WEEK 13: NONFERMENTERS hemolysis, was catalase positive, and was motile.

1. Based on carbohydrate utilization, what is the The most likely organism is:
difference between nonfermentative and a. Bacillus anthracis
fermentative organisms? b. Nocardia nova
c. Bacillus cereus
2. What is the typical natural habitat of most d. Tsukamurella spp.
nonfermenters?

3. Bacillus cereus is most noted for causing:

3. What types of infections do nonfermenters
cause? a. Food poisoning
b. Meningitis

4. What risk factors are associated with infections c. Sexually transmitted disease
caused by nonfermentative, gram-negative d. UTIs
bacilli?

4. Which forms of infection are caused by Bacillus

5. What are the four most common anthracis?
nonfermentative, gram-negative bacilli isolated a. Injectional
in the clinical laboratory?
b. Inhalation
c. Gastrointestinal
6. What are the typical susceptibility patterns of
d. Cutaneous
the most commonly isolated nonfermenters?
e. All of the above

7. What initial clues indicate that an isolate is a

nonfermenter? 5. The functionality of lethal factor requires the
presence of what other protein from Bacillus
anthracis to form an active toxin?
8. How would you differentiate P. aeruginosa from
a. Cyclic adenosine monophosphate (CAMP)
other fluorescent group pseudomonads?
b. Edema factor
c. D-Glutamic acid
9. What are the identifying characteristics of
Acinetobacter spp.? d. Protective antigen

10. Which nonfermenters are most commonly 6. Describe the appearance of spore-forming
isolated from patients with cystic fibrosis? bacteria seen with the spore stain.

WEEK 14: GRAM + RODS 7. Corynebacterium species often appear as

______________ on Gram staining.
1. An isolate with the appropriate colony and
microscopic morphology may be suspected to a. Pleomorphic, gram-positive, club-shaped
be Bacillus anthracis if it is: bacilli that appear in
a. β-Hemolytic on SBA palisades or in ―V‖ and ―L‖ formations
b. Nonmotile b. Branching gram-positive bacilli that appear as
fine, intertwining,
c. Catalase negative
delicate filaments
d. Gram negative, non–spore forming
c. Short, thin gram-positive bacilli that appear in
chains
2. An aerobic, gram-positive, spore-forming
d. Large square-ended, gram-positive or gram-
bacillus was isolated from raw vegetables that
variable bacilli in
were associated with an outbreak of
gastroenteritis. The organism produced β-

1
chains where the ends of the single cells fit snugly  Gram-positive bacilli, no spores observed
together  Catalase positive
 Hydrogen sulfide negative
 Motile at room temperature
8. The biochemical tests performed on a gram-
positive bacillus were consistent with a. Erysipelothrix rhusiopathiae
Corynebacterium diphtheriae. As a definitive test, b. Listeria monocytogenes
the laboratory scientist should now: c. Corynebacterium ureilyticum
a. Perform Gram staining of the isolate and d. Gardnerella vaginalis
observe this for its pleomorphic morphology
b. Prepare a methylene blue stain and examine it
for metachromatic granules 13. A commercial fisherman with red sores on his
hands was seen by his physician. Biopsy and
c. Perform an Elek test to determine whether the culture of one of the lesions grew an organism
organism produces exotoxin with the characteristics listed below: What is the
d. Subculture the organism to cystine-tellurite most likely identification?
blood agar and examine this for black colonies  Nonhemolytic on SBA
 Gram-positive bacilli, no spores observed
9. Diphtheria is uncommon in the United States  Catalase negative
because:  Hydrogen sulfide production positive
 Growth in gelatin resembled a test-tube
a. The insect vector has been eliminated brush
b. Of vaccination of the animal reservoir a. Rhodococcus equi
c. Of routine use of an effective human vaccine b. Listeria monocytogenes
d. Aggressive antimicrobial therapy has nearly c. Lactobacillus acidophilus
eliminated the bacteria
d. Erysipelothrix rhusiopathiae

