8.4 - Miscellaneous and Fastidious Gram-Negative Rods

A visitor to South America who returned with diarrhea is suspected of being infected with V. cholerae. Select the best medium for recovery and identification of this organism.

A. MacConkey agar

B. Blood agar

C. TCBS agar

D. XLD agar

C. TCBS agar

Key Distinguishing Features of Vibrio cholerae

• Clinical presentation: Causes cholera, marked by profuse watery diarrhea after exposure to contaminated water or seafood.

• Selective medium: Best isolated on TCBS agar (Thiosulfate Citrate Bile Salts Sucrose)—formulates high pH and bile salts to inhibit enterics.

• Identification on TCBS: V. cholerae ferments sucrose, producing yellow colonies, whereas V. parahaemolyticusforms green colonies.

• Other traits: Oxidase positive, motile curved GNR, grows in alkaline peptone water.

Summary
V. cholerae is best recovered on TCBS agar, where its yellow sucrose-fermenting colonies confirm the connection between this selective medium and the agent of cholera.

A curved gram-neg, rod-producing, oxidase pos colonies on blood agar was recovered from a stool culture. Given the following results, what is the most likely identification?

Lysine decarboxylase +

Arginine decarboxylase neg

Indole +

KIA: Alk/Acid

VP neg

Lactose neg

Urease +/-

String test neg

TCBS agar: green colonies

A. Vibrio cholerae

B. Vibrio parahaemolyticus

C. Shigella spp

D. Salmonella spp

B. Vibrio parahaemolyticus

Key Distinguishing Features of Vibrio parahaemolyticus

• Clinical presentation: Causes gastroenteritis from raw or undercooked seafood, especially shellfish.

• Growth traits: Curved GNR, oxidase positive, lactose negative, forms green colonies on TCBS (non–sucrose fermenter).

• Biochemical profile: Lysine +, arginine –, indole +, KIA: Alk/Acid, VP –, urease ±.

• Differentiation: V. cholerae forms yellow colonies (sucrose +) and is string test positive.

Summary
V. parahaemolyticus forms green colonies on TCBS and is the leading seafood-associated Vibrio causing diarrhea.

A gram-neg S-shaped rod recovered from selective media for Campylobacter species gave the following results:

Catalase +

Oxidase +

Nitrate Reduction +

Motility +

Hippurate Hydrolysis +

Growth at 42C: +

Nalidixic Acid: S

Pigment: neg

Grape odor: neg

Cephalothin: R

The most likely ID is:

A. Pseudomonas aeruginosa

B. Campylobacter jejuni

C. Campylobacter fetus

D. Pseudomonas putida

B. Campylobacter jejuni

Key Distinguishing Features of Campylobacter jejuni

• Clinical presentation: Leading cause of bacterial gastroenteritis, often from undercooked poultry or contaminated water.

• Morphology/growth: Gram-negative S-shaped rod, motile, oxidase+, catalase+, grows at 42 °C (thermophilic).

• Biochemical profile: Hippurate hydrolysis positive, nitrate reduction positive, nalidixic acid susceptible, cephalothin resistant.

• Differentiation: C. fetus grows best at 25–37 °C, is hippurate negative, and associated with systemic infections, not enteritis.

Summary
Campylobacter jejuni is a thermophilic, hippurate-positive, S-shaped rod that grows at 42 °C, confirming its role in enteric disease.

Which atmospheric condition is needed to recover Campylobacter spp from specimens inoculated onto a Campy-selective agar at 35C to 37C and 42C?

A. 5% O2, 10% CO2, and 85% N2

B. 20% O2, 10% CO2, and 70% N2

C. 20% O2, 20% CO2, and 60% N2

D. 20% O2, 5% CO2, and 75% N2

A. 5% O2, 10% CO2, and 85% N2

Key Distinguishing Features of Campylobacter spp Atmospheric Requirements

• Optimal growth conditions: Require microaerophilic environment — low O₂, increased CO₂.

• Specific composition: 5% O₂, 10% CO₂, and 85% N₂ supports growth at 35–42 °C.

• Growth media: Cultured on Campy-selective agar (e.g., Skirrow or Campy-BAP).

• Differentiation: Cannot grow in ambient air (too much O₂) or anaerobic conditions.

Summary
Campylobacter spp grow best under microaerophilic conditions—5% O₂, 10% CO₂, 85% N₂—which mimic their intestinal habitat.

Which groups of tests best differentiates Helicobacter pylori from C. jejuni?

A. Catalase, oxidase, and Gram stain

B. Catalase, oxidase, and nalidixic acid sensitivity

C. Catalase, oxidase, and cephalothin sensitivity

D. Urease, nitrate, and hippurate hydrolysis

D. Urease, nitrate, and hippurate hydrolysis

Key Distinguishing Features of Helicobacter pylori vs Campylobacter jejuni

• Clinical presentation: H. pylori → gastritis and peptic ulcers; C. jejuni → enteritis and diarrhea.

• Biochemical traits: H. pylori is urease positive, nitrate positive, and hippurate negative.

• C. jejuni is urease negative, nitrate positive, and hippurate positive.

• Differentiation: The combination of urease, nitrate, and hippurate hydrolysis best separates the two.

Summary
H. pylori is distinguished from C. jejuni by its strong urease activity and negative hippurate test.

Which of the following tests should be done first to differentiate Aeromonas spp from the Enterobacteriaceae?

