CHARACTERISTICS OF LEGIONELLA:

• They can infect and multiply within some free-

living amoebe/amoeba (species of Hartmanella,
Acanthamoeba and Naegleria), ciliated protozoa
(Tetrahymena), and biofilms.
• Isolated from lakes, rivers, hot springs and mud
• They can tolerate up to 3 mg/L of chlorine, and
thus resist water disinfection treatments.
• Cannot grow on routine media: BAP

LEGIONELLA PNEUMOPHILA o Selective Media: BCYE with L-cysteine,

• Medium: BCYE w/ L-cysteine (pH 6.9) ferric salt and a-ketoglutatrate
• Agent of Legionnaire’s disease (1,4 and 6) and • Serological Test
Pontiac fever. o Indirect Fluorescent Antibody (IFA) –
• Invades the bronchoalveolar macrophages, most common method for Legionnaire’s
which is a facultative intracellular pathogen. disease.
• Isolated in air-conditioned units, cooling towers, o Direct Fluorescent Antibody:
humidifiers and nebulizers. detecting common Legionella species in
• Serogroups: 1 to 7 LRT. (+) yellow or green-colored bacilli.
• Culture: blue-green, glistening • Rapid methods
• Primary Clinical Manifestation: o DNA test (PCR)
o Legionnaire’s disease o Urine antigen test: detects L.
▪ Aka: legionelliosis (febrile and pneumophila as early as 3 days from
pneumonic illness) infection.
▪ MOT: Airborne spread, LESSON 2: AEROBIC GRAM-POSITIVE
inhalation of aerosol BACILLI
▪ Symptoms: High fever, non-
productive cough, headache,
neurological and severe
bronchopneumonia
o Pontiac fever
▪ Non-fatal respiratory infection
▪ Main symptom is pneumonia.
o Wound abscess and encephalitis

LEGIONELLA SP.
LABORATORY DIAGNOSIS:
• Staining
o Faint staining, undetectable in Gram
staining.
o Prolonging smear for 10 minutes
o Legionella: intracellularly and
extracellularly located in phagocytes
o L. micdadei: weakly acid-fast
• Culture

TUAZON A. 13
 • ”string of pearl appearance” – when penicillin is
used (Susceptible).
• Related disease: Anthrax
LABORATORY DIAGNOSIS
• Specimen: Malignant pustule, sputum and
blood
• B. anthracis: isolated from normally sterile site
such as blood, lung tissue and CSF.
• Staining:
o Used of old culture in Gram staining:
Gram variable
o Spore stains: Malachite green and
McFadyean stain
o Capsule stain: India ink (blood and
CSF)
• Large, ‘boxcar’ shaped, ± spores
o Fluorescent microscopy: rapid
o Bacillus, Clostridium
presumptive diagnosis
• Small, pleiomorphic, angular arrangements
• Culture Media:
o Corynebacterium, Listeria,
o Culture media: BAP, CAP, egg yolk
Cutibacterium, Lactobacillus
agar (EYA), Phenylethyl alcohol (PEA),
• Beading, filamentous Polymyxin-lysozyme-EDTA-thallous
o Nocardia, Actinomyces acetate (PLET), bicarbonate agar, and
BACILLUS nutrient broth.
• Spore-forming, aerobic or facultatively o Enrichment and Selective technique:
anaerobic, rod-shaped bacteria and isolated application of heat or alcohol shock
from the soil technique before plating on media
o Bacillus anthracis o PEA: recommended for identification of
o Bacillus cereus B. anthracis in fecal specimen.
o Bacillus thuringiensis o PEA and PLET: used in isolating
o Bacillus mycoides Bacillus from contaminated specimen
• Species only form endospores aerobically o EYA: determine B. anthracis has
• Motile with peritrichous flagella except for produced lecithinase
Bacillus anthracis and Bacillus mycoides o Gelatine Medium: Inverted pine tree
• Survive in extreme environment appearance.
• Microscopy: large, boxcar-shaped, gram-positive
rods with clear, unstained, central spores or
“empty spaces”
• Biochemical test:(+) catalase; ferments
glucose; hydrolyzes starch
BACILLUS ANTHRACIS
• Causative agent of anthrax • Medusa head colony of Bacillus anthracis. (left
• Grow aerobically or anaerobically photo)
• Not part of normal flora and not highly • Bamboo fishing rods/pole appearance of
contagious Bacillus anthracis. (right photo)
• Non-motile, halophilic organism can withstand
up to 7% NaCl
• Used as biological weapon of mass destruction
• Grows in low pH (<6.0) environment, produce
lecithinase and ferments glucose.
• Virulence factor: D-glutamic acid capsule and
protein exotoxins
• Microscopy: Gram-positive, large,
encapsulated and square-ended rod; has
“bamboo fishing rods” appearance on the
unstained central spore
• Culture: BAP –colonies have “Medusa head” • Beaten egg yolk appearance of B. anthracis
appearance with swirling projections; are non- ANTHRAX
haemolytic; and “beaten egg white” appearance • Disease primarily affects: goats and sheep, by
when inoculating loop is used. feeding on plants that are contaminated with the
• Growth Factor: Thiamine (B1) spores.

TUAZON A. 14
• MOA: inhalation of spores during exposure to o IP = Incubation period
infected animals and contaminated animal • Emetic Type
products, or cuts through skin. o Heat stable enterotoxins
• Exotoxins: cause signs and symptoms. o Ingestion of improperly cooked rice
THREE FORMS OF ANTHRAX:
• Cutaneous anthrax
o Acquired thru skin cuts and abrasions
o Small papule 2-5 days after exposure
o Appearance: “black eschar”, black,
necrotic and painless central area that
does not produce pus.
• Pulmonary anthrax/Woolsorter’s disease
o inhalation of spores into the pulmonary
parenchyma
o resembles URTI with mild fever,
dyspnea
o this is usually acquired when getting
sheep’s fur.
• Gastrointestinal anthrax
o spores are inoculated into a lesion on
the intestinal mucosa following their
ingestion.
o Signs and symptoms: Abdominal pain,
nausea, anorexia, vomiting and bloody
diarrhea.
DIAGNOSTIC TEST
• Ascoli Test (Precipitin test)
o Detects thermostable anthrax antigens o B. Cereus type I; IP: 1 to 5 hrs
o (+): formation of precipitin band after
less than 15 minutes
• Catalase Test
o Bacillus (catalase +); Clostridium
(catalase -)
• Oxidase Test
o Variable (anthrax)
• DFA Test (Direct Fluorescent Antibody Test)
o (+) cell wall polysaccharide and capsule
antigen.
BACILLUS CEREUS (“FRIED RICE” BACILLUS)
• Causes food poisoning due to the ingestion of • Frosted glass appearance of B. cereus
contaminated cooked rice dishes or other food OTHER BACILLUS
products • Bacillus subtilis (Hay Bacillus)
• Commonly encountered Bacillus in opportunistic o Most commonly encountered laboratory
infections that causes eye and ear infection. contaminant
• Exhibits motility and resistant to penicillin o Halophilic organisms, up to 7% NaCl
• BAP: large and feathery; spreading growth; o Source of Bacitracin antibiotics
have a frosted-glass appearance and b- o Cause: Eye infection among prohibited
haemolytic drug users
• Biochemical Test: Ferments salicin; (+) o BAP: large, flat, and dull with ground
lecithinase
• Virulence factors: Enterotoxins (heat-stable
and heat-labile), cerelysin, phospholipase C and
pyogenic toxin.
• Best specimen for testing: Suspected food
(>105cells/gram)
TWO TYPES OF FOOD POISONING OR
GASTROENTERITIS:
• Diarrheal Type
o Heat labile enterotoxin
o Ingestion of contaminated meat, poultry
and vegetables. IP: 8 to 16 hrs.

TUAZON A. 15
 glass appearance; maybe b-haemolytic • “club shape” or coryneform
and exhibit pigment (pink, yellow, • Cells are arranged singly, in “palisades” of
orange or brown) parallel cells or in pairs of cells connected after
o Biochemical test: Ferment mannitol, cell division to form V or L shapes: “Chinese
xylose and arabinose letters” appearance
• Bacillus pumilus • Diphtheroid, meaning “diphtheria-like”
o biological indicator: sterilization method • Specie causing human infections: C.
o BAP: large and moist; blister-like diphtheriae, C. jeikeium, C. pseudotuberculosis,
appearance and b-haemolytic C. pseudodiphthericum, C. ulcerans, and C.
• Bacillus thuringiensis urealyticum
o insect pathogens
o produces parasporal crystals that can
be utilized as pesticide.
CORYNEBACTERIUM
• Catalase positive, gram-positive rods
• Non–acid-fast, non–spore-forming, and mostly
non-branching rods
• Lipophilic Corynebacteria
o C. jeikeiumand C. urealyticum
• Non-Lipophilic Corynebacteria CORYNEBACTERIUM DIPHTHERIAE
o C. diphtheriae, C. pseudotuberculosis, • AKA: diphtheria bacillus or Kleb-Loffler
C. pseudodiphtheriticum, C. striatum, C. bacillus
• Facultative anaerobe, Inhabits the human
nasopharynx in carrier
• MOA: inhalation of contaminated respiratory
droplets or direct contact with infected
cutaneous lesions.
• Readily killed by heat and by most of the usual
disinfectants.
• Glucose and maltose fermenter
• Virulence factor: Diphtheria toxin
• Preferred medium: Enriched medium with
serum, cysteine and potassium tellurite
• Microscopy:
o Rounded ends and “club-shaped
swelling”
o highly pleomorphic cells are arranged in
ulceransand C. xerosis pairs and create X, V, Y and L formation
that closely resembles Chinese letters.
• C. diphtheriae growing on sheep blood agar.
• Biochemical Test: (-) Urease; (+) Nitrate
CORYNEBACTERIUM
reduction.
• Corynebacteria can be divided into non lipophilic
• Best specimens: Nasopharyngeal and throat
and lipophilic species
swabs
• Majority of the species are found as indigenous
THREE BIOTYPES OF CORYNEBACTERIUM
microbiota of the skin and mucous membrane of
humans and animals. Species are pathogenic to DIPHTHERIAE
animals, plants, and humans. • Intermedius
• Species are found in fresh water. o Very small, flat, dry, and grayish-black
Corynebacterium are also found in salt water, colonies; non-haemolytic
soil, and air. • Mitis
• Species are non-motile, non-encapsulated, non- o Small, black and convex that have
spore forming and highly theomorphic rods. “fried-egg appearance”
• Specie are glucose and maltose fermenters • Gravis
except for C. pseudodiphthericum and C. o Large, flat and dark gray colonies
urealyticum. “daisy-head appearance”
• Corynebacteria are slightly curved, gram-
positive rods

TUAZON A. 16
 • Albert stain used for the visualization of
metachromatic granules which is also known as
Much granules.
DIPHTHERIA TOXIN
• Heat-labile
• Produced by strains with a lysogenic b-phage
that carries the TOX gene.
• Tissue necrosis and exudates formation
(psedomembrane lining) over the tonsils, larynx
and pharynx
• Alkaline pH (7.8 to 8.0), aerobic environment
and sufficient amount of iron in the medium.
o Necrosis caused by exotoxin
RESPIRATORY DIPHTHERIA o Adherent, bleeds if scraped
• Acute, infectious disease characterized by o Severity correlates with disease severity
production of systemic toxin and false o May lead to airway obstruction
membrane lining (Pseudomembranous • Management
formation) of the throat mucous membrane → o Airway support if needed
respiratory obstruction. o Hospitalization and isolation
• S/Sx: Low grade fever, Thick mucopurulent o Equine serum diphtheria antitoxin
nasal discharge and cough (consider)
• Control: Immunization (DPT vaccine o Antibiotics (penicillin or erythromycin)
[Diphtheria, pertussis, tetanus vaccine]) o Immunize close contacts
• Cutaneous or Skin Diphtheria (Aka Veldt sore
or Barcoo rot):
o Slow healing ulcer and membrane
formations; less severe.

• Pseudomembrane (left photo)

• Veldt sore or Barcoo rot (right photo)
C. UREALYTICUM
• Most frequently isolated and most clinically
significant.
• Urinary pathogen, strict aerobe and lipophilic
• Do not ferment glucose and maltose
• Microscopy: V shaped and palisades
Corynebacterium diphtheriae • Culture: BAP –pinpoint, white smooth, non-
• Club looking bacteria haemolytic
• Causes diphtheria • Urease producer
o Infection with a characteristic tough • Most frequent isolated and most clinically
leathery membrane that forms in the significant among them all.
pharynx. C. PSEUDODIPHTHERITICUM
• Four main subspecies • Normal flora of nasopharynx
o C. diphtheriae mitis • Respiratory infection, UTI, cutaneous wound
o C. diphtheriae intermedius infection in immunocompromised.
o C. diphtheriae gravis • Microscopy: arranged in parallel rows or
o C. diphtheriae belfanti palisades and do not exhibit any other
* bold font subspecies are the most common characteristics “pleomorphism” that is similar to
other.
• Urease and nitrate positive
• Pseudomembrane C. JEIKEIUM

TUAZON A. 17
• Skin normal flora in inguinal, axillary and rectal • Microscopic Loeffler methylene blue stain of
sites. Corynebacterium spp. (right)
• Obligate aerobe and antibiotic resistant
• Immunocompromised, Common cause of
diphtheroid prosthetic valve endocarditis in
adults.
• Microscopy: Pleomorphic, club-shaped and
arranged in V-shaped
• Urease and Nitrate negative
C. ULCERANS
• Animal contact and unpasteurized dairy
products.
• Skin ulcers and exudative pharyngitis
• Associated with diphtheria-like sore throats. LABORATORY DIAGNOSIS OF
• Culture: BAP-narrow zone of b-hemolysis CORYNEBACTERIUM
• CTBA - brown halo CULTURE
• Loeffler’s serum agar: Exhibit growth • Cystine Tellurite Blood Agar
• (+): Urease and gelatinase; (-) nitrate reduction. o Preferred medium
C. PSEUDOTUBERCULOSIS for isolation and
• Animal pathogens that human can contract thru identification of
direct contact with infected animals. crynebacterium
• Dermonecrotic toxin causes death of various cell o Modification of
types the Tinsdale agar
• Diphtheria toxin (causative agent) o Selective and
Differential
• Culture: CTBA –black color and surrounded by
o Contains sheep blood, bovine serum,
brown halo
cysteine
• BAP: small and yellowish-white
o (+): Colonies of corynebacterial exhibit a
• (+) urease; (-) gelatinase black or brown color after 48 hours
LABORATORY DIAGNOSIS o (+): C. diphtheriae, C. ulcerans and C.
• Specimen: Nasopharyngeal and throat swabs, pseudotuberculosis.
urine, blood and wound discharge • Tinsdale Agar
• Calcium alginate swab: Preferred for o Sheep blood,
collection. cystine, potassium
• Staining tellurite and sodium
o Gram positive, short or slightly curved thiosulfate
rods. o (+) Black color
o Various arrangement of C. diphtheriae surrounded by halo
are due to incomplete fission during o (+) all bio types of C. diphtheriae
multiplication. • Christensen Urea Slant
o Methylene blue staining: beaded o Urea production: C. urealyticum
formation • Pai’s Slant or Loeffler’s Serum Agar
o Neisser and Albert stain: o Microscopic and
Methachromatic granules metachromatic
• Culture: granules of C.
o BAP, CAP, CTBA, Tinsdale agar and diphtheriae.
Loeffler serum agar o (+): C. diphtheriae
o Tinsdale agar and CTBA: incubated for exhibit “poached-
atleast 48 hours at 35C to 40C egg” appearance
o Metachromatic granules of C.
diphtheriaeare called Babes-Ernst
bodies
• Schick Test
o Used to determine the susceptibility of a
person to diphtheria.
o Involves the intradermal introduction of
a small amount of the diphtheria toxin
into the arm of the suspected individual.
o (+): Redness and Swelling around the
• Microscopic Gram stain of diphtheroid (left) site.