10. True infections with nondiphtheria

Corynebacterium spp., such as C. jeikeium or C. 14. A 42-year-old man from Guatemala cuts his
striatum, are often in immunocompromised bare feet on thorns while walking. A
patients or patients who have had: subcutaneous abscess develops, and when the
patient is seen by a physician, his foot is swollen.
a. Insertion of hardware or prosthetic devices When the wound is pressed by the physician,
b. Coronary artery bypass surgery purulence is expressed along with some soft,
c. Vitamin B12 deficiency white granules. A filamentous organism that is
partially acid fast is recovered from the granules.
d. A lengthy hospital stay
This is most likely an __________ mycetoma
caused by ____________.
11. A(n) _________ test can help to differentiate a. Actinomycotic; Pseudallescheria boydii
which clinically significant Corynebacterium spp.
b. Actinomycotic; Nocardia brasiliensis
recovered from urine samples?
c. Eumycotic; Madurella mycetomatis
a. Gelatin hydrolysis; C. ulcerans
d. Eumycotic; Gordonia bronchialis
b. Reverse CAMP; C. pseudotuberculosis
c. Alkaline phosphatase; C. amycolatum
15. What other organisms can give similar clinical
d. Urease; C. urealyticum
and laboratory findings as those for Listeria
monocytogenes? How are these organisms
12. A newborn female becomes febrile and will differentiated from L. monocytogenes?
not feed for about an hour after birth. A gram-
positive rod is recovered from blood cultures from
16. A 17-year-old male presented to an
the newborn. The isolate has the characteristics
emergency department with a history of multiple
listed below: What is the most likely identity of the
episodes of febrile pharyngitis followed in 10 to 14
isolate?
days by extensive desquamation of his hands
 Weakly β-hemolytic on SBA and feet. The reoccurrences have followed

2
several courses of antimicrobial therapy, 2. The porphyrin test for H. influenzae would be
including amoxicillin and cephalosporins. Rapid __________ because the organism __________
group A streptococci screens and cultures have biosynthesize heme. The fluorescence result of
been consistently negative for Streptococcus the test would be __________.
pyogenes. A specimen with a request for an a. Negative; cannot; negative
alternative agent is submitted to a reference
laboratory, and the results listed below are b. Positive; cannot; positive
observe: c. Negative; can; positive
The patient was subsequently treated d. Positive; can; negative
with erythromycin, and he recovered. What was
the etiologic agent? SBA: small, slightly hemolytic
colony, which at 48 hours is a dark spot sunken in 3. Infections caused by β-lactamase–positive H.
the agar influenzae should be treated with which of the
following?
 Catalase negative
a. Ampicillin
 Nitrate negative
 Reverse CAMP test positive b. Penicillin
a. Corynebacterium diphtheriae c. Either of the above
b. Arcanobacterium haemolyticum d. None of the above
c. Listeria monocytogenes
d. Rhodococcus equi 4. Describe the optimal growth conditions for the
recovery of H. ducreyi.

