the infections with
combined approaches.

10.5.4.3  Corynebacterium spp. – Corynebacterium spp. are diphtheroid,

gram-positive, and facultative anaerobic mycobacterium, which constitute
approximately 50% of skin microflora (Blaise et al., 2008). The skin micro-
flora majorly constitute two species of Corynebacterium namely, C. diph-
theriae and nondiphtheriae corynebacteria (diphtheroids). C. diphtheriae
are reported to be witnessed in cutaneous ulcers of drug abusers, alcohol-
ics, and individuals exposed to poor hygienic environment. The nondiph-
theriae cornynebacteria or diphtheroids include totally 17  species, which
are diversely distributed in humans and other animals (Cogen et al., 2008).
The diphtheroid majorly found in human epithelium is C. jeikeium. It acts
as microbial barrier and protects the host from pathogenic attack by the
production of bacteroids. The enzyme superoxide dismutase produced by
C. jeikeium for shielding against the attack of free radicals, confers protec-
tion to host against free radicals (Storz and Imlay, 1999).

However, C. jeikeium is capable of developing papular eruptions in patients

who are immunocompromised and individuals with skin abrasions or trau-
mas. C. jeikeium harbors numerous virulence traits such as siderophores,
invasins, adhesins, biofilm formation, and superoxide dismutase (Joh et al.,
1999; Ton-That and Schneewind, 2004; Blaise et  al., 2008). The sidero-
phores mediates iron and manganese sequestration and helps to evade host
attack by surviving superoxide radicals. Adhesins and invasins promote the
virulence by aiding adhesion to host epithelium and host invasion. Also, the
 Role of Bacteria in Dermatological Infections     307

cell envelope of C. jeikeium contains corynomycolic acid, which helps it to

resist multiple antibiotic treatments (Cogen et al., 2008).

The biofilm formation together with other virulence traits affords resistance
toward many antibiotics. However, C. jeikeium remains susceptible to glyco-
peptides group of antibiotics such as vancomycin. Currently, erythromycin is
used as first-line treatment for skin infections instigated by Corynebacterium
spp. and is administered continuously for 3 to 4 weeks. Fusidic acid is also used
in the treatment. The emerging bacterial resistance debilitates the conven-
tional antibiotic treatments. Yet, reduction of hyperkeratosis using kerolytic
agent and lessening of sweat production using topical aluminum hydroxide
could upsurge the efficacy of prevailing strategies (Blaise et al., 2008).

10.6  Conclusion and future prospects

Bacterial role as commensal and pathogen still remains cryptic. Though,

skin microflora play numerous protective role and constitute microbial
barrier for shielding the host from pathogenic attack, their role in certain
infectious diseases still remains ambiguous. Thus, unpinning the role of skin
commensals in SSSIs, alterations in physiology of commensals during infec-
tions, interactions between commensals and pathogens, factors influencing
the severity of infections, molecular mechanisms underlying infectious role
of pathogens, as well as commensals and role of commensals in antibiotic
resistance of pathogens remains quintessential for safeguarding the man-
kind from the advent of new MDR strains and infectious pathogens.

Recently, many researches have been dedicated in reconnecting the tradi-

tional medical practices to the current scenario through the use of advanced
technologies for identifying the specific bioactive molecules present in the
natural sources to outstrip the deceiving resistance mechanisms of bacterial
pathogens. However, after documenting the in vitro anti-infective poten-
tial of active lead(s) against infectious pathogen(s), many researches halt
midway and do not prolong the research toward the in vivo analysis of
their efficacy. Albeit many researchers take their innovation to next level by
deciphering the molecular mechanisms underlying the anti-infective poten-
tial of active lead and assessing their activity in various in vivo models, the
analysis of aptness of active lead(s) for clinical applications has to be still
carried out. The proper formulation of active lead(s) with less cytotoxicity,
high stability, and bioavailability, followed by appropriate clinical trials for
employing the identified natural-based therapeutic molecules in clinical use
for eluding the global burden of antibiotic resistance and betterment of
human life will serve as the future endeavors.
308    Pocket Guide to Bacterial Infections

Acknowledgments

The authors thankfully acknowledge the support extended by Department

of Science and Technology, Government of India through PURSE [Grant
No. SR/S9Z-23/2010/42 (G)] & FIST Level I, Phase II (Grant No. SR/FST/LSI-
639/2015(C)), and University Grants Commission (UGC), New Delhi, through
SAPDRS1 [Grant No. F.3-28/2011 (SAP-II)]. The authors also gratefully thank
the Bioinformatics Infrastructure Facility (BIF) funded by Department of
Biotechnology, Government of India [Grant no. BT/BI/25/015/2012].

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11
Bacteriology of
Ophthalmic Infections
Arumugam Priya and Shunmugiah Karutha Pandian

Contents

11.1 Introduction 320

11.2 Eye—The photoreceptive and the infection prone organ 320
11.3 Ocular microbiota 321
11.4 Antibacterial protections in ocular surface 322
11.5 Ocular infections 322
11.5.1 Extraocular infections 322
11.5.1.1 Blepharitis 322
11.5.1.2 Dacryocystitis 326
11.5.1.3 Bacterial conjunctivitis 327
11.5.1.4 Hordeolum 328
11.5.1.5 Chalazion 329
11.5.1.6 Bacterial keratitis 330
11.5.1.7 Cellulitis 331
11.5.2 Intraocular infections 331
11.5.2.1 Endophthalmitis 331
11.5.2.2 Uveitis 332
11.6 Infectious bacteria in ocular diseases 333
11.6.1 Staphylococcus spp. 334
11.6.1.1 Virulence factors 335
11.6.1.2 Antibiotic resistance 337
11.6.1.3 Therapeutic interventions 337
11.6.2 Streptococcus spp. 338
11.6.2.1 Virulence factors 339
11.6.2.2 Antibiotic resistance 340
11.6.2.3 Therapeutic interventions 342
11.6.3 Pseudomonas aeruginosa 342
11.6.3.1 Virulence factors 343
11.6.3.2 Antibiotic resistance 344
11.6.3.3 Therapeutic interventions 345

319
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11.6.4 Coryneform bacteria 346

11.6.4.1 Virulence factors 347
11.6.4.2 Antibiotic resistance 347
11.6.4.3 Therapeutic interventions 348
11.7 Role of bacterial biofilm in ocular infections 348
11.8 Conclusion and future perspectives 350
Acknowledgments 351
References 351

11.1 Introduction

Despite the apparent sturdiness of the ocular surface and structure, the eye
is constantly exposed to the external microbial communities. The existence
of ocular microflora divulges the interplay of microbes in the ocular health
and disease. The crosstalk between the ocular microbial flora and the ocular
defense system coordinates the furtherance of ocular surface homeosta-
sis and health. The disparities in the host factors, environmental stipula-
tions, nutritional, and disease status of an individual may arbitrate ocular
microfloral shift (Miller and Iovieno, 2009). Pathological shift in the indig-
enous microbial community can cause hostile immune reactions or ocular
surface cellular damage and exert a pathogenic effect. Through numerous
earlier reports, native ocular flora has been shown to be predominantly
Staphylococcus epidermidis, Staphylococcus aureus, and Corynebacterium
spp. along with other less common organisms such as Propionibacterium,
Haemophilus, Pseudomonas, and so on (Capriotti et  al., 2009). Bacterial
ophthalmic infections vary greatly in severity from common conjunctivi-
tis to endophthalmitis, an adverse infectious postoperative complication.
Instantaneous detection and appropriate medication for ocular infections
will lessen the incidence of visual impairment and ocular morbidity (Muthiah
and Radhakrishnan, 2017). Hence, deciphering the relationship between
the normal microflora and etiology of ocular infections is imperative.

This chapter is aimed at describing the characteristics and clinical manifes-

tations of common bacterial intra- and extraocular infections and the role
of diverse microflora in infectious diseases associated with ocular surfaces.

11.2 Eye—The photoreceptive and the infection
prone organ

The eye, the organ of visual perception, is structurally comparted into three
layers or tunics, organized as internal segment that is composed of anterior
and posterior chambers, the iris, the lens, the vitreous cavity, the retina,
 Bacteriology of Ophthalmic Infections     321

the ciliary body, the choroid and the intrinsic ocular muscles, and enclosed
by external segments (i.e., the conjunctiva, the cornea, the sclera, and the
tear film). The internal segment of the eye presents a sterile environment by
efficient blood-retinal barrier (BRB), whereas the external segment, which is
exposed to the environment, is subjected to numerous microbial challenges
(Lu and Liu, 2016). Infections may occur in almost any part or tissue of the
ocular surface, orbit, and adnexa. The transference of the infection from a
site to the other may be ensued by the direct contact or indirectly through
blood vessels and nerves (Rumelt, 2016). The conjunctiva, the eyelid, and
the cornea are the frequently infected sites of eye because they act as a first
line preventive barrier against foreign bodies (Alfonso and Miller, 1990).
Prolonged infection or infection to delicate tissue like the cornea may lead
to the impairment of normal vision and can extend to loss of sight.

11.3  Ocular microbiota

Perceptive features of the ocular microflora are fundamental in understand-

ing the ocular diseases and infections. Axenfeld (1908) stated that the
microbiota of eyelid and conjunctiva are similar to that of the skin and upper
respiratory tract. Since then, microbial flora of the ocular surface has been
subjected to numerous studies to investigate the indigenous flora of the
healthy eyes, as a comparative analysis to interpret the microbial shift during
diseased state, to assess the microbial community before intraocular sur-
geries, or to review the prophylactic strategies in postoperative infections.
Axenfeld founds that Staphylococcus albus and Corynebacterium were fre-
quently isolated organisms, whereas Staphylococcus aureus, Streptococcus
spp., and few other gram-negative bacteria were found with least incidence.
The classification of ocular microbiota based on the culture-dependent
methods was alleged to be predominantly conquered by gram-positive
species such as Staphylococcus, Streptococcus, Propionibacterium, and
Corynebacterium; gram-negative species such as Neisseria, Haemophilus,
and few fungal species (Miller and Iovieno, 2009). Culture-based character-
ization significantly surpassed cultivable and fastidious growing organisms.
With the advent of molecular techniques, (Dong et  al., 2011) instigated
the genome based detection of ocular microbiota and revealed diverse
microbial community including commensal, environmental, and oppor-
tunistic pathogens (Dong et  al., 2011). The 12 genera, Pseudomonas,
Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter,
Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas,
Streptococcus, Streptophyta, and Methylobacterium, were represented
as core microbiome of the conjunctiva. Based on the sequencing of 16S
322    Pocket Guide to Bacterial Infections

rDNA V3–V4  hypervariable  segments of bacteria from conjunctival swab,

Huang et al. (2016) linked additional genera such as Millisia, Anaerococcus,
Finegoldia, Simonsellia, and Veillonella to the core conjunctival microbiota.
Numerous studies have evidenced that the use of contact lenses (Hovding,
1981; Larkin and Leeming, 1991; Fleiszig and Efron, 1992; Iskeleli et al., 2005;
Shin et al., 2016), the eyes that endured surgeries (Jabbarvand et al., 2016)
and patients with prolonged hospital stays (Sahin et al., 2017) presented varia-
tions in the microbial diversity and abundance. Moreover, variation in the
ocular microbiota between eyes of an individual and between individuals has
also been affirmed (Hovding, 1981).

11.4  Antibacterial protections in ocular surface

The association of indigenous microflora and the ocular mucosal and

immune epithelial cells maintains the ocular surface homeostasis through

• barrier preservation,
• inhibition of apoptosis and inflammation,
• producing inhibitory substances such as bacteriocins,
• eliminating harmful pathogens,
• accelerating wound healing and tissue regeneration,
• maintenance of immune tolerance, and
• linkage to adaptive immunity (Miller and Iovieno, 2009).

11.5  Ocular infections

Infections in the eye can be broadly classified into intra- and extraocular infec-
tion based on the site of infection origin. Intraocular infections may involve
different intraocular structures such as uveal tissues (e.g., choroid, ciliary
body, and iris), the retina, and the vitreous chamber. The extraocular infec-
tions may emerge in the following surfaces: eyelids, lacrimal sac, conjunctiva,
cornea, and the adnexal structures (Table 11.1) (Figure 11.1a and b).

11.5.1  Extraocular infections

11.5.1.1 Blepharitis – The infectious and inflammatory conditions of

the lid margin, including the eyelash follicles and sebaceous and apocrine
glands are generally described as blepharitis, the most encountered eye
infection. It typically occurs bilaterally and exists as a recurrent chronic con-
dition. Blepharitis is a multifactorial complex disease, which institutes sev-
eral overlapping signs and symptoms (Jackson, 2008). Meibomian gland
 Bacteriology of Ophthalmic Infections     323

Table 11.1   Diagnosis and Treatment Strategies for Intra- and Extraocular
Infections
Infection Diagnosis Treatment Strategies
Blepharitis • Clinical diagnosis with • Topical antibiotics
presenting signs and • Warm compress
symptoms
Dacryocystisis • Clinical diagnosis • Local massage over
• Differential diagnosis lacrimal sac
with sinusitis, punctual • Probing and syringing
ectropion, sebaceous • Dacryocystorhinostomy
cyst, cellulitis • Warm compress
• Systemic broad-spectrum
antibiotics
Bacterial • Clinical diagnosis with • Topical application of
Conjunctivitis presenting signs and broad spectrum
symptoms antibiotics –
• Conjunctival scraping aminoglycosides,
• PCR sulfacetamide solution,
• Differential diagnosis fluoroquinolones,
including viral, chlamydial tetracycline, and
and allergic conjunctivitis, chlroamphenicol
superficial keratitis,
blepharitis, acute angle
closure glaucoma
Hordeolum • Typically, no diagnostic • Lesions drain spontaneously
testing • Warm compress can reduce
• Imaging required in case the abscess
of further complications • Lid massage/lid scrub with
like periorbital cellulitis saline or mild shampoo
For persistent and large
lesions
• Erythromycin ophthalmic
ointment
• Incision and drainage under
local anesthesia
Chalazion • Ophthalmic examination • Warm compress
• Clinical diagnosis with • Topical antibiotic ointment
the presenting signs and • Systemic tetracycline
symptoms • Subcutaneous injection of
• Biopsy steroid triamcinolone
• CT scan of the face and acetonide
orbits • Incision and curettage
(Continued)
324    Pocket Guide to Bacterial Infections

Table 11.1 (Continued)  Diagnosis and Treatment Strategies for Intra- and
Extraocular Infections

Infection Diagnosis Treatment Strategies

Bacterial • Complete ophthalmic • Application of topical
Keratitis examination (visual antibiotics/corticosteroids
acuity, slit-lamp • Corneal transplantation
examination, intraocular
pressure)
• Corneal scraping and
pathological
examination
Preseptal and • CT • Warm compress
orbital cellulitis • Ultrasound • Intramuscular
cephalosporin injections
• Endoscopical drainage of
abscess
• Surgical interventions
Endophthalmitis • Pathological examination • Intravitreal antibiotics
from intraocular • Intravitreal steroids
specimen (vitreous or • Vitrectomy
aqueous humor), blood
culture, lumbar puncture
• Diagnostic PCR
Uveitis • Clinical examination • Systemic steroid/
• Laser Flare Cell Meter immunosuppressive/
• Ultrasound anti-inflammatory regimen
biomicroscopy • Intravenous polyclonal
• PCR-based molecular immunoglobulin treatment
detection of etiological
agents
• HLA-B27 typing
(for nongranulomatous
cases)
• Local antibody titre
determination
For Uveitis associated with
systemic diseases
• Serum lysozyme test
• Chest X-ray
• Gallium Scintigraphy
• Treponemal
hemagglutination and
Nontreponemal test

CT, computed tomography; PCR, polymerase chain reaction.

 Bacteriology of Ophthalmic Infections     325

Figure 11.1  (a) Extraocular infections and (b) Intraocular infections.

326    Pocket Guide to Bacterial Infections

dysfunction, conjunctival redness, crusting, hyperkeratinization and red-

ness of the eyelid, ocular itching, burning and irritation, dry or watery eyes,
and photophobia are typical symptoms of blepharitis (McCulley and Shine,
2000; Favetta, 2015). Surplus colonization of lid-margin microbes, abnormal
lid-margin secretion, or dysfunctional tear film will prompt the infection.

11.5.1.1.1  Classification and etiological agents – Anatomically, blepha-

ritis can be classified as anterior and posterior forms. Infections affecting the
anterior lid margin and eyelashes result in anterior blepharitis and the infec-
tions of Meibomian gland and proximal tissue lead to posterior blepharitis.
Apart from the anatomical site-based classification, blepharitis can also be
categorized as Staphylococcal-mediated blepharitis, seborrheic blepharitis,
mixed Staphylococcal, and seborrheic blepharitis (McCulley et  al., 1981).
The frequent organisms associated with blepharitis infection are S. epider-
midis, S. aureus, P. acnes, and Corynebacterium (Dougherty and McCulley,
1984; Groden et al., 1991). Infection is frequently observed in the contact
lens wearers and patients who underwent refractive, cataract, and other
ocular surgeries.

11.5.1.1.2  Pathophysiology – The exoenzymes produced by the caus-

ative organisms, in particular S. epidermidis, initiates irritation in the
eyelid and surrounding ocular surface that recruits the inflammatory
mediators to the site of infection (Lemp and Nichols, 2009). Lipolytic
exoenzymes such as triglyceride lipase, cholesterol esterase, wax ester-
ase, and sterol esters with the deliverance of irritating fatty acids will
result in the disruption of tear film integrity (Bron and Tiffany, 2004).
Alternatively, the variations in the secretion of Meibomian gland with
increased lipid secretion may offer provision for the proliferation of
microbes (Nichols et al., 2011). The implication of quorum sensing in the
bacterial load rise can increase the signs and symptoms of blepharitis
(Haque et al., 2010).

11.5.1.2  Dacryocystitis – Dacryocystitis is a painful inflammatory disor-

der of the lacrimal sac which occurs because of obstruction in the naso-
lacrimal duct. The obstruction may be due to primary acquired obstruction
or secondary to the trauma, infection or inflammation, or mechanical
obstruction. The blockage in the nasolacrimal duct causing stillness in the
tears within pathologically congested lacrimal duct will lead to dacryocys-
titis (Iliff, 1996). It is prevalent among the infants and the middle-aged
women. The clinical presentation may be mild as irritation and discomfort
to sight threatening. The signs and symptoms vary greatly according to the
etiology of the infection. Dacryocystitis was most frequently observed as
a unilateral infection.
 Bacteriology of Ophthalmic Infections     327

11.5.1.2.1 Classification and etiological agents – Dacryocystitis may

either present in acute or chronic form and congenital in rare instances.
Acute dacryocystitis is an acute inflammation of the lacrimal sac due to
infection by S. aureus or beta hemolytic Streptococcus, which results in ten-
derness and erythema and one fourth of the eye may be present with the
lacrimal abscess. If untreated, the infection may propagate to surrounding
tissues and cause periorbital or orbital cellulitis. The chronic dacryocystitis
exhibits epiphora, mucoid discharge, conjunctival hyperemia, and chronic
conjunctivitis (Ali et al., 2013, 2015). The congenital dacryocystitis will be
seen since infancy and the presentation will be usually epiphora with muco-
purulent discharge. The complications may further lead to orbital cellulitis
or even brain abscess and meningitis (Babar et al., 2011). The severity of the
infection may range from partial to total obstruction of the nasolacrimal
duct. No proper medication or therapy exists. Even the systemic antibiotic
therapy will diminish the condition in much slower pace (Cahill and Burns,
1993). The obstruction in the nasolacrimal sac can be relieved by exter-
nal or endonasal dacryocystorhinostomy (DCR) but leaves a potential risk
for endophthalmitis. Hence, chronic dacryocystitis should be cured before
intraocular surgeries.