TUAZON A. 18
 o Give 0.1 ml of diluted (1/50 MLD) LISTERIOSIS
diphtheria toxin → inject intradermally → • Infection affects neonates, pregnant women and
check reaction after 2-4 days (presence immunocompromised.
of redness and swelling) • Meat products should be thoroughly cooked or
• Toxigenicity heated before consumption
o Immunodiffusion Test - Elek’s Test • After ingesting contaminated dairy products like
▪ (+): 4 mm to 5 mm fine precipitin cheese
lines at 45 angle to the streaks. • Infected pregnant women may pass the
o Guinea pig lethal Test organisms onto the fetus
▪ AKA: diphtheria antitoxin test (in
vivo test) TYPES OF LISTERIOSIS:
o Tissue Culture Test • Maternal Disease (Pregnancy)
▪ detection of diphtheria antitoxin o During 3rdtrimester of pregnancy
(in vitro) o Miscarriage or stillbirth
o Polymerase Chain Reaction o S/Sx: Flu-like illness, fever, myalgia,
▪ used for detection of toxin • Neonatal Disease
o Associated with an intrauterine infection
due to the aspiration of infected amniotic
fluid
o Infected infants are at full term and
appear healthy at birth.
o Lead to meningitis that is usually seen in
3rd week of life
genes
• Disease Of Immunocompromised
LISTERIA o Develops through the ingestion of
• Coccobacillary in form and are arranged singly contaminated dairy products and
or in short chains that resemble streptococci processed meat products.
• Culture: small, smooth, translucent, grayish- LABORATORY DIAGNOSIS
blue and are surrounded by a narrow zone of b- • Specimen: Blood, CSF, and swabs of lesion
hemolysis.
• Motility Test:
LISTERIA MONOCYTOGENES o Wet mount/hanging drop method:
• Both human and animal pathogens Exhibits a “tumbling motility” at room
• Aerobic or facultatively anaerobic and non-spore temperature
forming o SIM: Has an “umbrella-shaped” or
• Motile with peritrichous flagella and exhibits a “inverted-Christmas-tree” pattern at 25C
characteristic “tumbling motility” but not at 35C
• Grow high salt medium with up to 10% NaCl • Culture Test:
• Optimal growth: 30C to 37C but can grow at 4C o BAP, CAP, BHI, Thioglycolate medium,
(cold temp) McBride agar and Nalidixic acid medium
• Recovered from the soil, dust, water, dairy o Enrichment technique: Cold enrichment
products and processed meats (4C using broth)
• Causes miscarriage or stillbirth in humans o Colonies and haemolytic pattern may be
• Virulence factor: confused with those of group
o Listeriolysin O (hemolytic and cytotoxic) streptococci
o Catalase, o Growth: 0.5C to 45C
o Superoxide dismutase (SOD), o Requires: Slightly increased amount
o Phospholipase C and of CO2
o p60 (surface protein) • Biochemical Test:
• MOA: ingestion of contaminated food such as o Positive: Glucose fermentation,
meat, chicken, dairy products and vegetables. catalase and motility, CAMP reaction –”
• Infection: Listeriosis block-type” hemolysis, Hippurate and
Bile esculin hydrolysis, growth in 6.5%
NaCl, VP, MR
o Negative: H2S, nitrate, urease
• (Left photo) Listeria with b-hemolysis
• (Right photo) Block hemolysis using the CAMP
test.
• Umbrella motility and the inverted christmas tree

TUAZON A. 19
 appearance Listeria + + + + +
• Left tube is positive, right tube is negative. monocytog
enes
Corynebact
+ - V V V
ERYSIPELOTHRIX RHUSIOPATHIAE erium spp.
• Thin, pleomorphic rods with the tendency to Streptococ - - - + V
form long filaments that are arranged in single, cus
short chains or in a V-shaped formation. agalactiae
• Facultatively anaerobic, non-motile and non- Enterococc
-* + - V +
spore forming, Gram positive, rod-shaped us spp.
bacterium and positive H2S. • Listeria (left)
• Isolated from wild and domestic animals like • Erysipelothrix (right)
birds and fish. ARCANOBACTERIUM
• Major reservoir: Domestic swine • Classified as pleomorphic, non-motile, rod-
• Gelatin stab culture: Has a pattern of a “pipe-
cleaner” or a “test tube brush” at 22C
• Related infections and disease: Endocarditis,
septicemia and erysipeloid (red-skin infection).
• MOA: Direct contact with infected excreta, blood
and flesh of animals through skin breaks.
• Predisposed: Veterinarians and fish handlers
• Lab Diagnosis:
o Best specimen: Tissue biopsies or
aspirates from the skin.
o Localized in the deeper layer of shaped and Gram-positive
subcutaneous tissue • BAP: colonies have a narrow zone of b-
• Culture hemolysis; exhibit pitting of the agar with a black
o BAP, nutrient broth, Columbia CAN opaque dot.
agar. • Species: A. haemolyticum, A. pyogenesand A.
o BAP: pinpoint with a-haemolytic or non- bernardiae
haemolytic zone. • Biochemical: Catalase Positive
• Biochemical Test: • A. haemolyticum: lipase and lecithinase
o (+): H2S production in a TSI medium, positive and Positive reverse CAMP reaction
glucose and lactose fermentation. due to Phospholipase D.
o (-): Catalase, oxidase, esculin
hydrolysis, nitrate reduction, VP and

KURTHIA
urease. • Obligate aerobe, gram
• Erysipeloid (red-skin infection) (left photo) positive rods, Motile by
• Test tube brush or pipe-cleaner appearance peritrichous flagella
(right photo) • Culture:
SUMMARY o BAP: Large
“medusa-head” appearance
o Nutrient agar: Exhibit a Rhizoid growth
• Biochemical Test: Catalase Positive; Negative:
Gelatinase and Oxidase
GARDNERELLA VAGINALIS
DIFFERENTIATION OF LISTERIA MONOCYTOGENES • Short, pleomorphic gram-positive rod or
AND OTHER GRAM-POSITIVE BACTERIA coccobacillus that often stains gram-variable or
Gro gram-negative.
Esculi
Catal n Motil
b- wth • G. vaginalis (causative agent) → bacterial
Organism hemol in vaginosis (BV) in humans
ase Hydrol ity
ysis 6.5 • BV is characterized by a malodorous discharge
ysis
NaCl and vaginal pH greater than 4.5.

TUAZON A. 20
• BV reduction in the Lactobacillus in the vagina, • Found in the soil and organic materials
followed by an increase in vaginal pH • Can cause disease to humans and many
• The observation of “clue cells,” large squamous animals
epithelial cells with gram-positive and gram- • Culture: Cells elongate to form branching,
variable bacilli and coccobacilli clustered on the filamentous forms while some organisms form
edges, aids the diagnosis of BV hyphae on the agar surface or into the agar
• Lab Diagnosis: o Hyphae is usually present only in fungi.
o Best specimen: Vaginal discharge o Aerobic actinomycetes is considered
collected from suspected BV. before as fungi.
o G. vaginalis grows best in 5% to 7% ACID-FAST AEROBIC ACTINOMYCETES:
CO2 at a temperature of 35°C to 37C. NOCARDIA
o The medium of choice for G. vaginalis • Partially acid-fast, obligate aerobic, non-motile
is human blood bilayer Tween (HBT) and catalase positive
agar. • Microscopy: Gram negative bacilli with long,
o V (vaginalis) agar also contains human thin, beaded, branching filaments; old cultures
blood and is used for recovery of this may fragment into short, rod-shaped, coccoid
organism. forms
o When cultured on human blood, • Cell wall contains peptidoglycan, meso-
colonies are b-hemolytic, small, gray, diaminopimelic acid (DAP) and sugars such as
arabinose and galactose
• Grow on media that are used to recover fungi
• Culture: Exhibit wrinkled, chalk-like and orange-
tan pigmentation
• Resistant to lysozyme
NOCARDIA
• Related Infection:
o Acquired thru the inhalation of the
and opaque. organisms that are present in dust and
SUMMARY soil.
o Nocardia: cause invasive pulmonary
infections with hematogenous
dissemination throughout the body.
AEROBIC ACTINOMYCETES
• Actinomycetoma (Actinomycotic mycetoma)
• Gram positive bacilli, Aerobes with a branching
o Chronic, localized, painless
filamentous growth that extends along the agar
subcutaneous infection that is
due to the substrate hyphae and into the agar
characterized by the presence of sulfur
granules in the affected tissue
o Nocardia brasiliensis
• Pulmonary disease
o Confluent bronchopneumonia where the
sputum is thick and purulent although
the encapsulation of the abscess is
absent.
o Affected tissue do not have sulfur
granules.
o Nocardia cyriacigeorgica and Nocardia
farcinica.

• (left photo) Appearance of sulfur granule

collected from draining sinus tracts. These
granules contain masses of filamentous
organisms with pus materials. The arrow points
out eosinophilic projections (clubs) characteristic
due to the aerial hyphae. of sulfur granules from gram-positive bacteria.
• Filamentous rods with beaded appearance

TUAZON A. 21
• (right photo) Colony morphology of Nocardia on • Immunocompromised individuals (HIV patient)
chocolate agar. and cause slowly, progressive, granulomatous
LABORATORY DIAGNOSIS pneumonia.
• Specimen: Biopsy or drainage material • Microscopy: Coccobacilli with a ”zigzag pattern”
• Pus and tissue specimen: best samples in and a filamentous form
performing a wet mount and staining. • Culture: BAP: colonies exhibit a pale pink or
• Staining Technique: yellow color
o Modified Ziehl-Nielsen or Kinyoun stain • Differential Test:
o Gomori’s methenamine-silver stain Susceptible to lysozyme
• Culture: GORDONIA
o BAP, CAP, BHI, TMA, LJ medium, SDA • Genus varies from Gram
without Chloramphenicol, Middlebrook positive to Gram
media, Litmus milk variable rods.
o Nocardia sp. are b-haemolytic • Partially acid-fast, non-motile and catalase
o Incubation at 10% CO2 promotes the positive
growth of Nocardia species. • Culture: Smooth and slimy with irregular edges;
o May not always survive the but may appear as dry or rough; and exhibit the
decontamination procedures for presence of mycelia.
mycobacteria. • Differential Test: Susceptible to lysozyme
o Tap Water Agar: observe the TSUKAMURELLA
morphology of actinomycetes and • Genus are Gram positive and shaped like long
differentiate branching Nocardia sp. rods that fragments into three parts.
from non-branching Rhodococcus sp. • Members are Slightly acid-fast when the
Kinyoun method is used
• Culture: Colonies are circular with rhizoid
edges; has no aerial hyphae ad exhibit white or
orange pigmentation.
NON-ACID FAST, AEROBIC GRAM-POSITIVE
ACTINOMYCETES
• (left photo) Gram staining of Nocardia
demonstrating irregular staining.
STREPTOMYCES
• Gram-positive and rod-shaped with branching
• (right photo) Acid-fast staining of Nocardia
showing partially acid-fast appearance filaments
• Found in soil
RHODOCOCCUS, GORDONIA AND
• Human pathogens: Streptomyces somaliensis
TSUKAMURELLA
(Agent of Actinomycotic mycetoma)
• Aerobic, catalase-positive, branching,
• Differential Test: Susceptible
filamentous, Gram-positive bacteria that can
to lysozyme
fragment into rods and cocci
• Culture Media: BHI agar and
• Isolated in soil, fresh and marine water and
organic matter
• Primarily acquired through inhalation
• Grow on most of the non-selective media for
bacterial, mycobacterial and fungal isolation
• Infection and disease: Skin infections,
pneumonia, peritonitis, catheter-associated
sepsis
RHODOCOCCUS EQUI SDA
• Non-motile and • Culture: Colonies are dry to chalky and heaped;
partially acid-fast and some exhibit a grayish white color and “musty
is composed of basement odor”
mycolic acid with ACTINOMADURA
longer carbon chains • Microscopic and colony
• Can persist and morphology of the
replicate within species of this genus is
macrophages very similar to that of
• Acquired through respiratory route and exposure the Nocardia species.
to infected animals
• Fungal wound infection that is known as eumycetoma.
• Most common form of eumycetoma is known as mycetoma pedis → Madura foot
• Species: Actinomadura madurae and A. pelletieri

TUAZON A. 22
 • Culture: “Molar tooth” appearance
TROPHERYMA WHIPPLEI
• Gram positive actinomycete and facultative, intracellular pathogen
• Etiologic agent: Whipple’s disease
• Affects the gastrointestinal tract, joint and muscles and is characterized by abdominal pain and diarrhea.
• Isolated from human feces, saliva and gastric secretions
• Diagnostic Test: (+) Periodic Acid-Schiff Staining (PAS) macrophages.
WHIPPLE’S DISEASE
1. Encephalopathy (inflammation of brain)
2. Lymphadenopathy
3. Malabsorption and diarrhea
4. Arthritis
5. (+) PAS stain (Periodic acid–Schiff)
6. Demonstrating a tropherphyma whippli bacilli within the macrophages