A. Urease

B. OF glucose

C. Oxidase

D. Catalase

C. Oxidase

Key Distinguishing Features of Aeromonas spp vs Enterobacteriaceae

• Clinical context: Aeromonas spp cause gastroenteritis, wound infections, and sepsis, often linked to freshwater exposure.

• Primary distinction: Aeromonas are oxidase positive, while Enterobacteriaceae are oxidase negative.

• Other traits: Aeromonas are facultatively anaerobic, motile, and glucose fermenters (may resemble enterics on MAC).

• Testing note: Oxidase is the first test performed to separate these groups.

Summary
An oxidase test is the key initial step — Aeromonas are oxidase positive, distinguishing them from the oxidase-negative Enterobacteriaceae.

Which is the best rapid test to differentiate P. shigelloides from a Shigella species on selective enteric agar?

A. Oxidase

B. Indole

C. Triple-sugar iron agar

D. Urease

A. Oxidase

Key Distinguishing Features of Plesiomonas shigelloides vs Shigella spp

• Clinical overlap: Both may cause acute diarrheal disease and appear similar on enteric media (non–lactose fermenters).

• Primary distinction: P. shigelloides is oxidase positive, while Shigella spp are oxidase negative.

• Other traits: P. shigelloides is motile, indole positive, and decarboxylase positive; Shigella is nonmotile and biochemically inert.

• Testing note: Oxidase is the most rapid and reliable test for differentiation.

Summary
An oxidase test quickly separates P. shigelloides (oxidase positive) from Shigella (oxidase negative) on enteric media.

Which are the best two tests to differentiate A. hydrophilia from P. shigelloides?

A. Oxidase and motility

B. DNase and Voges-Proskauer test

C. Indole and lysine decarboxylase

D. Growth on MacConkey and blood agar

B. DNase and Voges-Proskauer test

Key Distinguishing Features of Aeromonas hydrophila vs Plesiomonas shigelloides

• Clinical overlap: Both are oxidase-positive, motile, gram-negative rods found in freshwater and cause gastrointestinal or wound infections.

• Biochemical traits: A. hydrophila is DNase positive and VP positive.

• P. shigelloides is DNase negative and VP negative.

• Differentiation: DNase and Voges–Proskauer tests are the most reliable for separation.

Summary
A. hydrophila is DNase+ / VP+, while P. shigelloides is DNase– / VP–, making these tests key for differentiation.

Which genus (in which most species are oxidase and catalase positive) of small GNCB is associated mainly with animals but may cause endocarditis and bacteremia, as well as wound and dental infection in humans?

A. Aggregatibacter (formerly Actinobacillus spp)

B. Pseudomonas

C. Campylobacter

D. Vibrio

A. Aggregatibacter (formerly Actinobacillus spp)

Key Distinguishing Features of Aggregatibacter (formerly Actinobacillus) spp

• Clinical association: Normal flora of animals and humans; can cause endocarditis, wound, and dental infections (especially following animal contact or bites).

• Morphology/traits: Small gram-negative coccobacilli (GNCB) that are oxidase positive and catalase positive.

• Growth pattern: Often part of the HACEK group, requiring enriched CO₂ environments for optimal growth.

• Differentiation: Distinguished from Pseudomonas, Campylobacter, and Vibrio, which are motile rods and not typical oral flora.

Summary
Aggregatibacter spp are oxidase- and catalase-positive GNCB linked to animal-associated and oral infections, including endocarditis.

Which of the following tests may be used to differentiate Cardiobacterium hominis from Aggregatibacter spp (formerly Actinobacillus spp)?

A. Gram stain

B. Indole

C. Anaerobic incubation

D. Oxidase

B. Indole

Key Distinguishing Features of Cardiobacterium hominis vs Aggregatibacter spp

• Clinical relevance: Both are HACEK group organisms that cause endocarditis.

• Gram stain: C. hominis shows pleomorphic GNRs often with rosette or filamentous forms; Aggregatibacterappears as small, uniform GNCB.

• Biochemical traits: C. hominis is indole positive, while Aggregatibacter spp are indole negative.

• Differentiation: The indole test is the most reliable for distinguishing between the two.

Summary
Cardiobacterium hominis is indole positive, separating it from indole-negative Aggregatibacter spp in HACEK-associated endocarditis.

A mixture of slender GNRs and coccobacilli w/ rounded ends was recovered from blood cultures after a pt's root canal surgery. Given the following results after 48 hours, what is the most likely organism?

Catalase neg

Urease neg

Oxidase +

Indole neg

Ornithine decarboxylase +

Lysine decarboxylase +

X & V requirement neg

Carbohydrates neg (no acid produced)

Growth on blood & choc agar + (w/ pitting of agar)

Mac agar neg

A. Eikenella corrodens

B. Aggregatibacter

C. Cardiobacterium hominis

A. Eikenella corrodens

Key Distinguishing Features of Eikenella corrodens

• Clinical context: Part of the HACEK group; commonly associated with dental procedures, human bites, and endocarditis.

• Morphology/growth: Slender GNRs and GNCB that cause pitting or “corroding” of blood and chocolate agar; no growth on MacConkey.

• Biochemical traits: Oxidase positive, catalase negative, urease negative, indole negative, carbohydrate inert, ornithine and lysine decarboxylase positive.

• Differentiation: Distinguished from Aggregatibacter and Cardiobacterium by pitting agar and lack of carbohydrate utilization.