The bacteriology of acute and chronic dacryocystitis has a vast differ-

ence. Gram-negative rods dominate in the acute infections, whereas, both
gram-negative and -positive isolates were found in the chronic infections.
Staphylococcus spp., S. pneumoniae, P. aeruginosa, E. coli, (Hartikainen
et al., 1997; Iliff, 1996), Peptostreptococcus, Propionibacterium, Prevotella,
Fusobacterium (Brook and Frazier, 1998), Streptococcus pyogenes, and
Streptococcus viridans (Sarkar et al., 2015) are the predominant causative
organisms reported till date.

11.5.1.2.2  Pathophysiology – Under normal conditions, the lacrimal sac

is resistant to the infectious organisms. The lacrimal sac usually drains the
tear from eye into the nasal cavity. The obstruction in the nasolacrimal duct
may result in improper drainage or accumulation of the tears, desquamated
cells, and mucoidal secretions. This provides residence for secondary bacte-
rial infection (Iliff, 1996). Bacterial overload at the lacrimal sac will recruit
the anti-infective response which leads to acute or chronic dacryocystitis
infection (Pinar-Sueiro et al., 2012).

11.5.1.3  Bacterial conjunctivitis – The inflammation of the conjunctiva,

the transparent membrane that covers the sclera is termed as conjunctivi-
tis. Conjunctivitis forms the most common cause of red eye and most fre-
quently observed eye infection worldwide. Conjunctivitis can be a cause of
328    Pocket Guide to Bacterial Infections

bacterial or viral origin. Ocular allergy, other extraocular infections such as

blepharitis, dacryocystitis, dry eye, use of contact lenses, ophthalmic solu-
tions, and medications are stated as frequent causes of conjunctivitis. The
symptoms include tearing, burning, or stinging sensation, sticky eyelids in
the morning, mucopurulent secretions with distinct or severe pain.

The infection begins unilaterally. However, within 1–2 days the fellow eye
becomes infected. The condition is also found to be contagious, caused by
one or more bacterial species (Bartlett and Jannus, 2008).

11.5.1.3.1 Classification and etiological agents – Conjunctivitis can be

classified as hyperacute, acute, and chronic (Morrow and Abbott, 1998).
Neisseria gonorrhoeae is the common cause of hyperacute conjunctivitis with
the characteristic symptoms of abrupt onset, yellow-green purulent secretion,
redness, and irritation of the conjunctiva, tenderness and palpation, conjunc-
tival chemosis, conjunctival injection, lid swelling and so on. If hyperacute
gonococcal conjunctivitis is left untreated, the corneal involvement and endo-
phthalmitis is inevitable. N. meningitis is the second-most common cause
for hyperacute bacterial conjunctivitis (Hovding, 2008). Acute bacterial con-
junctivitis lasts for 3–4 weeks with burning and irritating sensation obviously
followed by purulent discharge. The common bacterial pathogens involved
in acute conjunctivitis are S. aureus, S. pneumoniae, and Haemophilus influ-
enza. Prompt medication will lower the contagiousness and reduces further
complications like corneal ulceration. The chronic conjunctivitis last for at least
4 weeks with similar signs of itching and burning sensation along with flaky
debris, erythema, and bulbar conjunctival injection. The chronic conjunctivitis
is commonly caused by Staphylococcus species and Moraxella lacunata with
occasional involvement of other bacteria (Thielen et al., 2000).

11.5.1.3.2 Pathophysiology – Pathophysiology of the conjunctivitis is

not well documented. The inflammation caused in the conjunctiva may be
induced due to the exogenous and endogenous infections and toxic agents
produced by the pathogens (Thielen et al., 2000).

11.5.1.4  Hordeolum – Hordeolum is an acute bacterial infection causing

inflammation on the eyelid margin. The infection presents the painful, ery-
thematous, and swollen furuncle. The onset of infection is spontaneous and
is dependent on the influence of lid hygiene. It is one of the most common
infections of the eye. Hordeola may be associated with various complica-
tions such as diabetes, blepharitis, seborrheic dermatitis, and individuals
with high levels of lipid secretion. Chalazion and hordeolum frequently
presents similar signs and are often misdiagnosed. The hordeolum affects
 Bacteriology of Ophthalmic Infections     329

the oil glands of the eye either internally (inside the eyelids) or externally (on
the eyelid, near eye lashes) (Lindsley et al., 2017).

11.5.1.4.1  Classification and etiological agents – The internal inflam-

mation affects the Meibomian gland, whereas the external inflammation
affects the Zeis or Moll gland (Wald, 2007). The external hordeola is more
common and are referred to as styes. In most instances, the lump resolves
spontaneously over a period even left untreated. However, the inflamma-
tion might spread to other ocular glands and cause secondary infections
like cellulitis. The incomplete elimination of bacteria may result in recurrent
hordeolum. Internal hordeola tend to be more painful than the external hor-
deolum (Lindsley et al., 2013). Internal hordeolum if untreated may lead to
development of chalazion. In both the forms, the size of the lesion is directly
proportionate to the severity of the infection (Lebensohn, 1950). The infec-
tion is usually caused by the Staphylococcus spp. that infects the eyelash
hair follicles (Mueller and Mcstay, 2008).

11.5.1.4.2 Pathophysiology – The deceptive secretory functions of

the ocular glands such as Zeis or Moll and Meibomian gland may result
in hordeolum. The Zeis gland secrets sebum with antiseptic properties
whereas Moll gland secrets IgA, mucin and lysosomes, which act as
immune barrier against the pathogenic organisms. The obstructions in
these glands lead to impaired defense system that is prone to infectious
organisms. Further bacterial infection with S. aureus, the most common
pathogen will activate the immune system and elicit immediate localized
inflammatory response followed by purulent or abscess development
(Bragg, 2017).

11.5.1.5 Chalazion – Chalazion, also known as a Meibomian cyst, is a

common eyelid disorder of all age groups. It is lipogranulomatous inflam-
mation of the ocular glands caused due to retention of the Meibomian
secretion in the sebaceous gland. Inflammation and irritation of the eyelid
and ocular surface with the formation of cyst are the common clinical pre-
sentations. The cyst formation usually does not affect the normal visual per-
ception. However, the size of the cyst may have an impact. Larger chalazion
cyst may interrupt the normal vision or induce astigmatism which can lead
to eye morbidity (Park and Lee, 2014). The predisposing factors associated
with chalazion include, Meibomian gland dysfunction, chronic blepharitis,
dry eye, seborrheic dermatitis, gastritis, and smoking (Nemet et al., 2011).
Other factors such as exposure to ultraviolet (UV) light, poor lid hygiene,
use of cosmetic products, and stress also contribute to cyst development,
but their role in disease is poorly understood.
330    Pocket Guide to Bacterial Infections

11.5.1.5.1  Etiological agents – Bacteria, especially S. aureus, is known to

be the primary causative agent of chalazion. However, the severity and eye
morbidity due to chalazion is dictated by the secondary bacterial superim-
posed infections (Otulana et al., 2008). Incidence of other bacterial agents
has not been encountered in years.

11.5.1.5.2  Pathophysiology – Retention of the Meibomian gland secre-

tion leads to the accumulation of lipid which further develops into a
lipogranulomatous form. Histological sections of chalazion have shown
to be composed of histiocytes, mononuclear granulocyte cells, lympho-
cytes, plasma cells, polymorphonuclear cells, and eosinophils (Mustafa
and Oriafage, 2001). Presence of a pseudo-capsule connective tissue was
often observed around the lesion (Ozdal et al., 2004). Liberation of lipids
provide hosting environment for the infectious organisms, which worsens
the case.

11.5.1.6  Bacterial keratitis – Keratitis is a complicated ocular infectious

disease of the cornea that can potentiate unilateral or bilateral ocular mor-
bidity. It is of microbial origin and predominantly due to bacteria. The infec-
tion of the cornea will lead to defect in the corneal epithelium, eventual
corneal scaring and perforation followed by stromal inflammation. The
common risk factors include contact lens wear, previous ocular surgeries,
presence of surgical sutures, ocular trauma, persistent topical corticosteroid
usage, and certain ocular surface diseases like blepharitis. Several systemic
diseases such as diabetes mellitus, rheumatoid arthritis, immunodeficiency,
and smoking can also contribute to keratitis (Bourcier et  al., 2003; Keay
et al., 2006)

11.5.1.6.1  Etiological agents – P. aeruginosa, S. aureus, coagulase nega-

tive Staphylococcus, and S. pneumoniae are considered prime causative
organisms (Green et al., 2008). Few other organisms including S. epidermi-
dis, Moraxella, S. marcescens, Bacillus, Corynebacterium, H. influenza, and
Alcaligenes xyloxidans are reported for their association with keratitis (Dart
et al., 2008).

11.5.1.6.2  Pathophysiology – Cornea acts as a preventive barrier against

the invading pathogen. In addition to this, host defensive mechanism will
prevent the corneal tissue from bacterial infections. Failure of the host
defense mechanism or corneal breaching will allow bacterial invasion
(Andrew et al., 2003). Inflammatory mediators infiltrate rapidly at the site
of corneal damage and results in corneal cloudiness around the infected
tissue. Various immune secretions will further damage the corneal tissue for
instance, IL-8 secretion will lead to neovascularization. Corneal damage and
 Bacteriology of Ophthalmic Infections     331

blood vessel formation will interfere the normal vision and if left untreated
fallouts in ocular morbidity (Schaefer et al., 2001).

11.5.1.7  Cellulitis – Cellulitis is generally described as the connective tissue or

subcutaneous tissue inflammation caused majorly due to infectious pathogens.
Cellulitis in the orbital surface is the infection in the soft tissues of the orbit which
predominantly affects the children. It is potentially a sight-threatening infec-
tion, which can also cause several systemic and life-threatening illnesses such
as cavernous sinus thrombosis, meningitis, cerebritis, endophthalmitis, and
brain abscess (Donahue and Schwartz, 1998; Georgakopoulos et al., 2010).
Common signs include cutaneous tenderness, erythema, severe pain, leuko-
cytosis, and so on. Several pediatric orbital cellulitis has been reported as a
secondary infection of paranasal sinus infection.

11.5.1.7.1  Classification and etiological agents – Partition of the soft

tissues of eyelid and orbit by orbital septum creates preseptal and postsep-
tal space. Infections at the preseptal space which is anterior to the orbital
septum is defined as the preseptal or periorbital cellulitis (Nageswaran
et  al., 2006). In orbital cellulitis, the infection is restricted to the poste-
rior of orbital septum (Mawn et  al., 2000). Trauma, sinusitis, and bacte-
remia are the major routes of orbital infections. Clinical manifestations of
periorbital cellulitis include erythema, induration, tenderness, chemosis,
proptosis, limited ocular motility, optic neuritis, hypesthesia, sensory dis-
tribution, and so on. Infection of the periorbital cellulitis is restricted to
the preseptal eyelid tissue. H. influenza, beta hemolytic Streptococcus spp.,
S.  aureus, S.  epidermidis, and S. pyogenes are major etiological agents.
Other rare causative agents are P. aeruginosa, N. gonorrhoeae, Treponema
pallidum, and Mycobacterium tuberculosis among others (Ambati et  al.,
2000; Carlisle and Fredrick, 2006).

11.5.1.7.2  Pathophysiology – Paranasal sinuses are the cavities that sur-

round the orbit of the eye. Infections in the sinuses will also contribute
to the pathophysiology of orbital cellulitis. In the thin paranasal sinuses,
several natural perforations occur to pass the valveless blood vessels and
nerves. These perforations are the major route of infections. Additionally,
due to thin architect of the orbital bones, formation of abscess from the
adjacent sinusitis is probably high (Lee and Yen, 2011).

11.5.2  Intraocular infections

11.5.2.1 Endophthalmitis – Endophthalmitis is an inflammatory disease

of the posterior eye segment (vitreous/aqueous humor) due to intraocular
bacterial or fungal infections (Callegan et al., 1999; Durand, 2017). It occurs
infrequently, but the infection results in devastated eye state and irreversible
332    Pocket Guide to Bacterial Infections

vision loss. The clinical presentation usually initiates with the blurred vision
and causes mild to severe pain, redness, absence of fundus, hypopyon, vitri-
tis, and inflammation in the anterior chamber (Jackson et al., 2014).

11.5.2.1.1  Classification and etiological agents – Based on the route

of infection, endophthalmitis is classified into endogenous bacterial or
exogenous postoperative endophthalmitis. Exogenous form occurs fol-
lowing the introduction of pathogens directly after ocular surgeries such
as cataract surgery, penetrating keratoplasty, or placement of keratopros-
thetics. Infection for endogenous endophthalmitis occurs through the
bloodstream after crossing the blood retinal barrier (Jackson et al., 2014).
In addition to the previously specified clinical presentations, endogenous
endophthalmitis will present systemic infections such as fever or influ-
enza-like symptoms. The clinical features and bacteriology of these cat-
egories vary relatively. In recent years, the postoperative endophthalmitis
has been reported in almost every type of ocular surgery, but predomi-
nantly following cataract surgery. The major etiological agents caus-
ing endophthalmitis includes S. aureus, S. epidermidis, Bacillus cereus,
Enterococcus faecalis, K. pneumoniae, P. aeruginosa, N. meningitides, S.
pneumoniae, Group B streptococci, and Nocardia spp. (Callegan et  al.,
1999; Jackson et al., 2014).

11.5.2.1.2 Pathophysiology – In endogenous endophthalmitis, the

organisms from the bloodstream enter the ocular surface either through
retina or uvea and invades the tissue after transecting through the blood
ocular barrier. With subsequent establishment, the pathogens reside in the
aqueous or vitreous humor. Bacterial load and the infiltration of inflamma-
tory cells will initiate destruction of the tissue and physiological imbalance
resulting in the loss of function in the anterior segment. If the pathogen
enters via retinal artery, the dissemination of bacteria along the retinal ves-
sels will cause irreversible tissue damage due to toxins produced by the
pathogens and by the activity of inflammatory cells (Greenwald et al., 1986).

11.5.2.2  Uveitis – Uveitis, the inflammation of the uveal tissue that encom-
passes iris, choroid, and ciliary body is a major blinding disorder (Biziorek
et  al., 2001). It is usually found in all age groups, but the severity of the
disease is much higher in pediatrics than adult disease. Pediatric uveitis is
usually asymptomatic, which results in inability to detect the disease in ear-
lier stage subsequently and leads to permanent vision loss (Curragh et al.,
2017). Neither simple clinical examination nor the noninvasive investigation
clearly state the causation of disease and the etiology remains unknown
in maximum cases. The association of trauma, infection, systemic diseases
such as tuberculosis, sarcoidosis, spondylarthropathies, Bechet’s disease,
 Bacteriology of Ophthalmic Infections     333

Whipple’s disease, Koyanagi-Harada syndrome, and inflammation can criti-

cally lead to uveitis (Biziorek et al., 2001).

Though usually non-symptomatic, certain types of uveitis may cause dis-

comfort, pain, photophobia accompanied with lacrimation, congestion, and
iridocyclitis which often leads to keratitis or keratopathy (Bartlett and Jannus,
2008). As the disease is related with the vision accomplishing pathway,
severe inflammation of the uveal tissue often leads to unilateral or bilateral
ocular morbidity based on the origin of the disease (Hogan et al., 1959).

11.5.2.2.1 Classification and etiological agents – Based on the ocu-

lar site of inflammation, uveitis can be classified into four major types as
anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis. The
inflammation of anterior chamber or the iris lesion or keratic precipitates are
usually demarcated as anterior uveitis. Intermediate uveitis can be defined as
the inflammation of the vitreous chamber with or without the involvement
of peripheral retina. Inflammation affecting retina, choroid, retinal vessels,
or posterior vitreous humor is defined as the posterior uveitis. Combination
of inflammation in all three described sites is collectively termed panuveitis
(Bodaghi et al., 2001).

Predominant infectious agents of uveitis include certain parasites such as

Toxoplasma gondii, Streptococcus spp., and fastidious bacteria such as
spirochetes, intracellular bacteria such as Chlamydia spp., Rickettsia spp.,
Coxiella burnetiid, and the gram-positive pathogen Tropheryma whipplei.
Bartonella henselae and B. grahamii are also reported as causative organ-
isms of uveitis (Drancourt et al., 2008; Terrada et al., 2009).

11.5.2.2.2  Pathophysiology – Bacterial products such as cell wall com-

ponents, proteins, endo- and exotoxins are suspected to be the triggers
of uveitis. The ocular surface synthesis proteins such as toll-like receptors
(TLR) and nod-like receptors (NLR) which respond well to the bacterial prod-
ucts and initiate inflammation on the infected site. NLRs are reported to be
closely associated with uveitis than TLRs. Though the association of these
proteins are known to cause the infection, the exact mechanism is not well
understood, which is essential in understanding the disease state and to
develop the treatment strategies for uveitis (Rosenbaum et al., 2008).

11.6  Infectious bacteria in ocular diseases

Microorganisms, specifically bacteria, are the leading causal mediators of

numerous infectious diseases, among which ophthalmic infections cannot
be ruled out certainly. Though, fungal (e.g., keratitis and endophthalmitis)
334    Pocket Guide to Bacterial Infections

and other parasitic (e.g., Acanthamoeba keratitis, Chagas disease, giardiasis,

Leishmaniasis, and Toxoplasmosis) infections are common, the prevalence
of bacterial ocular infections are exponential (Nimir et al., 2012; Squissato
et al., 2015). The organisms that cause ocular infections are predominantly
exogenous that find route into eye during surgeries through contaminations
from instruments and infusing fluids. Bacterial infections of the ocular
surface can be of mono- or polymicrobial origin. Several predisposing
factors such as contact lens wear, ocular surgeries, poor hygiene of the eye,
obstruction of nasolacrimal duct, diminished immune status, previous ocular
infections, and certain environmental factors make the eye susceptible
to bacterial infections. Both gram-positive and -negative bacteria can
cause ocular infections. Gram-positive pathogens are more predominant
in causing eye infections than the gram-negative organisms. S. aureus,
Coagulase negative Staphylococcus, S. pneumoniae, and P. aeruginosa are
the predominant bacterial isolates found in ocular infections. S. epidermidis,
N. gonorrhoeae, K. pneumoniae and H. influenza are less common isolates
from infected eyes. Several reports evidence the occurrence of Enterobacter,
Corynebacterium, Acinetobacter, Propionibacterium, and B.  subtilis in
various ocular infections. The types of bacteria, their distribution, load,
and the site of infection determine the severity of the ocular disease. Initial
treatment strategy include course of topical or systemic antibiotic regimen,
which upon regular use can promote antibiotic resistance mechanisms.
Emergence of antibiotic resistance may have serious consequences
such as development of sight-threatening complications (e.g., keratitis,
endophthalmitis, orbital cellulitis, retinitis, and dissemination of infections
to other major organs like brain). Moreover, quorum sensing and bacterial
biofilm play a critical role in recurrent infections.