LESSON 3: MYCOBACTERIA ii. Group II – scotochromogens, produces

A. Mycobacterium tuberculosis complex pigment with and without the presence
i. Mycobacterium tuberculosis of light.
ii. Mycobacterium bovis iii. Group III – non-photochromogens, does
iii. Mycobacterium africanum not produce any kind of pigment.
iv. Mycobacterium canetti ➢ Group I, II, III are slow
v. Mycobacterium microti growers.
B. Non-tuberculous mycobacteria iv. Group IV – rapid growers
i. Group I – photochromogens, produces MYCOBACTERIA
pigment when there is light, no light = no • Slender, slightly curved or straight, rod-shaped
pigment (photo – light, chromo – organisms
pigment) • Non-motile and do not form spores

TUAZON A. 23
 • The cell wall has extremely high lipid content; o Almost any organ of the body can be
thus, mycobacterial cells infected by M. tuberculosis
• Resist staining with commonly used basic • Pleurisy, an unexplained pleural effusion with
aniline dyes, such as those used in the Gram mononuclear pleurocytosis, manifests as cough,
stain, at room temperature. fever, and chest pain, resembling the
• Take up dye with increased staining time or presentation of bacterial pneumonia.
application of heat but resist decolorization with o water or pleural effusion in the lungs
acid-ethanol and upon examination through
• This characteristic is referred to as acid paracentesis (getting fluid in the lungs
fastness—hence, the term AFB—and is and observe the presence of
• Mycobacteria are strictly aerobic, but increased mononuclear pleurocytosis)
carbon dioxide (CO2) will enhance the growth of • Lymphadenitis – inflammation of the lymph
some species. nodes
• Cause pulmonary tuberculosis. • Genitourinary TB
• Studied using AFS (Acid Fast Staining) • Skeletal TB of the spine is referred to as Pott
• Important component is the Mycolic acid disease.
(targeted by the antibiotics that are used in o Positive in TB, skinny, and not able to
Mycobacteria) stand up
• Antibiotics are Isoniazid, Rifampin • Meningitis
MYCOBACTERIUM TUBERCULOSIS COMPLEX • Cerebrospinal fluid (CSF) examination usually
• Consists of M. tuberculosis, M. bovis reveals an elevated protein level, decreased
• (including the vaccination strain bacillus glucose level, and a predominance of
Calmette-Guérin), M. africanum, M. canettii, and lymphocytes.
M. microti. M. africanum • LJ medium (malachite green,
MYCOBACTERIUM TUBERCULOSIS egg white)
• Mycobacterium tuberculosis
• TB is usually a disease of the respiratory tract.
is characterized as cauliflower
• Tubercle bacilli are acquired from persons with
appearance.
active disease who are excreting viable bacilli by
sneezing or talking. • Colonies are typically raised,
with a dry, rough appearance.
• Hard tubercle or granuloma may be formed
• The colonies are
• The granuloma is an organization of
nonpigmented and classically
lymphocytes, macrophages, fibroblasts, and
described as being buff-colored
• capillaries.
• Elaboration of cord factor can result in
• With granuloma formation, healing occurs, as
characteristic cord formation.
well as fibrosis, encapsulation, and calcification,
• Optimal growth occurs at 35° C to 37° C.
with scar formation as a reminder of the past
infection. • Test results:
o Positive for niacin accumulation
o Granuloma treatment is from 9-12
o reduction of nitrate to nitrite
months
o Production of catalase
• In infected individuals, there is a potential for
o Grows on thiophene-2-carboxylic acid
reactivation of TB.
hydrazide (T2H)
TESTS USED IN PULMONARY TUBERCULOSIS
TREATMENT
• Clinical diagnosis of primary TB: Screening
• For pulmonary TB, treatment typically involves a
test or the positive PPD (purified protein
9-month course of therapy with isoniazid and
derivatives) skin test also known as Mantoux
rifampin, usually once per day the first month
skin test.
and twice a week thereafter.
• Other tests are X-ray, culture of the sputum,
o for severe/chronic TB, therapy is 12
AFS using the sputum.
months
• Diagnosis is confirmed by stained smear and o isoniazid and rifampin are the first life of
culture of sputum, gastric aspirates, or drugs for Pulmonary Tuberculosis.
bronchoscopy specimens. o Isoniazid inhibits mycolic acid.
• Extrapulmonary Tuberculosis Rifampin inhibits RNA polymerase.
o Aside from your lungs, it can also infect • Regimens also include a 2- to 8-week initial
other organs through hematogenous course of streptomycin or ethambutol
spread (spread through blood
• Pyrazinamide (PZA) may be added to the
circulation)
regimen if there is a suspicion of lowered cellular
• Miliary TB refers to the seeding of many organs immunity and a need to obtain bactericidal levels
outside the pulmonary tree with AFB through of antimycobacterial activity intracellularly in
hematogenous spread. (sesame seeds) macrophages.

TUAZON A. 24
• MDR-TB (multi drug resistant TB) is defined as MYCOBACTERIUM AVIUM SUBSP.
resistance to at least isoniazid and rifampin PARATUBERCULOSIS
o Resistance to ethambutol and • Causative agent of Johne disease, an intestinal
pyrazinamide only is not considered infection occurring as a chronic diarrhea in
MDR. cattle, sheep, goats, and other ruminants.
• Extensively drug-resistant TB (XDR-TB) is • very slow growth rate (3 to 4 months)
defined as resistance to isoniazid and • Needs mycobactin-supplemented medium for
rifampin plus resistance to any primary isolation.
fluoroquinolone and at least one of three MYCOBACTERIUM GENAVENSE
injectable second-line anti-TB drugs—the • cause of disseminated infections in patients with
aminoglycosides amikacin, kanamycin, or AIDS
capreomycin.
• enteritis and genital and soft tissue infections
o Primary drugs: RIPES (Rifampin,
• Middlebrook 7H11 agar supplemented with
Isoniazid, Pyrazinamide, Ethambutol,
mycobactin.
Streptomycin)
o Secondary drugs: Aminoglycosides • heat-stable catalase, pyrazinamidase, and
Amikacin, Kanamycin, or Capreomycin. urease
MYCOBACTERIUM BOVIS MYCOBACTERIUM HAEMOPHILUM.
• TB primarily in cattle but also in other ruminants, • Submandibular lymphadenitis, subcutaneous
as well as in dogs, cats, swine, parrots, and nodules, painful swellings, ulcers progressing to
humans. (domestic animals) abscesses, and draining fistulas are often the
clinical manifestations.
• The disease in humans closely resembles that
caused by M. tuberculosis and is treated • A unique characteristic of this organism is its
similarly. requirement for hemoglobin or hemin for growth.
• 21 days of incubation at 37° C. • Chocolate (CHOC) agar, Mueller-Hinton agar
with 5% Fildes enrichment, and Löwenstein-
• Test results:
Jensen (LJ) medium containing 2% ferric
o niacin-negative
ammonium citrate.
o do not reduce nitrate
o do not grow in the presence of (T2H) • Optimal growth temperature is 28° C to 32° C;
thiophene-2-carboxylic acid hydrazide little or no growth occurs at 37° C. cells are
strongly acid-fast, short, occasionally curved
MYCOBACTERIUM AFRICANUM
bacilli without banding or beading, and arranged
• Causes TB in Tropical Africa
in tight clusters or cords
MYCOBACTERIUM CANETTI • M. haemophilum is under group III
• Associated with AIDS patients MYCOBACTERIUM KANSASII
MYCOBACTERIUM MICROTI • M. kansasii strains have been isolated from
• Causes TB for both immunocompromised and water
competent • Infections are not normally considered
NONTUBERCULOUS MYCOBACTERIA contagious from person to person
• Slowly Growing Species • Susceptible to rifampin and ethambutol,
MYCOBACTERIUM AVIUM COMPLEX partially resistant to isoniazid and streptomycin,
• Mycobacterium avium and M. intracellulare are and resistant to pyrazinamide
part of the Mycobacterium avium complex • A multidrug regimen of isoniazid, rifampin, and
(MAC). ethambutol is currently recommended
o Also includes M. avium subsp. • Long rods with distinct crossbanding
paratuberculosis and M. avium subsp. • Colonies appear smooth to rough, with
silvaticum characteristic wavy edges and dark centers
• environmental saprophytes and have been when grown on Middlebrook 7H10 agar
recovered from soil, water, house dust, • Colonies are photochromogenic
• and other environmental sources • With prolonged exposure to light, most strains
• M. avium is a cause of disease in poultry and form dark red crystals of B-carotene on the
swine surface of and inside the colony.
• Zoonotic microorganisms • strongly catalase-positive
• the cells are short, coccobacillary, and uniformly • hydrolyze Tween 80 in 3 days
stained, without beading or banding • strong nitrate reduction
• production of a heat-stable catalase and the • Pyrazinamidase production
ability to grow on media containing 2μg/ mL of • Under Group I
T2H
MYCOBACTERIUM MALMOENSE
• Chronic pulmonary disease and cervical
lymphadenitis

TUAZON A. 25
• Resistant to isoniazid, streptomycin, p- • Young colonies grown on cornmeal agar have a
aminosalicylic acid, and rifampin and bird’s nest appearance, with characteristic
susceptible to ethambutol and cycloserine. sticklike projections.
• Short coccobacillus without cross bands on acid- • Group II
fast–stained smears. RAPIDLY GROWING SPECIES
• Colonies are smooth glistening, and opaque, MYCOBACTERIUM CHELONAE–
with dense centers MYCOBACTERIUM ABSCESSUS GROUP
MYCOBACTERIUM SCROFULACEUM • M. abscessus subsp. abscessus (formerly M.
• Cervical lymphadenitis in children abscessus), M. chelonae, and M. fortuitum.
• Group II • M. chelonae have been associated with a variety
MYCOBACTERIUM SIMIAE of infections of the skin, lungs, bone, central
• Isolated from the lymph nodes of monkeys nervous system, and prosthetic heart valves.
• Colonies on Middlebrook 7H10 agar are thin, • M. abscessus seen in patients with cystic
transparent or tiny, and filamentous fibrosis (CF)
• Group I • tap water is an important reservoir
MYCOBACTERIUM ULCERANS • positive 3-day arylsulfatase test, no reduction of
• Mycobacterium ulcerans is a rare cause of nitrate, and growth on MacConkey agar without
mycobacteriosis, also referred to as Buruli ulcer crystal violet
• Acid-fast cells are long, without beading or MYCOBACTERIUM FORTUITUM GROUP
crossbanding • M. fortuitum, M. peregrinum, and an unnamed
• Group III third species.
MYCOBACTERIUM MARINUM • Isolated from water, soil, and dust
• Mycobacterium marinum has been implicated in • Associated with localized cutaneous
diseases of fish and isolated from aquariums. infections
• Cutaneous infections in humans occur when • Middlebrook 7H11 agars after 1 to 2 days of
traumatized skin comes into contact with salt incubation colonies with branching
water or inadequately chlorinated fresh water • Filamentous extensions and rough colonies with
containing the organism. short aerial hyphae.
• Tender red or blue-red subcutaneous nodule, • Pleomorphic, ranging from long and tapered to
or swimming pool granuloma, usually occurs on short, thick rods partially acid-fast
the elbow, knee, toe, or finger • Positive 3-day arylsulfatase test and
• Susceptible to rifampin and ethambutol, reduction of nitrate
resistant to isoniazid and pyrazinamide, and MYCOBACTERIUM SMEGMATIS GROUP
partially resistant or intermediate to streptomycin • The M. smegmatis group contains two species,
• Cells of M. marinum are moderately long to long M. smegmatis and M. goodie.
rods with cross barring. • M. smegmatis has been implicated in rare cases
• M. marinum is photochromogenic; young of pulmonary, skin, soft tissue, and bone
colonies infections.
• Colonies grown in or exposed to light develop a • Cells are long and tapered or short rods with
deep yellow color. irregular acid fastness. Occasionally rods are
• Growth is optimum at incubation temperatures of curved with branching or Y-shaped forms;
28° C to 32° C. swollen, with deeper staining, beaded, or ovoid
• None reduces nitrate or produces heat-stable forms are sometimes seen.
catalase • Colonies appearing on egg medium after 2 to 4
• The organisms hydrolyze Tween 80 and days are usually rough, wrinkled, or coarsely
produce urease and pyrazinamidase. folded; smooth, glistening, butyrous colonies
• Group I may also be seen.
MYCOBACTERIUM XENOPI • Negative arylsulfatase reaction, positive iron
• Recovered from hot and cold water taps uptake, ability to reduce nitrate, and growth in
(including water storage tanks of hospitals) and the presence of 5% NaCl and on MacConkey
birds (hotspings) agar without crystal violet
• Susceptible to the quinolones (ciprofloxacin, MYCOBACTERIUM LEPRAE
ofloxacin); some isolates are susceptible to • Causative agent of Hansen disease (leprosy),
vancomycin, erythromycin, or cefuroxime an infection of the skin, mucous membranes,
• Colonies on Middlebrook 7H10 agar are small, and peripheral nerves
with dense centers and filamentous edges. • The two major forms of the disease are
• Cornmeal-glycerol agar reveals distinctive tuberculoid leprosy (localized & benign) and
round colonies with branching and filamentous lepromatous leprosy (disseminated &
extensions; aerial hyphae are usually seen in malignant)
rough colonies.

