Chapter 8.6 - Study of Aerobic Gram-Positive Rods, Spirochetes, Mycoplasmas, Ureaplasma, and Chlamydiae

Large, gram-positive, spore forming rods growing on blood agar as large, raised B-hemolytic colonies that spread and appear as frosted green-gray glass are most likely:

A. Pseudomonas spp

B. Bacillus spp

C. Corynebacterium spp

D. Listeria spp

B. Bacillus spp

Key Distinguishing Features

• Large gram-positive rods

• Spore-forming

• β-hemolytic, spreading colonies

• “Frosted gray-green glass” appearance on blood agar

• Often exhibit swarming or spreading edges

Summary
A spore-forming, β-hemolytic large rod with a frosted-glass colony morphology is characteristic of Bacillus species, typically non-anthracis Bacillus.

Bacillus anthracis and Bacillus cereus can best be differentiated by which tests?

A. Motility and B-hemolysis on a blood agar plate

B. Oxidase and B-hemolysis on a blood agar plate

C. Lecithinase and glucose

D. Lecithinase and catalase

A. Motility and B-hemolysis on a blood agar plate

Key Differentiating Features

• Bacillus anthracis

  • Nonmotile

  • Non-hemolytic (γ-hemolysis)

• Bacillus cereus

  • Motile (swarming/spreading colonies)

  • β-hemolytic on blood agar

Summary
Motility and hemolysis patterns on blood agar are the best simple tests to distinguish B. anthracis from B. cereus.

Which is the specimen of choice for proof of food poisoning by B. cereus?

A. Sputum

B. Blood

C. Stool

D. Food

D. Food

Key Point
To confirm Bacillus cereus food poisoning, the laboratory must demonstrate the organism or its toxin in the implicated food.

• Food → Best specimen for proof of foodborne intoxication

• Stool or vomitus may support the diagnosis but don’t prove the source

Summary
For definitive confirmation of B. cereus food poisoning, testing the suspected food is required.

A suspected B. anthracis culture obtained from a wound specimen produced colonies that had many outgrowths (Medusa-head appearance), but were not B-hemolytic on sheep blood agar. Which test should be performed next?

A. Penicillin (10-unit) susceptibility testing

B. Lecithinase test

C. Glucose test

D. Motility test

A. Penicillin (10-unit) susceptibility testing

Rationale

• A presumptive culture of Bacillus anthracis often exhibits penicillin susceptibility → a clearing of the zone around a penicillin disk supports the identification.

• Other Bacillus species (e.g., B. cereus) are typically β-hemolytic and resistant to penicillin.

• Because you already have a nonhemolytic, medusa-head colony suspect, the penicillin disk test serves as a quick confirmatory step before full identification.

Summary
In a presumptive B. anthracis isolate, a 10-unit penicillin disk test that shows susceptibility is a key rapid confirmatory tool.

Which of the following tests should be performed for initial differentiation of L. monocytogenes from Group B streptococci?

A. Gram stain, motility at room temperature, catalase

B. Gram stain, CAMP test, H2S/TSI

C. Oxidase, CAMP test, glucose

D. Oxidase, bacitracin

A. Gram stain, motility at room temperature, catalase

Key Differentiation

Feature	Listeria monocytogenes	Group B Strep (S. agalactiae)
Gram stain	Small GPR (coccobacilli)	GPC in chains
Motility (RT)	Positive – tumbling motility	Non-motile
Catalase	Positive	Negative

Why not CAMP?
Both Listeria and Group B Strep are CAMP-positive, so it does not separate them initially.

Summary
A Gram-positive rod, catalase-positive, motile at room temperature isolate is consistent with Listeria monocytogenesrather than Group B streptococcus.

Culture of a finger wound specimen from a meat packer produced short gram-positive, non-spore-forming bacilli on a blood agar plate showing no hemolysis. Given the following test results at 48 hours, what is the most likely identification?

Catalase neg

Motility (wet prep) neg

Motility (gel media 22C) + bottle-brush growth in stab culture

H2S/TSI +

A. Bacillus cereus

B. Listeria monocytogenes

C. Erysipelothrix rhusiopathiae

D. Bacillus subtilis

C. Erysipelothrix rhusiopathiae

Key Distinguishing Features

• Clinical clue: Meat handlers / animal exposure → erysipeloid infections

• Gram stain: Short G+ non–spore-forming bacilli

• Catalase: Negative (rules out Bacillus and Listeria)

• Motility:

  • Non-motile on wet prep

  • “Bottle-brush” pattern in gelatin stab → characteristic

• TSI: H₂S positive (unique among non-spore-forming GPRs)

• Hemolysis: Typically nonhemolytic

Summary
A catalase-negative, H₂S-positive, bottle-brush motility organism from a meat packer’s wound fits Erysipelothrix rhusiopathiae.