11.6.1 Staphylococcus spp.

S. aureus and coagulase negative staphylococci (CoNS) are the most pre-
dominantly isolated pathogens of infected eye and a major cause of
nosocomial eye infections. Among the heterogenous group of CoNS, S. epi-
dermidis, and S. saprophyticus are the frequently encountered organisms
(Mshangila et al., 2013). Despite their normal existence as commensal ocu-
lar microbiota, S. aureus and CoNS are frequent causative agents of most
of the eye infections with increasing frequencies over the course of time.
The incidence of Staphylococcus species in postoperative cases are even
higher, especially in cataract surgeries. In a clinical study, conducted with
the conjunctival culture of the patients who underwent cataract surgery,
around 80% of the cultures were found to be Staphylococcus spp., 45.2%
and 35% of CoNS and Staphylococcus spp., respectively (Lin et al., 2017).
 Bacteriology of Ophthalmic Infections     335

A 20-year retrospective study of posttraumatic endophthalmitis showed the

predominance of S. epidermidis (21.8%) and S. saprophyticus (12%) (Long
et al., 2014). Other than the aforementioned CoNS, S. cohnii, and S. hae-
molyticus were observed with lesser incidence (Sherwal and Verma, 2008).
In other clinical cases, methicillin-sensitive Staphylococcus aureus (MSSA)
and methicillin-resistant Staphylococcus aureus (MRSA) were found to be
associated with ocular infections. MRSA isolates were principally found in lid
and lacrimal disorders (Chuang et al., 2012). Community-associated MRSA
(CA-MRSA) and healthcare associated MRSA (HA-MRSA) are emerging
healthcare risk factors of various ocular infections (Hsiao et al., 2012; Wong
et al., 2017). Yet another report states that 50%–66% of hospital workers
are found to be infected with Staphylococcus (Rashid et al., 2012).

Staphylococcus infections can range from mild infections such as blephero

conjunctivitis, corneal ulcer to sight-threatening diseases such as keratoco-
nus, orbital cellulitis, endophthalmitis, keratitis, dacryocystitis, chorioretini-
tis, scleritis, and so on.

11.6.1.1  Virulence factors – S. aureus secretes numerous virulence fac-

tors, most of which are part of defense mechanism against the host immu-
nity. The secretory proteins of S.  aureus include alpha-toxin, beta-toxin,
gamma-toxin, panto-valentine leucocidin, and so on. In addition, S. aureus
also produces proteases, lipases, leucocidin, and exfolatin (O’Callaghan
et al., 1997).

The two major barriers of the cornea are the mucin layer and the intra-
cellular tight junction of corneal epithelium, which make the binding and
penetrance of infectious agents an impossible event. But the disruption of
these barriers increases the susceptibility of cornea to staphylococcal infec-
tion, which may lead to keratitis. The binding of S. aureus to the corneal
cell is achieved through two bacterial proteins, fibronectin-binding protein
and collagen-binding adhesin, which leads to significant tissue damage by
penetration of bacteria to the corneal epithelium (Rhem et al., 2000; Jett
and Gilmore, 2002).

The alpha-toxin binds to the protease receptor ADAM 10; thereby it can form
pores on the cell membrane, allowing small molecules to pass through. The
binding of alpha-toxin with the immune cells such as neutrophils, mono-
cytes, T-cells, and platelets allow calcium ions to pass through the pores and
thus causing cellular dysregulation. It can also cleave the E-cadherin mol-
ecules, which are associated with the attachment of cells. Thus, the alpha-
toxin binding to the corneal epithelium will eventually disrupt the epithelial
336    Pocket Guide to Bacterial Infections

layer and initiate the corneal ulceration. In endophthalmitis, the alpha-toxin

can cause inflammation in the retina, which can terminate in loss of retinal
function (Berube and Juliance, 2013; Kumar and Kumar, 2015).

Beta-toxin, a sphingomyelinase, acts on the scleral epithelium and not the

cornea. The F and S component of the gamma toxin, when bound to the
cell membrane, can penetrate and lyse the cell, which subsequently leads
to infiltration of neutrophils to cornea and iris, conjunctival reddening, che-
mosis, and fibrin accumulation in the anterior chamber. The gamma-toxin is
also toxic to the vitreous chamber (Callegan et al., 1994).

The bacterial surface antigens such as poly-N-acetylglucosamine (PNAG) and

lipoproteins, which act on TLRs can mediate release of proinflammatory cyto-
kines such as IL6 and IL8 and other antibacterial molecules, hBD-2, LL-37, and
iNOS that can trigger local inflammation and tissue destruction (Li et al., 2008).

The secreted proteases of S. aureus can inactivate beta-crystalline protein,

which prevents apoptosis in retina. Hence destruction of beta-crystalline
often leads to loss of retinal cells (Whiston et al., 2008).

PVL toxin is produced mainly by CA-MRSA strains. It is a two-component

toxin system composed of F and S protein, which are likely to produce more
severe form of keratitis than non-PVL producing strains. This toxin signifi-
cantly contributes to corneal virulence but the mechanism is poorly under-
stood (Sueke et al., 2013).

In conjunctivitis, the goblet cells of the conjunctiva when exposed to the

S. aureus toxin will activate the caspase 1 resulting in production of cytokine
IL-1 β which is an efficient inducer of inflammation (McGilligan et al., 2013).

In blepharitis, the lipid accumulation on eyelid forms a cyst. The lipid accu-
mulated is the cleavage product of cholesterol and fatty acids by the action
of enzyme lipase. The growth of S. aureus is stimulated by the presence of
cholesterol, which results in excessive colonization of bacteria on the eyelid.
On the other hand, the overgrowth of S. aureus and S. epidermidis on the
nasolacrimal duct potentiate the blockage of duct. This infection can spread
to the cornea, resulting in a corneal ulceration (Shine et al., 1993).

In addition to these virulence factors, S. aureus produces elastase for its

defense against the host immune system, which can form corneal ulcers
ultimately leading to keratitis (Wu et al., 1999).

Though the ocular immune system produces multiple components to pro-

tect from S. aureus infections, the corneal or retinal involvement will result in
adverse effects like corneal scarring, reduced visual acuity, or even loss of vision.
 Bacteriology of Ophthalmic Infections     337

11.6.1.2 Antibiotic resistance – The emerging antibiotic resistance

and production of numerous virulence factors potentiates the infectious
nature of Staphylococcus spp. in ocular infections. Staphylococcus spp.
can evolve very rapidly against a range of antibiotics. S. aureus isolates,
which are known as MRSA that are resistant to methicillin, are commonly
resistant to other beta-lactam antibiotics such as penicillin, carbapenem,
cephalosporin, and monobactam (Rayner and Munckhof, 2005). The
antibiotic susceptibility of CoNS is unpredictable. Penicillin resistance is
extremely common worldwide. Hence, penicillinase-resistant beta-lactam
antibiotics such as flucloxacillin and oxacillin are commonly used for first-
line therapy. Most of the strains of CoNS, CA-MRSA, and HA-MRSA were
found to be multiresistant. In an antibiotic susceptibility study conducted in
the year 2012, MRSA strains were found to be susceptible to vancomycin,
teicoplanin, and gentamicin (Kotlus et al., 2006; Hsiao et al., 2012). Ocular
MRSA strains were highly resistant to fluoroquinolones including the
fourth generation (Kotlus et al., 2006). CA-MRSA and HA-MRSA strains
were found to be highly sensitive to cotrimoxazole, rifampicin, fusidic acid,
and minocycline. The frequently used topical antibiotic chloramphenicol
was relatively found to be sensitive to almost all Staphylococcus spp.
(Wong et al., 2017). S. aureus develops resistance against antibiotic with
various mechanisms including enzymatic inactivation of antibiotics with
penicillinase and aminoglycoside-modifying enzymes, altering the target
and thereby decreasing the affinity of antibiotics, trapping of antibiot-
ics and efflux pumps. Horizontal gene transfer, spontaneous mutations,
and positive selection can also confer antibiotic resistance (Pantosti et al.,
2007) (Figure 11.2).

11.6.1.3 Therapeutic interventions – The use of antibiotics results in

resistance mechanisms over a period. The combinations of different antibi-
otics, for which resistance has not been evolved or use of natural antibac-
terial agents will potentiate the therapeutic strategies. Dajcs et  al. (2001)
reported the effectiveness of lysostaphin in treating MRSA-mediated endo-
phthalmitis and keratitis. Lysostaphin is a zinc metalloproteinase enzyme
isolated from S. simulans, which can potentially slay down S. aureus by
rapid cell wall digestion. Hence, topical application or intravitreal injec-
tion of lysostaphin will be an effectual therapy for ocular staphylococcal
infections.

Bacteriophage therapy, antibody therapy targeting the virulence factors

of Staphylococcus, will be the effective alternates to antibiotics. Caballero
et al. (2015) produced a high-affinity monoclonal antibody against S. aureus
alpha-toxin and proved its effectiveness as a therapy for S. aureus–mediated
keratitis (Caballero et al., 2015). Bacteriophage-derived lytic proteins such as
338    Pocket Guide to Bacterial Infections

Figure 11.2  Virulence factors of Staphylococcus spp.

endolysins have been investigated as antimicrobials. Hence, endolysin technol-

ogy for killing of S. aureus has been reported in the recent past (Schmelcher
et al., 2012; Roach and Donovan, 2015). In addition to this, antimicrobial blue
light therapy has been reported as a potential alternative or a combinatorial
therapy for treatment of keratitis-mediated by S. aureus (Zhu et al., 2017).

With these emerging therapeutic procedures, the staphylococcal infections

of the eye could be minimized in the near future.

11.6.2  Streptococcus spp.

Streptococci are gram-positive bacteria belonging to the phylum

Firmicutes, which encompass numerous species that either exist as a
commensal or human pathogen. Based on the surface antigens, strepto-
coccal species are classified into various groups. Group A streptococcus
(GAS)–S. pyogenes and Group B streptococcus–S. agalactiae (GBS) are
beta hemolytic, whereas S. pneumoniae are alpha-hemolytic. GAS and
GBS can cause invasive diseases. Though infrequently observed, contribu-
tions of Streptococcus spp. in various ocular infections have been reported
extensively. Streptococcus spp. were found in both intra- and extraocular
infections including non-sight-threatening conditions such as conjuncti-
vitis, blepharitis, dacryocystitis to sight-threatening preseptal and orbital
 Bacteriology of Ophthalmic Infections     339

cellulitis, keratitis, and endophthalmitis. S. pneumoniae was reported to be

the frequent pathogenic isolate from pediatric conjunctivitis (Friedlaender,
1995; Buznach et al., 2005; Patel et al., 2007). In some bacterial conjunc-
tivitis cases, S. viridians were observed as the sole positive culture (Cavuoto
et  al., 2008). S. pneumoniae is considered one among the three-most
common pathogens of bacterial conjunctivitis. Along with S. aureus,
S. pneumoniae forms the most common gram-positive isolate of dacryo-
cystitis followed by GAS and viridian streptococcus (Pinar-Sueiro et  al.,
2012; Sarkar et al., 2015). Streptococcus spp. are predominant in preseptal
and orbital cellulitis (Donahue and Schwartz, 1998; Georgakopoulos et al.,
2010). Among them, the adult with periorbital cellulitis are more likely to
be infected with GAS, whereas, younger children are likely to be infected
with ­S. ­pneumoniae (Schwartz and Wright, 1996). After S. pneumoniae,
S. pyogenes is frequently observed in ocular cellulitis and in rare instances
with other Streptococcal spp. such as S. sanguinis and S.  ­milleri (Ambati
et al., 2000; Howe et al., 2004). Endophthalmitic infections were found to
be caused by Streptococcus spp., namely S. pneumoniae, S. dysgalactiae,
and GBS (Jackson et al., 2014). Due to routine use of pneumococcal vac-
cine against pneumoniae, Hauser et al. (2010) reported that the incidence
of S. pneumoniae in ocular infections are on decline. Yet, among other
Streptococcus spp., S. pneumoniae is f­ requently being reported in various
eye infections.

11.6.2.1  Virulence factors – Streptococcus spp. remains a major patho-

gen of man despite advent antibiotic therapy. The pathogenicity of
Streptococcus has been conferred by numerous virulence traits. Bacterial
components such as capsule, cell wall components, secretary proteins, and
enzymes are thought to be the key virulent factors of Streptococcus spp.
Major virulence factors and their mechanisms are now detailed.

Pneumolysin (PLY) is a potent 53  KDa pore-forming cytotoxin synthesized

and located in the cytoplasm of pneumococci. Release of this toxin occurs
immediately after spontaneous autolysis of the bacterial cell. Upon release,
they interact with the cholesterol and bind the lipid bilayer followed by
transmembrane pore formation and lysis of the cell. The pore-forming abil-
ity of the PLY can potentially disrupt the corneal epithelium upon infection.
In addition to the cytotoxic property, it can directly activate the classical
complement system. Activation of complement can either result in produc-
tion of chemotactic molecules or direct the complement-mediated mem-
brane attack on the host cell. The initiation of proinflammatory cytokines
will mediate inflammation and tissue damage. Hence, the release of PLY
on the corneal surface eventually leads to corneal ulceration followed by
keratitis (Paton, 1996).
340    Pocket Guide to Bacterial Infections

Though the capsular polysaccharide of S. pneumoniae, also known as

pneumococcal PS capsule, does not essentially involve in the inflammatory
response but contributes to the disease progression by effectively defend-
ing the complement mediated phagocytic attack (Mitchell et  al., 1997).
In addition to this, secretory proteases cleave the capsule-specific human
antibodies, immune components, and blocks the inflammatory responses
induced by the cell wall components (Rubins and Janoff, 1998). Surface pro-
tein A and adhesins mediate the attachment of bacterial cell surface com-
ponents to the host cells. Cell wall components can also generate an array
of inflammatory mediators, which are more likely to initiate tissue damage.

Autolysin is a cell wall degrading enzyme found on the cell envelop. It is

activated under conditions such as nutrient starvation and blockage of cell
wall synthesis and autolyzes the bacterial cell to release the cytoplasmic
content into the surrounding. The cell wall degradation products and the
virulent components from cytoplasmic content will exert inflammatory and
toxic effect on the host tissue (Mitchell et al., 1997).

The enzyme neuraminidase assists in bacterial colonization and facilitate

adherence on the populated surface. This is achieved by the cleavage of
sialic acid residues on the epithelial layer. During conjunctivitis, the produc-
tion of neuraminidase by ocular surface colonization of S. pneumoniae will
damage mucins, which are made up of sialic acid residues and considered
a major component of the mucus layer of cell surface epithelia, which leads
to further complications (Williamson et al., 2008).

The enzyme hyaluronidase is produced by wide range of gram-positive

organisms including most of the Streptococcus spp. This enzyme degrades
the hyaluronic acid, a major component of connective tissue. Cornea and
sclera, which are connective tissues of eye, are prone to tissue damage by
hyaluronidase enzyme. Hence, the damaged cornea will lead to invasion of
pathogens and mark in infectious conditions such as keratitis, endophthal-
mitis, and so on (Mitchell et al., 1997).

Superoxide dismutases (SOD) are metalloenzymes that act as an oxidative

stress defense mechanism. There are two types of SOD in S. pneumoniae,
MnSOD and FeSOD. MnSOD has been reported to play a major role in viru-
lence of pneumococcus (Yesilkaya et al., 2000; Mitchell, 2000).

11.6.2.2 Antibiotic resistance – S. pneumoniae and S. pyogenes have

been reported to show resistant against a wide range of antibiotics includ-
ing penicillin, cephalosporin, macrolide, and lincosamide. The alteration of
penicillin-binding protein (PBP) confers resistance to penicillin. The efficiency
 Bacteriology of Ophthalmic Infections     341

of other antibiotics such as beta-lactam, cephalosporin, and carbapenem

are also reliant on PBP. Hence, the activity of these antibiotics is reduced in
penicillin resistant Streptococcus spp. (Baquero et  al., 1991; Doern et  al.,
1996). Most of the clinical isolates are now being found resistance to treat-
ment with single or combination of antibiotics, an indication of multidrug
resistance.

Macrolide resistance has been reported as early as in 1990s. The macro-

lide resistance is conferred by efflux genes (mef, mel) and target-modifying
methylase genes (erm). The macrolide efflux is mediated by the gene product
of mef (A) in both S. pneumoniae and S. pyogenes. The most common form
of target site modification is mediated by rRNA methylase, ­di-methylating
a specific adenine residue on the 23S rRNA (Hyde et al., 2001; Farrell et al.,
2002). In addition to this, secretary enzymes confer resistance through
inactivating the drug molecule (Leclercq et al., 2002). The dissemination of
macrolide resistance genes through a mobile genetic element has also been
reported. In addition to transformation and recombination, conjugative
mobile elements transmit the resistance genes among the pneumococcal
and other Streptococcus spp. (Chancey et al., 2015) (Figure 11.3).

Figure 11.3  Virulence factors of Streptococcus spp.

342    Pocket Guide to Bacterial Infections

11.6.2.3 Therapeutic interventions – Though antibiotic resistance is

widely reported, antibiotics remain the first-line therapy for infections medi-
ated by Streptococcus spp. For patients allergic to penicillin, macrolides are
the suggested antibiotics. Macrolides and lincosamides are recommended
in combinations with beta-lactams for invasive infections (Silva-costa
et  al., 2015). From various case studies, it is evident that macrolides and
lincosamides are the better alternatives to penicillin. In a clinical study,
Gregori et  al. (2015) used intravitreal injection of antivascular endothelial
growth factor (anti-VEGF) for the treatment of infectious endophthal-
mitis caused by Streptococcus and Staphylococcus spp.; it lessened the
recurrent infection and was found to be a better treatment in preventing
vision loss. The ­treatment of streptococcal infection remains challenging
due to their potent virulence factor and antibiotic resistance mechanisms.
Hence in recent years, natural sources have been widely explored as an
alternate to the e­ xisting antibiotics. Viszwapriya et  al. (2016; Viszwapriya
and Subramenium, 2016) reported betulin, a naturally available triterpe-
noid and 2,­4 -Di-tert-butylphenol from seaweed surface-associated bacte-
rium Bacillus subtilis as effective anti-infective agents against S. pyogenes.
Therefore, natural biomolecules alone or in combination with antibiotics
will be an effective therapeutic strategy for Streptococcus infections.