TUAZON A. 26
 • Symptoms of tuberculoid leprosy include skin o Lepromatous lion appearance
lesions and nerve involvement that can produce • Combination of diaminodiphenylsulfone
areas with loss of sensation. (dapsone), clofazimine, and rifampin
• Patients with lepromatous leprosy if untreated, • M. leprae has not been grown on artificial
life-threatening media.
o It is characterized by skin lesions and • mouse footpad or foot pad of armadillo
progressive, symmetric nerve damage. • the length of the bacillus is at least five times the
o Lesions of the mucous membranes of width of the bacillus
the nose may lead to destruction of the • Skin test:
cartilaginous septum, resulting in nasal o Lepromin test: (+) tuberculoid
and facial deformities.
CLASSIFICATION ORGANISM
M. tuberculosis
TB complex M. africanum
M. bovis
Photochromogens (Group I) M. asiaticum
M. kansasii
M. marinum
M. simiae
M. flavescens
M. gordonae
Scotochromogens (Group II)
M. scrofulaceum
M. szulgai
Nonchromogens (Group III) M. avium complex
M. celatum
M. haemophilum
M. gastri
M. genavense
M. malmoense
M. nonchromogenicum
M. shimoidei
M. terrae
M. trivale
M. ulcerans
M. xenopi
M. abscessus
M. fortuitum group
M. chelonae group
Rapid growers (Group IV)
M. phlei
M. smegmatis
M. vaccae

SPECIES ALWAYS CONSIDERED PATHOGENS

Pulmonary and disseminated tuberculosis; millions of cases
M. tuberculosis Humans
annually in the world
M. leprae Humans Leprosy
Tuberculosis-like disease rear in North-America; M. bovis is
M. bovis Humans, cattle
closely related to M. tuberculosis
SPECIES POTENTIALLY PATHOGENIC IN HUMANS
Moderately common causes of disease
M. avium complex Soil, water, birds, fowl, Disseminated, pulmonary; very common in AIDS patients; occurs
swine, cattle, in other immunocompromised patients; uncommon in patients with
environment normal immune system
M. kansasii Water, cattle Pulmonary, other sites
Rapid growers
Cutaneous lesions most common, subcutaneous abscesses,
M. fortuitum & M. Soil, water, animals,
disseminated infection; grow in ≤ 7 days; M. fotuitum is more
chelonae marine life
susceptible to antibiotics
SAPROPHYTIC SPECIES THAT VERY RARELY CAUSE DISEASE IN HUMANS
M. gordonae Water These saprophytic Mycobacterium species are very uncommon
M. flavescens Soil, water causes of disease in humans. Positive cultures for these

TUAZON A. 27
M. fallax Soil, water mycobacteria usually represent environmental contamination of
M. gastri Gastric washings specimens and not disease. Many of the saprophytic mycobacteria
grow best at temperatures ≤ 33°C. There are many other
saprophytic Mycobacterium species not listed here that seldom if
ever appear in cultures of patients’ specimens.
UNCOMMON TO VERY RARE CAUSE OF DISEASE
M. africanum Humans, monkeys Pulmonary cultures; resembles M. tuberculosis; rare
Blood in AIDS patients; grows in liquid medium (BACTEC) and on
M. genavense Humans? pet birds?
solid medium supplemented with mycobactin j; grows in 2-8 weeks
M. haemophilum Unknown Subcutaneous nodules and ulcers primarily in AIDS patients;
requires hemoglobin or hemin; grows at 28-32°C; rare
Pulmonary tuberculosis-like (adults), lymph nodes (children); most
reported cases are from Sweden but organism may be much more
M. malmoense Unknown, environment
widespread; M. malmoense is closely related to M. avium-
intracellulare; Takes 8-12 weeks to grow
M. marinum Fish, water Subcutaneous nodules and abscesses, skin ulcers
Cervical lymphadenitis; usually cured by incision, drainage, and
M. scrofulaceum Soil, water, moist food
removal of involved lymph nodes
M. simiae Monkeys, water Pulmonary, disseminated in AIDS patients; rare
M. szulgai Unknown Pulmonary, tuberculosis-like; rare
M. ulcerans Humans, environment Subcutaneous nodules and ulcers; may be severe; M. ulcerans is
closely related to M. marinum; takes 6-12 weeks to grow; optimal
growth at 33°C suggests environmental source; rare
M. xenopi Water, birds Pulmonary, tuberculosis-like with preexisting lung disease; rare
LESSON 3: MYCOBACTERIA • Biochemical Test: (-) Niacin and Nitrate
MYCOBACTERIUM TUBERCULOSIS COMPLEX reduction
• Mycobacterium tuberculosis (Koch’s bacillus)
• Mycobacterium bovis
• Mycobacterium africanum
• Mycobacterium canetti
• Mycobacterium microti
MYCOBACTERIUM TUBERCULOSIS
• Longest replication time
• Virulence factor: Cord factor
• Culture: slow-growing; buff color; raised and
dry; “CAULIFLOWER-LIKE APPEARANCE”.
Rough colonies exhibit “cording” (Curve strands
bacilli)
• Biochemical Test: (+) Niacin and Nitrate
reduction • Vaccine given after birth (right leg) BCG, (left
• Replication time: 20 to 22 hrs leg) Hepa
MYCOBACTERIUM AFRICANUM
• Associated human cases
of tuberculosis in tropical
Africa
• Detection of organism
requires the use of
spoligotyping (spacer oligotyping)
MYCOBACTERIUM CANETTI
• Smooth strain of M.
tuberculosis
• Grows more rapidly
than M. tuberculosis (6
MYCOBACTERIUM BOVIS days)
• Tuberculosis in human and animals (cattle, • Isolated from an AIDS patient with mesenteric
dogs, cat and swine) tuberculosis
• Attenuated strain used for vaccination (Bacillus- • Biochemical Test: (+) Niacin and Nitrate
Calmette-Guerin or BCG vaccine) reduction
• Culture: Slow-growing, small, granular, rounded
and non-pigmented

TUAZON A. 28
 MYCOBACTERIUM MICROTI • Cause: Buruli ulcer (painless
• Isolated from TB patients both nodule under the skin after
immunocompromised and previous trauma)
immunocompetent individuals. • Microscopy: Moderately
long rods without cross-
banding
NON-TUBERCULOUS MYCOBACTERIA • Culture: smooth, rough and non-pigmented (6-
• Group I: Photochromogens 12 weeks incubation
• Group II: Scotochromogens • Biochemical Test: (+) Heat stable catalase
• Group III: Non-Photochromogens MYCOBACTERIUM GORDONAE (TAP WATER
• Group IV: Rapid Growers BACILLUS)
NON-TUBERCULOUS MYCOBACTERIA: SLOW • Contaminates the tap
GROWERS water used by the patients
MYCOBACTERIUM AVIUMCOMPLEX in rinsing their mouths
• Specie: M. avium, M. prior to the procedure for
intracellulare, M. avium sputum collection.
subsp. Paratuberculosis • Rarely cause infection to
and M. avium subsp. human
silvaticum (wood pigeon • Culture: Smooth and yellowish-orange colored
bacillus) • Biochemical: (+) Tween 80 hydrolysis and heat-
• Most common cause of Pulmonary infection to stable catalase, (-) nitrate reduction
human, pathogen in AIDS. MYCOBACTERIUM XENOPI
• GI tract: most common site of colonization and • Recovered from hot and cold-
dissemination in AIDS. water taps, hospital storage
• Microscopy: Pleomorphic, short coccobacilli tanks
without beading; (+) PAS • Potential pathogen of
• Biochemical: (+) Heat stable catalase pulmonary infection in adults
MYCOBACTERIUM KANSASII (YELLOW • Non-photochromogenic and
BACILLUS) scotochromogenic
• Second to M. avium • Microscopy: Long and filamentous
complex to cause NTM lung • Culture: MB7H10: small with filamentous edges
disease (Chronic cavitary • Cornmeal glycerol agar: branching filaments
pulmonary lesion) • Growth: 42°C
• Not contagious from person • Biochemical: (+) heat stable catalase,
to person pyrazinamidase
• Microscopy: Long rods with distinct • M. xenopi has been classified with non-
crossbanding photochromogenic group however, colonies are
• Culture: MB7H10: smooth to rough with dark frequently bright yellow.
centers and waxy edges • Once incubated in absence of light, they will be
• Photochromogens: Dark red crystals of 10-B- scrotochromogens with yellow pigment at 42°C
carotene MYCOBACTERIUM TERRAE COMPLEX
MYCOBACTERIUM MARINUM • Normally saprophytic and rarely causes human
• Disease of fishes and isolated from aquariums infections
• Causative agent: “Swimming pool granuloma” • Species: M. terrae, M. triviale and M.
red or bluish red nodule on the elbow, knee, toe nonchromogenicum
or finger. Occurs when an open wound comes in • Microscopy: Short to medium coccobacilli
contact with contaminated chlorinated fresh • Culture:
water or salt water o M. triviale: rough and dry
• Natural Reservoir: Fresh water and salt water o M. terrae: smooth
• Microscopy: Long rods with cross barring o M. nonchromogenicum: smooth to
• Culture: smooth to tough, wrinkled, yellow rough and white to buff
• Biochemical test: (+) Tween 80 hydrolysis and
heat stable catalase, (+) growth in 5% NaCl (M.
terrae)

MYCOBACTERIUMULCERANS
• Third most common Mycobacterium species
after M. tuberculosis and M. leprae • Left: M. triviale. Right: M. terrae

TUAZON A. 29
 DISTINGUISHING
SPECIE MICROSCOPY CULTURE BIOCHEMICAL TEST
FEATURES
Saphrophytes; rarely
Dysgonic smooth and (+) High level of heat
M. asiaticum Acid-fast coccoid cells cause human
pigmented colonies stable catalase
infections
M. genavense Distinct acid-fast cells Dysgonic colonies; (+) semi-quantitative Fastidious; Do not
requires an extended and heat stable grow on routine
incubation (6-8 catalase; media; recovered in
weeks) (+) Pyrazinamidase BACTEC culture
Rough to smooth and
non-pigmented; The
Distinct acid-fast cells;
recommended media
short or curved,
include CA, MHA, Requires hemoglobin
M. haemophilum without beading,
with 5% Fildes or hemin for growth
appears clusters or
enrichement, LJ
cords
medium with 2% ferric
ammonium citrate
M. malmoense Short coccobacilli Non-pigmented, (+) Tween 80 Growth at 22C
without crossbands smooth, glistening hydrolysis, heat- requires 7 to 12
and opaque colonies stable catalase and weeks of incubation
with dense centers pyrazinamidase

OTHER NTM-SLOW GROWERS

M. simiae Short coccobacilli Filamentous colonies; (+) Niacin and high One of the very few
yellow and smooth level of heat stable NTM that produces
colonies after catalase niacin
extended incubation

Light yellow to deep (+) High-level, heat

M. scrofulaceum Medium to long rods orange colonies with stable catalase and
dense centers urease

M. szulgai Medium to long rods Yellow to orange and (+) Heat stable
with cross barring smooth to rough catalase
colonies

NON-TUBERCULOUS MYCOBACTERIA • Exhibits greater

• Group IV: Rapid Growers / Fast growers resistance to
MYCOBACTERIUM FORTUITUM antimicrobial agents
• Most common, rapidly • Microscopy: Strongly
growing mycobacteria acid-fast with
that are associated with pleomorphism in young
localized cutaneous and cultures
soft tissue infections • Culture: Rough to smooth, non-pigmented and
• Microscopy: have no filamentous branching
Pleomhorpic–long to • MAC: growth without crystal violet
short, thick rods MYCOBACTERIUM ABSCESSUS SUBSP.
• Old cultures: partially AFB ABSCESSUS
• Cultures: • Reservoir: Tap water
o MB7H11: branching and filamentous • Related infection: Chronic lung
with aerial hyphae disease and Otitis media
o MAC: growth in media without • Culture: MAC –exhibit growth in a
CRYSTAL VIOLET medium without CRYSTAL
MYCOBACTERIUM CHELONAE VIOLET
• Associated with cutaneous infections in • Biochemical: (+) 3-day
immunocompromised persons arylsulfatase; (-) Nitrate

TUAZON A. 30
 MYCOBACTERIUM SMEGMATIS o Claw-shaped hands
• Related infections: o Pendulous ear lobes
Pulmonary, skin and bone o Saddle nose
infections • Suppressed (low resistance)
• Microscopy: Long and
tapered rods with partial
acid-fastness; maybe
beaded or ovoid in form
• CULTURE:
o MB7H10: smooth or rough
o Culture: MAC –exhibit growth in a
medium without CRYSTAL VIOLET
• Biochemical: (-) 3-day arylsulfatase; (+) Nitrate
reduction
MYCOBACTERIUM LEPRAE
• Chronic skin disease, mucous membrane and
nerve tissue
• Not considered contagious disease
• MOT: Person to person contact through
inhalation (nasal secretions), Contact with
infected skin, arthropod bites and ingestion of
breast milk and transplacental transmission for
infants
• Forms: Tuberculoid (localized & benign) and
Lepromatous Leprosy (disseminated &
malignant)
• Microscopy: Rod shaped and exhibit “cigar-
pocket” or “pocket-fence”
• Skin test: Fernandez and Mitzuda Reaction
LABORATORY DIAGNOSIS
• Acid-Fast Staining (Biopsy)
• Culture: Footpads of Mice, definitive Tests
• Serological Test:
o Fluorescent leprosy antibody absorption
test
o DNA amplification
o ELISA
• Heat stable catalase: Negative

TUBERCULOID LEPROSY ((TT)

• Well demarcated, dry patch
• Minimal disfigurement
o No leonine facies
o No claw-shaped hands
o No pendulous earlobes
• Good immune response (high resistance)

LEPROMATOUS LEPROSY (LL)

• Disfigurement is there
o Leonine facies

TUAZON A. 31
 LESSON 4: ANAEROBIC BACTERIA AND RICKETTSIA AND CHLAMYDIA
ANAEROBIC BACTERIA

ENDOGENOUS ANAEROBES COMMONLY INVOLVED IN HUMAN INFECTIONS

INFECTION ANAEROBE
Actinomycosis Actinomyces israelii, other Actinomyces spp.
Antibiotic-associated diarrhea;
Clostridioides (Clostridium) difficile
• Pseudomembranous colitis
Bacteremia Bacteroides fragilis group, fusobacteria, clostridia,
peptostreptococci
Bacteroides spp., Prevotella spp., Porphyromonas spp.,
Brain abscess Fusobacterium spp., Clostridium spp. (these infections are
often polymicrobial)
Infections of the female genitourinary tract Peptostreptococci, Bacteroides spp., Clostridium spp.,
Prevotella bivia, Prevotella disiens, Actinomyces israelii
(IUD associated)
B. fragilis group, other Bacteroides spp., Fusobacterium
Intraabdominal infections, liver abscess, peritonitis, spp., Clostridium perfringens, other
perineal and perirectal infections Clostridium spp., peptostreptococci (frequently
polymicrobial)
Myonecrosis C. perfringens, Clostridium novyi, Clostridium septicum
(80%–95% of the cases)
Peptostreptococci, Porphyromonas spp., Fusobacterium
Oral, sinus, dental infections
spp. (often polymicrobial)
Aspiration pneumonia, pleuropulmonary infections Porphyromonas spp., Fusobacterium nucleatum,
peptostreptococci, B. fragilis group, Actinomyces spp.