17. A sample from a female patient suspected to

have bacterial vaginosis is sent to the laboratory. 5. Compare the pathogenesis of H. aegyptius
Which of the following would be an appropriate with H. influenza biogroup aegyptius.
medium for this specimen?
a. Loeffler medium 6. A cervical culture for possible gonococcal
b. HBT agar infection is sent to the microbiology laboratory.
After 24 hours of incubation, the modified Thayer-
c. CTBA
Martin (MTM) plate has small opaque colonies
d. BCYE agar that adhere slightly to the medium. Microscopic
examination reveals gram-negative
18. A 57-year-old man from New York City coccobacilli, many with square ends. The
presents to the emergency department with organism ferments glucose and is superoxol and
diarrhea, arthralgia, abdominal pain, catalase negative. The most likely identification
malabsorption, and weight loss of 10 lb (4.5 kg) is:
over the last month. A duodenal biopsy is a. N. gonorrhoeae
performed, but no infectious agent is recovered b. K. denitrificans
on culture media. However, on Gram staining, a
c. M. catarrhalis
gram-positive rod is observed in macrophages in
the biopsy tissue. The organism is identified by d. H. ducreyi
16S rRNA gene sequencing. What is the most
likely identity of this organism?
7. A 52-year-old man who had recently received
a. Bacillus anthracis a kidney transplant was admitted to the hospital
b. Nocardia asteroides with a low-grade fever, a heart murmur, and
neutropenia. He had a history of periodontal
c. Tropheryma whipplei
disease and recently had had two teeth
d. Erysipelothrix rhusiopathiae extracted. Blood cultures were positive after 48
hours. The isolate grew on chocolate (CHOC)
agar and sheep blood agar (SBA) in 5% CO2. The
WEEK 15: HACEK, LEGIONELLA & BORDETELLA
colonies were nonhemolytic, slightly adhered to
1. Describe the appearance of H. influenzae with the surface of the media, and had a slight yellow
the X and V strip test. appearance when removed. The isolate was

3
catalase, indole, and oxidase negative. b. Stool
Microscopic morphology indicated gram- c. Urine
negative fusiform bacilli. The most probable
identification is: d. Cerebrospinal fluid (CSF)

a. A. aphrophilus
b. K. kingae 8. Are adults immune to Bordetella pertussis
infection? Explain.
c. C. hominis
d. Capnocytophaga spp.
9. What are the clinical samples of choice for the
diagnosis of B. pertussis infection?
8. An isolate from an infected cat bite is oxidase,
catalase, ornithine decarboxylase, indole, and
urease positive. After 48 hours of incubation at 10. What transport media are appropriate for
35° C, growth on SBA was described as mucoid maximum recovery of B. pertussis?
colonies exhibiting a greenish-brown halo. A
MAC agar plate shows no growth. What organism 11. Which method is preferred for the detection of
is the mostly likely cause of the infection? Bordetella in nasopharyngeal smears?

9. Which microbiologic tests are most useful in 12. Compare the diseases caused by B. pertussis
differentiating B. melitensis from H. influenzae? and B. parapertussis.

10. A patient is complaining of a painful cervical 13. Is serology a good method to identify and
lymph node following a case of pharyngitis. respond to pertussis outbreaks in real time?
Further investigation reveals that the patient
consumed a medium-cooked wild rabbit in a
restaurant in Germany 2 months earlier. What is 14. The antimicrobial agent of choice for the
the most likely cause of the infection? treatment of pertussis is:
a. Ampicillin
1. What risk factors contribute to the more severe b. Amoxicillin
form of legionellosis? c. Erythromycin
d. Penicillin
2. What environmental factors contribute to
infection caused by Legionella spp.?
15. B. bronchiseptica is considered normal oral
biota in:
3. What is the culture medium of choice for the a. Humans
recovery of Legionella spp.?
b. Dogs
4. What factors of Legionella can contribute to
c. Cows
the colonization of human-made water supplies?
d. Rats

5. What is the best nonrespiratory specimen for

rapid detection of Legionella? WEEK 16: CHLAMYDIA, RICKETTSIA
1. What organisms should be considered as
possible causes of neonatal conjunctivitis?
6. What presumptive identification methods are
currently used to identify Legionella spp. in
culture? 2. What stains should be performed on
conjunctival scraping for microscopic
examination for the diagnosis of inclusion
7. Besides respiratory tract specimens, what
conjunctivitis?
clinical specimen is useful for the sensitive
detection of Legionella antigen? 3. For the neonate described in the Case in Point,
what other clinical conditions could have
a. Blood

4
resulted from infection with the causative
organisms? 8. Where are most cases of leptospiroses
contracted within the United States, and why is
4. What STD is caused by Chlamydia trachomatis this important when considering the typical
serotypes L1, L2, L2a, L2b, and L3? incubation period of the infection?