11.6.3  Pseudomonas aeruginosa

P. aeruginosa is a gram-negative, opportunistic bacterial pathogen, which
is a major etiological agent of numerous infectious diseases affecting the
eye and surrounding tissues. P. aeruginosa ocular infections are most fre-
quently observed as from an exogenous origin from an environmental
source. However, endogenous infection as a result of metastasis has also
been reported. The predisposing factors associated with P. aeruginosa–
mediated ocular infections includes (1) corneal trauma or ulceration, (2)
preexisting ocular infections or diseases, (3) immunosuppressive chemo-
therapy, (4) immunodeficiency in infants, (5) corneal aberrations caused by
contact lens wear, (6) application of contaminated eye area cosmetics, and
(7) systemic infections (Wilson and Ahearn, 1977; Reid and Wood, 1979;
Kreger, 1983; Wu et al., 2015). In addition to this, implantation of intraocu-
lar lens with Pseudomonas contamination often leads to endophthalmitis.
P. aeruginosa can affect almost every part of the eye, namely, diseases
affecting the surrounding tissue of the eye, such as dacryocystitis, blephari-
tis, orbital cellulitis; extraocular infections including conjunctivitis, keratitis,
and corneoscleral ulceration; intraocular infection such as endophthalmitis
(John et al., 1996; Brito et al., 2003; Delia et al., 2008; Bhattacharjee et al.,
2016). P. aeruginosa is a common cause of postoperative endophthalmitis.
 Bacteriology of Ophthalmic Infections     343

Corneal damage and invasion of the bacteria to intraocular regions are

threatening to sight by development of secondary glaucoma or cataract.
Though P. aeruginosa is the most recurrently reported Pseudomonas spe-
cies in ophthalmic infections, P. acidovorans, P. stutzeri, and P. fluorescens
also have been less frequently reported as causative organisms of human
ocular infections. Dissemination of bacteria from ocular site to bloodstream
has not been reported in adults. However, in premature infants, fatal sep-
ticemia can develop from the spread of Pseudomonas from the eye (Burns
and Rhodes, 1961). The presence of various cell-associated and secreted
extracellular virulence factors expands the pathogenicity of P. aeruginosa
in various infections.

11.6.3.1  Virulence factors – Virulence factors of P. aeruginosa and their

associated mechanisms in ophthalmic infections are detailed next.

Sialic acid specific adhesin based N-acetylneuraminic acid receptor on the

cell surface of P. aeruginosa mediates the binding to the ocular epithelium
which is subsequently followed by colonization and infection (Hazlett et al.,
1986). The establishment of adherence to the host surface progresses the
infection by damaging the underlying tissue with the extracellular toxic sub-
stances and evokes the host immune system. This sequential process will
aggravate the tissue damage.

Among all other virulence factors, the ability to invade the host cell is con-
sidered the most crucial virulent trait in the pathogenicity of organism.
Invasion of P. aeruginosa inside the corneal epithelium is reported with two
suggestive mechanisms (i.e., transcellular migration and invasion through
destruction of corneal epithelium). Fleiszig et al. (1996) reported the inva-
sion and intracellular survival of P. aeruginosa within corneal epithelial cells.
Because intracellular bacteria can evade the host immune system and anti-
biotic treatment, P. aeruginosa infection can lead to serious consequences
like ocular morbidity (Fleiszig et  al., 1994). In addition to invasion, where
both the bacterial and host cell is viable up to 24 hours, some ocular clinical
strains of P. aeruginosa are reported as exerting cytotoxic effect on the host
cell (Fleiszig et al., 1996).

Exotoxin A (ExoA), the most toxic extracellular product of P. aeruginosa,

contributes to the corneal damage on infection. It can directly act on epi-
thelial, endothelial, and stromal cells of the cornea and cause necrosis by
inhibiting protein synthesis. Exo A is nonproteolytic in nature. However,
it inhibits the protein synthesis by transferring adenosine 5’diphosphate
ribose (ADPR) of nicotinamide adenine dinucleotide to mammalian elonga-
tion factor 2 (Ohman et al., 1980; Lyczak, 2000).
344    Pocket Guide to Bacterial Infections

Protease enzymes of P. aeruginosa acts as a crucial pathogenic trait in ocu-

lar infections. Proteoglycan matrix of the cornea is degraded by this enzyme.
P. aeruginosa strains usually produce three proteases: protease I, elastase
(protease II), and alkaline protease (protease III) (Wretlind and Pavlovskis,
1983). Secretion of elastase (LasB) results in the cleavage of elastin, fibrin,
and collagen, which provides mechanical strength to the connective tis-
sue such as cornea. The integrity of the corneal matrix is impaired by the
action of elastase. Elastase interferes with the host defense mechanism by
cleaving immunological factors such as IgG, IgA, IFNγ, TNF-α, and so on
(Willcox, 2007).

The flagella and pili of Pseudomonas acts as adhesins in corneal infection

mediating cell invasion and cytotoxicity. These extracellular appendages
bind specifically to the glycosphingolipidasialo-GM1 on the host cell. This
binding event eventually contributes to the corneal damage (Lyczak, 2000).

11.6.3.2 Antibiotic resistance – Extensive treatment of P. aeruginosa

with antibiotics generates selective pressure that initiates antibiotic resis-
tance. P. aeruginosa confers resistance against wide range of antibiotics
such as aminoglycosides (i.e., gentamicin, tobramycin, and amikacin) that
inhibits the protein synthesis; beta-lactams (i.e., piperacillin and ceftazi-
dime) that inhibit the peptidoglycan layer; and polymyxins (i.e., colomycin
and colistin) that bind with the phospholipids of cytoplasmic membrane.
Antibiotic resistance in P. aeruginosa is imparted through following fac-
tors: It is intrinsically resistant to antibiotics because of low permeability
of the cell wall, expresses a wide group of resistance mechanisms, attains
resistance through mutations in genes that regulate resistant genes, and
acquires additional resistance genes from plasmids, transposons, and bac-
teriophages (Lambert, 2002).

To date, four different efflux systems have been discovered in P. aeruginosa

mexAB-oprM, mexXY-oprM, mexCD-oprJ, and mexEF-oprN. Extrusion of
beta-lactams and quinolones are mediated by mexAB-oprM, aminoglyco-
sides are extruded by mexXY-oprM, and mexEF-oprN extrudes carbapen-
ems and quinolones (Ziha-zarifi et al., 1999).

Inactivation or modification of antibiotics by secretory enzymes and chang-

ing the target by spontaneous mutations also contribute to antibiotic resis-
tance. In the current scenario, to combat multidrug resistant Pseudomonas
infections, effective natural bioactive molecules such as curcumin from
Curcuma longa will effectively attenuate the virulent traits and lessens the
infection without conferring antibiotic resistance (Sethupathy et al., 2016)
(Figure 11.4).
 Bacteriology of Ophthalmic Infections     345

Figure 11.4  Virulence factors of Pseudomonas aeruginosa.

11.6.3.3 Therapeutic interventions – Multidrug efflux pump is a major

mechanism by which pathogenic organisms exhibit antibiotic resistance. Hence,
inhibition of such efflux pumps appears as a promising therapeutic strategy.
In search of efflux pump inhibitors, numerous natural sources, synthetic mol-
ecules, and existing antibiotics have been screened in recent years. The family
of efflux pump that mediates significant antibiotic resistance in P. aeruginosa
belongs to resistance nodulation division (RND) family. Adamson et al. (2015)
demonstrated that the putative efflux pump inhibitors trimethoprim and ser-
traline alone or in combination with antibiotic, namely levofloxacin, resulted in
enhanced therapeutic assistance. Phe-Arg-β-naphthylamide (PAβN) has been
reported to be the most effective and broad-spectrum inhibitor of P. aeru-
ginosa RND efflux pump. PAβN is now shown to encompass impact on the
virulence of P. aeruginosa. It is evident that efflux pump inhibitors can have
potential anti-virulent property (Rampioni et al., 2017).

Martins et al. (2008) used a unique therapeutic strategy for the treatment
of infectious keratitis with the combination of riboflavin and ultraviolet-A
(UVA; 365 nm). The UVA-induced riboflavin, efficiently exhibited antimicro-
bial property against bacterial and fungal isolates of keratitis, including the
multidrug resistant P. aeruginosa.

Blue light treatment of P. aeruginosa exhibited strong antimicrobial activity

with inactivation of an array of Pseudomonas virulence factors. Blue light
treatment in synergy with antibiotics also efficiently decreased the patho-
genicity of P. aeruginosa (Fila et al., 2017).
346    Pocket Guide to Bacterial Infections

Phage therapy is one more potential therapeutic remedy for P. aeruginosa

infections. Currently, there are 137  completely sequenced Pseudomonas
phage genomes available in the databases (Pires et  al., 2015). The treat-
ment of Pseudomonas infections with the combination of phage and other
antimicrobial agents as an effectual therapy has also been reported (Torres-
Barcelo et al., 2014).

With the advent of these therapeutic approaches, there are high possibili-
ties that infections caused by Pseudomonas will decline over time.

11.6.4  Coryneform bacteria

Coryneform bacteria comprises a group of aerobic, asperogenous, gram-

positive, rod-shaped bacteria, which includes the following genera:
Corynebacterium, Turicella, Arthrobacter, Brevibacterium, Dermabacter,
Propionibacterium, Rothia, Exiguobacterium, Oerskovia, Cellulomonas,
Sanguibacter, Microbacterium, Aureobacterium, Arcanobacterium, and
Actinomyces (Funke et  al., 1997). Though less frequently encountered,
Corynebacterium and Propionibacterium are the most-common coryne-
form bacteria associated with several systemic and ocular infections. The
species C. macginleyi, isolated from an eye specimen, was the first lipophilic
coryneform bacteria reported (Riegel et  al., 1995). C. macginleyi resides
as an ocular flora but acts as an opportunistic pathogen in conjunctivitis
and other ocular infections. It also has been described as a conjunctiva-
specific pathogen because it is predominantly isolated from conjunctivitis
eyes and infrequently found in sight-threatening infections such as keratitis
and endophthalmitis (Joussen et  al., 2000; Ly et  al., 2006; Suzuki et  al.,
2007). Ruoff et al. (2010) reported corneal ulceration and scaring caused by
C. macginleyi as a sole causative agent, suggesting that C. macginleyi can
cause ocular infection even when it is not associated with other opportunis-
tic or pathogenic organisms. Detection and identification of C. macginleyi
species is problematic as it is fastidious, requires enriched media, and being
sequestered by other organisms. Culture-independent techniques like PCR
will enumerate the presence of C. macginleyi. In a case report, Ferrer et al.
(2004) encountered sterile endophthalmitis followed by cataract surgery.
However, the sequence analysis showed C. macginleyi as the causative
agent of endophthalmitis. Mycobacterium keratitis is often misdiagnosed
with Corynebacterium keratitis (Garg et  al., 1998). C. pseudodipthericum
was found in conjunctivitis in extremely rare instances (Joussen et al., 2000).

P. acnes and other Propionibacterium spp. normally inhabit the conjunc-

tiva. Postoperative Propionibacterium endophthalmitis has been reported
scarcely. P. acnes are commonly associated with chronic blepharitis, chala-
zion, dacryocystitis, endophthalmitis, and keratitis. Predisposing factors for
 Bacteriology of Ophthalmic Infections     347

P. acnes infections include existence of foreign bodies, systemic infections,

and diseases such as diabetes, previous ocular surgery, immunodeficiency,
and steroid therapy. P. acnes infection can appear shortly after surgery or
after longer period because they can reside intracellularly and remain dor-
mant for prolonged duration.

11.6.4.1 Virulence factors – Low oxidation-reduction potential within

eye provides suitable environment for the growth of anaerobes especially
P. acnes. Persistent intracellular localization of P. acnes and its secretory
metabolites and enzymes will result in tissue damage (Csukas et al., 2004).
Hemolytic and cytotoxic activity of P. acnes has been reported. Various
enzymes such as Chondroitin sulfatase, hyaluronidase, gelatinase, phos-
phatase, lecithinase, and hemolysin production are found in P. acnes, which
potentiate its virulence (Hoeffler, 1977). The enzyme chondroitin ­sulfatase
initiates hydrolysis of sulfate groups from N-acetyl-D-galactosamine
­6 -sulfate and keratan sulfate, which are structural carbohydrates of corneal
tissue. Hyaluronidase hydrolyses the nonsulfated glycosaminoglycan, the
hyaluronic acid of connective tissues. The enzyme lecithinase is a type of
phospholipase, which can act on lecithin and cause hemolysis (Figure 11.5).

11.6.4.2 Antibiotic resistance – Corynebacterium spp. have developed

resistance against antibiotics such as fluoroquinolones, chloramphenicol,
cefazolin, vancomycin, sulbenicillin etc., C. macginleyi are extremely resistant
to fluoroquinolones and macrolides (Joussen et al., 2000; Eguchi et al., 2008),

Figure 11.5  Virulence factors of Coryneform bacteria.

348    Pocket Guide to Bacterial Infections

whereas P. acnes are highly resistant to macrolides and tetracyclines (Aubin

et al., 2014). Corynebacterium spp. attain antibiotic resistance through gen-
eral resistance mechanisms such as drug efflux, target modification, inactiva-
tion, or modification of drug molecule.

11.6.4.3  Therapeutic interventions – As C. macginleyi and P. acnes are

resistant to commonly available topical antibiotics in ophthalmology, use
of natural sources offers a potential therapeutic value. Koday et al. (2010)
investigated the antimicrobial efficacy of various medicinal plants against
C. macginleyi and reported the bioactive potential of methanolic extracts
of 36 medicinal plants. Terminalia catappa, Terminalia chebula, Rosa indica,
Albizia lebbeck, and Butea monosperma were reported to have significant
bactericidal activity.

11.7  Role of bacterial biofilm in ocular infections

Biofilm is a consortium of microorganisms which are structurally enclosed

within the exopolymeric conglomerate matrix that generally encompass
extracellular DNA, protein, and polysaccharide of both bacterial and host
components. Biofilm thus forms a three-dimensional complex architect
of microbial community achieved through cell-to-cell communication
(Flemming et  al., 2016). Biofilm can form on either natural surfaces such
as teeth, heart valve, or on abiotic surfaces such as medical implants, cath-
eters, and contact lenses, among others. Cells within biofilm are physiologi-
cally heterogenous and that results in decreased susceptibility to antibiotics.
Bacteria residing on biofilm matrix are 1000-fold less sensitive to antibiotics
than their planktonic counterparts. Biofilm formation in ocular infections
is achieved by persistent colonization of bacteria either on abiotic surfaces
that are in contact with the ocular tissue or implanted within ocular surface
or formation of biofilm on biotic surfaces of the eye. Prosthetic devices
of eye range from contact lens to lens implant, glaucoma tubes, punctual
plugs, stents, corneal sutures, and scleral buckles (Hou et al., 2012). Among
this array of prosthesis, contact lens present the most frequent and severe
form of biofilm-related ocular infections. Improper handling of contact
lens attributes to contamination. Around 80% of contact lens wearers are
reported for bacterial contamination either without presenting signs and
symptoms or bacterial keratitis (McLaughlin-Borlace et al., 1998). Extended
contact lens wear is a major predisposing factor for keratitis. During surgi-
cal cataract removal, bacteria can enter the eye surface and reside on the
implanted intraocular lens, leading to endophthalmitis. Biofilm formation
of punctal plug, a prosthetic for the treatment of tear-deficient dry eye,
has been reported for acute conjunctivitis (Yokoi et al., 2000). In addition
 Bacteriology of Ophthalmic Infections     349

to direct effect of biofilm, the secretory components such as endotoxins

or lipopolysaccharides from organisms residing in the biofilm matrix has a
major role in pathogenicity of the biofilm-associated infections. Endotoxins
can exert numerous effect on host tissue by eliciting cytokines, inflam-
matory components, and complement cascade of host immune system.
Diffuse lamellar keratitis is a major complication of corrective surgery laser
assisted in situ keratomileusis (LASIK), which is initiated due to the con-
tamination of sterilizer reservoirs with gram-negative pathogens. The use
of contaminated instruments recruits several inflammatory mediators and
the endotoxin produced by gram-negative organisms from bacterial biofilm
will worsen the condition (Holland et  al., 2000). Bacteria residing in bio-
film matrix of chronic keratitis condition have been implicated in infectious
crystalline keratopathy (ICK). Biofilm formation on biotic ocular surface is
extremely difficult and infrequent. In certain cases of ICK, biofilm forma-
tion in the absence of prosthetic materials has been noted. Mihara et al.
(2004) reported a case with movable mass of bacterial biofilm on the ocular
surface, which was initially considered calcification. Yellowish white calci-
fication was present on the nasal sclera and corneal region, which moved
on blinking. Pathological examination of the calcified mass revealed the
formation of biofilm by numerous gram-positive bacilli with neutrophils.
Hence the authors suggested that unusual mass on ocular surface without
the involvement of biomaterial should not be deliberated merely as calci-
fication. Possibilities of infection, involvement of infectious bacteria, and
biofilm formation should be considered before recommending treatment
strategies. Biofilm formation by S. aureus, S. epidermidis, P. acnes, and
P. aeruginosa on ocular surfaces with or without the contribution of bio-
material have been profusely reported (Leid et al., 2002; Hou et al., 2012).

Infections caused by biofilm are massively resistant to antimicrobial agents.

Hence, the treatment of biofilm-related infections are extremely difficult to
eradicate. Pathogenic bacteria residing on the biofilm matrix employ various
tolerance and resistance mechanisms to withstand antibiotics. Production
of extracellular DNA, exopolysaccharide matrices, stress response, presence
of various biofilm-specific genetic determinants, efflux pumps, intracellu-
lar communications such as quorum sensing ,and horizontal gene transfer
afford the survival of biofilm on treatment with antimicrobial agents (Hall
and Mah, 2017).

Detailed research on biofilm-specific antibiotic resistance and tolerance

will help in improvising treatment for biofilm-related infectious diseases.
Moreover, due to increased resistance to antimicrobial agents, research on
alternates to antibiotics are in surge. Augmentation of antibiotic efficacy
with combinations of antibiotics or natural biomolecules, phage therapy,
350    Pocket Guide to Bacterial Infections

Figure 11.6  Biofilm formation on abiotic surface (i.e., contact lens) and its associ-
ated antibiotic resistance and tolerance mechanisms. (1) Abiotic surface (i.e., contact
lens); (2) biofilm cells embedded in a matrix; (3) exopolysaccharides; (4) extracellular
DNA; (5) stress responses; (6) genetic determinants that are specifically expressed
in biofilm cells; (7) multidrug efflux pumps; (8) intracellular interactions (horizontal
gene transfer); (9) persister cells; (10) biologically active molecules; and (11) bacteria
shed to the environment.

use of bacteriocins, photodynamic therapy, and antibiofilm and antiquorum

sensing agents are widely discussed and reported as convincing therapeutic
strategies at this time (Allen et al., 2014) (Figure 11.6).