ENDOGENOUS ANAEROBES OF VARIOUS ANATOMIC SITES

SITE ANAEROBES
Skin Propionibacterium, peptostreptococci
Peptostreptococci, Actinomyces, Propionibacterium,
Upper respiratory tract
Campylobacter, Fusobacterium, Prevotella, Veillonella
Actinomyces, Eubacterium/Eggerthella, peptostreptococci,
Oral cavity Campylobacter, Fusobacterium, Prevotella,
Bifidobacterium, Porphyromonas, Veillonella
Intestine Bifidobacterium, Eubacterium/Eggerthella, Clostridium,

TUAZON A. 32
 peptostreptococci, Bacteroides fragilis group,
Parabacteroides, Bilophila, Campylobacter, Fusobacterium,
Porphyromonas, Prevotella, Sutterella, Veillonella
Peptostreptococci, Bifidobacterium, Fusobacterium,
Genitourinary tract
Lactobacillus, Mobiluncus, Prevotella, Veillonella

POTENTIAL VIRULENCE FACTORS OF ANAEROBIC BACTERIA

ANAEROBES KNOWN OR
POTENTIAL VIRULENCE FACTOR POSSIBLE ROLE
THOUGHT TO POSSESS
Polysaccharide capsules Promotes abscess formation; Bacteroides fragilis, Porphyromonas
antiphagocytic function gingivalis
Fimbriae, fibrils enable organisms to
Adherence factors B. fragilis, P. gingivalis
adhere to cell surfaces
CLOSTRIDIAL TOXINS, EXOENZYMES
Collagenases Catalyze the degradation of collagen Certain Clostridium spp.
Cytotoxins Toxic to specific types of cells C. difficile
DNases Destroy DNA Certain Clostridium spp.
Enterotoxin Toxic to cells of the intestinal mucosa C. difficile
Lyse red blood cells liberating
Hemolysins Certain Clostridium spp.
hemoglobin
Hyaluronidase Catalyzes the hydrolysis of hyaluronic Certain Clostridium spp.
acid, the cement substance of tissues
Catalyze the hydrolysis of ester
linkages between fatty acids and
Lipases Certain Clostridium spp.
glycerol of triglycerides and
phospholipids
Neurotoxins (e.g., botulinum toxin, Destroy or disrupt nerve tissue C. botulinum, C. tetani
tetanospasmin)
Catalyze the splitting of host
Phospholipases Certain Clostridium spp
phospholipids (lecithinase)
Proteases Split host proteins by hydrolysis of Certain Clostridium spp
peptide bonds
o Non-encapsulated; except C.
GRAM-POSITIVE ANAEROBIC SPORE-FORMING perfringens.
BACILLI o Single haemolytic reaction; except C.
• Clostridum spp. perfringens
o Specie: C. perfringens, C. novyi, C. CLOSTRIDIUM PERFRINGENS (GAS
histolyticum, C. bifermentans, C. GANGRENE BACILLUS)
sordellii, C. innocuum, C. botulinum and • Most commonly isolated member
C. tetani • Virulence factor: a-toxin and enterotoxin
o Histotoxic: C. perfringens, C. novyi, C. • Microscopy: “Boxcar-shaped” bacilli with
histolyticum, C. septicum, C. subterminal spores
bifermentans • Biochemical:
• Most commonly causes myonecrosis is C. o (+) Lecithinase – EYA (egg yolk agar)
perfringens o (+) Nagler test – Lecithovitellin
CLOSTRIDIUM o (+) Reverse CAMP Test –arrowhead-
• Obligate anaerobes, catalase negative, Gram- shaped zone of hemolysis towards test
positive, spore-forming bacilli organism
• Toxins: acquired through ingestion or open • Culture:
wounds that have been contaminated with soil o BAP: dome-shaped and grayish white
• Virulence contributors: Collagenase, with double zones of hemolysis
hyaluronidase, lecithinase (soil destruction) and ▪ Alpha and beta zones (double
phospholipase zones)
• Carbohydrate fermenter; except: C. tetani and o Litmus milk: Stormy fermentation of milk
C. histolyticum • Related Disease:
• Characteristics: o Gas gangrene (myonecrosis)
o Form endospores anaerobically ▪ Blister that has water inside,
o Motile; except: C. perfringens, C. tissue necrosis
ramosum and C. innocuum. o Clostridial necrotizing enteritis or
o Have swollen sporangia except: C. Enteritis necroticans
perfringens and C. bifermentans

TUAZON A. 33
 ▪ Ingested beta enterotoxin in a • BOTULISM: double or blurred vision, impaired
contaminated food. speech, difficulty in swallowing, weakness and
▪ Bloody diarrhea, abdominal pain paralysis.
CLOSTRIDIUM TETANI (TACK HEAD o Two types of botulism:
BACILLUS) ▪ Foodborne botulism
• Endospore is usually in dust, soil, or dirt/fecal(?) • Usually due to ingestion
of animals in the farm of the preform toxin in
• Virulence factor: Tetanospasmin (neurotoxin) preserved or meat-
• Microscopy: Drumstick or tennis-racket based food or canned
appearance (terminal spores) goods.
• Culture: • Commonly caused by
o BAP: slow, anaerobic, heavy, smooth, botulism toxin A.
and swarming growth, narrow zone of b- ▪ Infant botulism
hemolysis • Actual infection caused
• Biochemical: (+) gelatinase and indole; (-) by ingesting the
Lecithinase and lipase organism from the raw
• Infection: Tetanus, Tetanus neonatorum honey or through
• Tetanospasmin: endopeptidase cleaves the breastfeeding for
synaptic vesicle membrane protein, infants.
Synaptobrevin. CLOSTRIDIUM DIFFICILE
o Cause tension or cramping and twisting • Most Common cause of antibiotic-associated
in skeletal muscles that surrounds the diarrhea and pseudomembranous colitis (bloody
wound and tightness of jaw muscles. diarrhea with necrosis of colonic mucosa).
• Tetanus: Characterized by trismus or lock jaw • Acquired in hospitals by individuals who are
and risus sardonicus or distorted grin. IP: 3 to 21 receiving antibiotics
days. • “Infection control dilemma” among hospitalized
o Symptoms: muscle rigidity, difficulty of patients.
swallowing, rigidity of the abdomen, • Ferments fructose-producing formic acid that
chest, back & limbs changes the color of medium to pink to yellow.
• Virulence factor: Toxin A (enterotoxin) and
Toxin B (cytotoxin).
• Microscopy: Chains up to 6 cells that are
aligned from end to end with oval subterminal
endospores.
• Culture:
o Cycloserine-cefoxitin-fructose agar
• (left) Gramstained appearance of terminal (CCFA) – colonies exhibit yellow color
spores of Clostridium tetani and a “ground-glass” appearance.
• (right) Gram-stained appearance of subterminal o BAP: “Horse stable” odor; non-
spores of Clostridium sordellii haemolytic and produce fluorescent
CLOSTRIDIUM BOTULINUM (“CANNED FOOD” chartreuse. (under UV)
BACILLUS)
• Usually found in soil and aquatic sediments. LABORATORY DIAGNOSIS
• Potential bioterrorism agent COLLECTION, TRANSPORT AND STORAGE:
• Presence of subterminal spores • If cannot processed immediately: should be kept
• Virulence factor: Botulism toxin in room temp.
• Culture: BAP-b-haemolytic colonies • Transport promptly to the laboratory under
• Infection: Botulism anaerobic condition or with minimal O2
• Botulism toxin: neurotoxin that is considered exposure.
as one of the most potent natural toxins known RECOMMENDED SPECIMEN FOR ANAEROBIC
to man. CULTURE
o Cleaves the synaptic vesicle membrane • Specimen must be collected at the actual site;
protein, synaptobrevin. swabbing of mucosal surface is insufficient.
o Preventing exocytosis and the release • Needle aspiration: best specimen for anaerobic
of the neurotransmitter, Acetylcholine. culture
o Small amount to produce paralysis and • Swabs: only be used when performing
death aspiration is not possible or if a biopsy specimen
o Botulism antigens/agent: A to G (7 is not available.
antigenic types) • Swab should be placed into a 0.5 mL sterile
o Cause human disease: A, B and E thioglycolate broth.

TUAZON A. 34
 • Food and fecal specimens that are suspected of DIFFERENTIAL TEST:
C. perfringens food poisoning should be • Catalase Test:
transported at 4°C. o Catalase negative; Rgt: 15% H2O2
UNACCEPTABLE SPECIMENS FOR • Direct Nagler Test:
ANAEROBIC CULTURE: o Using EYA plate and C. perfringens type
• Swabs, Sputum, Bronchial washings, Feces and A antitoxin.
effluents from ileostomy and colostomy, and o (+): Inhibition of the lecithinase reaction
gastric and small bowel contents. that is produced by C. perfringens.
GRAM STAIN • Mouse Neutralization Test
• Spores are not observed in Gram-stained o Definitive identification test for C.
smears of clinical specimens that contain botulism
clostridia, unless incubated for many days. • Reverse CAMP Test:
• C. ramnosum and C. clostridioforme: Gm (-) o Confirm the presence of C. perfringens.
o (+) Arrowhead-shaped zone of
CULTURE:
hemolysis at the intersection of the two
• Culture media: Anaerobic blood agar, streaks towards clostridium isolates,
thioglycollate, EYA, CCFA, PYG, brucella blood • Cell Culture Cytotoxicity Test
agar, PEA and CNA. o Gold standard test for detection of C.
• Transport media: pre-reduced anaerobically difficile toxin. Requires 2-3 days to
sterilized (PRAS) medium and Amies medium. achieve positive result.
• Cycloserine and Cefoxitin in CCFA: Inhibit gram- • Lipid/Lipase And Lecithinase Test
negative coliforms. o (+) Lecithinase: opaque zone
• Neutral red: pH indicator in CCFA ▪ C. perfringens, C. bifermentans,
• EYA: detect lecithinase and lipase activity. C. novyi
o (+) Lipase “Mother of pearl” app. Or
gasoline on water appearance.
▪ C. botulinum, C. novyi
PRESUMPTIVE IDENTIFICATION OF GRAM-POSITIVE ANAEROBES
COLONY MORPHOLOGY ON BLOOD SPOT
IDENTIFICATION CELLULAR MORPHOLOGY
AGAR INDOLE
Clostridium difficile Large, flat colonies; barnyard odor, Thin rods, rare spores Negative
chartreuse fluorescence
Large, irregular-shaped, double zone of β-
C. perfringens Boxcar, large, square rods Not done
hemolysis
C. septicum Smoothly swarming Thin rods, subterminal spores Negative
C. sordellii Very large, lobate, irregular, flat Thin rods, subterminal spores Positive
C. tetani Smoothly swarming but slow growing Swollen terminal spores Positive
“Peptostreptococci” Small, peaked, circular Cocci, pairs and chains Not done
Cutibacterium acnes Small, opaque, enamel white, circular Coryneform rods Positive
(catalase-positive)

ACCEPTABLE SPECIMENS FOR ANAEROBIC BACTERIOLOGY

ANATOMIC SOURCE SPECIMENS AND RECOMMENDED METHODS OF COLLECTION
Central nervous system Cerebrospinal fluid, aspirated abscess material, tissue from biopsy or autopsy
Dental, ear-nose-throat specimens Aspirated abscess material, biopsied tissue
Localized abscesses Needle and syringe aspiration of closed abscesses
Decubitus ulcers Aspirated pus
Sinus tracts or draining wounds Aspirated material
Specimens obtained during surgery from depths of wound or underlying bone
Deep tissue or bone
lesion
Pulmonary Aspirate obtained by direct lung puncture; pleural fluid obtained by
thoracentesis; open lung biopsy; sulfur granules from draining fistula
Intraabdominal Aspirate from abscess, ascites fluid, peritoneal fluid, tissue
Urinary tract Suprapubic bladder aspiration
Female genital tract Aspirate from loculated abscess; culdocentesis specimen
Other Blood, bone marrow, synovial fluid, biopsied tissue from any normally sterile
site

TUAZON A. 35
 DIFFERENTIAL CHARACTERISTICS OF ANAEROBIC NON-SPORE FORMING BACILLI AND COCCI
DISTINGUISHING
ORGANISM GRAM STAIN REACTION
CHARACTERISTICS
Actinomyces Anaerobic, straight or slightly curved, Gram- Young colonies –spider-like or wooly
positive rods that are banded or beaded appearance
Old colonies – “molar tooth”
appearance
Pale-staining, pleomorphic, Gram-negative rods Grayish-white, circular, smooth, and
Bacteroides fragilis
with a “safety pin” appearance non-hemolytic
Bacteroides ureolyticus Pale-staining, thin, Gram-negative rods Colonies corrode (pit) the agar
Gram-positive diphtheroids; that are coccoid or
Small, white, shiny and convex
Bifidobacterium spp. pointed in shape with bifurcated (forked) ends
colonies
which resemble a shape of a “dog bone”
Clostridium septicum Gram positive rods in young culture that turn Formation of Rhizoid margins that
Gram negative with age; Have subterminal resemble “medusa head”
spores
Pleomorphic, Gram-positive rods that are
Eubacterium Fluorescent chartreuse color
seagull wing-shaped
Fusobacterium nucleatum Spindle-shaped, Gram-negative rods that The medium exhibits a green color
resemble a Capnocytophaga upon air exposure; colonies have
“breadcrumb-like” appearance
Gram variable rods or short coccobacilli that
Lactobacillus Pinpoint colonies
resemble streptococci
Leptotrichia Large-fusiform Gram-negative rods “Raspberry-like” colonies
Gram positive cocci that are paired singly, pairs
Peptococcus niger Small black and shiny colonies
or in tetrads
Peptostreptococcus Large, Gram-positive coccobacilli in chains Grayish-white colonies that emits foul
anaerobius odor.
Brown, mucoid colonies with brick red
Porphyromonas Gram negative coccobacilli
fluorescence
Propionibacterium Diphtheroids-like, Gram-positive rods; that have Small, grayish-white colonies
(anaerobic diphtheroids) a palisade arrangement
White, Shiny colonies with a brick-red
Prevotella Gram negative rods
fluorescence
Veillonella parvula Tiny Gram-negative diplococci Red fluorescence

PRESUMPTIVE IDENTIFICATION OF GRAM-NEGATIVE ANAEROBES

COLONY
COLONY MORPHOLOGY ON CELLULAR SPOT
IDENTIFICATION MORPHOLOGY ON
BLOOD AGAR OR KVLB AGAR MORPHOLOGY INDOLE
BBE AGAR
Bacteroides fragilis Large (>1 mm) Large, convex, black Regular Not done
group gray
Campylobacter Tiny rods or
Translucent, pitting (some) No growth Negative
ureolyticus coccobacilli
Bilophila Tiny, translucent Translucent, with Regular to filamentous Negative
wadsworthia black center at 72 h
Fusobacterium Fusiform, thin with
Ground glass or breadcrumb No growth Positive
nucleatum pointed ends
Porphyromonas Small, translucent or opaque, No growth Tiny coccobacilli Positive
brick red fluorescence on blood
agar, no growth on KVLB agar
Small, translucent or opaque,
Prevotella brick red
No growth Tiny coccobacilli Negative
intermedia fluorescence on blood agar and
KVLB agar
Veillonella Small, translucent or opaque, No growth Tiny diplococci Negative
brick red
fluorescence on blood agar, no
growth on
KVLB agar

TUAZON A. 36
 CHARACTERISTICS OF SOME CLINICALLY ENCOUNTERED ANAEROBIC COCCI

OTHER MICROORGANISMS PROPIONIBACTERIUM ACNE

BACTEROIDES FRAGILIS • Indigenous microbiota of the skin
• Most commonly isolated anaerobes from blood • Frequently isolated from blood culture
cultures LACTOBACILLUS
• B-Lactamase producer, non-motile and • Pleomorphic, Gram-positive rods
saccharolytic. • Non-motile, aerotolerant
• Cause: intra-abdominal abscesses. • Species: L. acidophilus, L. fermentum, L.
• Culture: Bacteroides bile esculin (BBE) agar – vaginalis and L. salivarius
exhibit a gray color and growth with 20% bile
and cause the blackening of the originally LACTOBACILLUS ACIDOPHILUS
yellow-colored agar. • Part of indigenous microbiota of the mouth, GIT
• Biochemical: (+) Esculin hydrolysis and vaginal canal.
ACTINOMYCES ISRAELLI • Protects female genital tract from urogenital
• Most common cause of infections
actinomycosis • Related infection: Bacterial vaginosis
• Indigenous microbiota of oral • Differential medium: Tomato juice agar (pH 3 to
cavity. 4)
• Molar tooth appearance • Biochemical Test: (-) catalase, H2S and esculin
hydrolysis.