5. How does LGV differ from other sexually 9. Differentiate between treponemal and
transmitted diseases caused by C. trachomatis? nontreponemal serologic tests for syphilis.

6. With what types of infections are associated 10. What is the recommended method for the
with Chlamydophila pneumoniae? laboratory diagnosis of Lyme borreliosis?

7. What is psittacosis or ornithosis? 1. From what source did the neonate described in
the Case in Point likely acquire the infection?

8. What is the most common laboratory method

used to diagnose rickettsial diseases? Explain. 2. Would routine prenatal culture of the mother
have yielded this organism?

9. What cells do the Ehrlichia and Anaplasma

species typically infect in humans? 3. Why was the Gram stain result negative?

10. How does Coxiella burnetii differ from the 4. How does primary atypical pneumonia caused
Rickettsia spp.? by M. pneumonia differ from pneumonia caused
by S. pneumoniae?

WEEK 17: SPIROCHETES, MYCOPLASMA &

UREAPLASMA 5. List the four common species of mollicutes
associated with the genitourinary tracts of
humans.
1. What are the general characteristics of
spirochetes?
6. What special stain is used on suspected
colonies of Mycoplasma?
2. What risk factors are associated with Borrelia
spp. endemic relapsing fever?
7. What culture media are used to isolate M.
pneumoniae, M. hominis, and U. urealyticum?
3. Which tickborne species of Borrelia is
associated with a rash or skin lesion?
8. What is the significance of isolating M. hominis
from a vaginal specimen?
4. What is the significance on infectious disease
transmission of finding partially engorged ticks
attached to skin? 9. What current serologic assays are available to
demonstrate M. pneumoniae antibodies?

5. What is the test of choice for the laboratory

diagnosis of relapsing fever (borreliosis)? 10. Why are the mollicutes universally resistant to
penicillin?

6. Name the four strains within the genus

Treponema that are pathogenic for humans.
7. What are the stages of a T. pallidum subsp.
pallidum infection? Is the final stage usually seen
in developed countries?

