Micro 8.4 Miscellaneous and Fastidious Gram-Negative Rods

Question 1

1. 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

Answer 1

C. TCBS agar

The growth of yellow or green colonies on the selective TCBS agar is dependent on whether the organism ferments sucrose (producing yellow colonies). Vibrio spp. also grow well on 5% sheep blood, chocolate, and MacConkey agars. Enrichment with alkaline peptone broth, pH 8.4, helps recover Vibrio spp. from stool specimens.

Question 2

2. A curved gram-negative, rod-producing, oxidase-positive 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.

Answer 2

B. Vibrio parahaemolyticus

V. parahaemolyticus appear as green colonies on TCBS agar, whereas V. cholerae appear as yellow colonies on TCBS. V. cholerae is the only Vibrio species that causes a positive string test result. In the test, a loopful of bacterial colonies is suspended in sodium deoxycholate, 0.5%, on a glass slide. After 60 seconds, the inoculating loop is lifted out of the suspension. V. cholerae forms a long string resembling a string of pearls. Salmonella spp. and Shigella spp. are oxidase negative.

Question 3

3. A gram-negative S-shaped rod recovered from selective media for Campylobacter species gave the following results:
   Catalase = +
   Oxidase = +
   Nitrate Reduction = +
   Motility = +
   Hippurate hydrolysis = +
   Growth at 42°C = +
   Nalidixic acid = Susceptible
   Pigment = Neg
   Grape odor = Neg
   Cephalothin = Resistant
   The most likely identification is:

A. Pseudomonas aeruginosa
B. Campylobacter jejuni
C. Campylobacter fetus
D. Pseudomonas putida

Answer 3

B. Campylobacter jejuni

The only Campylobacter spp. that hydrolyze hippurate are C. jejuni and subsp. doylei. However, some strains of P. aeruginosa grow on agar selective for Campylobacter at 42°C. C. fetus usually will not grow at 42°C but will grow at 25°C and 37°C.

Question 4

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

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

Answer 4

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

Campylobacter spp. are best recovered in a microaerophilic atmosphere (reduced O2). The use of a CO2 incubator or candle jar is not recommended because the amount of O2 and CO2 do not permit any but the most aerotolerant Campylobacter to survive. Cultures for Campylobacter should be incubated for 48 to 72 hours before reporting no growth.

Question 5

5. Which group 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

Answer 5

D. Urease, nitrate, and hippurate hydrolysis

H. pylori is found in specimens from gastric secretions and biopsies and has been implicated as a cause of gastric ulcers. It is found only in the mucous-secreting epithelial cells of the stomach. Both H. pylori and C. jejuni are catalase and oxidase positive. However, Helicobacter spp. are urease positive, which differentiates them from Campylobacter spp.

Question 6

6. 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

Answer 6

C. Oxidase

Aeromonas hydrophilia and other Aeromonas spp. have been implicated in acute diarrheal disease as well as cellulitis and wound infections. Infections usually follow exposure to contaminated soil, water, or food. Aeromonas growing on enteric media are differentiated from the Enterobacteriaceae species by demonstrating that colonies are oxidase positive. The Aeromonas are sometimes overlooked as pathogens because most strains grow on selective enteric agar as lactose fermenters.

Question 7

7. 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

Answer 7

A. Oxidase

P. shigelloides is an NLF that will resemble Shigella spp. on MacConkey agar. Both are TSI Alk/Acid and urease negative. Plesiomonas produces indole and Shigella usually causes delayed production of indole. However, Plesiomonas is oxidase positive, whereas Shigella spp. are oxidase negative. P. shigelloides has been added to the Enterobacteriaceae family through the use of nucleic acid–based methods and is the only member that is oxidase positive.

Question 8

8. 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

Answer 8

B. DNase and Voges-Proskauer test

Both these bacteria cause diarrhea, grow well on enteric agar, and may be confused with other pathogenic gram-negative rods. Both organisms are positive for oxidase, motility, indole, and lysine decarboxylase.