FLUORESCENCE UNDER LONG-WAVE ULTRAVIOLET LIGHT

ORGANISM COLOR OF FLUORESCENCE
Prevotella (pigmented) Brick red
P. bivia, P. disiens Light orange to pink (coral)
Porphyromonas Brick red (some no fluorescence)
Fusobacterium Chartreuse (yellow/green)
Veillonella Red but fades rapidly
Clostridium ramosum Red
C. innocuum, C. difficile Chartreuse
Eggerthella lenta Red or no fluorescence

PRIMARY SETUP MEDIA RECOMMENDED FOR RECOVERY OF ANAEROBES

MEDIUM ORGANISMS COMMENTS
Anaerobic blood agar (CDC) Supports growth of almost all obligate An enriched medium containing sheep
and facultative anaerobes, best for blood for enrichment and detection of
anaerobic gram-positive cocci hemolysis, vitamin K (required by
some Porphyromonas spp.), and yeast
extract
Bacteroides bile esculin agar Supports growth of bile-tolerant A selective medium containing

TUAZON A. 37
 Bacteroides spp., some strains of gentamicin (which inhibits most
Fusobacterium mortiferum, Klebsiella aerobic organisms), 20% bile (which
pneumoniae, enterococci, and yeasts inhibits most anaerobes), and esculin;
may grow to a limited extent used primarily for rapid isolation and
presumptive identification of members
of the B. fragilis group, which grow
well on Bacteroides bile esculin agar
(because of their bile tolerance) and
turn the originally light-yellow medium
to black (because of esculin
hydrolysis)
Brucella blood agar Supports growth of almost all obligate An enriched medium containing sheep
and facultative anaerobes, best for red blood cells for enrichment and
gram-negative bacteria detection of hemolysis, casein
peptones, dextrose, yeast extract,
vitamin K, and hemin
A selective medium containing
kanamycin (which inhibits most
facultative gram-negative bacilli),
vancomycin (which inhibits most gram-
positive organisms and vancomycin-
Supports growth of Bacteroides and
sensitive strains of Porphyromonas
Kanamycin–vancomycin–laked Prevotella spp.; yeasts and
spp.), and laked blood (which
blood agar kanamycin-resistant, facultative, gram-
accelerates production of brown-black
negative bacilli will also grow
pigmented colonies by certain
Prevotella spp.); used primarily for
rapid isolation and presumptive
identification of pigmented species of
Prevotella
Phenylethyl agar Supports growth of almost all obligate A selective medium containing sheep
anaerobes (gram-positive and gram- red blood cells and phenylethyl
negative) and gram-positive, alcohol; used primarily to suppress the
facultative anaerobes growth of any facultative, gram-
negative bacilli (e.g.,
Enterobacteriaceae) that might be
present in the clinical specimen,
especially swarming Proteus spp.
A selective medium containing sheep
red blood cells and the antimicrobials
Supports growth of almost all obligate colistin and nalidixic acid; used
Colistin–nalidixic acid blood agar anaerobes (gram-positive and gram- primarily to suppress the growth of any
plate negative) and gram-positive, facultative, gram-negative bacilli (e.g.,
facultative anaerobes Enterobacteriaceae) that might be
present in the clinical specimen,
especially swarming Proteus spp.
Anaerobic broth (e.g., thioglycollate Supports growth of almost all types of Because obligate anaerobes can be
and chopped or cooked meat) bacteria; in thioglycollate broth, overgrown by more rapidly growing
obligate aerobes and microaerophiles facultative organisms present in the
grow near the top, obligate anaerobes specimen and killed by their toxic,
at the bottom, and facultative metabolic by-products, thioglycollate
anaerobes throughout the broth broth serves only as a
backup source of culture material
(e.g., in case there is no growth on
plated media because of a jar failure
or presence of antimicrobial agents in
the specimen); chopped meat
carbohydrate broth can be used in
place of thioglycollate broth; broth
cultures should never be relied on
exclusively for isolating anaerobes
from clinical material

TUAZON A. 38
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
LESSON 4.1: RICKETSSIACEAE A ND andersoni)
RELATED ORGANISMS Dog ticks
RICKETTSIA (Dermacentor
variabilis)
• Simplest bacterial form and considered as
Brown Dog
transitional organism between bacteria and
ticks
virus.
(Rhipicephalu
• Fastidious bacteria and obligate, intracellular
s sanguineus
parasites.
and
• Gram-negative cell wall, motile, will not grow in Amblyomma
cell-free media
cajennense
• Multiply: Binary fission TRANSITIONAL GROUP
• Survive briefly outside of their host Mouse mite
• Microscopy: Small, pleomorphic, gram-negative Rickettsia akari Rickettsial pox (Liponyssoide
bacilli. They do not undergo any intracellular s sanguineus)
developmental cycle. Rickettsia felis Flea-borne spotted Flea bite or
• Culture: require living cells for growth fever feces
• MOA: Human become infected following the bite TYHPUS GROUP
of an infected arthropod vector. (lice, tick, mites) Rickettsia Epidemic Body louse
• Mode of prevention: Avoid contact with prowazekii typhus/Brill- (Pediculus
respective vector Zinsserdisease humanus
• Accidental host: Humans corporis)
• Agents of bioterrorism: R. prowazekii and R. Squirrel flea
rickettsii. (Orchopeas
• Rickettsia is divided into three groups: howardi)
o Spotted fever group Squirrel louse
o Transitional group (Neohematopi
o Typhus group nus
PATHOGENSIS OF RICKETTSIA sciuriopteri)
SPOTTED FEVER GROUP: Rat flea
Endemic murine
• Rocky mountain spotted fever (RMSF) most Rickettsia typhi (Xenopsylla
typhus
serious rickettsial infection. cheopis)
• For RMSF, humans are the accidental hosts and SCRUB TYPHUS GROUP
ticks are the main vector and reservoir. Chigger
• Rashes developed on the palms of the hands Orientia (Leptotrombid
Scrub typhus
and soles of the feet. tsutsugamuchi ium deliense)
• Boutonneuse fever: Rashes that are similar to bite
RMSF but on face. ANAPLASMATACEAE
• Tache noires (black spot): present in BF Lone star tick
Ehrlichia Human Monocytic
• Rickettsialpox: rashes face and extremities (Amblyomma
chaffeensis ehrlichiosis
americanum)
TYPHUS GROUP
Anaplasma Human Deer tick
• Endemic typhus characterized by rashes on phagocytophila granulocytoropic (Ixodes
the face, palms, and soles of feet of the sick. anaplasmosis scapularis
• Rashes are not commonly observed and Ixodes
• Inhalation of aerosol from dried infected flea pacificus)
feces is also a mode of transmission of OTHER ORGANISMS
Rickettsia typhi infection. Coxiella Q fever Inhalation of
INFECTION/DISEAS VECTOR/MO burnetti aerosol and
ORGANISM
E T infected
SPOTTED FEVER GROUP animals
Rickettsia Boutonneuse fever Ticks Feces of body
conorii or (Rhipicephalu louse
Mediterranean s sanguineus) Bartonella
Trench fever (Pediculus
spotted fever quintana
humanus
Rickettsia Rocky Mountain Wood ticks corporis)
rickettsii Spotted Fever (Dermacentor Bartonella Cat scratch disease Kitten scratch

TUAZON A. 39
henselae Bacillary or bite • MOA: inhalation of contaminated aerosols from
angiomatosis dried animal feces and ingestion of
Sandfly contaminated unpasteurized milk
Bartonella Oroya fever and
(Lutzomyia) • Animal reservoir: Cattle, goats and sheep
bacilliformis verruga peruana
bite EHRLICHIA
ORIENTIA TSUTSUGAMUSHI • Genus belongs to the family Anaplasmataceae
• Belong to family Rickettsiaceae • Species of the genus are Gram-negative
• Categorized as a separate genus due to the coccobacilli that undergo an intracellular
absence of lipopolysaccharide and development cycle following the infection of
peptidoglycan and the presence of 54 to 58 kDa circulating WBC’s (replicate ion occurs in the
major surface protein leukocytes)
• It replicates in the cytoplasm of its host cell and • Microscopy: Presence of intravacuolar
is released through a process that involves microcolony that resembles “mulberries” or a
“pinching off” the host cell morula.
• MOA: Bite of an infected arthropod vector THREE DEVELOPMENTAL STAGES
• Vector: Leptotrombidum delicense (Chigger) • Elementary body (infective form)
• Accidental host: Humans and rats • Initial bodies
ANAPLASMA PHAGOCYTOPHILA • Morulae
• It causes human ➢ Natural hosts: Human and animals (dog and
granulocytotropic deer)
anaplasmosis ➢ Primary vector: Amblyomma americanum
• Vector: Ixodes pacificus ➢ Species: E. chaffeensis and E. ewingii
(Western black-legged ➢ Infection: Human monocytic ehrlichiosis
tick) and Ixodes scapularis (deer tick) LABORATORY DIAGNOSIS FOR RICKETTSIAEAE
• Reservoir: Peromyscus leucopus (white-footed DIRECT METHODS
mouse) • Immunohistology (Immunofluorescence and
BARTONELLA immunoenzyme stains)
• The species of this genus are facultative, o Skin biopsy is utilized or usually used.
intracellular and Gram-negative bacilli. • Giemsa stain
• They live within the RBC in their natural CULTURE
mammalian hosts • Culture media: Yolk sacs of embryonated eggs
• Species can be cultivated in CAP with 5% CO2 and tissue culture
or in charcoal yeast extract agar. • Rickettsia, Ehrlichia and Anaplasma can be
• Some species exhibit a “twitching motility in wet isolated from human in an antibiotic-free,
mounts (B. bacilliformis and B. henselae). centrifugation-enhanced shell vial cell culture
INFECTIOUS AND • Columbia blood agar with 5% defibrinated blood
SPECIES
DISEASES for B. bacilliformis
Trench fever (louse-borne • Lung tissue cells are the preferred medium for
B. quintana
disease) C. burnetti
B. henselae Cat scratch disease SEROLOGICAL TEST
B. elizabethae Infective endocarditis
B. bacilliformis Oroya fever (chronic • Only test preferred for diagnosis of rickettsial
verruga peruana) and disease.
febrile acute haemolytic • It is used to confirm rickettsiosis/ricketsioses
anemia during convalescence stage.
Cat scratch disease • Antibodies to rickettsia can be detected until at
B. clarridgeiae least 2 weeks after the infection.
(secondary agent)
COXIELLA BURNETTI • Rickettsiosis are seldom diagnosed serologically
during the acute stage of the illness due to the
• Only specie in the genus Coxiella
absence of an early antibody response.
• Causative agent of Q (Query) fever which is a
systematic infection of the lungs
INDIRECT IMMUNOFLUORESCENT ANTIBODY
• Extremely contagious and can be considered as (IFA) ASSAY
a potential bioterrorism agent • Gold standard serologic test or reference
• Can survive extracellularly because of its method for rickettsioses and Q fever.
endospore-like body WEIL-FELIX REACTION
• Can infect birds and rodents, which in turn • Presumptive test for rickettsioses
excrete the organisms via their urine, feces and • Agglutination of certain strains of Proteus
birth products vulgaris by serum from patient.
• Not transmitted by arthropod vectors

TUAZON A. 40
 • Individual with Q fever, ehrlichiosis and CHLAMYDIA TRACHOMATIS
rickettsial pox do not produce Weil-Felix • Major sexually transmitted pathogens
antibody • Principal cause of: PID (pelvic inflammatory
MICROIMMUNOFLUORESCENT DOT TEST disease) and ocular trachoma
• Excellent for detecting antibodies to Rickettsia • Travel through the birth canal where infants can
• Used for early diagnosis of RMSF be infected during birth
NUCLEIC ACID AMPLIFICATION (PCR) • Associated within fertility and ectopic pregnancy
• Cat scratch disease can be performed through • Natural host: Humans
PCR testing of lymph nodes. • Unique features:10 stable plasmids
• PCR a diagnostic tool for ehrlichiosis • Biovars: Lymphogranuloma venereum (LGV),
CHLAMYDIACEAE mouse and pneumonitis trachoma
• Genus are non-motile, small (0.2to1.5um) and • Serovars of C. trachomatis based on MOMP
have Gram-negative cell wall (major outer membrane protein) antigenic
differences:
• Obligate, intracellular organisms that requires
living cells for growth • Serotypes A, B, Ba, C: endemic trachoma
• Serotypes L1, L2, L2a,
• Do not possess cytochromes and cannot
synthesize their own ATP. L2b, L3 – LGV
• They are called “energy parasites” because they • Serotypes D to K, Da,
depend on the eukaryotic cells of their host for Ia, Ja – PID, urethritis,
metabolism, growth and reproduction. cervicitis, epididymitis
and inclusion
• Replicate by binary fission in the cytoplasm of
conjunctivitis
infected cells.
TWO MORPHOLOGIC FORMS: RELATED INFECTION:
o Trachoma
RETICULATE BODY (RB) o LGV
• replicative and non-infectious form o Inclusion conjunctivitis
• Intracellular and metabolically active form of TRACHOMA
Chlamydia.
• Chronic inflammation of the
• Multiply: Binary fission conjunctiva that lead to
ELEMENTARY BODY (EB) blindness
• Infectious form. • Cause distortion of the eyelids
• Extracellular from a Chlamydia and is spherical (eyelashes become
in shape. misdirected and turned-in)
• Resembles Gram-negative bacilli with a rigid cell • MOT: Contact with contaminated objects, hand
wall to hand contact with
• Infects cells through inducing active carriers and through
phagocytosis vectors (flies)
LYMPHOGRANULOMA
VENEREUM (LGV)
• Sexually transmitted
disease which has a multi-system involvement
• Small, painless ulcer or papule appears initially
and then nodules (buboes) develop after several
weeks
INCLUSION CONJUNCTIVITIS
• Characterized by an abundant eye discharge
and swollen conjunctiva
• Affects infants who acquired it through aspiration
or ocular exposure during birth