5
POINTS TO REMEMBER: CAMP inhibition reaction or reverse CAMP
WEEK 13: test.
 G. vaginalis is part of the normal biota of the
Nonfermenters urogenital tract but might play a role in BV. It
 Nonfermenters will not acidify the butt of TSIA is weakly β-hemolytic on HBT agar and stains
or KIA. as a gram-variable rod.
 Most nonfermenters are oxidase positive, a  Aerobic actinomycetes are generally soil
key test in differentiation from most members inhabitants. They are weak pathogens
of the family Enterobacteriaceae. sometimes associated with wounds after
 Nonfermenters are environmental isolates, traumatic implantation into subcutaneous
which rarely cause disease in healthy tissue.
humans.  Nocardia spp. are gram-positive, filamentous
 Nonfermenters can be more resistant to organisms that can grow on nutritionally
antimicrobial agents than members of the simple media and are partially acid fast.
family Enterobacteriaceae. There are  To provide accurate identification and
specific methods and breakpoints available speciation of actinomycetes, 16S rRNA gene
for many of the nonfermenters in the CLSI sequencing is often required; however, this is
standards. unavailable in most laboratories.
 The most common nonfermenters isolated in  Members of the genus Bacillus are aerobic,
clinical microbiology laboratories are P. gram-positive, catalase- positive, rod-shaped
aeruginosa, A. baumannii complex, S. organisms that form endospores.
maltophilia, and B. cepacia complex.  Many aerobic gram-positive spore-forming
 Acinetobacter spp. may retain the crystal bacilli are rarely associated with human
violet stain resembling gram-positive infections. The most important pathogen in
bacteria. this group is B. anthracis.
 Phenotypic identification will not identify all  anthracis is generally susceptible to penicillin
nonfermenters; MALDI-TOF or 16S rRNA DNA and is nonmotile and nonhemolytic on SBA,
sequencing will be needed for confirmation, features important to differentiate the
if needed. organism from B. cereus.
 Specimens from patients with CF often  cereus is a common cause of food poisoning
demonstrate a wide variety of and opportunistic infections. Food poisoning
nonfermentative bacilli and may require that caused by B. cereus and occurs in two forms:
more definitive identification methods be diarrheal and emetic.
used. The most common, however, is P.
aeruginosa.
WEEK 15:
HACEK
WEEK 14:
 The genus Haemophilus consists of gram-
Corynebacterium negative coccobacilli or bacilli. They are
 C. diphtheriae causes serious disease in facultative anaerobes that are generally
populations of countries where the oxidase and catalase positive.
diphtheria vaccine is not available.  Haemophilus spp. require preformed growth
 Nondiphtheria Corynebacterium species can factors, X and V factors, present in blood.
be opportunistic pathogens. Testing for these factors with X and V strips
 L. monocytogenes can be differentiated and the porphyrin test are important aspects
from streptococci and enterococci on the of laboratory identification procedures.
basis of Gram stain morphology, catalase  The advent of the Hib vaccine has
reaction, and motility. decreased infection by H. influenza type b in
 E. rhusiopathiae differs from L. children in the United States by 99%, but
monocytogenes in catalase reaction, serotype b remains prevalent in developing
hydrogen sulfide production, and lack of countries.
ability to grow at 4° C.  H. ducreyi causes the STD chancroid. In
 haemolyticum can be differentiated from contrast to many other Haemophilus spp., it is
other non–spore-forming gram-positive bacilli not considered part of the normal oral biota
and β-hemolytic streptococci by Gram stain of humans.
morphology, catalase activity, and the

6
 HACEK is an acronym for Haemophilus spp.,  B. pertussis and B. parapertussis cause
Aggregatibacter actinomycetemcomitans, pertussis, or whooping cough, and are
Cardiobacterium hominis, Eikenella primary human pathogens of the respiratory
corrodens, and Kingella spp. The HACEK tract.
group is an important cause of endocarditis,  Bordetella spp. are tiny, gram-negative
specifically related to poor oral hygiene or coccobacilli and might become elongated
dental procedures. if recovered from media containing
 Pasteurella, Brucella, and Francisella spp. are cephalexin.
important causes of zoonotic infections.  Nasopharyngeal aspirates or swab
 The most common Pasteurella species specimens should be plated directly onto
isolated from humans, P. multocida, most culture media or transferred to an
frequently causes wound infections after cat appropriate transport system (casamino acid
or dog bites. or Amies transport medium) at the bedside.
 Brucella and Francisella spp. are fastidious  Contemporary laboratory diagnosis of
gram-negative coccobacilli causing pertussis generally employs culture isolation
zoonoses and are considered potential with or without PCR testing.
bioterrorism agents.  B. bronchiseptica can be part of the normal
oral biota of dogs and cats, and infections in
humans typically follow bites by these
Legionella & Bordetella animals.
 Legionella spp. are pleomorphic, weakly  Antimicrobial susceptibility testing is not
staining gram-negative bacilli, which are normally performed on B. pertussis. The drug
ubiquitous in aquatic environments and of choice in the treatment of pertussis is
infect humans through the respiratory route. erythromycin.
 Legionella spp. are transmitted to humans
primarily via aerosolized particles. Sources
include contaminated potable water WEEK 16:
distribution systems, respiratory therapy Chlamydia, Ricekttsia, & other orgs
equipment, and recreational waters.  Chlamydiae and rickettsiae are obligate
Transmission between humans has not been intracellular organisms.
demonstrated.  Chlamydia trachomatis is the most common
 Legionnaires’ disease is a febrile disease with sexually transmitted bacterial pathogen, and
pneumonia that is often associated with certain serovars are associated with
travel. Pontiac fever is a milder febrile disease trachoma, which can result in blindness.
resembling influenza that may be caused by  The LGV strains of C. trachomatis are more
inhalation of bacterial toxin. invasive, producing a more serious infection
 On BCYE agar, colonies appear convex and and pronounced antibody response.
glistening. The central portion of young  NAATs are better assays for the diagnosis of
colonies have a ―ground glass‖ appearance, C. trachomatis infections compared with
light gray and granular, whereas the cultures.
periphery of the colony has pink or light blue  Chlamydophila pneumoniae is a relatively
or bottle-green bands. common respiratory tract pathogen
 Using a combination of nonselective and considered responsible for many cases of
selective BCYE agar is the best strategy for community acquired pneumonia.
isolation of Legionella organisms. Acid  Chlamydophila psittaci is the cause of
treatment of specimens contaminated with psittacosis, also known as parrot fever or
other bacteria before inoculation enhances ornithosis. This bacterium produces lower
isolation of Legionella spp. respiratory tract infections in humans.
 Rapid assays useful for the detection of  The Rickettsia spp. are important human
Legionella spp. are the urine antigen pathogens responsible for a number of
detection tests. diseases, including RMSF, rickettsialpox, and
 Infections caused by Bordetella spp. are typhus.
acquired through the respiratory tract via  The Rickettsia, Orientia, Ehrlichia, and
respiratory droplets or direct contact with Anaplasma are typically transmitted to
infectious secretions. humans by the bites of arthropods.