Question 9

9. Which genus (in which most species are oxidase and catalase positive) of small gram-negative coccobacilli is associated mainly with animals but may cause endocarditis and bacteremia, as well as wound and dental infections in humans?

A. Aggregatibacter (formerly Actinobacillus spp.)
B. Pseudomonas
C. Campylobacter
D. Vibrio

Answer 9

A. Aggregatibacter (formerly Actinobacillus spp.)

Aggregatibacter spp. (formerly Actinobacillus spp.) and formerly Centers for Disease Control and Prevention (CDC) groups HB-3 and HB-4 share many biochemical characteristics of the Haemophilus spp. Infections most often associated with this gram-negative coccobacillus are subacute bacterial endocarditis and periodontal disease (its main habitat is the mouth). The most common human isolate is Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, which grows slowly on chocolate agar. It is positive for catalase, nitrate reduction, and glucose fermentation. It does not grow on MacConkey agar and is negative for oxidase, urease, indole, X, and V requirements.

Question 10

10. 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

Answer 10

B. Indole

C. hominis (indole positive) is a gram-negative coccobacillus biochemically similar to
Aggregatibacter (Actinobacillus) spp. (indole negative). Like Aggregatibacter (formerly Actinobacillus) spp., it is a cause of endocarditis. However, Cardiobacterium spp. are positive for cytochrome oxidase and negative for nitrate reduction and catalase, whereas most Aggregatibacter (formerly Actinobacillus) spp. are negative for oxidase and positive for nitrate reduction and catalase. C. hominis will grow on blood agar after 48 to 72 hours in 5% CO2 at 35°C, but Aggregatibacter (formerly Actinobacillus) requires chocolate agar.

Question 11

11. A mixture of slender gram-negative rods and coccobacilli with rounded ends was recovered from blood cultures after a patient’s root canal surgery. Given the following results after 48 hours, what is the most likely organism?
    Catalase = Neg
    Ornithine decarboxylase = +
    Urease = Neg
    Lysine decarboxylase = +
    Oxidase = +
    X and V requirement = Neg
    Indole = Neg
    Carbohydrates = Neg (no acid produced)
    Growth on blood and chocolate agar = + (with pitting of agar)
    Growth on MacConkey agar = Neg

A. Eikenella corrodens
B. Aggregatibacter (formerly Actinobacillus) spp.
C. Cardiobacterium hominis
D. Proteus spp.

Answer 11

A. Eikenella corrodens

E. corrodens is a part of the normal flora of the upper respiratory tract and the mouth. It is often seen after trauma to the head and neck, dental infections, and human bite wounds. It requires blood for growth. The organism causes pits in the agar, where colonies are located. The smell of bleach may be apparent when the plates are uncovered for examination. Aggregatibacter (formerly Actinobacillus) spp. and C. hominis both utilize several carbohydrates, and Proteus spp. are oxidase negative.

Question 12

12. 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 deoxycholate agar

Answer 12

A. Sheep blood agar

Both K. kingae and E. corrodens are gram-negative rods that are oxidase positive and catalase negative. Both grow well on blood and chocolate agars and cause pitting of the media, and neither grows on MacConkey or XLD agar. However, K. kingae strains produce a narrow zone of β-hemolysis on sheep blood agar similar to that of group B streptococci.

Question 13

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

A. Middle ear infection
B. Endocarditis
C. Meningitis
D. Urogenital infection

Answer 13

B. Endocarditis

Kingella spp. are gram-negative coccobacilli or plump-looking rods. They are part of the normal flora of the upper respiratory and urogenital tracts of humans. Infection is seen primarily in patients having underlying heart disease, poor oral hygiene, or
iatrogenic mucosal ulcerations (e.g., radiation therapy), in whom the organism is recovered from blood cultures.