Nonspore-forming, slender, gram-positive, rod-forming palisades and chains were recovered from a vaginal culture and grew well on tomato juice agar. The most likely identification is:

A. Lactobacillus spp

B. Bacillus spp

C. Neisseria spp

D. Streptococcus spp

A. Lactobacillus spp

Key Distinguishing Features

• Gram-positive rods in palisades/chains

• Non–spore-forming → rules out Bacillus

• Normal vaginal flora → dominant protective organism

• Grows well on tomato juice agar → selective for Lactobacillus

• Produces lactic acid to maintain low vaginal pH

Summary
A slender GPR from a vaginal culture with good growth on tomato juice agar is classic for Lactobacillus species.

A Corynebacterium species recovered from a throat culture is considered a pathogen when it produces:

A. A pseudomembrane of the oropharynx

B. An exotoxin

C. Gray-black colonies with a brown halo on Tinsdale agar

D. All of these options

D. All of these options

Key Pathogenic Features of Corynebacterium diphtheriae

• Pseudomembrane formation in the throat → hallmark of diphtheria

• Produces diphtheria exotoxin → systemic effects (myocarditis, neuropathy)

• Tinsdale agar: gray-black colonies with a brown halo due to cystinase activity

Summary
A Corynebacterium isolate from a throat is considered pathogenic when it shows toxin production, pseudomembrane involvement, and classic morphology on Tinsdale agar — all of the above.

A presumptive diagnosis of Gardnerella vaginalis can be made using which of the following findings?

A. Oxidase and catalase tests

B. Pleomorphic bacilli heavily colonized on vaginal epithelium

C. Hippurate hydrolysis test

D. All of these options

D. All of these options

Key Points for Presumptive Gardnerella vaginalis Identification

• Pleomorphic gram-variable bacilli coating vaginal epithelial cells → clue cells

• Hippurate hydrolysis positive in most isolates

• Oxidase and catalase negative to weak → part of typical screening panel

Summary
A combination of characteristic microscopy, biochemical reactions, and hippurate positivity supports a presumptive ID of Gardnerella vaginalis in cases of bacterial vaginosis.

A gram-positive branching filamentous organism recovered from a sputum specimen was found to be positive with a modified acid-fast stain method. What is the most likely presumptive identification?

A. Bacillus spp

B. Nocardia spp

C. Corynebacterium spp

D. Listeria spp

B. Nocardia spp

Key Distinguishing Features

• Gram-positive branching filamentous rods

• Partially acid-fast with a modified acid-fast stain (e.g., Kinyoun with weaker decolorizer)

• Often recovered from respiratory specimens → causes nocardiosis (cavitary pneumonia, abscesses)

• Aerobic actinomycete, differentiating it from anaerobic branching bacteria like Actinomyces

Summary
A branching, partially acid-fast GPR from sputum is presumptively Nocardia.

Routine laboratory testing for Treponema pallidum involves:

A. Culturing

B. Serological analysis

C. Acid-fast staining

D. Gram staining

B. Serological analysis

Key Points

• Treponema pallidum cannot be cultured on standard media — requires host tissue.

• It does not stain well with Gram stain or acid-fast methods.

• Diagnosis relies on serologic testing:

  • Nontreponemal tests (RPR, VDRL) → screening

  • Treponemal tests (FTA-ABS, TP-PA) → confirmation

Summary
Routine testing for syphilis is based on serologic detection of antibodies, not culture or conventional staining.

Spirochetes often detected in the hematology laboratory while scanning a blood film, even before the physician suspects the infection, are:

A. Borrelia spp

B. Treponema spp

C. Campylobacter spp

D. Leptospira spp

A. Borrelia spp

Key Points

• Borrelia are the only common spirochetes visible on routine Wright- or Giemsa-stained peripheral blood smears

• Seen in relapsing fever caused by tick or louse vectors

• They appear as loosely coiled spirochetes circulating in blood

Why not the others?

• Treponema and Leptospira → too thin to be seen on routine blood smears

• Campylobacter → not a spirochete

Summary
If a blood smear reveals spirochetes, think Borrelia until proven otherwise.

Which of the following organisms is the cause of Lyme disease?