HUMAN DISEASES CAUSED BY CHLAMYDIACEAE SPECIES

SPECIES SEROVARS DISEASE HOST
Chlamydia trachomatis A, B, Ba, C Trachoma Humans
Inclusion conjunctivitis (adult
and newborn)
D, Da, E, F, G, H, I, Ia, J, Nongonococcal urethritis
Humans
Ja, K Cervicitis
Salpingitis
Pelvic inflammatory disease

TUAZON A. 41
 Endometritis
Acute urethral syndrome
Proctitis
Epididymitis
Pneumonia of newborns
Perihepatitis (Fitz-Hugh–
Curtis syndrome)
L1, L2, L2a, L2b, L3 Lymphogranuloma
venereum
Pneumonia, bronchitis
Chlamydophila
1 Pharyngitis Humans
pneumoniae
Influenza-like febrile illness
Chlamydophila psittaci 10 Psittacosis Birds
Endocarditis
Abortion

LABORATORY DIAGNOSIS o Complement fixation: family-reactive

• Specimen: Urethra and cervical secretions, antibodies. Used to diagnosed LGV.
conjunctiva discharge, nasopharynx and rectal Genus specific antigen. (+) Titer: > 1:64
swabs and material aspirated from fallopian o Microimmunofluorescence (MICRO-
tubes and epididymis IF) Assay: Used for type-specific
• Use Dacron and rayon-tipped swabs is antibodies of C. trachomatis
preferred. ▪ Preferred method for
CULTURE: identification of C. trachomatis
• Culture media: Buffalo green monkey kidney ▪ It can be used for the diagnosis
cells, He La 299 cells, Hep-2 cells, McCoy cells of LGV, trachoma, inclusion
and Cyclohexamide-treated McCoy cells. conjunctivitis, and chlamydial
neonatal infection.
CYTOLOGIC EXAMINATION:
▪ (+): IgM titer of 1:32
• Cell scrapings from conjunctiva of newborns or
persons with ocular trachoma.
IMMUNOASSAY
• Enzyme immunoassay (EIA): Most commonly
• Direct fluorescent antibody (DFA)
used rapid antigen assay
o Fluorescein-isothiocyanate-conjugated
monoclonal antibofy • Detects the outer membrane LPS chlamydial
antigen or MOMP antigen
ANTIGEN DETECTION AND NAAT
o Major outer membrane protein
• Nucleic acid amplification is the most sensitive
method for detection of C. trachomatis.
• LPS antigen major antigen that is detected
• Specimen: Endocervical and urethral swabs.

CHLAMYDOPHILA PSITTACI
• Causative agent of psittacosis or ornithosis
• Endemic pathogen of birds’ specie such as
SERODIAGNOSIS parrots, parakeets, chicken and ducks
• Extractable LPS and elementary body with keto- • MOA: Inhalation of infected aerosols from dried
deoxyoctonate is the primary antigen that can be bird excreta or handling of infected birds
identified in genus specific test. • Commonly used test: Complement fixation
• Negative test: exclude chlamydial infection. with titer of >1:32

TUAZON A. 42
 • Sensitive method: Direct • Human pathogen that is transmitted through
microimmunofluorescence aerosol droplets.
• Precautionary measures: Only laboratories • One of the major causes of infectious respiratory
with biosafety level 3 facilities can perform the disease
isolation and cultivation of the specimens • Associated with pneumonia, bronchitis,
CHLAMYDOPHILA PNEUMONIAE pharyngitis and sinusitis.
• Formerly known as the Chlamydia pneumonia • Specimen for isolation: Nasopharyngeal
strain TWAR. aspirates, sputum and throat swabs.
o TWAR – Taiwan Acute Respiratory • Culture media: He LA cells or Hep-2 cell lines
Chlamydia • Preferred method: Microimmunofluorescent
assay.
PROPERTIES C.TRACHOMATIS C. PSITTACI C. PNEUMONIAE
Host range Humans Birds Humans
Elementary body Round Round Pear-shaped
Round, vacuolar
Inclusion morphology and Variable, dense Levinthal-
Halberstaeder-Prowazek Round, dense
inclusion body Cole-Lillie bodies
bodies
Stain used Macchiavello stain and
Lugol’s Iodine Giemsa stain
Giemsa stain
Glycogen-containing
Present Absent Absent
inclusions
Susceptibility to
Sesceptible Resistant Resistant
sulfonamides
Trachoma, LGV and
Disease Psittacosis Pneumonia, Pharyngitis
inclusion conjunctiva
Number of Serovars 20 10 1

COMPARATIVE PROPERTIES OF MICROORGANISMS

• Zoonotic disease acquired by humans through

inhalation of dried excreta of animals or infected
birds.
• Causes: mild flu, lung infection
• Also known as Ornithosis or Chlamydiosis.
LESSON 5: CELL WALL-DEFICIENT &
SPIROCHETES AND MISCELLANEOUS
BACTERIA
CELL WALL DEFICIENT BACTERIA
• Mycoplasma
• Ureaplasma
• Specie: M. pneumoniae, M. hominis, M.
fermentans, M. pirum, M. penetransand U.
PARROT DISEASE urealyticum
• Psittacosis (1929) MYCOPLASMA AND UREAPLASMA
o Ducks, pigeon, hen, sparrow, cockatiels, • Both mollicutes
macaws, budgerigars

TUAZON A. 43
 • Smallest free-living organisms that are capable • Swab: Calcium alginate and Dacron swabs.
of growing in artificial media. • Transport medium: SP4 (sucrose phosphate
• Pleomorphic organisms that lack cell wall buffer, horse serum and neural red).
• They found mainly in the oropharyngeal, upper • Mycoplasma and Ureaplasma grow slowly
respiratory and genitourinary tracts. than most of the other bacteria
• Slow growing, fastidious and facultative • M. hominisis the only species that is capable of
anaerobes that replicate by binary fission. growing on BAP and CAP
• Requires sterols (cholesterol) for membrane • M. Pneumoniae requires biphasic culture
function and growth system and incubation of up to three weeks in
• Common parasites of the genital tract and their chamber with 5% to 10% CO2
transmission is related to sexual activity. • Urea and/or arginine: incorporated into the
• Etiologic agent: Primary atypical pneumonia or media to detect the presence of U. urealyticum
walking pneumonia and M. hominis and produce alkaline reaction.
• Extremely fastidious • Mycoplasma in Shepard’s 10B arginine
• MOA: Inhalation of contaminated aerosol medium: red color. Blood culture: not
droplets recommended
• Initiation of Disease: Attachment to respiratory • Serodiagnosis
mucosal cells, evasion from phagocytosis and o ELISA: Most commonly used
modulation of the immune system. serological method for diagnosis of
• Culture: SP4 broth: Yellow color Mycoplasma and Ureaplasma.
ATYPICAL PNEUMONIA • Manganese Chloride Urea Test
o Rapid identification test for U.
• Can occur as separate incidents or as outbreaks
urealyticum
in closed population such as in school, military
o Reaction for the test is observed under
camps and within family members.
a dissecting microscope.
MYCOPLASMA HOMINIS & UREAPLASMA o (+) results: dark brown precipitate of
UREALYTICUM(GENITAL MOLLICUTES) manganese oxide around the colonies.
• Recovered from the genital tract of healthy o U. urealyticum utilizes manganese
adults. chloride in the presence of urea.
• Cause prostatitis and pelvic inflammatory
disease. • Dienes stain of
• Colonization among infants occurs during Mycoplasma spp. colonies
passage through an infected birth canal and demonstrating typical fried
results in the isolation of these organisms from egg appearance
their nose and throat.
• M. hominis: Causative agent of postabortal
fever and postpartum fever in women. • Typical mixed sizes of
• Culture: Mycoplasma spp. on
o M. hominis: “Fried-egg” appearance on primary isolation
plated medium media, Mycoplasma
o U. urealyticum: “dark-brownish lumps” salivarium.
on a A7 or A8 Agar media.
LABORATORY DIAGNOSIS
• Specimen: Throat swab, serum,
bronchoalveolar lavage, sputum and lung tissue
for M. pneumoniae
• Genital mycoplasma: urethral, vaginal or
endocervical swab, blood, urine, prostatic
secretions and semen.
• No direct method or gram staining can be
used for identification since mycoplasma
and ureaplasma lacks cell wall.
CULTURE:
• Culture media: Beef or Soybean protein with
serum, fresh yeast extract, biphasic SP4
medium, pleuropneumonia-like organism
(PPLO) broth or agar with yeast extract and
horse serum, A8 agar (usually used for
ureaplasma), Shepard’s 10B arginine broth and
modified New York City medium.

TUAZON A. 44
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
SUMMARY OF ASSOCIATION OF GENITAL MOLLICUTES WITH UROGENITAL AND NEWBORN DISEASES
Disease, Target Mycoplasma Mycoplasma
Ureaplasma spp. Comments
Population hominis genitalium
Nongonococcal Ureaplasma spp.
urethritis cause some cases,
None Weak Strong
but the proportion is
unknown
An association with a
few cases of chronic
Prostatitis None Weak None disease has been
reported; a causal
relation is unproven
Epididymitis Mycoplasma spp. are
None None Weak not an important
cause
M. hominis is often
Vaginitis and associated with
None None None
cervicitis disease, but a causal
relation is unproven
Pelvic inflammatory M. hominis causes
disease Strong Strong None some cases, but the
proportion is unknown
Recent studies
indicate that M.
Postpartum fever Strong None Weak
hominis may be a
major cause
Urinary calculi Ureaplasma spp.
cause calculi in male
rats, but no
None None Weak convincing evidence
exists that they cause
natural human
disease
M. hominis causes
Pyelonephritis Strong None None
some cases
Involuntary infertility Ureaplasma spp. are
associated with
Weak None
altered motility of
sperm
An association exists,
Chorioamnionitis Strong None Strong but a causal relation
is unproven
Low birth weight An association exists,
None None Strong but a causal relation
is unproven
Further clarification is
Neonatal infections,
needed, but
including sepsis,
Strong Strong importance is growing
pneumonia,
in a selected prenatal
meningitis
population
Neonatal period, These findings need
particularly preterm further clarification
delivery, very low birth because most
weight; clinical signs Strong Weak Strong neonatal infections
compatible with resolve without
meningitis (CSF), therapy, but in low
pneumonia (trachea), socioeconomic

TUAZON A. 45
sepsis (blood) groups the diagnostic
workup of newborns
should include CSF
and blood cultures for
detection of
mycoplasmas. This
includes low-birth-
weight and preterm
newborns, in whom
traditional CSF cell
counts and cultures
would be negative

• Identification of Mycoplasma-infected cell culture using DNA fluorochrome

stain (Hoechst 33258 stain). A, Mycoplasma orale. B, Uninfected Vero cell culture
highlighting the DNA-rich nucleus. C, Mycoplasma salivarium. The mycoplasma
appear as small, pinpoint, fluorescent bodies throughout the background.

• Mixed isolation of Mycoplasma hominis and Ureaplasma urealyticum showing

why U. urealyticum was originally called “T” for “tiny strain”.

COMPARATIVE FEATURES OF VARIOUS LABORATORY METHODS USED TO DETECT MYCOPLASMA

PNEUMONIAE, MYCOPLASMA HOMINIS, AND UREAPLASMA UREALYTICUM
Detection Method Mycoplasma pneumoniae Mycoplasma hominis Ureaplasma urealyticum
NONSEROLOGIC
Method of choice using
Method of choice, but must
urease detection, but must
Culture Traditionally difficult differentiate infection
differentiate infection from
from colonization
colonization
Indirect Respiratory antigen for
immunofluorescence early-stage infection
Research use only but is
promising
Polymerase chain reaction Assays are being evaluated Assays are being evaluated Assays are being evaluated
SEROLOGIC
Traditional assay but <50%
seroconvert; need fourfold
rise between acute-phase
Complement fixation
and convalescent sera; >32
single titer may be
suggestive
Immunofluorescent Measures IgG and IgM Measures IgG and IgM
antibody separately separately; not
recommended
Latex agglutination IgM/IgG IgG only
Enzyme immunoassay Method of choice
Reactive IgM, IgG, and IgA,
but IgM level may remain
elevated for 1 year