11.8  Conclusion and future perspectives

The bacterial pathogens causing acute or chronic infections on either

extraocular or intraocular surfaces increase the incidence of ocular mor-
bidity and are often sight threatening. Proper maintenance of ocular
hygiene is the initial precautionary measure for the management of ocular
microbial infections. Bacteria of both endogenous and exogenous origin
can cause mild to severe forms of ophthalmic infections. Antibiotic treat-
ment and surgery are prevailing therapeutic regimens. Persistent bacterial
infections are a result of antibiotic resistance that are caused by long-term
topical or systemic antibiotic therapy and the ability of the bacteria to
 Bacteriology of Ophthalmic Infections     351

form biofilm on biotic or abiotic surfaces. In addition to this, complica-

tions of postoperative infections further deteriorate the condition and fail
to provide absolute remedy from ocular infections. Even though there are
lot of emerging therapeutic interventions such as phage therapy, photo-
dynamic therapy, bacteriocins, and antibiofilm agents, permutation with
phytomolecules will intensify the treatment procedures. Further insight on
predisposing factors and appropriate prognosis is imperative and should
be explored in detail.

Acknowledgments

The authors thankfully acknowledge the support extended by

Department of Science and Technology, Government of India through
PURSE [Grant No. SR/S9Z-23/2010/42 (G)] & FIST (Grant No. SR-FST/
LSI-087/2008), and University Grants Commission (UGC), New Delhi,
through SAP-DRS1  [Grant No. F.3-28/2011 (SAP-II)]. The authors also
thankfully acknowledge the Bioinformatics Infrastructure Facility funded
by Department of Biotechnology, Government of India [Grant No. BT/
BI/25/015/2012 (BIF)].

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12
Role of Bacteria in Urinary
Tract Infections
Gnanasekaran JebaMercy, Kannan Balaji,
and K. Balamurugan

Contents

12.1 Introduction 365

12.2 Urease 366
12.3 Proteus spp. 366
12.3.1 Virulence factors 366
12.3.2 Interaction with host 368
12.4 Staphylococcus aureus 368
12.4.1 Lipoteichoic acid 369
12.4.2 Interaction with host 370
12.5 Treatment and future prospective 370
References 370

12.1 Introduction

Microorganisms are the first forms of life to develop on Earth, approximately

4 billion years ago (Altermann & Kazmierczak, 2003). Microorganisms are
beneficial in many aspects to the society, but at the same time, they are also
highly harmful in regard to infections and diseases. Severe public health
problems like urinary tract infections (UTIs) are caused by a range of patho-
gens, most importantly Proteus spp. and Staphylococcus spp. The social
costs of these infections were about USD 3.5 billion per year in the United
States. It is an important cause of morbidity in infants and older people. UTI
can be caused by both gram-positive and -negative bacteria. In this chapter,
we are going study the virulence factors of UTI pathogens Proteus mirabilis
and Staphylococcus aureus.

Proteus spp. belongs to the group of opportunistic pathogens and is widely

distributed in the natural environment. Under favorable conditions, they
can cause severe infections in individuals who are immunocompromised.
These bacteria can also be the main agents of nosocomial infections and

365
366    Pocket Guide to Bacterial Infections

infections of the urinary tract, respiratory tract, and wound infections

(Tiwana et  al., 1999; Warren, 1996). Gram-positive bacterium S. aureus
leads to serious complications in many animal models (Lindsay, 2010). It can
colonize in the Caenorhabdtis elegans gut and invade into other organs by
disrupting the gut epithelial cells further leading to mortality (Garsin et al.,
2001; Sifri et al., 2003).

12.2 Urease

Urease is an enzyme used for the hydrolysis of urea. A healthy individual

produces about 30 g of urea per day (Newsholme & Leech, 2011). Most of
the UTI pathogens have urease activity as a part of its virulence character.
The urease-dependent process is associated with bacterial UTI, including
those caused by Proteus and Staphylococcal spp. Urease produced by these
bacteria can lead to the formation of stones because of the precipitation of
the minerals struvite and carbonate apatite. The stone formation is used to
protect the pathogen (Griffith et al., 1976). These stones can block the flow
of urine and lead to tissue damage (Schaffer & Pearson, 2015) Ammonia
produced during the above process can damage the glycosaminoglycan
surface of the urothelium, and it will allow the entry for the other bacte-
rial infections (Rutherford, 2014). It was shown that urease from Proteus
mirabilis can hydrolyze urea several times faster than urease synthesized by
other species of bacteria (Jordan & Nicolle, 2014).

12.3  Proteus spp.

The opportunistic pathogens, Proteus genus, are commonly encountered

in nosocomial infections, and it is comparatively more resistant to antibiotic
agents and is, therefore, one of the most difficult organisms to deal clini-
cally. During the introduction of ampicillin and nalidixic acid into the army
of antibiotics, it was found that these treatments were satisfactory in the
control of Proteus spp. infections (Stratford, 1964). It is a gram-negative,
motile, urease-positive, lactose-negative, and indole negative organism. It is
an important cause for the UTIs in patients who are hospitalized.

12.3.1  Virulence factors

Gram-negative Proteus rods are widely distributed in the natural environ-

ment, and they can especially be found in polluted water and soil. They
are also the inhabitant of human and animal intestines. Proteus bacilli are
opportunistic pathogens; under favorable conditions, they can cause severe
infections in individuals who are immunocompromised. These bacteria can
 Role of Bacteria in Urinary Tract Infections     367

also be the main agents of nosocomial infections, infections of the respira-

tory tract, wound infections, and rheumatoid arthritis (Tiwana et al., 1999;
Warren, 1996; Różalski et al., 1997). Proteus have developed several viru-
lence determinants such as fimbriae, flagella, urease, amino acid deami-
nases, proteases, hemolysins, swarming growth, and LPS which enable
them to colonize, survive, and multiply in the host (Coker et al., 2000). One
of the mechanisms which support the survival of gram-negative bacteria
in the host is resistance to the bactericidal action of normal human serum
(Kumar et al., 1997; Grzybek-Hryncewicz et al., 1981).

Proteus mirabilis causes 90% of all Proteus infections. It is facultatively anaer-

obic, rod-shaped bacterium. It shows swarming motility, and urease activity.
P. mirabilis usually isolated from the urine of patients and individuals with
the urinary catheters. Infection can lead to serious complications including
acute pyelonephritis, stone formation in the bladder and kidney, encrusta-
tion, and obstruction of the catheter, fever, and bacteremia (Griffith et al.,
1976; Mobley & Warren, 1987; Rubin et al., 1986). P. mirabilis is the second-
leading cause of bacteremia (Setia et al., 1984). It has a many virulence fac-
tors like urease (Griffith et al., 1976; Mobley & Warren, 1987; Jones et al.,
1990; Johnson et al., 1993), fimbriae (Mobley & Belas, 1995), flagella (Belas
& Flaherty, 1994; Allison et al., 1994), hemolysin (Mobley et al., 1991), IgA
protease (Loomes et al., 1990; Wassif et al., 1995), and amino acid deami-
nase (Massad et al., 1995). And it has a distinct type of protease ZapA metal-
loprotease, which is capable of degrading antibacterial immune peptides
(Allison et al., 1992; Belas et al., 2004) (Figure 12.1). In different from other
pathogens, P. mirabilis exhibits a unique kind of character called swarming
migration, vegetative rods undergo differentiation at the colony border into
long, aseptate filaments that have 50-fold more flagella per unit cell surface
area (Allison et  al., 1992; Williams et  al., 1978). Another member of this

Figure 12.1  Virulence determinants of P. mirabilis.

368    Pocket Guide to Bacterial Infections

opportunistic group, Proteus vulgaris is also known to cause urinary, blood,

and wound infections. The P. vulgaris strains isolated from patients with UTI
are often insensitive to the bactericidal action of normal human serum which
poses a severe clinical problem (Kwil et al., 2013).

12.3.2  Interaction with host

Interaction of Proteus spp. with C. elegans was reported recently and the
study established C. elegans as a model for studying phenotypic changes
and regulation of MAP kinase immune pathway in the host against the
bacterial infection. Being in the group of opportunistic pathogens, Proteus
spp. causes large number of nosocomial infections. The Proteus spp. does
not cause death in wild type C. elegans (JebaMercy & Balamurugan, 2012).
Proteus spp. can cause mortality in MAP kinase pathway mutant C. elegans.
And the author showed the involvement of innate immune pathways spe-
cific players at the mRNA level against Proteus spp. infections. In addition,
the lipopolysaccharides (LPS) from P. mirabilis treated with mutant C. ele-
gans showed structural changes compared with wild type worms exposed,
and it clearly indicates P. mirabilis changes its internal system according to
the specific host for effective infection (JebaMercy et al., 2013).

12.4  Staphylococcus aureus

Staphylococci are gram-positive, cluster-forming coccus, non-motile, non-

spore-forming facultative anaerobes. Not only UTIs, but S. aureus can also
cause a variety of illnesses from minor infections to life-threatening dis-
eases. It can cause superficial skin lesions UTIs. S. aureus is one of the
major causes of nosocomial infection of surgical patients and infections
related with indwelling medical procedures. Enterotoxin and super anti-
gens from S. aureus cause food poisoning and toxic shock syndrome
(Lowry et al., 1998).

The ability of S. aureus to cause a wide spectrum of disease has been attrib-
uted to its ability to produce a broad array of pathogenicity factors. These
factors can be subdivided into three general groups: cell-associated prod-
ucts, secreted exoproteins, and regulatory loci. Cell-associated products,
including adhesins of the microbial surface components recognizing adhe-
sive matrix molecules (MSCRAMM) family and capsular polysaccharide,
facilitate binding to host tissue and help resist host immune responses. The
exoproteins like cytolysins and extracellular proteases are known to fight
against host immunity and results in tissue invasion and nutrient absor-
bance (Lowy et al., 1998; Sifri et al., 2003).
 Role of Bacteria in Urinary Tract Infections     369

S. aureus produces a large number of virulence factors, and these include

extracellular toxins like alpha-toxin and cell-wall-associated proteins, which
are important for colonization, immune evasion, and tissue destruction.
Treatment of S. aureus infections has become complicated by the emer-
gence of widespread antimicrobial resistance (Garvis et al., 2002).

In 1959, Methicillin was introduced to treat penicillin-resistant S. aureus–

related diseases. Methicillin-resistant S. aureus (MRSA) was emerged in the
UK around 1961 and spread around European countries and then to Japan,
Australia, and the United States. The methicillin-resistance gene (mecA)
encodes a methicillin-resistant penicillin-binding protein that is not present
in susceptible strains and is believed to have been acquired from a dis-
tantly related species. mecA is carried on a mobile genetic element, the
staphylococcal cassette chromosome mec (SCCmec), of which four forms
have been described that differ in size and genetic composition. Many
MRSA isolates are multiresistant and are susceptible only to glycopeptide
antibiotics such as vancomycin and investigational drugs. MRSA isolates
that have decreased susceptibility to glycopeptides (glycopeptide interme-
diately susceptible S. aureus [GISA]), reported in recent years, are a cause
of great public health concern (Martin et  al., 2002). Community-related
MRSA is associated with increased disease severity, ranging from cutaneous
abscesses to deadly necrotizing pneumonia. USA300 and USA400 are the
two dominant CA- MRSA strains. The USA400 strain causes deadly infec-
tions but in less frequency, whereas USA300 was widespread and mainly
related with community infections and life-threatening infections like nec-
rotizing pneumonia (Wu et al., 2010).

12.4.1  Lipoteichoic acid

Lipoteichoic acid (LTA) is a surface-associated adhesion amphiphile from

gram-positive bacteria made up of a polymer of repetitive 1,3-phosphodi-
ester-linked glycerol-I-phosphate units with a glycolipid anchor (Leopold &
Fischer, 1992). It gets released from the gram-positive bacterial cells during
bacteriolysis induced by host factors like lysozyme, cationic peptides from
leucocytes, or during the antibiotic treatment. It binds to target cell recep-
tors like CD14 or toll-like receptors. LTA can interact with antibodies and
activate the passive immune kill phenomenon. LTA is similar to the endo-
toxin lipopolysaccharide and shares several of its pathogenetic properties
(Dishon et al., 1967). Chemotaxis of human neutrophils and phagocytosis
were also inhibited by LTA (Raynor et al., 1981; Card et al., 1994). LTA also
induced expression of macrophage inflammatory protein 1  alpha. These
findings suggest that LTA may have a role in the regulation, recruitment, and
activation of leukocytes in inflammatory sites (Nonogaki et al., 1995). LTA,
370    Pocket Guide to Bacterial Infections

teichoic acid, and peptidoglycan each inhibit proliferation of fibroblasts by a

still undefined mechanism and also act on T cells to activate nuclear factor
kappa B. LTA, therefore, seems to be a versatile immunomodulator that can
alter cell responses in inflammatory conditions (Elgavish et al., 2000).

12.4.2  Interaction with host

According to Sifiri et al. (2003). S. aureus infects C. elegans, ultimately leading

to worm death, and key aspects of S. aureus pathogenesis and interaction
with the innate immune system have been mechanistically conserved from
nematodes through vertebrates. C. elegans will be a great model to study the
novel staphylococcal genes required for the mammalian pathogenesis and
host innate immune defense systems (Sifri et al., 2003). Death in C. elegans is
mainly due to the disruption of the gut epithelium and assault of the internal
organs by the invading pathogen (Garsin et al., 2003; Sifri et al., 2003; Irazoqui
et al., 2010). Deformed anal region (Dar) was identified in S. aureus infection
and is mainly relying on both β-catenin and MAPK pathways (Irazoqui et al.,
2010). S. aureus requires protease and alpha-hemolysin for the pathogenesis
in C. ­elegans (Sifri et al., 2003). The p38 MAPK and the catenin pathways are
required for the host immune system against S. aureus. The significance of this
pathway in S. aureus pathogenesis is comparable in higher vertebrates, where
catenin activates NFκB-mediated immune gene expression.

12.5  Treatment and future prospective

Bacterial UTIs are recurrent infections in the normal and the nosocomial envi-
ronments. Two types of antimicrobial treatment required for all the UTIs: (1)
rapid and effective response to therapy and prevention of recurrence (2) preven-
tion of emergence of resistance (Wagenlehner & Naber, 2006). The important
disadvantage of antibiotic therapies is the development of antibiotic resistance.
There is no major improvement in the UTI treatment for the last decade except
antibiotic therapy (Nickel, 2002). Development of vaccines to prevent the UTIs
in susceptible persons is an exciting emerging project. Recent papers report-
ing the oral and vaginal substance preparations used to stimulate the patients’
immune system against the UTI pathogens. The suitable formulation with
appropriate clinical trials will remove the burden of nosocomial UTI infections.

References

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Elgavish, A. 2000. NF-kappaB activation mediates the response of a
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13
Role of Bacteria in
Blood Infections
Kannan Balaji, Gnanasekaran JebaMercy,
and K. Balamurugan

Contents

13.1 Introduction 376

13.1.1 Blood 376
13.1.2 Red blood cells 376
13.1.3 White blood cells 377
13.1.4 Platelets 377
13.1.5 Plasma 377
13.2 Bacterial blood infections 377
13.2.1 Bacteremia 377
13.2.1.1 Epidemiology 378
13.2.2 Sepsis 378
13.2.2.1 Epidemiology 378
13.2.3 Meningitis 379
13.2.3.1 Epidemiology 379
13.2.4 Pericarditis 379
13.2.4.1 Epidemiology 379
13.2.5 Endocarditis 380
13.2.5.1 Epidemiology 380
13.2.6 Osteomyelitis 380
13.3 Predominant bacteria in blood infections 380
13.3.1 Streptococcus spp. 380
13.3.1.1 Streptococcus pyogenes 380
13.3.2 Staphylococcus aureus 381
13.3.3 Escherichia coli 381
13.3.4 Klebsiella pneumonia 381
13.3.5 Neisseria meningitides 382
13.3.6 Other bacteria 382
13.3.7 Polymicrobial infections 382

375
376    Pocket Guide to Bacterial Infections

13.4 Pathogenesis and virulence mechanisms 382

13.4.1 Emergence of resistance 382
13.4.2 Membrane proteins 383
13.4.3 Capsules 383
13.4.4 Extracellular virulence products 383
13.4.5 Superantigens 385
13.5 Conclusion and future prospects 385
References 386

13.1 Introduction

Blood is a vital human body fluid that plays several important roles like
circulation of oxygen to various parts of the body, cleaning of metabolic
wastes from cells, regulation of body, and immunity. Human body harbors
millions of microorganisms in various parts of the body like oral region,
gut, skin, and respiratory tract from birth to death. These microbes exist in
several forms like commensal, normal flora, carriers, and pathogens. When
it enters the bloodstream, bacteria confers with the host immune system
and is compelled to exhibit virulence factors for its survival. Blood infections
caused by bacteria are a global burden because of the increase in virulent
and drug-resistant strains. Blood-related infections associated with bacteria
are in increasing trend in the rate of mortality because drug-resistant strains
remain intractable to treat. This chapter focuses on components of blood,
blood infections caused by bacteria, bacterial species involved, and their
virulence mechanisms.

13.1.1 Blood

Blood is a specialized fluid which supplies oxygen and nutrients to cells pres-
ent in various parts of the body and carry away metabolic wastes from the
cells. Human blood is composed of red blood cells (RBCs), white blood cells
(WBCs), platelets, and plasma.

13.1.2  Red blood cells

RBCs or erythrocytes are the most abundant cells in the blood, which
comprise about 40% to 45% in whole blood. They are large microscopic
disc-like structured cells without nucleus. The red color of the RBCs is by
a special protein called hemoglobin, which carries oxygen from lungs to
other cells in the body, and in return, it carries away carbon dioxide from
cells to lungs.
 Role of Bacteria in Blood Infections     377

13.1.3  White blood cells

WBCs or leukocytes are much less abundant than RBCs and account for
about 1% in whole blood volume. They are nucleated, and apart from blood,
these WBCs are also present in other body organs like the liver, spleen, and
lymph nodes. WBCs are the protecting agent in a human body from infec-
tions. They comprise neutrophils, lymphocytes, monocytes, basophils, and
eosinophils. Among these cells, the lymphocytes are of two types, T and B.

13.1.4 Platelets

Platelets are not cells like RBCs or WBCs; they are cell fragments without a
nucleus that involved in the blood-clotting process known as coagulation.
Platelets, along with several factors, gather at the site of injury and create
a fibrin clog to prevent blood flow. Platelets are also called thrombocytes.

13.1.5 Plasma

Plasma is a clear, yellow-tinted liquid that carries RBCs, WBCs, and plate-
lets. Plasma are the major component in blood, which comprises about
55% of the total blood volume. It contains sugars, proteins, vitamins, lipids,
enzymes, and salts. Plasma carries away the metabolic wastes from the cells
and helps the flow of blood through vessels to various body parts.

13.2  Bacterial blood infections

Blood is a nutrient-rich human body fluid and is more prone to bacterial

encounters during blood transfusion, through open wounds, burn wounds,
and other bacterial infections.