Summary
Eikenella corrodens is an oxidase-positive, agar-pitting GNR linked to oral flora infections and post–dental-procedure bacteremia.

Kingella kingae can best be differentiated from E. corrodens by using which medium?

A. Sheep blood agar

B. Chocolate agar

C. MacConkey agar

D. Xylose lysine deoxychocolate agar

A. Sheep blood agar

Key Distinguishing Features of Kingella kingae vs Eikenella corrodens

A. Sheep blood agar ✅
B. Chocolate agar
C. MacConkey agar
D. Xylose lysine deoxychocolate agar

Answer: A. Sheep blood agar

• Clinical overlap: Both belong to the HACEK group, causing endocarditis and oropharyngeal infections.

• Growth traits: K. kingae produces β-hemolysis on sheep blood agar, while E. corrodens shows no hemolysis but pits the agar surface.

• Both may grow on chocolate agar but differ in colony appearance on blood agar.

Summary
Sheep blood agar best differentiates the two—K. kingae is β-hemolytic, whereas E. corrodens is nonhemolytic with pitting colonies.

K. kingae is usually associated with which type of infection?

A. Middle ear infection

B. Endocarditis

C. Meningitis

D. Urogenital infection

B. Endocarditis

Key Distinguishing Features of Kingella kingae

• Clinical relevance: Member of the HACEK group; most often causes endocarditis, particularly in children and young adults.

• Other infections: Can produce septic arthritis and osteomyelitis in pediatric patients.

• Growth traits: β-hemolytic on sheep blood agar, oxidase positive, and grows on chocolate agar.

Summary
Kingella kingae is primarily associated with endocarditis, occasionally extending to bone and joint infections in young children.

Cultures obtained from a dog bite wound produced yellow, tan, and slightly pink colonies on blood & chocolate agar, with a margin of fingerlike projections appearing as a film around the colonies. Given the following results at 24 hours, which is the most likely organism?

Oxidase +

Catalase +

Growth on MacConkey: neg

Motility: neg

A. Aggregatibacter (formerly Actinobacillus) spp

B. Eikenella spp

C. Capnocytophaga spp

D. Pseudomonas spp

C. Capnocytophaga spp

Key Distinguishing Features of Capnocytophaga spp

• Clinical relevance: Found in the oral flora of dogs and cats; causes wound infections, sepsis, and cellulitisfollowing animal bites.

• Morphology/growth: Yellow-tan to pink colonies with fingerlike or spreading projections forming a thin film on blood and chocolate agar; no growth on MacConkey.

• Biochemical traits: Oxidase positive, catalase positive, nonmotile, capnophilic (enhanced by CO₂).

• Differentiation: Eikenella pits agar but lacks spreading projections; Pseudomonas grows on Mac and is oxidase positive with a grape odor.

Summary
Capnocytophaga spp are oxidase- and catalase-positive, nonmotile GNRs producing film-like spreading colonies, classically isolated from dog or cat bite wounds.

Smooth gray colonies showing no hemolytic activity were recovered from an infected cat scratch wound culture; the colonies grew on blood & chocolate agar (a must odor was noted) but failed to grow on MacConkey agar. The organisms were gram-negative pleomorphic rods that were both catalase and oxidase positive and strongly indole positive. The most likely organism is:

A. Haemophilus spp

B. Pasteurella spp

C. Proteus spp

D. Pseudomonas spp

B. Pasteurella spp

Key Distinguishing Features of Pasteurella spp

• Clinical relevance: Commonly causes soft tissue infections after cat or dog bites or scratches.

• Morphology/growth: Gram-negative pleomorphic rods forming smooth gray colonies with a musty odor; grow on blood and chocolate agar, no growth on MacConkey.

• Biochemical traits: Oxidase positive, catalase positive, and strongly indole positive.

• Differentiation: Haemophilus requires X and/or V factors; Proteus is motile and produces a swarming pattern; Pseudomonas grows on MacConkey and produces pigment/odor.

Summary
Pasteurella spp are oxidase-, catalase-, and indole-positive pleomorphic GNRs with a musty odor, typically isolated from cat bite or scratch wounds.

Which media should be used to recover B. pertussis from a nasopharyngeal specimen?

A. Chocolate agar

B. Blood agar

C. MacConkey agar

D. Bordet-Gengou agar

D. Bordet-Gengou agar

Key Distinguishing Features of Bordetella pertussis

• Clinical relevance: Causative agent of whooping cough, transmitted via respiratory droplets; isolated from nasopharyngeal specimens.

• Selective media: Best recovered on Bordet-Gengou agar, containing potato infusion, glycerol, and blood, which supports growth and inhibits contaminants.

• Alternative media: Regan-Lowe agar (charcoal–blood medium) can also be used.

• Colony appearance: Small, smooth, mercury-drop-like colonies after 3–7 days of incubation.

Summary
B. pertussis is best recovered on Bordet-Gengou agar, the classic selective medium for isolating the agent of whooping cough.

Which medium is recommended for the recovery of Brucella spp from blood and bone marrow specimens?

A. Biphasic Castenada bottles with Brucella broth

B. Blood culture bottles with Brucella broth

C. Bordet-Gengou agar plates and THIO broth

D. Blood culture bottles with THIO broth

A. Biphasic Castenada bottles with Brucella broth

Key Distinguishing Features of Brucella spp Recovery

• Clinical relevance: Brucella spp cause brucellosis, a systemic zoonosis acquired from animal exposure or unpasteurized dairy.