Question 14

14. Cultures obtained from a dog bite wound produced yellow, tan, and slightly pink colonies on blood and 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 agar = Neg Motility = Neg

A. Aggregatibacter (formerly Actinobacillus) spp.
B. Eikenella spp.
C. Capnocytophaga spp.
D. Pseudomonas spp.

Answer 14

C. Capnocytophaga spp.

The Capnocytophaga species C. gingivalis, C. sputigena, and C. ochracea are part of the normal oropharyngeal flora of humans; however, C. canimorsus and C. cynodegmi (formerly CDC groups DF-2 and DF-2-like bacteria) are associated with infections resulting from dog bite wounds and cat bites as well as scratch wounds.

Question 15

15. Smooth gray colonies showing no hemolytic activity were recovered from an infected cat scratch wound culture; the colonies grew on blood and chocolate agar (a musty 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.

Answer 15

B. Pasteurella spp.

Pasteurella multocida (P. canis) is part of the normal mouth flora of cats and dogs and is frequently recovered from wounds inflicted by them. It produces large amounts of indole and therefore an odor resembling that of colonies of E. coli. Pseudomonas spp. are also catalase and oxidase positive but can be ruled out because it grows on MacConkey agar and does not produce indole.

Question 16

16. 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

Answer 16

D. Bordet-Gengou agar

B. pertussis is an oxidase-positive, nonmotile, gram-negative coccobacillus and appears as small, round colonies resembling droplets of mercury on Bordet-Gengou media with sheep blood agar. It is fastidious and does not grow on chocolate or MacConkey agar. However, B. pertussis adapts to blood agar, growing within 3 to 6 days. This organism is the cause of whooping cough, which can be prevented by immunization with the diphtheria, tetanus, pertussis (DPT) vaccine. The DPT vaccine contains diphtheria and tetanus toxoids and killed whole-cell B. pertussis.

Question 17

17. 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

Answer 17

A. Biphasic Castenada bottles with Brucella broth

Although blood agar will support the growth of Brucella spp., Castenada bottles are the medium of choice. Castenada bottles contain a slant of enriched agar medium that is partially submerged and surrounded by an enriched broth medium. As the specimen is injected into the bottles and mixed, the agar slant is simultaneously coated with the blood (or bone marrow). Brucella is the cause of undulant fever (acquired by ingesting infected, unpasteurized milk products) and is responsible for many cases of fever of unknown origin. Brucella spp. are facultative intracellular (multiply in phagocytic
cells) organisms and grow very slowly, usually requiring 4 to 6 weeks for recovery. Brucella melitensis is the most frequently recovered species.

Question 18

18. 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

Answer 18

A. Basic fuchsin and thionin

Question 19

19. 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

Answer 19

C. Cysteine and cystine

Question 20

20. Which medium is best for recovery of Legionella pneumophila from clinical specimens?

A. Chocolate agar
B. Bordet-Gengou agar
C. New yeast extract agar
D. Buffered charcoal–yeast extract (CYE) agar

Answer 20

D. Buffered charcoal–yeast extract (CYE) agar

Question 21

21. 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

Answer 21

A. Indole and xylose

Question 22

22. Haemophilus species that require the V factor (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

Answer 22

C. Chocolate agar

Question 23

23. 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

Answer 23

A. NAD and Hemin

Staphylococcus growing on sheep blood agar produce NAD (V factor) as a metabolic by-product and β-hemolysins which lyse sheep RBCs, releasing hemin (X factor). This satellite feature allows pinpoint-sized colonies of Haemophilus spp. to grow around Staphylococcus colonies. Sheep blood agar alone does not support the growth of Haemophilus spp., which require V factor because of the presence of V factor–inactivating enzymes that are present in the agar.

Question 24

24. 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

Answer 24

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

Production of β-hemolysis is used to distinguish these two species from other Haemophilus with the same X and V requirements. Horse blood agar furnishes X factor and, when supplemented with yeast extract, supports the growth of Haemophilus spp. Sheep blood agar is not used because it contains growth inhibitors for some Haemophilus spp. The chart at the top of the page summarizes the characteristics of the Haemophilus spp.