13.2.1 Bacteremia

Presence of bacteria in the bloodstream is termed bacteremia. Despite anti-

biotic treatment, bloodstream infections by bacteria leads to deleterious
effects and is associated with high mortality rates (Salomão et al., 1999).
Staphylococcus aureus, Escherichia coli, and Streptococcus pneumoniae are
the most common cause of bacteremia (Siegman-Igra et al., 2002; Kollef
et  al., 2011; Vallés et  al., 2013). Based on the case of origin, bacteremia
are differentiated into hospital-acquired bacteremia (HAB) and community-
acquired bacteremia (CAB).

In HAB, the infection is caused by several ways, such as contaminated blood

transfusion, organ or stem cell transplants, medical procedures like dental
treatments, and contaminated catheters and needles. CAB is defined as the
bacteremia associated with several factors like a particular population age
378    Pocket Guide to Bacterial Infections

group; geographical location; climatic condition; and along with other dis-
ease conditions like diabetes, endocarditis, pneumonia, urinary tract infec-
tions, and HIV (Christaki & Giamarellos-Bourboulis, 2014).

13.2.1.1 Epidemiology – Extensive use of medical procedures and the

emergence of drug-resistant pathogens and nosocomial infections leads
to an increasing incidence of bacteremia. The occurrence rate of CAB per
year in 100,000  individuals varies according to the geographic locations
and reported as 153 episodes in Olmsted County in the United State, 101.2
cases in Victoria, Canada, 92  episodes in Denmark, and 31.1  episodes in
Thailand (Viscoli, 2016). The etiology varies depending on the age group,
geographical location, climate, environment, and other associated illness
(Friedman et al., 2002; Siegman-Igra et al., 2002). Increasing incidence of
multidrug resistant bacteremia in after transplants (Oliveira et al., 2007) will
be a challenge to medical treatments.

13.2.2 Sepsis

Sepsis is a severe clinical complication by host inflammatory response trig-

gered against any infection. Sepsis is an irregulated host immune response
that leads to organ dysfunction (Singer et al., 2016). It is one of the most
common causes of hospitalization, and untreated sepsis cases are poten-
tially fatal. Anyone can have sepsis, but it is prevalent in individuals who are
older or immunocompromised. Some of the common causes of sepsis are
pneumonia, kidney infection, and bacteremia. It ranges from mild sepsis to
septic shock syndrome. Untreated sepsis leads to septic shock syndrome,
which results in difficulty in breathing and decrease in platelet counts, urine
output, and blood pressure. In addition, septic shock causes blood clots
that cause organ failure and ultimately leads to death.

13.2.2.1  Epidemiology – Sepsis is a critical illness and the leading cause of

mortality worldwide. The United States spends more than $20 billion (5.2%)
of its total medical costs on sepsis treatment (Torio & Andrews, 2011). The
reported cases of sepsis are increasing, especially with greater recognition
and in aging populations with more comorbidities. In the United States, the
reports of severe sepsis are estimated to be 300 cases per 100,000 pop-
ulation (Iwashyna et  al., 2012; Gaieski et  al., 2013). Studies on patients
admitted to the Intensive Care Unit in Europe and China states that 37.4%
and 37.3%, respectively, were diagnosed with severe sepsis (Vincent et al.,
2006; Zhou et al., 2014). Despite advanced medical treatments, one in four
patients who develop severe sepsis will die during their hospitalization. In
the case of septic shock, the mortality rate is approaching an alarming rate
of 50% (Singer et al., 2016).
 Role of Bacteria in Blood Infections     379

13.2.3 Meningitis

Meninges are the membrane layers present in brain and spinal cord. Fluid
present in the meninges get infected by bacteria or viruses and cause
inflammation in the meninges. This condition is called meningitis. A range
of pathogenic bacteria can cause bacterial meningitis. Streptococcus pneu-
monia is the most common causative pathogen. Other bacterial pathogens
like Neisseria meningitides, Haemophilus influenza, Listeria monocyto-
genes, and Group B Streptococcus also causes meningitis.

13.2.3.1  Epidemiology – Bacterial meningitis is a life-threatening infection

in the central nervous system and is a leading cause of mortality (Matthijs
et al., 2010); 13,974 cases of bacterial meningitis were reported in 27 states
in the United States in late 1970s (Schlech et  al., 1985). Until the clinical
use of pneumococcal vaccine, every year almost 6000 people reported the
development of meningitis (Chávez-Bueno & McCracken, 2005). Bacterial
meningitis was even more a serious problem in many developing countries.
Sub-Saharan Africa is referred to as the meningitis belt for its meningococ-
cal meningitis prevalence (Campagne et al., 1999; de Gans & van de Beek,
2002). In Dakar, Senegal, 50 cases of bacterial meningitis were reported out
of 100,000 individuals, where 1 in 250 children get infected within 1 year
from birth (Greenwoods, 1987). With the advent of proper diagnostic
methods and effective vaccines against the causative pathogens, significant
reductions have been seen in the disease burden. However, differentiation
between acute bacterial meningitis and acute viral meningitis has always
remained a challenge because of its prognostic significance.

13.2.4 Pericarditis

The pericardium is a thin protective membrane present around the heart,

and inflammation in this membrane is called pericarditis. There are sev-
eral factors that cause pericarditis include bacteria, virus, parasite, or fungal
infection and trauma from injury or any surgery. In the case of bacterial
pericarditis, pathogenic bacteria enters and causes infection in the peri-
cardium. The most common causative pathogens are Staphylococcus spp.,
Streptococcus spp., and Pneumococcus spp. Bacterial infections from other
parts of the body like pneumonia and bloodstream infections and after
surgery are some of the routes that cause bacterial pericarditis.

13.2.4.1  Epidemiology – Bacterial pericarditis has become rare with an

incidence of 1 of 18,000 individuals by the arrival of antibiotics and effective
diagnostic methods. But pericarditis is a rapid-progressive, high-risk infec-
tion with a mortality rate of 100% if left untreated. Mostly diagnosed after
death during a postmortem examination, the mortality rate remains 40%,
380    Pocket Guide to Bacterial Infections

even with treatment because of the associated sequelae such as severe

sepsis, septic shock, and cardiac constriction and tamponade (Klacsmann
et al., 1977; Sauleda et al., 1993).

13.2.5 Endocarditis

Endocarditis is an infection inside the heart lining, heart valves, or blood

vessels. Bacteria present in the bloodstream is called bacterial endocarditis
or infective endocarditis. Heart valves are devoid of the immune response
system.

13.2.5.1  Epidemiology – Infective endocarditis is associated with long-

term hospitalization and requires surgery for treatment (Moreillon & Que,
2004). In the United States, hospitalization of patients rose from 28,195
in 1998 to 43,419 in 2009 (Bor et al., 2013). Increasing rate of transplant
surgeries, prosthetic valves, and cardiovascular implantable electronic
devices in recent years revised the trend worldwide. In addition, nosoco-
mial infections and the postsurgery scenario makes endocarditis a seri-
ous issue; the mortality rate remains unchanged as approximately 25%
(Ambrosioni et al., 2017).

13.2.6 Osteomyelitis
Infection in bone regions are termed osteomyelitis. Bacteria present in
bloodstream infections or open wounds of fractured bones or bone surgery
are the common mode of infection. S. aureus is the most common caus-
ative bacteria. Untreated osteomyelitis causes impaired blood circulation,
which ultimately leads to bone death (i.e., osteonecrosis). In some cases,
osteomyelitis spreads to nearby bone joints and causes septic arthritis.

13.3  Predominant bacteria in blood infections

13.3.1  Streptococcus spp.

The genus Streptococcus comprises of gram-positive bacteria, which are

found to inhabit a wide range of hosts. In humans, streptococci are often
found to colonize the mouth and pharynx. However, in certain circum-
stances, they may also inhabit the skin, heart, or muscle tissue. S. pyogenes,
S. pneumonia, and Group B Streptococcus are the important blood infec-
tion-causing pathogens.

13.3.1.1  Streptococcus pyogenes – S. pyogenes or Group A Streptococcus

is a gram-positive, nonmotile, non-spore-forming coccus that occurs in
chains. S. pyogenes colonizes the throat or skin and produces a variety of
 Role of Bacteria in Blood Infections     381

pyogenic infections in mucous membranes, tonsils, skin, and deeper tissues.

S. pyogenes causes blood-related infections, including toxic streptococcal
syndrome, bacteremia, sepsis, pneumonia, and meningitis (Cunningham,
2000). They are the common cause of puerperal sepsis or childbed fever.
S. pyogenes is also responsible for streptococcal toxic shock syndrome,
and it has gained notoriety as the “flesh-eating bacterium,” which invades
skin and soft tissues, and in severe cases, leaves infected tissues or limbs
destroyed (Stevens, 1999).

S. pyogenes delivers a number of blood infection-related postinfection

sequelae, including acute glomerulonephritis and reactive arthritis and is
associated with disability and death in children worldwide. S. pyogenes
have become such a serious pathogen by means of its improved virulence
factors and superantigens. As a blood-infective pathogen, it has developed
complex virulence mechanisms to avoid host defenses.

13.3.2  Staphylococcus aureus

S. aureus is a gram-positive cocci that causes a range of pathogenicity

from normal commensal to invasive infection. S. aureus is one of the lead-
ing causes of blood infections like bacteremia and sepsis. Nosocomial and
­medical device implant-related infections, especially with multidrug resis-
tant S. aureus, are of serious concern. S. aureus bacteremia is the most
common cause of sepsis in pediatric population, which is the leading cause
for hospital visits and admission to intensive care units (Schlapbach et al.,
2015; Munro et al., 2018).

13.3.3  Escherichia coli

E. coli is a gram-negative bacilli, colonized in the human intestine as com-

mensal, and causes urinary tract infections. E. coli is the leading cause of
bloodstream infections and a report states about 53,000  senior citizens
older than 65 years of age are affected by E. coli bacteremia every year.
In addition, emergence of extended-spectrum beta-lactamases producing
E. coli strains showed resistance to antibiotic treatment and cause very high
mortality rate in senior adults (Tumbarello et al., 2010).

13.3.4  Klebsiella pneumonia

K. pneumonia is a gram-negative, rod-shaped, nonmotile bacteria with

a protective polysaccharide capsule and is part of the Enterobacteriaceae
family. This capsule helps the bacteria to escape against many host defense
mechanisms. K. pneumonia is the cause of bloodstream-associated infec-
tions like bacteremia, sepsis, and pneumonia.
382    Pocket Guide to Bacterial Infections

13.3.5  Neisseria meningitides

N. meningitides is a gram-negative diplococci that is colonized in the human

respiratory tract as a commensal. N. meningitides is the important cause of
meningitis globally (Hill et al., 2010). It also causes life-threatening menin-
gococcemia, sepsis, and disease-associated bloodstream infections.

13.3.6  Other bacteria

In addition to the pathogens already discussed, there are several other bac-
teria that are involved in bloodstream infections and sepsis. Some of them
are Pseudomonas aeruginosa, Listeria monocytogenes, Haemophilus influ-
enza, Salmonella spp., and Shigella spp.

13.3.7  Polymicrobial infections

The ancient paradigm that one microbe causes one infection has become
entrenched by our understanding of microbiology since the time of Robert
Koch. In addition to single species infections, blood infections will cause
serious effects and complicate the treatment process in polymicrobial infec-
tions. Two different species in combination with virus or fungi form a kind
of microbial team called a polymicrobial infection. Complex microbial inter-
actions lead to the emergence of drug resistance, disease manifestations,
and quorum sensing. According to Del Pozo et al. (2007), biofilm communi-
ties in most circumstances, including human infections, tend to be polymi-
crobial communities. Combining multiple bacteria with other microbes like
fungal species or viruses in a single community provides numerous advan-
tages such as passive resistance (Weimer et al., 2011), metabolic teamwork
(Fischbach & Sonnenburg, 2011; Elias & Banin, 2012), by-product influence
(Carlsson, 1997), quorum sensing systems (Elias & Banin, 2012), and many
more competitive advantages. A review by McCuller (2013) discusses the
existence of polymicrobial interactions between the influenza virus and spe-
cific bacteria in pneumonia.

13.4  Pathogenesis and virulence mechanisms

13.4.1  Emergence of resistance

The impact of antibiotic-resistant strains is an alarming scenario in the case

of bacterial pathogenesis. In the case of bloodstream infection, the encoun-
ters of antibiotic-resistant strains is mostly by nosocomial contaminations
in patients in the Intensive Care Unit (Tabah et al., 2012). The emergence
of methicillin-resistant S. aureus (MRSA), extended-spectrum beta-­
lactamases producing E. coli, metallo-beta-lactamase producing E. coli and
 Role of Bacteria in Blood Infections     383

K. pneumoniae, and carbapenemase-producing Enterobacteriaceae are the

challenges in treating bloodstream infections (Laupland and Church, 2014).

13.4.2  Membrane proteins

In S. pyogenes, the major virulence factor is M protein present in the mem-

brane, which shows antiphagocytic effect by interfering with opsoniza-
tion via the alternative complementary pathway (Bisno, 1991). M protein is
composed of two polypeptide chains complexed in an alpha-helically coiled
configuration anchored in the cell membrane and appears as fibrils on the
cell surface. The chains comprise four repeat blocks (labeled A–D), each
differing in size and amino acid sequence, within which there are seven-
residue repeats of nonpolar amino acids. These streptococcal M proteins
have been used to divide S. pyogenes into different M serotypes. Beall et al.
(1996) introduced the polymerase chain reaction (PCR)-based M protein
serotyping method where a specific primer pair was employed for ampli-
fication and identification of the emm allele with the help of Genebank-
submitted sequences. In K. pnemoniae, outer membrane protein A is one
of the major pathogenesis factors involved in suppression of host immune
responses (March et al., 2011).

13.4.3 Capsules

The S. pyogenes contains an encapsulation called a hyaluronic acid c­ apsule

(Figure 13.1). It is composed of a high-molecular-weight polymer ­consisting
of alternating residues of N-acetylglucosamine and glucuronic acid. This
capsule protects S. pyogenes by resisting phagocytosis. An acapsular
­isogenic mutant S. pyogenes lost its ability to resist phagocytic killing
and showed decreased virulence up to 100-fold (Moses et al., 1997). Like
K.  pneumoniae, N. meningitidis  strains are characteristics of a protective
­polysaccharide capsule layer. In N. meningitides, these capsules are used
for serotyping, and a total of 13  serogroups have been described so far.
The majority of the invasive or bloodstream infections will be manifested by
strains belonging to only five specific serogroups.

13.4.4  Extracellular virulence products

S. pyogenes exerts several extracellular products, of which two distinct

hemolysins streptolysin O and streptolysin S have been well character-
ized (Hackett & Stevens, 1992; Fontaine et al., 2003; Harder et al., 2009).
Streptolysin O derives its name from its oxygen lability and is reversibly
inhibited by oxygen and irreversibly inhibited by cholesterol. It is a member
of a family of highly conserved pore-forming cytolysins (Bhakdi et al., 1985,
1993) and stimulates targeted autophagy (O’Seaghdha & Wessels, 2013).
384    Pocket Guide to Bacterial Infections

Streptolysins; NADase;
Pili M protein type antigen;
Pyrogenic exotoxins,
lipoteichoic acid;
Hyaluronidase;
R and T protein
Streptokinases;
Streptodornases

Extracellular
Hyaluronic acid capsule
substances
Peptidoglycon (Cell wall)
(Group carbohydrate
antigen: rhamnose,
-N acetylglucosamine)

i
cc
co Cell membrane
ed
ain
ch
es
en
yog
S.p

β-hemolytic S. pyogenes
in blood agar plate

Figure 13.1  Representative image of virulence factors present in a pathogen

Streptococcus pyogenes.

In addition to its effect on erythrocytes, extracellular products released

by such bacteria are toxic to a variety of cells and cell fractions, including
polymorphonuclear leukocytes, platelets, tissue-culture cells, lysosomes,
and isolated mammalian and amphibian hearts (Duncan & Schlegel, 1975;
Bisno et al., 2003). Streptolysin S is a hemolysin produced by S. pyogenes in
the presence of serum or several other substances such as serum albumin,
alpha-lipoprotein, and ribonucleic acid. It exists in intracellular, cell-surface-
bound, and extracellular forms, and it is one of the most potent cytotox-
ins known (Sierig et al., 2003; Datta et al., 2005; Sumitomo et al., 2011).
Other extracellular antigenically distinct enzymes are DNases A, B, C, and
D, which are involved in degradation of DNA, streptokinase, which causes
dissolution of clots by catalyzing the conversion of plasminogen to plasmin,
streptococcal pyrogenic exotoxin B (speB), a potent protease that cleaves
the PMN binding site (Ji et al., 1996; Bisno et al., 2003), streptococcal inhibi-
tor of complement, which inhibits lysis of the bacterium by binding to the
insertion site of complement (Fernie-King et al., 2002).

The pathogenesis of P. aeruginosa starts from adherence to epithelial cells

using adhesins and exoenzyme S. An exotoxin A causes tissue necrosis fol-
lowed by phospholipase C, which is a hemolysin. The exoenzyme S causes
 Role of Bacteria in Blood Infections     385

resistance to macrophages by disrupting the normal cytoskeletal organiza-

tion and destruction of immunoglobulin G and A (Kalifa et al., 2011).

The N. meningitides strain is characteristics for its successful immune evasion

strategies. Although vaccines have been developed against some serogroups,
a universal meningococcal vaccine remains a challenge because of their fre-
quent antigenic varying ability of the organism; it also mimics host structures.
N. meningitides is established as a successful pathogen with the help of a
number of virulence mechanism and includes the capsule layer, lipopolysac-
charides (LPS), and a number of surface-expressed adhesive proteins.

13.4.5 Superantigens

Superantigens are extracellular protein toxins released by gram-positive bac-

teria in the blood that are pyrogenic, increase host susceptibility to endotoxic
shock, suppress immunoglobulin production, and have mitogenic activity
for specific T-cell subset (Curtis, 1996; Commons et al., 2008). This produces
an extensive release of proinflammatory cytokines with massive immune
activation. These superantigens are likely to be involved in the pathogen-
esis of various invasive infections. A total of 11 superantigens have been
identified so far, including streptococcal pyrogenic exotoxin (spe) A, C, G,
H, I, J, K, L, M, streptococcal mitogenic exotoxin (sme) Z, and streptococcal
superantigen (ssa) (Commons et al., 2008). Except speG, speJ, and smeZ, all
of the superantigens-encoding genes are associated with bacteriophages
(Ferretti et al., 2001; Proft & Fraser, 2003; Proft et al., 2003). S. aureus strains
secrete around 24 superantigens, which involved in antiphagocytic activity
and establishment of toxic shock syndrome Type-1 (Spaulding et al., 2013).