7
 Ehrlichia and Anaplasma are intracellular the organisms are visible in blood with bright-
parasites of white blood cells—mononuclear field microscopy.
cells and granulocytes, respectively.  Laboratory diagnosis of Lyme disease caused
 Coxiella burnetii is the causative agent of Q by B. burgdorferi sensu lato is accomplished
fever. Infection is most often transmitted by by two-tiered serology. Initial positive or
inhalation of dried birthing fluids. The equivocal EIA results are confirmed with
ingestion of unpasteurized milk is also a risk Western blot.
factor.  Treponemes can cross the placenta and be
transmitted from an infected mother to her
fetus. Congenital syphilis affects many body
WEEK 17: systems and is therefore severe and
Mycoplasma & Ureaplasma mutilating. All pregnant women should have
 The mollicutes are minute organisms serologic testing for syphilis early in
characterized by the lack of a cell wall. pregnancy.
 The most clinically significant species of the
Mycoplasmataceae include M.
pneumoniae, M. hominis, and Ureaplasma
spp., although others are beginning to be
recognized as opportunistic pathogens.
 M. pneumoniae is an important cause of
community-acquired, atypical pneumonia.
 M. hominis and Ureaplasma spp. are genital
mycoplasma commonly diagnosed by
culture, although PCR technology is also
available.
 Because of the lack of a cell wall, the
mycoplasmas are inherently resistant to the
β-lactam antibiotics.

Spirochetes
 Spirochetes are slender, flexuous, helically
shaped, motile bacteria.
 Leptospires are most likely to enter the
human host through small breaks in the skin
or intact mucosa.
 The incubation period of leptospirosis is
usually 10 to 12 days but ranges from 3 to 30
days. The onset of clinical illness is generally
abrupt, with nonspecific, influenza-like
constitutional symptoms, such as fever, chills,
headache, severe myalgia, and malaise.
 The pathogenic borreliae are commonly
arthropod-borne (by a tick or louse) and
cause relapsing fever and Lyme disease.
 B. recurrentis and similar species cause
relapsing fever. The relapses are caused by
immune evasion, including antigenic
variation. During the course of a single
infection, borreliae systematically change
their surface antigens.
 During the febrile period, diagnosis of
relapsing fever is readily made by Giemsa or
Wright staining of blood smears. Relapsing
fever is the only spirochetal disease in which