• Optimal medium: Biphasic Castaneda bottles combine solid and liquid Brucella broth, improving recovery from blood and bone marrow specimens.

• Growth traits: Slow-growing, aerobic, and requires enriched media with extended incubation (up to 30 days).

• Differentiation: THIO broth or standard blood culture systems are less effective for isolation.

Summary
Brucella spp are best recovered using biphasic Castaneda bottles with Brucella broth, which enhance growth from blood and bone marrow cultures.

In addition to CO2 requirements and biochemical characteristics, B. melitensis and Brucella abortus are differentiated by growth on media containing which two dyes?

A. Basic fuchsin and thionin

B. Methylene blue and crystal violet

C. Carbol fuchsin and iodine

D. Safranin and methylene blue

A. Basic fuchsin and thionin

Key Distinguishing Features of Brucella melitensis and Brucella abortus

• Clinical relevance: Both cause brucellosis, but differ in animal reservoirs — B. melitensis (goats/sheep) and B. abortus (cattle).

• Differentiation method: Species are distinguished by growth on media containing basic fuchsin and thionin dyes at specific concentrations.

• Growth patterns:

  • B. melitensis → Inhibited by thionin, not inhibited by basic fuchsin.

  • B. abortus → Inhibited by basic fuchsin, not inhibited by thionin.

• Additional differentiation: B. suis and B. canis show different inhibition profiles.

Summary
B. melitensis and B. abortus are differentiated by dye sensitivity to basic fuchsin and thionin, aiding species-level identification in brucellosis.

Which of the following amino acids are required for growth of Francisella tularensis?

A. Leucine and ornithine

B. Arginine and lysine

C. Cysteine and cystine

D. Histidine and tryptophan

C. Cysteine and cystine

Key Distinguishing Features of Francisella tularensis

• Clinical relevance: Causes tularemia, a zoonotic infection transmitted by ticks, rabbits, or aerosols.

• Growth requirements: Requires cysteine or cystine for growth; will not grow on routine media.

• Preferred media: Cysteine-enriched chocolate agar or buffered charcoal yeast extract (BCYE) agar.

• Differentiation: Distinguished from other small gram-negative coccobacilli (Brucella, Pasteurella) by this unique amino acid requirement.

Summary
F. tularensis requires cysteine or cystine for growth, making this amino acid dependency diagnostic for tularemia.

Which medium is best for recovery of Legionella pneumophilia from clinical specimens?

A. Chocolate agar

B. Bordet-Gengou agar

C. New yeast extract agar

D. Buffered charcoal-yeast extract (CYE) agar

D. Buffered charcoal-yeast extract (CYE) agar

Key Distinguishing Features of Legionella pneumophila

• Clinical relevance: Causes Legionnaires’ disease and Pontiac fever, typically acquired from aerosolized water sources.

• Growth requirements: Requires L-cysteine and iron salts for growth; will not grow on blood or chocolate agar.

• Optimal medium: Buffered charcoal-yeast extract (BCYE) agar provides both nutrients and neutralizes toxic compounds.

• Colony morphology: Gray-white, ground-glass appearance after 3–5 days incubation.

Summary
L. pneumophila is best recovered on BCYE agar, which supplies the cysteine and iron essential for its growth.

Haemophilus aegyptius (formerly H. influenzae biogroup aegyptius) causes ocular infections ("pink eye") and requires X and V factors in the primary medium for growth. H. aegyptius and H. influenzae can further be identified and differentiated by which two tests?

A. Indole and Xylose

B. Glucose and urease

C. Oxidase and catalase

D. Aminolevulinic acid (ALA) test and oxidase

A. Indole and Xylose

Key Distinguishing Features of Haemophilus aegyptius vs Haemophilus influenzae

• Clinical relevance: H. aegyptius causes acute conjunctivitis ("pink eye"), while H. influenzae causes respiratory and systemic infections.

• Growth requirements: Both require X (hemin) and V (NAD) factors for growth.

• Differentiation tests:

  • H. aegyptius → Indole negative, xylose negative.

  • H. influenzae → Indole positive, xylose positive.

• ALA test: Not useful for distinguishing these two, as both require external hemin (X factor).

Summary
H. aegyptius and H. influenzae are best differentiated by indole and xylose tests, with H. influenzae positive for both.

Haemophilus species that require the V factors (NAD) are easily recovered on which primary agar plate?

A. Blood agar made with sheep RBCs

B. Blood agar made with horse RBCs

C. Chocolate agar

D. Xylose agar

C. Chocolate agar

Key Distinguishing Features of Haemophilus spp Growth Requirements

• Growth factors: Many Haemophilus spp require V factor (NAD) and sometimes X factor (hemin).

• Medium selection: Chocolate agar provides both factors because heating lyses RBCs, releasing hemin (X) and NAD (V).

• Blood agar limitation: Sheep blood agar does not release NAD, so Haemophilus requiring V factor will not grow unless “satelliting” near NAD-producing colonies (e.g., S. aureus).

• Differentiation: Horse blood may release some NAD, but chocolate agar remains the preferred medium.

Summary
Haemophilus spp that require V factor (NAD) are best recovered on chocolate agar, where both X and V factors are freely available.

Which of the following products is responsible for satellite growth of Haemophilus spp around colonies of Staphylococcus growing on sheep blood agar?