13.5  Conclusion and future prospects

The pathogenesis and the disease virulence caused by the bacteria in blood-
stream is still ambiguous. Though blood carries protective immune response
machinery, the evolving preventive and resistance mechanisms among bac-
teria still prevails. Emergence of multidrug resistance and extreme drug
resistance shows an alarming need for new treatment strategies. Recently,
many researchers focus on the use of bacteriophage therapy with geneti-
cally engineered phages to target specific pathogenic bacterial species. In
addition, the use of active principles from traditional medicines and anti-
quorum sensing molecules against drug-resistant pathogens are recent ini-
tiatives in the field of pharmacology.

Despite the recent medical advances, the upcoming challenges in blood-

stream infections include global increases in the older population; massive
386    Pocket Guide to Bacterial Infections

raise in transplantation surgeries; nosocomial infections; and multidrug and

extreme drug resistance and persisters, which keep the blood-related infec-
tions an increasing trend. To overcome these issues, a proper epidemio-
logical survey on disease prevalence, early diagnosis, and proper dosage of
antibiotics needs to be practiced to prevent the global burden of antibiotic
resistance and well-being of mankind.

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Index

Note: Page numbers in italic and bold refer to figures and tables respectively.

ABSSSI (acute bacterial SSSI) 280–1 autoimmunity 210

AB toxin, Clostridium perfringens 29 autoinducer-2 (AI-2) 232
accessory gene regulator (agr) locus 304 autoinducers 232, 303
acne vulgaris 305–6 autolysin 305, 340
acquired pellicle 173 autolysis 231
actin cytoskeleton mediate invasion 64–5 AVD see atherothrombotic vascular disease (AVD)
Actinobacillus actinomycetemcomitans 178
Actinomyces 177 Bacillus cereus 36, 36, 37, 38–9
Actinomycosis israelii 195 Bacillus welchii 36, 37
active ulcer 34 bacteremia 14, 16, 95, 152, 377–8
acute bacterial skin/skin structured infections bacteria: at bucal mucosa 108; colonization/
284–6, 285; cellulitis 288–9; adherence, oral cavity 173, 175;
cutaneous abscess 293–4; folliculitis commensal 115–16, 307; dental
287–8; impetigo 286–7; NF 294–5; caries 117–18; in dental plaque 109;
scarlet fever/scarlatina 289–90; SSSS ERU 204; identification, diagnostic
286, 290–1; wound infections 291–3 methods 44; in ocular diseases
acute bacterial SSSI (ABSSSI) 280–1 333–48; in saliva 108–9; uveitis 204
acute bronchitis 82 bacteria in skin infections 295–6, 296;
acute conjunctivitis 328 Pseudomonas aeruginosa 302–4;
acute dacryocystitis 327 Staphylococcus aureus 296–9;
acute diarrheal illness 5 Streptococcus spp. 299–301
acute otitis media 81 bacterial conjunctivitis 323, 325, 327–8, 336
acute rhinosinusitis 81 bacterial endocarditis 192, 380
adherens junction (AJ) 153 bacterial keratitis 324, 325, 330–1, 335
adhesins 152 bacterial oral microbiome 108–9
adhesion 152 bacterial plaque 109, 172
aEPEC (atypical EPEC) 7 bacteriocins 233–5, 304–5
Aeromonas 31 Bacteriodes fragilis 32
AFLP (amplified fragment length polymorphism) 241 Bacteriodes fragilis enterotoxin (BFT) 32
Aggregatibacter actinomycetemcomitans 168, 174 bacteriolysins 234
aggressive periodontitis 188 Bacterium lactis 225
allergies 251 Bacteroides 180
alpha-mangostin 305 β-galactosidase 245
alpha toxin 29, 335–6 beta toxin 29, 336
Alzheimer disease 121 betulin 342
amplified fragment length polymorphism (AFLP) 241 BFT (Bacteriodes fragilis enterotoxin) 32
anterior blepharitis 326 bifidobacteria 226–7
antibiotic resistance 236–7; blood infections 382–3; bioactive peptides 244–5
coryneform bacteria 347–8; biofilm 84, 89, 109, 172, 180, 191, 292, 306, 348;
Pseudomonas aeruginosa 303; in ocular infections 348–50, 350; oral
Staphylococcus spp. 337; 114–15; plaque 117, 173; skin 281
Streptococcus spp. 340–1 biomarker, oral microbiome as 120–1
antibiotics 236 bleeding-associated bacteria 177
anti-C precipitin test 300 blepharitis 322, 323, 325, 326, 336
apical periodontitis 119, 188 blood 376–7
aquaculture 254 blood–brain barrier 152–3
archae 108 blood infections 377; antibiotic resistance 382–3;
aspiration pneumonia (ASP) 94 bacteremia 377–8; bacteria in 380–2;
astrocytes 154 capsules 383; endocarditis 380;
astroglia 154 extracellular virulence products 383–5;
astrogliosis 154 membrane proteins 383; meninges
atherogenesis 141; bacterial pathogens 138; 379; osteomyelitis 380; pericarditis
inflammation 136–7; microbial 379–80; sepsis 378
component 142; “response to injury” bone-loss patterns 188
model 138, 141 bronchiolitis 81
atheromas 139–40, 142 bucal mucosa, bacteria at 108
atherosclerosis 135–6; antibiotic clinical buccal cellulitis 288
trials 140–1; defined 137; DNA bullous impetigo 286
evidence 139; microbiome 139–41; burn wounds 292
seroepidemiology 139
atherothrombotic vascular disease (AVD) CAB (community-acquired bacteremia) 377–8
135–7, 140; epidemiology 138–9 CAD (coronary artery disease) 136
atypical EPEC (aEPEC) 7 caecum, ulcer in 18

391
392    Index

CagA (cytotoxin-associated gene A) protein 33 CNS see central nervous systems (CNS)
calculus 172–3 coagulation 377
CAMBRA (caries management by risk cobalamin (vitamin B12) 230
assessment) 189 coli surface antigens 8
Campylobacter 19; clinical features 20–1; colonization: meningitis 152; nasopharyngeal 89;
diagnosis 21; incubation period 20; oral cavity 114–15; resistance 115–16
pathogenesis 19–20; prevention 22; colonization factor antigens (CFA) 8
treatment 21 commensal bacteria 115–16, 307
Campylobacter pylori 36, 37 community-acquired bacteremia (CAB) 377–8
CAMs (cell adhesion molecules) 158 complement activation pathway 158–9
cancer 249 complement system 158–9
carcinogenesis 196 complicated SSSIs (cSSSIs) 280, 284; cellulitis 285,
caries affected zone 184 288–9
caries, dental 116–18, 172–3, 180, 181; congenital dacryocystitis 327
development of 182; diagnostic conjugated linoleic acid (CLA) 228, 244
characteristics 186–7; microorganisms conjunctivitis 323, 325, 327–8, 336
in 176–7; treatment 188 conserved repetitive sequences 241
caries management by risk assessment contamination zone, peri-apical lesion 183, 183
(CAMBRA) 189 cornea 330–1, 335
caries, pathogenesis 180–2; extraradicular corneal ulceration 336, 339
infections 184–5; infection zones coronary artery disease (CAD) 136
182–3, 183; intraradicular infections Corynebacterium diphtheriae 306
184; remineralization-demineralization Corynebacterium jeikeium 306–7
cycle 182; root caries 183–4 Corynebacterium macginleyi 346
cataracts 215 Corynebacterium spp. 306–7
caveolae 67–8 coryneform bacteria 346–7; antibiotic resistance
caveolar endocytosis 67 347–8; therapeutic interventions 348;
caveolin-1 67 virulence factors 347, 347
cavitation 189 craters 188
CDI (Clostridium difficile infection) 24 C-reactive protein (CRP) 137
CDT (cytolethal distending toxin) 20 crevicular fluid 112
cell adhesion molecules (CAMs) 158 crevicular sulcus 112
cellulitis 285, 288–9, 324, 325, 331 Cronobacter sakazaki 40
cementum 170 CRP (C-reactive protein) 137
centor score technique 290 CSF (cerebrospinal fluid) 154–5
central nervous systems (CNS): bacterial entry into cSSSIs see complicated SSSIs (cSSSIs)
CNS 152–3; bacterial multiplication cusps 169
in CSF 154; complications of Shigella cutaneous abscess 285, 293–4
14–15; host response to 154–5; cyclic adenosine monophosphate (cAMP) 8
invasion 152–5; pathogens in CNS cyclic guanosine monophosphate (cGMP) 8
153–4 cytokine storm 158
cerebrospinal fluid (CSF) 154–5 cytolethal distending toxin (CDT) 20
cervical caries 186 cytotoxin 86
CFA (colonization factor antigens) 8 cytotoxin-associated gene A (CagA) protein 33
chalazion 323, 325, 329–30
chancre 194 dacryocystitis 323, 325, 326–7
chemokines 158 DAEC (diffusely adherent Escherichia coli) 6, 12
cholera 29 danger associated molecular patterns (DAMPs) 157
chronic conjunctivitis 328 daptomycin 86
chronic dacryocystitis 327 DDH (DNA-DNA hybridization) 239
circulatory system 111 demineralization 182
CLA (conjugated linoleic acid) 228, 244 dendritic cells 248
classification/etiological agents: blepharitis dental apparatus 169–70
326; cellulitis 331; chalazion 330; dental caries see caries, dental
conjunctivitis 328; dacryocystitis 327; dental plaque 109, 172
endophthalmitis 332; hordeolum 329; dentin 169, 181–2
keratitis 330; uveitis 333 dermatitis exfolitiva neonatorum 290
clinical features: Campylobacter 20–1; Clostridium diagnosis: Bacillus cereus 39; Campylobacter 21;
difficile 25–6; EHEC 10; EPEC 7; ETEC 8; cellulitis 289; Clostridium difficile 26;
Salmonella typhi 18; Yersinia 23 Clostridium perfringens 29;
Clostridium difficile 24, 36, 37; clinical features cutaneous abscess 293; dental caries
25–6; clinical/microbiological 186–7; EHEC 10–11; EPEC 7; ETEC 8;
properties 25; diagnosis 26; folliculitis 287–8; impetigo 287;
identification on culture medium leptospirosis 215–16, 216–17;
25; incidence of 25; non-laboratory- NF 294–5; periodontitis 187–8;
based tests 26–7; pathogenesis 26; Salmonella 17; Salmonella typhi
symptoms 25–6; treatment 27 18–19; scarlet fever 290; Shigella 15;
Clostridium difficile infection (CDI) 24 SSSS 291; Vibrio cholerae 30; wound
Clostridium perfringens 27, 36, 37; diagnosis 29; infections 292; Yersinia 23–4
incidence 28; pathogenesis 28–9; diarrhea 250
symptoms 28; treatment 29 diffuse lamellar keratitis 349
Clostridium tetani 195 diffusely adherent Escherichia coli (DAEC) 6, 12
 Index     393

diphtheroids 306 foodborne illness/food poisoning 35, 36; Bacillus

DNA-DNA hybridization (DDH) 239 cereus 36, 38–9; Cronobacter
dysbiosis 113–14, 117, 195 sakazaki 40; diagnosis, treatment,
and complications 37–8;
EAEC (enteroaggregative Escherichia coli) 6, 11–12 Listeria monocytogenes 39–40;
early-onset sepsis (EOS) 156 Mycobacterium tuberculosis 40–3;
ecthyma 286 Staphylococcus aureus 35–6;
EHEC (entero hemorrhagic Escherichia coli) 6, 9–11 Vibrio vulnificus 40
EIEC (enteroinvasive Escherichia coli) 6, 11 fragilysin 32
Ekiri syndrome 15 functional food 252–7
enamel 169–70 fungi 107–8
enamel de-/re-mineralization 117 “fusiform” Bacteroides 179
encapsulated Haemophilus influenzae 89
endocarditis 380 γ–aminobutyric acid (GABA) 243
endodontic infections 184; treatment 189 gamma-toxin 336
endodontic lesions 119–20 GAS (group A Streptococcus) 286, 289, 300–1
endodontic-periodontal lesions 120 gastroenteritis 4
endogenous endophthalmitis 332 gastrointestinal (GI) bacterial infections 3–5;
endophthalmitis 324, 325, 331–2, 342 Aeromonas 31; Bacteriodes fragilis
endotoxins 349 32; Campylobacter 19–22; Clostridium
enteric fever 17–19, 18, 59; symptoms 59–60 difficile 24–7; Clostridium perfringens
enteroaggregative Escherichia coli (EAEC) 6, 11–12 27–9; Escherichia coli 5–12;
enterohemorrhagic Escherichia coli (EHEC) 6, 9–11 Helicobacter pylori 32–5; Plesiomonas
enteroinvasive Escherichia coli (EIEC) 6, 11 31–2; Salmonella 16–19; Shigella
enteropathogenic Escherichia coli (EPEC) 5, 6, 7 12–16; symptoms 4; Vibrio 29–31;
enterotoxigenic Escherichia coli (ETEC) 6, 7–9 Yersinia 22–4
EOS (early-onset sepsis) 156 gastrointestinal tract (GIT) 4
EPEC (enteropathogenic Escherichia coli) 5, 6, 7 GBS (Guillain-Barré syndrome) 21
epidemiology: AVD 138–9; bacteremia 378; cellulitis genotypic classification, Leptospira 209
288–9; cutaneous abscess 293; GI see gastrointestinal (GI) bacterial infections
endocarditis 380; folliculitis 287; gingival crevice 112
impetigo 286; leptospirosis 210; gingival epithelium 106
meninges 379; NF 294; pericarditis gingivitis 118
379–80; scarlet fever 290; sepsis 378; gonorrhea 195
SSSS 291; wound infections 292 granulomas 91, 92
epithelium 106 green complex bacteria 178
EPS (exopolysaccharide) 229, 244 group A Streptococcus (GAS) 286, 289, 300–1
epsilon toxin, Clostridium perfringens 29 Guillain-Barré syndrome (GBS) 21
equine recurrent uveitis (ERU) 204, 211 gumma 195
erythrocytes 376
erythrogenic toxins 289 HAB (hospital-acquired bacteremia) 377
Escherichia coli 5; blood infections 381; DAEC 12; Haemophilus influenzae 88–91
EAEC 11–12; EHEC 9–11; EIEC 11; Haemophilus influenzae type b (Hib) disease
EPEC 5, 8; ETEC 7–9; types 5, 6 89–91
ETEC (enterotoxigenic Escherichia coli) 6, 7–9 Helicobacter pylori 32–3, 34, 36, 37, 193; diagnosis
etiology: leptospirosis 209–10; meningitis and sepsis 34–5; pathogenesis 33; symptoms
149–50; root caries 183 33–4; treatment 35
ExoA (exotoxin A) 343 hemoglobin 376
exoenzymes 326 hemolysin (HlyE) 67
exogenous postoperative endophthalmitis 332 hemolytic streptococci 300
exopolysaccharide (EPS) 229, 244 hemolytic uremic syndrome (HUS) 9–10, 14
exotoxin A (ExoA) 343 hepatic encephalopathy 251
exposome 113 heterofermentative bacteria 227
extensively drug-resistant TB (XDR-TB) 93 heteropolysaccharides 229, 244
external hordeolum 329 Hib (Haemophilus influenzae type b) disease 89–91
extracellular glycosyltransferase pathway 229 high-mobility group box-1 (HMGB-1) 157
extraocular infections 322, 323–4, 325; bacterial HlyE (hemolysin) 67
conjunctivitis 327–8, 336; blepharitis homofermentative bacteria 227
322, 326, 336; cellulitis 331; chalazion homopolysaccharides 229, 244
329–30; dacryocystitis 326–7; hordeolum 323, 325, 328–9
hordeolum 328–9; keratitis 330–1 horizontal transmission mode, meningitis 151
extraradicular infections, dental caries 184–5 hospital-acquired bacteremia (HAB) 377
eye 204, 212, 320–1; see also ocular infections host invasion pathways: caveolae-mediated
invasion 67–8; factors influencing
facial cellulitis 288 68–9; T3SS 63–7
fastidious microorganisms 231 human microbiome 107
Fauchard, P. 190 Human Microbiome Project 104, 121
fibroblasts 183 HUS (hemolytic uremic syndrome) 9–10, 14
fissures, tooth 169, 169, 170 hyaluronic acid capsule 383
folate (vitamin B11) 229–30 hyaluronidase 340
folliculitis 285, 287–8 hyperacute conjunctivitis 328
foodborne diarrheal illness 4 hypopyon 215
394    Index

immune-mediated resuscitation (IMR) 140 late-onset sepsis (LOS) 156

immune paralysis 158 leaky gut syndrome 137
immune response 110–11 LEE (locus for enterocytes effacement) 7
immune system: neonatal sepsis 156; Leptospira 204–5, 212; classification 207–9, 209;
of neonates 148; oral 109–11 cultural characteristics 206–7;
immunological reactions 103 genotypic classification 209; molecular
impetigo 285, 286–7 characteristics 207; serological
impetigo contagiosa 286 classification 208–9
IMR (immune-mediated resuscitation) 140 Leptospiraceae 207–8
incubation period: Campylobacter 20; EHEC 10; leptospiral ERU 214, 215
EPEC 7; ETEC 8; Plesiomonas 32; leptospiral genes 208
Salmonella 16; Shigella 13–14; typhoid leptospiral lipopolysaccharide (LPS) 205
fever 18; Yersinia 22 leptospiral uveitis 205, 214
infective endocarditis 380 leptospires: growth condition 206; history 204–5;
inflammation of atherogenesis 136–8 morphological characteristics 205–6
inflammatory bowel diseases 250 leptospirosis 204, 212; complications 215; diagnosis
integumentary system 281 215–16, 216–17; epidemiology
intermediate uveitis 333 210; etiology 209–10; infectious
internal hordeolum 329 cycle 210–13; morbidity and mortality
intestinal TB 40–3, 41 215; pathogenic mechanism 211–13;
intracellular pathogen 89 prevention 218–19; risk factors 213;
intraocular infections 322, 323–4, 325; treatment 217–18
endophthalmitis 331–2, 342; leukocytes 377
uveitis 332–3 lignocellulosics 231–2
intraradicular infections, dental caries 184 lipid metabolism 228
iNTS (invasive NTS) 60 Lipopolysaccharides (LPS) 155, 157
invasion: actin cytoskeleton mediate 64–5; lipoteichoic acid (LTA) 369–70
caveolae-mediated 67–8; CNS 152–5; Listeria monocytogenes 39–40
host invasion pathways see host locus for enterocytes effacement (LEE) 7
invasion pathways; meningitis 152; LOS (late-onset sepsis) 156
OMP PagN invasion 66–7; OMP Rck lower respiratory tract (LRT) 79, 80
invasion 66; T3SS-dependent 63–5; lower respiratory tract infections (LRTIs) 80
T3SS-independent 65–7 lower respiratory tract bacterial infections (LRTBIs)
invasive NTS (iNTS) 60 82–4, 83; Haemophilus influenzae
iota toxin, Clostridium perfringens 29 88–91; Klebsiella pneumoniae 93–5;
irreversible pulpitis 187 Mycobacterium tuberculosis 91–3;
irritation zone, peri-apical lesion 183, 183 Staphylococcus aureus 84–6;
Streptococcus pneumoniae 86–8
junctional epithelium 106, 112, 170–1, 171, 188 LPS see leptospiral lipopolysaccharide (LPS);
juvenile periodontitis 188 Lipopolysaccharides (LPS)
LRINEC (Laboratory Risk Indicator for NF score) 294–5
keratitis 324, 325, 330–1, 335 LRT (lower respiratory tract) 79, 80
Keyes tetrad 180, 180 LRTBIs see lower respiratory tract bacterial
keystone pathogen hypothesis 115 infections (LRTBIs)
Klebsiella pneumoniae 93–5, 381 LRTIs (lower respiratory tract infections) 80
Klebsiella pneumoniae carbapenemase beta- LTA (lipoteichoic acid) 369–70
lactamase (KPC) 95 lumen, oral cavity 109–10
Klebsiella rhinomatosis 195 luxS-mediated universal signaling system 232
lysostaphin 337
LAB see lactic acid bacteria (LAB)
labile toxin (LT), heat 8 macrolide resistance 341
Laboratory Risk Indicator for NF score (LRINEC) mannitol 230
294–5 MDR-TB (multidrug-resistant tuberculosis) 93
lactase 245 Meibomian cyst 329
lactic acid 231, 245 meningitis 148, 379; causative organisms 149, 150;
lactic acid bacteria (LAB) 225–6; beneficial 242–50; etiology 149–50; neonatal
biosafety assessment of 235–6; cancer see neonatal meningitis
treatment potential 249; in chemical Methicillin 369
industry 256–7; in cosmetics 256; methicillin-resistance gene (mecA) 369
future perspective 257; genomes methicillin-resistant Staphylococcus aureus
226–7; growth 231–3; identification (MRSA) 84, 85, 297–8, 335, 337, 369
238–41; medical application 250–2; methicillin-susceptible Staphylococcus aureus
morphology, cytology and physiology (MSSA) 84, 298
226–31; proteolytic system 228; microbial oral communities 108
taxonomy 237–41 microflora, skin 282–4, 283, 306
lactobacilli 176–7 microorganisms 253, 365
Lactobacillus 226 molecular-genetic techniques 239
Lactobacillus plantarum NC8 232 M protein 383
Lactococcus lactis 225 MRSA see methicillin-resistant Staphylococcus
lantibiotics 233, 298 aureus (MRSA)
laryngitis 81 MSSA (methicillin-susceptible Staphylococcus
las QS system 302–3 aureus) 84, 298
 Index     395