TUAZON A. 46
SPIROCHETES AND MISCELLANEOUS BACTERIA fresh blood, injuries from contaminated needle
• Treponema sticks and handling specimen
• Borrelia • Symptoms: Chancre (hard chancre), fever, sore
• Leptospira throat, headache, rashes (palm and soles) and
• Miscellaneous: Streptobacillus moniliformis, gummas on skin.
Spirillum minus, Klebsiella granulomatis, o Soft chancre: H. ducreyi
Capnocytophaga and Enterococci • Stages of syphilis:
GENERAL CHARACTERISTICS OF o Primary syphilis
SPIROCHETES o Secondary syphilis
o Latent stage
• Belongs to the order
o Tertiary stage or Late stage
Spirochaetales
• Unusual morphologic
PRIMARY SYPHILIS
features • Appearance of hunterian or hard chancre,
• Facultatively anaerobic • Painless, usually seen on the genitalia
or aerobic • Develops 10 to 90 days after infection
• Various types of motility • No systemic signs and symptoms
pattern SECONDARY SYPHILIS
• Have fibrils or axial filaments which are flagella- • Develops 2 to 12 weeks after appearance of
like organelles that wrap around the bacterial chancre
cell wall and facilitate motility (exhibiting a • All lesions that are observed seen in this phase
“corkscrew like” winding) are highly infectious
• General microscopy: slender, helical and • Chancre heals but organisms are still
unicellular bacteria disseminated via blood stream
• Genera: Treponema, Borrelia and Leptospira. • Symptoms: Fever, Sore throat, headache and
TREPONEMA rashes (palms and soles)
• Greek word: Trepein means “to turn” and nema LATENT STAGE
“thread”, “turning thread” • Disease becomes subclinical but not necessarily
• Darkfield microscope dormant
• Infects only human • Occurs within more than a year of infection
• Stain poorly with Gram or Giemsa stains • In this stage, diagnosis can be made only by
• Reproduction: Transverse fission serological test
• Microscopy: Thin, spiral organisms with three TERTIARY STAGE/LATE SYPHILIS
axial filaments. Cell end are pointed and
• Tissue-destructive phase
covered with a sheath.
• Appears 10 to 25 years after initial infection
TREPONEMA PALLIDUM SUBSP. PALLIDUM
• In this stage, individuals are not usually
• Causative agent of syphilis infectious
• Microaerophiles which survives longer in the • Complications: Central nervous disease
presence of 3% to 5% oxygen, (neurosyphilis), cardiovascular abnormalities,
• Killed rapidly at 42C eye disease and granuloma-like lesions
• Remains visible in whole blood or plasma for (gummas)
atleast 24hrs, which is potential importance in LABORATORY DIAGNOSIS
blood transfusion. • Specimen: Skin lesions (cleaned with saline)
• Can cross intact mucous membrane and • Oral lesions: should not be examined because
placenta non-pathogenic spirochetes will lead to false-
• Darkfield microscopy: White against a dark positive results.
background and long with fine spirals that have MICROSCOPIC EXAM
10 to 13 coils and three fibrils/periplasmic
flagella. • Direct examination of exudates is recommended
and motility
• Generationtime:30hours
• Definitive test: Darkfield microscopy
• Diagnostic Test:
o Treponemal reagin • Stain: Levaditi’s stain and Fontana-Tribondeau
o Non-treponemal reagin stains.
SYPHILIS • Direct detection in lesions: FITC (Fluorescein
isothiocyanate-labeled antibody)
• AKA: French disease / Italian disease / The SERODIAGNOSIS (SECONDARY AND
Great Pox
TERTIARY)
• Disease of blood vessels and perivascular areas
• Known as “great imitator” • Non-treponemal Test/Non-Specific Test –
• MOA: sexual contact, congenital transmission, Screening test. Detects the presence of non-
skin contact with active lesion; transfusion of specific antibody or antibody-like protein like

TUAZON A. 47
 your regain or Wassermann antibodies. This is GENERAL CHARACTERISTICS OF BORRELIA
used to monitor syphilis. • Slow growing sphirochetes that multiply by
o RPR - Rapid Plasma Reagin binary fission
o VDRL - Venereal Disease Research • Composed of 3 to 10 loose coils and is actively
Laboratory motile
o USR - Unheated Serum Reagin • Species have 15 to 20 axial filaments and two
o TRUST - Toluidine Red Unheated insertion disks
Serum Test • They stain well with Geimsa stain and can be
o ELISA - Enzyme-Linked Immunosorbent visualized by using brightfield microscopy.
assay • Species that have been grown in vitro are
• Treponemal Test/Specific Test – Confirmatory microaerophilic.
test. Usually used to detect the presence of BORRELIA SPP.
antibody to the treponemal antigens.
o FTA-ABS - Fluorescent Treponemal
BORRELIA RECURRENTIS
Antibody Absorption • Agent of louse-borne/ epidemic/ European
o MHA-TP - Microhemagglutination Assay relapsing fever
for Treponema Pallidum • Vector: Louse (Pediculus humanus)
(Microhemagglutination Test for • Reservoir: Humans
Treponema palli- dum) BORRELIA HERMSII, B. TURICATE, B. DUTONI
o TPHA - Treponema Pallidum AND B. PARKERI
Hemagglutination • These agents are tick-borne relapsing fever/
o TPPA - Treponema Pallidum Particle endemic/ American relapsing fever.
Agglutination Assay • Vector: Soft ticks (Ornithodoros)
MOLECULAR TEST BORRELIA BURGDORFERI, B. GARINII AND B.
• PCR used for neurosyphilis detection during AFZELII
tertiary syphilis • Agents of Lyme disease (B. burgdorferi)
• Western blot detection of congenital syphilis. • Vector: Hard ticks (Ixodes) – Ixodes pacificus,
TREPONEMA SPP. Ixodes scapularis (deer tick), Ixodes
TREPONEMA PALLIDUM SUBSP. PERTENUE persulcatus, Ixodes dammini
• Causative agent of Yaws or frembesia tropica • Transmission: Bite of the Ixodes ticks
• Acquired by direct contact through skin breaks • Natural hosts of ticks: Deer and Rodents
with an infected lesion. (Peromyscus leucopus or white-footed mouse)
• Non-venereal infection, chronic ulcerative sores • All stages of ticks (larval, nymph and adult) can
TREPONEMA PALLIDUM SUBSP. ENDEMICUM harbour the organisms and transmit disease
• Causative agent of endemic non-venereal RELATED DISEASES AND LABORATORY
syphilis or Bejel DIAGNOSIS
• Non-venereal syphilis, appearance of primary • Related diseases
lesion near mouth. Pimple-like lesions on trunk, RELAPSING FEVER
arms and legs • It is an acute infectious disease with recurring
TREPONEMA CARATEUM febrile episodes (2 to 10 relapses)
• Causative agent of pinta or carate • Symptoms: Fever, headache, myalgia (2 to 15
• Acquired by contact with infected skin days after infection)
• Pinta: skin infection characterized with primary LYME DISEASE
lesions or graduall enlarging papule with • Acute, recurring inflammatory infection involving
enlargement of regional lymph nodes. (Red to the large joints, like knees.
blue macular rash) • Hallmark of infection are erythema migrans
TREPONEMA DENTICOLA & TREPONEMA (bull’s eye lesion on the skin) and swelling
SOCRANSKI LABORATORY DIAGNOSIS
• Ulcerative gingivitis and chronic periodontitis. MICROSCOPIC EXAMINATION
BORRELIA (BLOOD SPIROCHETES) • Giemsa and Wright stain
• Borrelia recurrentis, Borrelia dutoni, Borrelia o Darkfield microscopy, Blood culture after
hermsii, Borrelia turicatae, Borrelia parkeri, 2 to 3 weeks of incubation at 35C.
Borrelia afzelii, Borrelia burgdorferi (most • Relapsing Fever
commonly encountered, causes lyme disease) o Specimen: Peripheral blood
and Borrelia garinii o Spirochetes in peripheral blood, stained
• B. recurrentis (arrows) in as blue colored.
blood. • Lyme Disease
o Specimen: Blood, CSF, and Biopsy
specimen

TUAZON A. 48
 o Tissue section: Warthin-Starry stain is o Entry through breaks in the skin,
used. mucous membranes or conjunctiva
CULTURE o Direct contact with the urine of carriers
• Culture media: Barbour-Stoenner-Kelly medium like rats
or Chick embryo o Contact with bodies of water that are
• Organisms are slow-grower and requires 7 to 14 contaminated with the urine of the
days at 35C carriers
o Upon entry, Leptospira rapidly invades
SERODIAGNOSIS
the bloodstream and spread throughout
• Relapsing fever: Serological test reveals the CNS and Kidneys.
increased titers to Proteus OXK antigens (up to TYPES OF LEPTOSPIROSIS
1:80)
ICTERIC LEPTOSPIROSIS OR WEIL
• Lyme disease: Serology is the standard method
SYNDROME
for the diagnostic testing of this disease.
• IGM and IGG antibodies are detected in the • Severe form of illness that affects the liver and
serum. kidneys and causes vascular dysfunction
• Serology: ELISA and IFA (Indirect • Death up to 10% of the cases
Immunofluorescent Assay) • Icteric – jaundice/yellowish discoloration
MOLECULAR TESTS (paninilaw)
ANICTERIC LEPTOSPIROSIS
• PCR is important in diagnosis of B. burgdorferi
DNA in urine. • Symptoms: Septicemic stage of infection, high
LEPTOSPIRA fever and severe headache (3 to 7 days)
• Leptospira interrogans (pathogenic species, followed by the immune stage
looks like a question mark)) and Leptospira • Hallmark of immune stage: Aseptic meningitis.
biflexa (non-pathogenic species) • Absence of yellowish discoloration of the skin
• Leptospira interrogans LABORATORY DIAGNOSIS
serotype • Specimen: Blood, CSF and tissues for the
bacterimic phase (first week); urine for the
immune phase (second week)
• Microscopic Examination
• Darkfield microscopy, can be used for the
detection of motile leptospires in the specimens.
• Culture
GENERAL CHARACTERISTICS OF o Culture media: Fletcher’s medium,
LEPTOSPIRA Ellinghausen-McCullough-Johnson-
• Genus belongs to the family Leptospiraceae Harris (EMJH) medium, Bovine serum
under the order of Spirochaetales. albumin, Stuart’s broth and Noguchi’s
• Species of this genus are obligate aerobes medium
which can be grown in artificial media. o Fletcher’s and EMJH media are semi-
• They are tightly coiled and are highly motile with solid media.
hooked ends. (looks like a question mark) • Serodiagnosis
• They live in the lumen of the renal tubules and o Commonly used methods for antigen
shed into the urine detection: ELISA, Radio immunoassay
(RIA) and immunomagnetic capturing
• Recommended animals for cultivation:
o Antigen detection:
Hamsters and guinea pigs
Immunofluorescence and
• Microscopy: Tightly coiled, Thin, flexible
immunohistochemistry
organisms with two long axial filaments that
o Reference method: microscopic
exhibit a spinning motility.
agglutination (MA) using living cells
• Virulence factor: Hemolysin (L. interrogans)
• Molecular Test
• Growth factor: Hemoglobin and thiamine o Detects leptospiral DNA in infected
• Animal Reservoir: Rats and Dogs patients
LEPTOSPIRA SPP. o Methods: Polymerase chain reaction
LEPTOSPIROSIS OR INFECTIOUS JAUNDICE and hybridization techniques.
• Zoonotic disease in humans caused by MISCELLANEOUS BACTERIA
Leptospira interrogans • Streptobacillus moniliformis
• Acquired in home and recreational settings • Spirillum minus/minor
(swimming pools) • Klebsiella granulomatis
• Symptoms: Fever, headache, myalgia, anorexia • Capnocytophaga
and vomiting. • Enterococci
• MOA: • Gardenella

TUAZON A. 49
 STREPTOBACILLUS MONILIFORMIS arthralgia; and lymphadenitis;
• Etiologic agent: Rat-bite fever and Haverhill recurrent fever if and recurrent
fever in humans untreated fever if untreated
• Gram-negative bacillus Diagnosis Culture and Dark group
• Normally found in oropharynx of wild and serology microscopy,
laboratory rats microscopy of
• Facultatively anaerobic Giemsa-stained
• Non-motile, non-encapsulated and non- blood smear,
haemolytic and animal
• Diene’s stain: required to demonstrate the L- inoculation
form colonies Penicillin (L
• Microscopy: Yeast-like shape, Chains of bacilli forms not
Penicillin: in case
sensitive to
• Broth: fluff or breadcrumbs appearance Antibiotic of endocarditis,
penicillin). Both
• BAP: Fried-egg appearance with dark center therapy addition of an
forms sensitive
SPIRILLUM MINUS/MINO aminoglycoside
to streptomycin
• Rat-bite fever known as sodoku in humans and tetracycline
• Grow non artificial culture media Mortality Higher if Lower if
• Strictly aerobic, closely related Neisseria untreated (10%) untreated (6%)
• Direct visualization of specimen (blood, CAPNOCYTOPHAGA
exudates or lymph node tissue) using Giemsa • GNR with long fusiform shape.
stain, Wright stain or darkfield microscopy is
• Fastidious facultative anaerobe that grows
recommended.
slowly and needs enriched agar.
• Microscopy: Thick, helical, gram-negative
• C. animorsus
bacilli with 2 to 3 coils and are motile by
o Most common cause of severe disease
polytrichous polar flagella.
in humans.
KLEBSIELLA GRANULOMATIS o Normal flora in oral cavity of dogs (and
• Formerly known as Calymmatobacterium cats)
granulomatis • Classic clinical scenario:
• Etiologic agent: Granuloma inguinal or o Septic shock with fever, rash,
donovanosis: Sexually transmitted disease hypotension, renal failure → can evolve
nodule enlarged with beefy, erythematous, to purpura fulminans or gangrene.
granulomatous and painless lesion that easily • Risk factor for severe disease
bleed. o Contacts with clog (bite/scratch),
• Culture: Yolk sac or fresh egg yolk medium. immunocompromised host, asplenia,
• Microscopy: Blue rods with “safety pin” app and cirrhosis.
is surrounded by a pink capsule, presence DONOVAN BODIES
Donovan bodies in mononucleated endothelial
cells.
CAPNOCYTOPHAGA
• Indigenous microbiota of the oral cavity of
humans and animals (dog and cats)
• Resembles HACEK group in their CO2
requirements for enhanced growth
• Gliding motility on solid surface
• Facultatively anaerobic with a negative reaction
to most biochemical tests
• Microscopy: Gram-negative rods to filamentous
or spindle-shaped bacteria
• Culture: BAP or CAP: slight yellow or orange DONOVANOSIS
pigmentation.
COMPARISON OF RAT-BITE FEVER CAUSED BY
STREPTOBACILLUS AND SPIRILLUM SPECIES
Streptobacillus
Spirillum minus
moniliformis
Incubation Short (10 days) Long (15 days)
period
High fever, Fever,
headache, chills, headache, chills,
Symptoms
myalgia, rash, rash,
arthritis, lymphangitis,

TUAZON A. 50
ENTEROCOCCI (GROUP D STREPTOCOCCI)
• Specie: Enterococcus faecalis, Enterococcus
faecium, Enterococcus avium, Enterococcus
gallinarum, Enterococcus durans, Enterococcus
raffinosus
ENTEROCOCCUS SPP.
• Belong to family Streptococcaceae
• Produce D antigen
• Indigenous microbiota of human and animals’
intestinal tracts
• Not highly pathogenic but are frequent causes of
nosocomial infections
• Resistant to multiple antimicrobial agents
• Most common isolates: E. faecalis
• Virulence factor: Extracellular serine protease,
gelatinase and cytolysin
• Related infections: UTI, endocarditis,
bacteremia, wound infections
• Non-haemolytic or maybe alpha or beta
haemolytic
• Laboratory test: (+) Bile esculin and PYR; (+)
growth in 6.5% Nacl (Halophilic)
GARDENELLA VAGINALIS
• Gram-variable
• Coccobacillus
• Low numbers in normal vaginal flora
o Mostly lactobacilli
o Keep vaginal pH <4.5
• Whiff Test or KOH Test
o Specimen: Vaginal secretions
o Reagent: 10% KOH
o (+) Result: Exhibits “fishy amine
odor
o Also look for clue cells

GOODLUCK!

- aly

TUAZON A. 51