A. NAD and Hemin

B. Lactose

C. Indole

D. Oxidase

A. NAD and Hemin

Key Distinguishing Features of Haemophilus Satellite Phenomenon

• Mechanism: Haemophilus spp require V factor (NAD) and sometimes X factor (hemin) for growth.

• Source: When grown on sheep blood agar, Haemophilus cannot access NAD because it is trapped in intact RBCs.

• Staphylococcus effect: Staphylococcus aureus releases NAD (V factor) and hemin (X factor) as byproducts of its metabolism and hemolysis, enabling Haemophilus to grow as satellite colonies around it.

• Diagnostic use: This satellite growth helps identify Haemophilus spp in mixed cultures.

Summary
Haemophilus spp exhibit satellite growth around S. aureus due to released NAD and hemin, which supply their essential growth factors.

Which of the following plates should be used to identify H. haemolyticus and H. parahaemolyticus?

A. Sheep blood agar and chocolate agar

B. Horse blood agar and Mueller-Hinton agar with X and V strips

C. Brain-heart infusion (BHI) agar with sheep red cells added

D. Chocolate agar and Mueller-Hinton agar with X factor added

B. Horse blood agar and Mueller-Hinton agar with X and V strips

Key Distinguishing Features of Haemophilus haemolyticus and Haemophilus parahaemolyticus

• Clinical relevance: Both are part of the normal upper respiratory flora but can cause opportunistic infections.

• Growth factors:

  • H. haemolyticus requires X and V factors.

  • H. parahaemolyticus requires V factor only.

• Medium use:

  • Chocolate agar supplies both X and V factors for primary recovery.

  • Sheep blood agar is used to demonstrate hemolysis—both species are β-hemolytic, unlike nonhemolytic H. influenzae and H. parainfluenzae.

Summary
Use chocolate agar for growth and sheep blood agar to observe β-hemolysis, confirming H. haemolyticus or H. parahaemolyticus.

The majority of H. influenzae infections are caused by which of the following capsular serotypes?

A. a

B. b

C. c

D. d

B. b

Key Distinguishing Features of Haemophilus influenzae Type b (Hib)

• Clinical relevance: Type b is the most virulent capsular serotype, responsible for the majority of invasive infections.

• Major diseases: Causes meningitis, epiglottitis, septicemia, and pneumonia, especially in unvaccinated children.

• Virulence factor: The polyribitol phosphate (PRP) capsule enhances resistance to phagocytosis and complement-mediated killing.

• Other serotypes: Types a, c–f, and nontypeable strains cause milder or localized infections (e.g., otitis media).

Summary
Most serious H. influenzae infections are caused by serotype b, due to its PRP capsule and high invasiveness.

Which of the following, usually recovered from blood cultures, is generally associated with subacute bacterial endocarditis?

A. Haemophilus influenzae

B. Haemophilus ducreyi

C. Aggregatibacter (Haemophilus) aphrophilus

D. Haemophilus haemolyticus

C. Aggregatibacter (Haemophilus) aphrophilus

Key Distinguishing Features of Aggregatibacter aphrophilus

• Clinical relevance: Member of the HACEK group, commonly isolated from blood cultures in cases of subacute bacterial endocarditis.

• Source: Normal flora of the oral cavity and upper respiratory tract; infection often follows dental proceduresor poor oral hygiene.

• Growth traits: Capnophilic, grows on chocolate and blood agar (requires CO₂ enrichment), and does not require X or V factors.

• Differentiation: Unlike H. influenzae or H. ducreyi, A. aphrophilus is non–X/V-dependent and rarely causes respiratory or genital infections.

Summary
Aggregatibacter (Haemophilus) aphrophilus is a HACEK organism most often associated with subacute bacterial endocarditis.

Which Haemophilus species is difficult to isolate and recover from genital ulcers and swollen lymph nodes?

A. Haemophilus aegyptius

B. Haemophilus ducreyi

C. Haemophilus haemolyticus

D. Haemphilus parahaemolyticus

B. Haemphilus ducreyi

Key Distinguishing Features of Haemophilus ducreyi

• Clinical relevance: Causes chancroid, a sexually transmitted infection characterized by painful genital ulcersand inguinal lymphadenopathy.

• Isolation difficulty: Fastidious and fragile, requiring enriched media (e.g., GC agar with hemoglobin and IsoVitalex) and high humidity with 5–10% CO₂ for recovery.

• Growth traits: Requires X factor (hemin) but not V factor (NAD); colonies are grayish-yellow and rough.

• Differentiation: Unlike H. aegyptius or H. haemolyticus, H. ducreyi is nonhemolytic and strictly associated with genital lesions.

Summary
Haemophilus ducreyi is a fastidious, X factor–dependent species that causes chancroid and is difficult to isolate from genital ulcers and swollen lymph nodes.

Which of the following is a characteristic of strains of H. influenzae that are resistant to ampicillin?

A. Production of beta-lactamase enzymes

B. Hydrolysis of chloramphenicol

C. Hydrolysis of urea

D. All of these options

A. Production of beta-lactamase enzymes

Key Distinguishing Features of Ampicillin-Resistant Haemophilus influenzae

• Mechanism of resistance: Resistance is due to the production of β-lactamase enzymes, which hydrolyze the β-lactam ring of ampicillin, rendering the drug inactive.

• Testing: Detected by nitrocefin (cefinase) disk test.

• Other drugs: Chloramphenicol resistance occurs by acetyltransferase production, not related to ampicillin resistance.