mucins 247 oral anatomy 105–6

mucosal immunity 110 oral biofilms 114–15
mucosa, oral 171–2 oral cavity 105–6, 114, 120; biodiversity within
multidrug efflux pump 345 115; colonization 114–15; future
multidrug-resistant (MDR) Salmonella 60 perspectives 195–7; junctional
multidrug-resistant tuberculosis (MDR-TB) 93 epithelium 170–1, 171; mucosa 171–2;
mutans streptococci 177 periodontal apparatus 170; structures,
Mycobacterium tuberculosis 195; GIT 40–3, 42; attributes 169–72; systemic diseases
RTI 91–3 and conditions 192–4; tooth/dental
apparatus 169, 169–70, 170
nasopharyngeal colonization 89 oral diseases 116, 168, 194–5; bacteria
necrotizing enteritis 29 colonization/adherence 173, 175;
necrotizing fasciitis (NF) 294–5 caries 116–18, 172–3; endodontic
Neisseria gonorrhea 195 lesions 119–20; periodontitis 118–19,
Neisseria meningitides 382, 385 172–3
neonatal meningitis: bacteremia 152; CNS invasion oral health 168–9
152–5; colonization and invasion oral immune system 109–11; crevicular flow 112–13;
152; infection 151–2; mode of saliva flow 111–12
transmission 151; pathophysiology oral microbiome 103–5; bacterial 108–9; behavior
151–5; risk factors 151 114–15; composition 107, 118;
neonatal sepsis: anti-inflammatory responses 158; to disease-associated microbiome
complement system 158–9; 115–16; geographic influences
defined 155; extrinsic factors 156; on 109; as health biomarker 120–1;
immune system 156; infection 156–7; healthy 107–9; imbalances 116–20;
influencing factors 156; intrinsic nonbacterial 107–8; oral anatomy
factors 156; microbiology 157; and 105–6
molecular and cellular events 157–9; oral microflora 195
onset of 156; pathophysiology 155–9; oral mucosa 171–2
proinflammatory response 158; oral tissues 106
risk factors 151 orange complex bacteria 177–8
neonates 148–9 orbital cellulitis 324, 325, 331
neuraminidase 340 osteoblasts 183
neurotropic pathogens 153 osteomyelitis 380
neutrophils 13, 17, 112–13, 211 outer membrane proteins (OMP) 66; PagN invasion
next-generation sequencing (NGS) techniques 66–7; Rck invasion 66
104–5, 108, 118
NF (necrotizing fasciitis) 294–5 PAβN (phe-Arg-β–naphthylamide) 345
nisin 234–5 PagN protein 66–7
NOD (nucleotide binding oligomerization PAI (pathogenicity associated island) 7
domain) 158 PAMPs (pathogen-associated molecular patterns) 157
nonbacterial oral microbiome 107–8 Panton–Valentine leucocidin (PVL) 297–8, 336
non-bullous impetigo 286 panuveitis 333
noncavitated lesions 186 paranasal sinuses 331
nondiphtheriae corynebacteria 306 pars plana vitrectomy (PPV) 218
nonencapsulated Haemophilus influenzae 89 pathogen-associated molecular patterns
nonpathogenic Leptospira 208, 209 (PAMPs) 157
nonspecific plaque hypothesis 117 pathogenesis: Campylobacter 19–20;
nontyphoidal salmonellosis/Salmonella (NTS) Clostridium difficile 26; Clostridium
16–17, 60 perfringens 28–9; dental caries
nosocomial infection 94 180–5; EHEC 9; EPEC 7; ETEC 8;
no-typeable Haemophilus influenzae (NTHI) 89 Haemophilus influenzae 89, 90;
NTS (nontyphoidal salmonellosis/Salmonella) Helicobacter pylori 33; Klebsiella
16–17, 60 pneumoniae 94; Mycobacterium
nucleotide binding oligomerization domain tuberculosis 91–2, 92; oral cavity
(NOD) 158 172; periodontitis/periodontal
nutritional supplements 252–7 disease 118–19, 185–6; Shigella 13;
Staphylococcus aureus 84–6, 85;
ocular diseases 333–4 Streptococcus pneumoniae 87, 87–8;
ocular infections 320, 322; bacterial biofilm in Yersinia 22–3
348–50, 350; extraocular 322–31, pathogenicity associated island (PAI) 7
323–4, 325; intraocular 322, 323–4, pathogenic Leptospira 208, 209, 213
325, 331–3 pathophysiology: blepharitis 326; cellulitis 331;
ocular infections, bacteria in 333–4; coryneform chalazion 330; conjunctivitis 328;
bacteria 346–8; Pseudomonas dacryocystitis 327;
aeruginosa 342–6; Staphylococcus endophthalmitis 332; hordeolum 329;
spp. 334–8; Streptococcus spp. keratitis 330–1; meningitis 151–5;
338–42 sepsis 155–9; uveitis 333
ocular manifestation 204, 213, 214 pattern recognition receptors (PRRs) 154; signaling
ocular microbiota 321–2 of 157
ocular surface 320; antibacterial protections in 322; PCV7 (7-valent pneumococcal conjugate vaccine) 88
synthesis proteins 333 pediatric uveitis 332
OMP see outer membrane proteins (OMP) penicillin 236
396    Index

peptidoglycan 226 red blood cells (RBCs) 376

perianal cellulitis 288 red complex bacteria 177
periapical abscess 187–8 refractory periodontitis 188
periapical lesion: development 185; zones 183, 183 Reiter’s syndrome 15
pericarditis 379–80 remineralization–demineralization cycle 182
pericardium 379 respiratory system 79–80
periodontal abscess 188 respiratory tract 79
periodontal apparatus 170 respiratory tract infections (RTIs) 80–1, 81–2
periodontal pathogens 121, 139 respiratory tree 82, 84
periodontal pocket 188 “response to injury” model, atherogenesis 138, 141
periodontitis/periodontal diseases 118–21, 137, restriction fragment length polymorphism
172–3; diagnostic characteristics (RFLP) 240
187–8; microorganisms 177–80; rhinoscleroma 195
pathogenesis 185–6; treatment 190–2 rhl QS system 302–3
periorbital cellulitis 288, 331 riboflavin (vitamin B2) 229
PFGE (pulsed field gel electrophoresis) 240 risus sardonicus 195
pharyngitis 81 Ritter’s disease 290
phe-Arg-β–naphthylamide (PAβN) 345 root caries 183–4, 186
phenylalanyl-tRNA synthase alpha subunit rRNA superfamily VI 19
(pheS) 240 RTIs (respiratory tract infections) 80–1, 81–2
pits, tooth 169, 169, 170
PLA (polylactic acid) 256–7 saliva 111–12; bacteria in 108–9
plaque biofilm 112, 173 salivary duct 111
plasma 377 salivary glands 111
plasmin 89 Salmonella 36, 37, 61; host invasion pathways
platelets 377 see host invasion pathways; incubation
pneumococcal PS capsule 340 period 16; intracellular survival 69–70;
pneumolysin (PLY) 339 invasion strategies 62; nontyphoidal
pneumonia 80–1, 82 salmonellosis 16–17; Salmonella typhi
polylactic acid (PLA) 256–7 17–19, 18; serotypes 61–2
polymicrobial infections 382 Salmonella enterica 61
polysaccharides 229 Salmonella-induced filaments (SIF) 69
Porphyromonas gingivalis 139, 168, 174, 178–9 Salmonella pathogenicity islands (SPIs) 63, 64
posterior blepharitis 326 Salmonella typhi 17–19, 18
PPV (pars plana vitrectomy) 218 scarlet fever/scarlatina 285, 289–90
prebiotics 252 SCCmec (staphylococcal cassette chromosome
primary enteropathogens 4 mec) 297
probiotic products 242 SCFA (short-chain fatty acids) 246
probiotics 252–4 SCVs (small colony variants) 69–70, 84
proinflammatory response 158 secondary impetigo 286
prophylaxis see treatment sepsis 148, 378; causative organisms 149, 150;
Propionibacterium acnes 305–6, 346–7 etiology 149–50; neonatal see
proteolytic system of LAB 228 neonatal sepsis
Proteus mirabilis 367 septic shock 148, 378; microbiology 157
Proteus spp. 365–6; interaction with host 368; seroepidemiology 139
virulence factors 366–8, 367 serological classification, Leptospira 208–9
protozoan 107 7-valent pneumococcal conjugate vaccine (PCV7) 88
PRRs see pattern recognition receptors (PRRs) Shiga enterotoxin (ShET) 13
pseudomembranous colitis 24, 27 Shiga toxin (Stx) 9, 13
Pseudomonas aeruginosa 302–4, 342–3; Shiga toxin-producing Escherichia coli
antibiotic resistance 344; therapeutic (STEC) see entero hemorrhagic
interventions 345–6; virulence factors Escherichia coli (EHEC)
343–4, 345 Shigella 12–13; clinical features 13–15; CNS
pulpal inflammation 187 complications 14–15; diagnosis 15;
pulpal lesions 185 incubation period 13–14; intestinal
pulsed field gel electrophoresis (PFGE) 240 complications 14; pathogenesis 13;
purple complex bacteria 178 prevention 16; symptoms 13–14;
purulent cellulitis 288 transmission mode 13; treatment 16
PVL (Panton–Valentine leucocidin) 297–8, 336 short-chain fatty acids (SCFA) 246
pyorrhea see periodontitis/periodontal diseases SIF (Salmonella-induced filaments) 69
pyrogenic exotoxins 289 single active ulcer 34
SipC (translocon) 64–5
quorum-sensing bacteria 232 16S rRNA gene 239
quorum sensing (QS) systems 302–3 skin 280–1; barrier functions of 281–2, 282;
infections see acute bacterial skin/skin
radicular cyst 187 structured infections; bacteria in skin
rampant caries 186 infections; microflora 282–4, 283, 306
random amplified polymorphism DNA (RAPD) 240–1 skin and skin structure infections (SSSIs) 280, 284
RBCs (red blood cells) 376 skin and soft tissue infections (SSTIs) 280
Rck invasin 62 skin commensals: Corynebacterium spp. 306–7;
Rck protein 66 Propionibacterium acnes 305–6;
reactive arthritis 21 Staphylococcus epidermidis 304–5
 Index     397

slide agglutination test 30 therapeutic interventions: coryneform bacteria

small colony variants (SCVs) 69–70, 84 348; Pseudomonas aeruginosa
Socransky, S. 177 345–6; Staphylococcus spp. 337–8;
SOD (superoxide dismutases) 340 Streptococcus spp. 342
sorbitol 230 thrombocytes 377
sphingomyelinase 336 tight junction (TJ) 153
SPI-1 effectors 64 toll-like receptors (TLRs) 154–5, 155, 157, 336
Spirochaetaceae 207–8 tonsillitis 81
Spirochaetales 205 tooth 169, 169–70, 170
Spirocheta interrogans 204 tracheobronchial tree 82, 84
spirochetes 173, 179 translocon 63
SPIs (Salmonella pathogenicity islands) 63, 64 treatment: Bacillus cereus 39; Campylobacter 21;
SptP (effector protein) 65 cellulitis 289; Clostridium difficile 27;
SSSIs (skin and skin structure infections) 280, 284 Clostridium perfringens 29;
SSSS (staphylococcal scalded skin syndrome) 285, cutaneous abscess 293–4; dental
286, 290–1 caries 188; EHEC 11; endodontic
SSTIs (skin and soft tissue infections) 280 infections 189; EPEC 7; ETEC 8–9;
stable toxin (ST), heat 8 folliculitis 288; Haemophilus
staphylococcal cassette chromosome mec influenzae 89–91; Helicobacter
(SCCmec) 297 pylori 34–5; impetigo 287; intestinal
staphylococcal scalded skin syndrome (SSSS) 285, TB 43; Klebsiella pneumoniae 94–5;
286, 290–1 leptospirosis 217–18; Mycobacterium
Staphylococcus aureus 286, 334–6; blood infections tuberculosis 93; NF 295; periodontitis
381; GIT 35–6, 36, 37; RTI 84–6; skin 119, 190–2; Salmonella 17; Salmonella
infections 296–9; UTIs 368–70 typhi 18–19; scarlet fever 290;
Staphylococcus epidermidis 304–5 SSSS 291; Staphylococcus aureus 86;
Staphylococcus spp. 334–5; antibiotic resistance 337; Streptococcus pneumoniae 88;
therapeutic interventions 337–8; UTIs 370; Vibrio cholerae 31; wound
virulence factors 335–6, 338 infections 292–3; Yersinia 24
staphyloxanthin 298 trigger mechanism 62
stimulation zone, periapical lesion 183, 183 Trojan horse mechanism 153
stratified squamous epithelium 171 tuberculosis 81, 82, 91, 93, 195
Streptococcus mutans 168, 176–7, 180–1, 196 two active ulcer 34
Streptococcus pneumoniae (Pneumococcus) 86–8 type III secretion system (T3SS) 63; actin cytoskeleton
Streptococcus pyogenes 380–1, 383; capsules mediate invasion 64–5; dependent
383; extracellular virulence products invasion 63–5; independent invasion
383–5; virulence factors 384 65–7; structure 63
Streptococcus sanguis 173 typhoid fever 17–19, 18
Streptococcus spp. 299–301, 338–9; antibiotic typical EPEC (tEPEC) 7
resistance 340–1; blood infections
380–1; therapeutic interventions 342; ulcer 41; active 34; in caecum 18
virulence factors 339–40, 341 uncomplicated SSSIs 280, 284; impetigo 285, 286–7
Streptococcus thermophilus 233 upper respiratory tract (URT) 79, 80
streptolysin O 383–4 upper respiratory tract bacterial infections (URTBIs)
streptolysin S 384 82–4, 83; Haemophilus influenzae
Stx (Shiga toxin) 9, 13 88–91; Klebsiella pneumoniae
styes 329 93–5; Mycobacterium tuberculosis
subgingival calculus 118, 172 91–3; Staphylococcus aureus 84–6;
sugars 227 Streptococcus pneumoniae 86–8
superantigens 385 upper respiratory tract infections (URTIs) 80
superoxide dismutases (SOD) 340 urease 366
supragingival calculus 172 urinary tract infections (UTIs) 365; future
symbiosis 113 prospective 370; Proteus spp. 366–8;
symptoms: Bacillus cereus 38; bacterial infections Staphylococcus aureus 368–70;
of GI 4; blepharitis 322, 326; treatment 370
Campylobacter 20; Clostridium difficile URT (upper respiratory tract) 79, 80
25–6; Clostridium perfringens 28; URTBIs see upper respiratory tract bacterial
conjunctivitis 328; Helicobacter pylori infections (URTBIs)
33–4; Listeria monocytogenes 39; URTIs (upper respiratory tract infections) 80
Salmonella typhi 18; Shigella 13–14; UTIs see urinary tract infections (UTIs)
Vibrio cholerae 30; Yersinia 23 uveal tract 213
syphilis 194 uveitis 204, 324, 325, 332–3
systemic inflammatory response syndrome (SIRS)
158; anti-inflammatory responses 158 vancomycin 86, 299
vancomycin-intermediate MRSA (VIMRSA) 298–9
T3SS see type III secretion system (T3SS) verotoxin producing Escherichia coli (VTEC) see
tagatose 230 entero hemorrhagic Escherichia coli
Tannerella forsythia 173, 174, 179 (EHEC)
teichoic acids 226 vertical transmission mode, meningitis 151
tEPEC (typical EPEC) 7 Vibrio cholerae 29–31
terminal ileum, ulcer in 18 Vibrio vulnificus 40
tetanus 195 VIMRSA (vancomycin-intermediate MRSA) 298–9
398    Index

virulence factors 8, 11, 174, 179; Aeromonas 31; weight of evidence 173, 174
coryneform bacteria 347, 347; Weil, A. 205
DAEC 12; Proteus spp. 366–8, 367; wheat germ agglutinin (WGA) 253
Pseudomonas aeruginosa 343–4, 345; white blood cells (WBCs) 377
Staphylococcus epidermidis 304–5; wound infections 285, 291–3
Staphylococcus spp. 335–6, 338; Wzy-dependent pathway 229
Streptococcus pyogenes 384; XDR-TB (extensively drug-resistant TB) 93
Streptococcus spp. 339–40, 341 xylitol 231
virulence genes 66 yellow complex bacteria 178
viruses 107 Yersinia 22, 36, 37; clinical features 23;
visceral fat accumulation 248 diagnosis 23–4; pathogenesis 22–3;
vitamin B2 (riboflavin) 229 treatment 24
vitamin B11 (folate) 229–30 Yersiniabactin (Ybt) 23
vitamin B12 (cobalamin) 230 Yersinia outer membrane proteins (Yops) 22
vitamin K 230 zipper mechanism 62
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