Question 25

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

A. a
B. b
C. c
D. d

Answer 25

B. b

The majority of H. influenzae infections occur in children under 5 years of age and are caused by capsular serotype b (Hib), one of six serotypes designated a through f. This capsular strain appears to contain a virulence factor that makes it resistant to phagocytosis and intracellular killing by neutrophils. The nontypable strains do not have a capsule and are part of the normal respiratory flora. Serotyping of Haemophilus is performed by mixing colonies with agglutinating antibodies available as commercial agglutination kits. Identification is also done by using PCR.

Question 26

26. 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

Answer 26

C. Aggregatibacter (Haemophilus) aphrophilus

A. (H.) aphrophilus does not require either X or V factor for growth and is differentiated from the other Haemophilus species by its ability to produce acid from lactose and a positive ALA test. H. influenzae and H. haemolyticus are incapable of synthesizing protoporphyrin from Δ-ALA and are negative for this test

Question 27

27. 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. Haemophilus parahaemolyticus

Answer 27

B. Haemophilus ducreyi

H. ducreyi requires exogenous X factor and causes genital lesions referred to as “soft chancres.” It is difficult to grow and requires commercial chocolate agar or gonococcus base medium containing 1% to 2% hemoglobin, 5% fetal calf serum, and 1% IsoVitaleX enrichment. The plates must be incubated at 33oC in a 3% to 5% CO2 moist environment for 2 to 3 days. Recovery of H. ducreyi from specimens constitutes a reportable sexually transmitted disease (chancroid).

Question 28

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

A. Production of β-lactamase enzymes
B. Hydrolysis of chloramphenicol
C. Hydrolysis of urea
D. All of these options

Answer 28

A. Production of β-lactamase enzymes

Roughly 20% of H. influenzae strains produce β-lactamase, which hydrolyses and inactivates the β-lactam ring of ampicillin (and penicillin).

Question 29

29. A small, gram-negative coccobacillus recovered from the CSF of a 2-year-old 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 identification?

A. Haemophilus parainfluenzae
B. Haemophilus influenzae
C. Haemophilus ducreyi
D. Aggregatibacter (formerly Haemophilus) aphrophilus

Answer 29

B. Haemophilus influenzae

H. influenza is the main cause of meningitis in young, unvaccinated children. Although several biotypes of H. parainfluenzae produce indole and urease, H. parainfluenzae does not require X factor for growth. H. ducreyi requires X factor, but not V factor. A. (H.) aphrophilus does not require either X factor or V factor for growth.

Question 30

30. 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

Answer 30

A. X factor requirement

The X factor requirement for growth is the cause of many inaccuracies when
identifying Haemophilus spp. requiring this factor. False-negative results have been attributed to the presence of small amounts of hemin in the basal media, or X factor carryover from colonies transferred from primary media containing blood. The ALA test determines the ability of an organism to synthesize protoporphyrin intermediates in the biosynthetic pathway to hemin from the precursor compound Δ-ALA. Haemophilus species that need exogenous X factor to grow are unable to synthesize protoporphyrin from Δ-ALA and are negative for the ALA test. These include H. influenzae, H. haemolyticus, H. aegyptius, and H. ducreyi.

Question 31

31. 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 gram-negative bacillus 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.

Answer 31

A. Bartonella spp.

Bartonella spp., frequently the cause of zoonoses infections, are difficult to grow on primary culture media because they are facultative intracellular organisms. Bartonella henselae is the organism found in domestic cats, the main reservoir. When CSD is suspected from the patient’s history, blood cultures should be smeared and Gram staining performed. Bartonella spp. are biochemically inert—that is, they are negative for oxidase, catalase, indole, and urease tests. Therefore, commercial identification systems, DNA amplification for various genes, and indirect immunofluorescence assays (IFAs) are used to identify these organisms.

Question 32

32. A 5-year-old nonimmunized male with 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 gram-negative coccobacillus after 3 days incubation. Carbohydrate and biochemical tests gave negative results. What is the most likely identification?