A. Treponema pallidum

B. Neisseria meningitidis

C. Babesia microti

D. Borrelia burgdorferi

D. Borrelia burgdorferi

Key Points

• Transmitted by Ixodes (deer ticks)

• Causes Lyme disease with erythema migrans rash, arthritis, neurologic and cardiac involvement

• A spirochete, similar morphology to Borrelia spp causing relapsing fever

Summary
Lyme disease is caused by Borrelia burgdorferi, a tick-borne spirochete.

The diagnostic method most commonly used for the identification of Lyme disease is:

A. Serology

B. Culture

C. Gram staining

D. Acid-fast staining

A. Serology

Key Points

• Borrelia burgdorferi does not stain well with Gram or acid-fast stains

• Culture is difficult and not routinely available

• Diagnosis relies on antibody detection using a two-tier system:

  • Screening: EIA/ELISA

  • Confirmation: Western blot (IgM/IgG)

Summary
Lyme disease is primarily diagnosed by serologic testing, not culture or microscopy.

Primary atypical pneumonia is caused by:

A. Streptococcus pneumoniae

B. Mycoplasma pneumoniae

C. Klebsiella pneumoniae

D. Mycobacterium tuberculosis

B. Mycoplasma pneumoniae

Key Points

• Causes primary atypical pneumonia → often called “walking pneumonia”

• Lacks a cell wall, so:

  • Not visible on Gram stain

  • β-lactams ineffective

• Often presents with dry cough, low fever, diffuse infiltrates

• Common in school-aged children and young adults

Summary
Primary atypical pneumonia is classically due to Mycoplasma pneumoniae.

Which organism typically produces "fried egg" colonies on agar within 1 to 5 days of culture obtained from a genital specimen?

A. Mycoplasma hominis

B. Borrelia burgdorferi

C. Leptospira interrogans

D. Treponema pallidum

A. Mycoplasma hominis

Key Points

• Produces “fried-egg” colonies: dense center with lighter periphery

• Appears in 1–5 days on specialized urogenital Mycoplasma media, such as:

  • SP4 agar

  • A7 or A8 agar (contains sterols and inhibitory antibiotics)

• No cell wall →

  • Not visualized on Gram stain

  • β-lactam antibiotics ineffective

• Associated with urogenital infections (e.g., PID, postpartum fever)

Summary
A genital isolate showing fried-egg colonies on SP4/A7/A8 agar is characteristic of Mycoplasma hominis.

The manganous chloride-urea test is used for the identification of which organism?

A. Mycoplasma pneumoniae

B. Ureaplasma urealyticum

C. Bacillus cereus

D. Borrelia burgdorferi

B. Ureaplasma urealyticum

Key Points

• Manganous chloride–urea test detects strong urease activity

• U. urealyticum rapidly hydrolyzes urea, producing manganese precipitate → color change

• Found in genitourinary tract infections

• No cell wall → resistant to β-lactams; requires specialized media containing urea + sterols

Summary
A positive manganous chloride–urea test is highly indicative of Ureaplasma urealyticum.

A gram-positive (gram-variable), beaded organism with delicate branching was recovered from the sputum of a 20-year-old patient with leukemia. The specimen produced orange, glabrous, waxy colonies on Middlebrook agar that showed partial acid-fast staining with the modified Kinyoun stain. What is the most likely identification?

A. Rhodococcus spp

B. Actinomadura spp

C. Streptomyces spp

D. Nocardia spp

D. Nocardia spp.

Why Nocardia?

• Gram-positive / gram-variable, beaded, delicate branching filaments

• Partially acid-fast with modified Kinyoun stain (key differentiator)

• Causes pulmonary infection in immunocompromised patients (e.g., leukemia)

• Can grow on Middlebrook agar — like other aerobic actinomycetes

Key Differentiation Points

• Nocardia → branching filaments + partial acid-fast

• Rhodococcus → more often coccoid, salmon-pink colonies

• Streptomyces / Actinomadura → not acid-fast

Summary
A branching, partially acid-fast GPR from sputum of an immunocompromised patient = Nocardia spp.