• Clinical note: β-lactamase–positive strains are common in respiratory and systemic infections.

Summary
Ampicillin-resistant H. influenzae produce β-lactamase, which inactivates the antibiotic’s β-lactam ring.

A small GNCB recovered from the CSF of a 2 yo unvaccinated child gave the following results:

Indole +

X requirement +

Urease +

Sucrose neg

Glucose + (acid)

V requirement +

Lactose neg

Hemolysis neg

Which is the most likely ID?

A. Haemophilus parainfluenzae

B. Haemophilus influenzae

C. Haemophilus ducreyi

D. Aggregatibacter (formerly Haemophilus) aphrophilus

B. Haemophilus influenzae

Key Distinguishing Features of Haemophilus influenzae

• Clinical relevance: Major cause of bacterial meningitis in unvaccinated children (particularly type b).

• Growth requirements: Requires both X factor (hemin) and V factor (NAD) for growth.

• Biochemical traits: Indole positive, urease positive, glucose fermenter, nonhemolytic, lactose and sucrose negative.

• Differentiation:

  • H. parainfluenzae requires only V factor.

  • H. ducreyi requires X factor only.

  • Aggregatibacter spp do not require X or V factors.

Summary
Haemophilus influenzae is X and V factor–dependent, indole and urease positive, and is the leading cause of meningitis in unvaccinated young children.

The ALA test (for porphyrins) is a confirmatory procedure for which test used for identification of Haemophilus species?

A. X factor requirement

B. V factor requirement

C. Urease production

D. Indole production

A. X factor requirement

Key Distinguishing Features of the ALA (Porphyrin) Test

• Purpose: The δ-aminolevulinic acid (ALA) test confirms whether an organism can synthesize hemin (X factor)from ALA.

• Interpretation:

  • Positive ALA test → organism does not require X factor (e.g., H. parainfluenzae).

  • Negative ALA test → organism requires X factor (e.g., H. influenzae).

• Mechanism: Detects porphyrin fluorescence (red under UV light) indicating hemin synthesis.

Summary
The ALA test confirms the X factor requirement—a negative result identifies species like H. influenzae that depend on external hemin.

An older woman who cared for several domestic cats was hospitalized with suspected cat scratch disease (CSD). Blood cultures appeared negative, but after several days, a small, slightly curved pleomorphic GNB grew on BHI agar (with 5% horse or rabbit blood). Other biochemical testing gave negative results. What is the most likely identification?

A. Bartonella spp

B. Brucella spp

C. Kingella spp

D. Haemophilus spp

A. Bartonella spp

Key Distinguishing Features of Bartonella spp

• Clinical relevance: Causes cat scratch disease (CSD), trench fever, and bacillary angiomatosis; transmitted via cat scratches or bites (B. henselae most common).

• Growth traits: Fastidious, pleomorphic, curved gram-negative bacillus that grows slowly on BHI agar with 5% horse or rabbit blood.

• Biochemical profile: Oxidase, catalase, and urease negative; minimal biochemical reactivity.

• Culture characteristics: Requires enriched media and prolonged incubation (up to 2–6 weeks).

Summary
Bartonella henselae is the slow-growing, pleomorphic GNB responsible for cat scratch disease, often requiring extended incubation on blood-enriched media for recovery.

A 5 yo nonimmunized male w/ a persistent cough, fever, and flulike symptoms was admitted to the hospital. Nasopharyngeal swabs were cultured on 15% blood, chocolate, Bordet-Gengou, and Regan-Lowe (with 10% charcoal) agars. All media grew a GNCB after 3 days incubation. Carbohydrate & biochemical tests gave negative results. What is the most likely ID?

A. Haemophilus influenza

B. Bordetella pertussis

C. Haemophilus parainfluenzae

D. Bordetella bronchiseptica

B. Bordetella pertussis

Key Distinguishing Features of Bordetella pertussis

• Clinical fit: Unimmunized child with paroxysmal cough/fever → classic whooping cough.

• Culture media: Slow growth (≈3 days) on Bordet–Gengou and Regan–Lowe (charcoal); small GNCB also seen on chocolate and 15% blood.

• Biochemical profile: Inert on routine carbohydrate/biochem tests; nonmotile, urease negative.

• Differentiation: B. bronchiseptica is typically urease positive and motile; Haemophilus spp need X/V factors and don’t characteristically grow on pertussis media.

Summary
A biochemically inert GNCB from nasopharyngeal swabs growing on Bordet–Gengou/Regan–Lowe in an unvaccinated child points to B. pertussis.

A 29 yo male who often hunted rabbits & spent a lot of time in the woods was admitted to the hospital with skin ulcers on his upper extremities. At 48 hrs, a small coccobacillus was recovered only from the aerobic blood culture bottle. The organism stained poorly with Gram stain but did stain with acridine orange. Cultures taken from the ulcers did not grow on primary media. What is the most likely ID?

A. P. aeruginosa

B. Pseudomonas fluorescens

C. Chryseobacterium spp

D. Francisella tularensis

D. Francisella tularensis

Key Distinguishing Features of Francisella tularensis

• Clinical fit: Rabbit exposure + skin ulcers → classic ulceroglandular tularemia.

• Microscopy: Tiny, faintly staining GNCB on Gram; acridine orange positive.

• Culture pattern: Fastidious; may grow only in aerobic blood culture early; no growth on routine primary media from ulcers.

• Nutritional needs: Requires cysteine/cystine (e.g., cysteine-enriched chocolate, BCYE).

Summary
A poorly staining small coccobacillus after rabbit contact, recovering mainly from blood culture and needing cysteinefor growth, is most consistent with F. tularensis.

A neonate was readmitted to the hospital with a diagnosis of meningitis. CSF revealed gram-negative straight rods. At 24 hrs, the organism grew on 5% sheep blood and chocolate agars displaying a yellow pigment. On MacConkey agar, it appeared as an NLF. Colonies were oxidase, DNase, and gelatinase positive, and oxidized glucose and mannitol. What is the most likely ID?

A. Haemophilus influenza

B. Elizabethkingia meningosepticum

C. Stenotrophomonas maltophilia

D. Acinetobacter spp

B. Elizabethkingia meningosepticum

Key Distinguishing Features of Elizabethkingia meningosepticum

• Clinical relevance: Causes neonatal meningitis and sepsis, especially in hospitalized or premature infants.

• Morphology/growth: Straight gram-negative rods producing yellow pigment on blood and chocolate agar; non-lactose fermenter on MacConkey.

• Biochemical traits: Oxidase positive, DNase positive, gelatinase positive, and oxidizes glucose and mannitol.

• Differentiation:

  • Stenotrophomonas maltophilia is oxidase negative.

  • Acinetobacter spp are oxidase negative and non-pigmented.

  • H. influenzae requires X and V factors and does not grow on Mac.

Summary
Elizabethkingia meningosepticum is an oxidase- and DNase-positive, yellow-pigmented nonfermenter that causes neonatal meningitis.

A 46 yo dog warden was admitted to the hospital with several puncture bite wounds encountered while wrangling a stray dog. Culture at 48 hrs produced small yellow colonies on 5% sheep blood & chocolate agars in 10% CO2, but no growth on MacConkey agar. Gram staining showed gram-negative curved, fusiform rods. Colonies were oxidase & catalase positive. What is the most likely ID?

A. Capnocytophaga canimorsus

B. Francisella tularensis

C. Legionella pneumophilia

D. Pseudomonas aeruginosa

A. Capnocytophaga canimorsus

Key Distinguishing Features of Capnocytophaga canimorsus

• Clinical relevance: Part of dog and cat oral flora; causes septicemia, cellulitis, and wound infections following animal bites, especially in dog handlers or immunocompromised patients.

• Morphology/growth: Gram-negative curved or fusiform rods producing small yellow colonies on blood and chocolate agar in 10% CO₂; no growth on MacConkey.

• Biochemical traits: Oxidase positive, catalase positive, and capnophilic.

• Differentiation: F. tularensis is tiny, poorly staining, cysteine-requiring; Legionella needs BCYE; Pseudomonasgrows on MacConkey and produces pigment.

Summary
Capnocytophaga canimorsus is an oxidase- and catalase-positive, fusiform GNR associated with dog bite wound infections.

The AACEK (formerly HACEK) group of organisms consisting of Aggregatibacter (Haemophilus) aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella spp) are known by this acronym to denote which type of infection?

A. Urinary tract infection

B. Subacute bacterial endocarditis

C. Pharyngitis

D. Tonsillitis

B. Subacute bacterial endocarditis

Key Distinguishing Features of the AACEK (formerly HACEK) Group

• Members: Aggregatibacter aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella spp.

• Clinical relevance: All are part of the oropharyngeal flora and are well-known causes of subacute bacterial endocarditis, especially following dental procedures or in patients with damaged heart valves.

• Growth traits: Slow-growing, capnophilic, and require enriched media (e.g., chocolate agar, CO₂ incubation).

• Differentiation: Distinct from more acute causes of endocarditis (e.g., Staphylococcus aureus) due to slower onset and chronic course.

Summary
The AACEK group is best known for causing subacute bacterial endocarditis, arising from oral flora organisms that enter the bloodstream.

A suspected case of Legionnaires disease was noted on the request form for a culture & sensitivity ordered on a sputum sample. The pt was a 70 yo male, who presented with a positive serological test result for Legionella spp. What is the most efficient way to confirm the infection using the submitted sample?

A. Culture the sputum on MacConkey agar

B. Gram stain of the sputum

C. Acid-fast staining

D. Direct immunofluorescent microscopy

D. Direct immunofluorescent microscopy

Key Distinguishing Features of Legionella pneumophila Confirmation

• Clinical relevance: Causes Legionnaires’ disease, a severe atypical pneumonia often linked to contaminated water sources.

• Diagnostic approach:

  • Serology suggests exposure but does not confirm active infection.

  • Direct immunofluorescent antibody (DFA) microscopy on sputum provides rapid and specific confirmation by detecting Legionella antigens.

• Culture: Requires BCYE agar for isolation, but results take several days.

• Other stains: Legionella stains poorly with Gram stain and is not acid-fast.

Summary
For rapid confirmation of Legionnaires’ disease, perform direct immunofluorescent microscopy on sputum to visualize Legionella pneumophila antigens.

Gastric biopsy material was obtained from a 35 yo male. A suspected case of H. pylori could presumptively be identified & then ultimately confirmed by which of the following methods?

A. Urea broth test and polymerase chain reaction

B. Gram staining and culture

C. Acid-fast staining and culture

D. Giemsa staining and culture

A. Urea broth test and polymerase chain reaction

Key Distinguishing Features of Helicobacter pylori Identification

• Clinical relevance: Major cause of chronic gastritis and peptic ulcer disease.