A direct smear from a nasopharyngeal swab stained with Loeffler methylene blue stain showed various letter shapes and deep blue, metachromatic granules. The most likely identification is:

A. Corynebacterium spp

B. Nocardia spp

C. Listeria spp

D. Gardnerella spp

A. Corynebacterium spp

Corynebacterium spp background/morphology
• Loeffler methylene blue stain shows metachromatic (Babes-Ernst) granules
• Club-shaped GPR arranged in V/L shapes or “Chinese letters”
• Respiratory specimen suggests C. diphtheriae as primary concern
• Produces diphtheria toxin that inhibits EF-2 → tissue necrosis, pseudomembrane
• Confirm with ELEK test or PCR for tox gene
• Cystine-tellurite agar → black colonies

Quick differential:
• Nocardia: branching, partial AFB stains
• Listeria: small rods, tumbling motility
• Gardnerella: clue cells in BV, not nasopharynx

Summary: Metachromatic granules plus “Chinese letter” rods from the throat means Corynebacterium.

Which of the following is the best, rapid, non-culture test to perform when G. vaginalis is suspected in a patient with vaginosis?

A. 10% KOH test

B. 3% H2O2 test

C. 30% H2O2 test

D. All of these options

A. 10% KOH test

Gardnerella vaginalis background/morphology
• Short, pleomorphic gram-variable rods associated with bacterial vaginosis
• Adheres to squamous epithelial cells → clue cells
• Produces amine odor when treated with 10% KOH (positive whiff test)
• Often catalase negative and nonmotile
• Typically no WBCs despite infection (disruption of normal flora, not inflammation)

Quick differential:
• Candida: budding yeast + pseudohyphae on KOH; pruritus common
• Trichomonas: motile flagellates on wet prep; ↑ WBCs
• Lactobacillus: gram-positive rods, maintains vaginal pH

Summary: The rapid whiff test with 10% KOH helps confirm Gardnerella in BV.

Which is the test of choice for the confirmation of Chlamydia trachomatis infection in urine?

A. Enzyme immunoassay antigen testing

B. PCR molecular testing

C. Culture using McCoy and Hela cells

D. Microimmunofluorescence (MIF) test

B. PCR molecular testing

Chlamydia trachomatis background/diagnostics
• Obligate intracellular pathogen; does not grow on routine media
• Nucleic acid amplification tests (NAATs), including PCR, are the gold standard
• Urine NAAT preferred for screening due to high sensitivity and noninvasive collection
• Culture (McCoy/HeLa cells) possible but slow, technically complex, and less sensitive
• MIF and EIAs used historically but inferior specificity/sensitivity

Quick differential:
• Neisseria gonorrhoeae: NAAT also test of choice from urine or genital swabs
• Mycoplasma/Ureaplasma: require special media or NAAT since no cell wall
• HSV: PCR or viral culture from lesions rather than urine

Summary: NAAT/PCR on urine is the most sensitive and rapid test for Chlamydia.

Which test is the most reliable for the detection of M. pneumonia in serum and for the confirmation of diagnosis?

A. EIA test and direct antigen testing

B. Cold agglutinin testing using group O RBCs

C. Culture on SP4 glucose broth with arginine

D. Complement fixation

A. EIA test and direct antigen testing

Mycoplasma pneumoniae background/diagnostics
• Preferred confirmation uses EIA/ELISA antigen or IgM antibody detection
• Cold agglutinins are unreliable and nonspecific
• Culture on SP4 media is slow and rarely done outside reference labs
• Complement fixation historically used but less reliable and slower than modern assays
• Causes atypical “walking pneumonia” with nonproductive cough

Quick differential:
• Chlamydia pneumoniae: serology or NAAT
• Legionella pneumophila: urine antigen (SG1) or PCR
• Viral atypical pneumonias: negative for cold agglutinins, PCR used

Summary: EIA/direct antigen testing is the preferred, reliable method for confirming M. pneumoniae infection.

Identify the following bacterium-specimen pairing that is mismatched (specimen not appropriate for isolation):

A. Chlamydia (Chlamydophila) psittaci-fecal swab

B. Chlamydia trachomatis - first voided urine

C. Chlamydia trachomatis - endocervical swab

D. Chlamydia pneumonia - throat swab or sputum

A. Chlamydia (Chlamydophila) psittaci-fecal swab

Chlamydia psittaci background/diagnostics
• Cause of psittacosis transmitted from birds
• Best specimens: respiratory samples (sputum, BAL), sometimes blood for serology
• Fecal swabs are inappropriate for clinical diagnosis in humans

Chlamydia trachomatis specimen notes
• First-void urine: excellent for NAAT screening
• Endocervical swab: traditional specimen of choice

Chlamydia pneumoniae specimen notes
• Respiratory pathogen; detected from throat swabs or sputum via NAAT/serology

Quick differential:
• C. pneumoniae: mild atypical pneumonia
• C. trachomatis: STI, neonatal conjunctivitis/pneumonia
• C. psittaci: bird exposure, flu-like illness, hepatosplenomegaly

Summary: C. psittaci requires respiratory specimens, not fecal swabs.

Which of the following organisms are transmitted to animals and humans after a tick bite?

A. Leptospira spp

B. Chlamydia and Mycoplasma spp

C. Neisseria sicca

D. Ehrlichia and Anaplasma spp

D. Ehrlichia and Anaplasma spp

Ehrlichia/Anaplasma background/diagnostics
• Tick-borne obligate intracellular bacteria
• Infect WBCs
– Ehrlichia: monocytes
– Anaplasma: neutrophils
• Causes flu-like illness, cytopenias, and elevated liver enzymes
• Diagnose by PCR, serology, or morulae on peripheral smear
• Transmitted by Ixodes or Amblyomma ticks

Quick differential:
• Leptospira: water/animal urine exposure, not ticks
• Chlamydia/Mycoplasma: person-to-person, no tick link
• Neisseria sicca: respiratory commensal, not vector-borne

Summary: Ehrlichia and Anaplasma are tick-borne infections targeting white blood cells.

Following a hike in the woods, a young male noted a tick on his ankle. He removed the tick, but 2 weeks later, he noticed a circular, "bull's eye" rash at the site of the bite. Which specimen(s) should be obtained to establish a diagnosis of Lyme borreliosis?

A. Lymph node biopsy, skin scraping

B. Blood, CSF, skin biopsy

C. Hair, fingernails

D. Saliva, sputum

B. Blood, CSF, skin biopsy

Borrelia burgdorferi background/diagnostics
• Transmitted by Ixodes ticks; classic erythema migrans (“bull’s-eye” rash)
• Two-tier serology recommended: EIA screen → Western blot confirmation
• Blood or CSF specimens can aid diagnosis in later disseminated disease
• Skin biopsy from rash edge useful for PCR or histopathologic confirmation
• Culture rarely performed clinically

Quick differential:
• Southern tick–associated rash illness (STARI): EM-like rash, different etiology
• Anaplasma/Ehrlichia: leukopenia, morulae in WBCs
• Rocky Mountain spotted fever: petechial rash wrists → trunk, serology/PCR needed

Summary: Blood, CSF, or skin biopsy provide appropriate sources for Lyme disease testing.

Partially acid-fast, gram-positive, filamentous-branching bacteria that fragmented into rods and cocci were seen in a sputum sample from a patient with HIV infection. Which of the following is the most likely presumptive identification?

A. Rhodococcus equi

B. Streptococcus spp

C. Bacillus subtillus

D. Corynebacterium spp (diphtheroids)

A. Rhodococcus equi

Rhodococcus equi background/morphology
• Partially acid-fast, GPR, filamentous-branching forms that fragment into rods/cocci
• Opportunistic pathogen; associated with HIV/immunocompromised patients
• Causes cavitary pneumonia resembling TB
• Salmon-pink colonies on culture (may take time to develop)
• Differentiated from Nocardia by more coccoid forms and lack of extensive aerial hyphae

Quick differential:
• Streptococcus: gram-positive cocci, not acid-fast, no branching
• Bacillus subtilis: spore-former, non–acid-fast rods
• Corynebacterium: club-shaped rods, no branching, diphtheroids in normal flora

Summary: Partially acid-fast branching bacteria that break into cocci in an HIV patient point to Rhodococcus equi.

An asymptomatic 25-year-old female patient with persistent nongonococcal urethritis (NGC) and pelvic inflammatory disease (PID) was suspected of being infected with Mycoplasma genitalium. Which category of testing should not be utilized to establish a final identification?

A. Nucleic acid amplification test (NAAT)

B. Real time PCR

C. PCR

D. Culture

D. Culture

Mycoplasma genitalium background/diagnostics
• Extremely slow-growing and fastidious—may take up to 6 months to culture
• Culture is not practical and not recommended for clinical diagnosis
• NAATs, PCR, and real-time PCR are the preferred methods for detection
• Organism lacks a cell wall, making it resistant to β-lactam antibiotics
• Causes nongonococcal urethritis (NGU), PID, and cervicitis

Quick differential:
• Mycoplasma hominis: culture possible, but not for M. genitalium
• Ureaplasma urealyticum: can be cultured on A8 agar, urea positive
• Chlamydia trachomatis: intracellular, detected via NAAT, not culture

Summary: Culture is not used for M. genitalium—molecular testing is the diagnostic standard.