• Presumptive test: Urea broth (rapid urease test)—H. pylori produces strong urease activity, rapidly hydrolyzing urea to ammonia and raising pH (color change to pink).

• Confirmatory test: Polymerase chain reaction (PCR) detects H. pylori–specific DNA sequences, providing definitive confirmation.

• Alternative methods: Histologic stains (e.g., Giemsa, Warthin–Starry) and culture can support diagnosis but are less rapid.

Summary
H. pylori is best presumptively identified by the urea broth test and confirmed by PCR detection of its genetic material.

Which acronym is used in reference to the slow-growing group of gram-negative bacteria indicated in subacute bacterial endocarditis?

A. HLACK

B. AACEK

C. ANBBC

D. NBCCBS

B. AACEK

Key Distinguishing Features of the AACEK Group

• Acronym meaning: Aggregatibacter aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella spp.

• Clinical relevance: Slow-growing, fastidious gram-negative bacteria that cause subacute bacterial endocarditis, typically following dental procedures or arising from oral flora.

• Growth traits: Capnophilic, requiring enriched CO₂ conditions and extended incubation on chocolate or blood agar.

• Historical note: Formerly known as the HACEK group before reclassification of Haemophilus species into Aggregatibacter.

Summary
The acronym AACEK refers to the slow-growing gram-negative organisms most often associated with subacute bacterial endocarditis.

A 20 yo male presented with soft chancres in the genital area and swollen lymph nodes. A culture specimen was taken but failed to grow after 4 days on chocolate agars, sheep blood agar, and MTM agar. Gram staining showed small, pleomorphic gram-negative rods. What is the most likely presumptive identification?

A. Neisseria gonorrhoeae

B. Haemophilus ducreyi

C. Haemophilus influenzae

D. Moraxella spp

B. Haemophilus ducreyi

Key Distinguishing Features of Haemophilus ducreyi

• Clinical relevance: Causes chancroid, a sexually transmitted infection characterized by painful genital ulcersand inguinal lymphadenopathy.

• Growth characteristics: Extremely fastidious; does not grow on routine chocolate, sheep blood, or MTM agarwithout supplementation. Requires X factor (hemin) but not V factor (NAD) and high humidity with 5–10% CO₂.

• Microscopy: Small, pleomorphic gram-negative rods often seen in “school of fish” or “railroad track”arrangements on direct smears.

• Differentiation:

  • N. gonorrhoeae and Moraxella are oxidase-positive diplococci, not rods.

  • H. influenzae requires both X and V factors and causes respiratory infections, not genital ulcers.

Summary
Haemophilus ducreyi is a fastidious, X factor–dependent GNR causing painful chancroid ulcers and swollen lymph nodes.

A suspected B. pertussis diagnosis relies on symptoms & growth of the organism on Regan-Lowe agar or Bordet-Gengou agar (displaying small, shiny colonies resembling mercury droplets), and the colonies take several days to grow. The most reliable serologic identification is with which method?

A. Enzyme-linked immunosorbent assay (ELISA) using paired samples

B. Agglutination

C. Compliment fixation

D. Enzyme immunoassay

A. Enzyme-linked immunosorbent assay (ELISA) using paired samples

Key Distinguishing Features of Bordetella pertussis Serologic Identification

• Clinical relevance: Causes whooping cough, characterized by paroxysmal coughing, especially in unvaccinated children.

• Culture characteristics: Slow-growing GNCB on Regan–Lowe or Bordet–Gengou agar, forming small, shiny “mercury drop” colonies after several days.

• Serologic testing: ELISA using paired acute and convalescent sera is the most reliable method for confirming infection by detecting a fourfold rise in antibody titers against pertussis antigens.

• Other methods: Agglutination and complement fixation are less sensitive and specific.

Summary
A paired-sample ELISA is the most reliable serologic method for confirming B. pertussis infection following culture or clinical suspicion.

a 7-year old female became ill after eating a chicken sandwhich from a fast food resaurant. After 24 hours of gastroenteritis, a stool swab was obtained for culture, with normal fecal flora growing on Mac and XLD agars at 18 hours. Furthermore, growth was observed on Camp-BA at 48 hours ( at both 42C and 37C incubation). Which test(s) differntiate C. Jejuni subsp. doylei from C. jejuni subsp. jejuni because both grow at 37C and 42 C

A. Urease

B. Hippurate hydrolysis

C. Cephalothin and nalidixic acid antibiotic disks

D. Growth at 25C

C. Cephalothin an nalidixic acid antibiotic disks

Key Distinguishing Features of Campylobacter jejuni subsp. doylei vs Campylobacter jejuni subsp. jejuni

• Clinical relevance: Both subspecies cause gastroenteritis, often from undercooked poultry or contaminated food.

• Growth traits: Both grow at 37 °C and 42 °C, unlike most other Campylobacter spp.

• Differentiation: Determined by antibiotic susceptibility:

  • C. jejuni subsp. jejuni → Nalidixic acid susceptible, cephalothin resistant.

  • C. jejuni subsp. doylei → Nalidixic acid resistant, cephalothin susceptible.

• Other features: Both are oxidase positive, motile curved GNRs.

Summary
Campylobacter jejuni subspecies are differentiated by cephalothin and nalidixic acid disk testing, not by temperature or hippurate results.