A. Haemophilus influenza
B. Bordetella pertussis
C. Haemophilus parainfluenzae
D. Bordetella bronchiseptica

Answer 32

B. Bordetella pertussis

B. pertussis, the cause of whooping cough, is highly contagious during the 5- to 10-
day period after acquisition. The incidence of whooping cough is greater in nonimmunized individuals, and therefore, is higher in children ages less than 1 year. B. bronchiseptica is only rarely found in humans, but may cause respiratory disease in animals. Unlike B. pertussis it is positive for nitrite, urease, and motility. A direct identification by DFA using polyclonal antibody to detect B. pertussis in smears from nasopharyngeal material is the quickest method and less costly compared with PCR.

Question 33

33. A 29-year-old male who often hunted rabbits and spent a lot of time in the woods was admitted to the hospital with skin ulcers on his upper extremities. At 48 hours, 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 identification?

A. Pseudomonas aeruginosa
B. Pseudomonas fluorescens
C. Chryseobacterium spp.
D. Francisella tularensis

Answer 33

D. Francisella tularensis

Persons handling samples suspected of containing F. tularensis must wear gloves and use a biological safety cabinet (follow biosafety Level [BSL]-2 controls). For cultures, BSL-3 controls must be followed. Tularemia (rabbit fever or deer fly fever) is one of the most common laboratory-acquired infections, and it is recommended that specimens be sent to a reference laboratory for identification and serological testing. F. tularensis requires cysteine and cystine to grow. It may grow on chocolate agar supplemented with IsoVitaleX and also on charcoal yeast extract agar used to isolate Legionellae.

Question 34

34. A neonate was readmitted to the hospital with a diagnosis of meningitis. CSF revealed gram-negative straight rods. At 24 hours, 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 identification?

A. Haemophilus influenza
B. Elizabethkingia (formerly Chryseobacterium) meningosepticum
C. Stenotrophomonas maltophilia
D. Acinetobacter spp.

Answer 34

B. Elizabethkingia (formerly Chryseobacterium) meningosepticum

Elizabethkingia (formerly Chryseobacterium) meningosepticum can cause septicemia and meningitis in neonates and immunocompromised adults. The ability to encapsulate, produce proteases, and survive in chlorinated tap water are factors that contribute to hospital-acquired infections with this bacterium.

Question 35

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

A. Capnocytophaga canimorsus
B. Francisella tularensis
C. Legionella pneumophila
D. Pseudomonas aeruginosa

Answer 35

A. Capnocytophaga canimorsus

C. canimorsus are part of the oral flora of dogs. The organisms require at least 5% CO2 for growth and grow slowly on blood and chocolate agars. Colonies can grow in 48 hours if cultured in high CO2 on BHI agar with 5% sheep blood.

Question 36

36. 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

Answer 36

B. Subacute bacterial endocarditis

Blood cultures growing small, gram-negative rods should alert the microbiologist to the possibility of infection with one of the five AACEK (formerly HACEK) organisms. Although responsible for less than 5% of bacterial endocarditis overall, greater than 50% of endocarditis cases caused by gram-negative rods result from one of them.

Question 37

37. A suspected case of Legionnaires disease was noted on the request form for a culture and sensitivity ordered on a sputum sample. The patient was a 70-year-old 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

Answer 37

D. Direct immunofluorescent microscopy

Legionella spp. stain poorly, if at all, with Gram stain. Legionella pneumophilia is not acid fast, although L. micdadei, which accounts for a small percentage of Legionella pneumonia infections, is acid-fast positive. Specimens suspected of containing Legionella spp. should be handled in a Class II biological safety cabinet. Legionella spp. require buffered charcoal–yeast extract (BCYE) agar for growth and will not grow on MacConkey agar. Because culture can take up to 10 days, rapid diagnosis by direct immunofluorescence (DIF) and DNA amplification are preferred. Direct fluorescent antibody (DFA) tests are not as sensitive as culture or PCR but are specific and can be used to rapidly confirm a positive serological test, which may be positive in the absence of disease. MALDI-TOF MS can be used to identify isolates, but this method cannot distinguish serotypes. PCR methods using respiratory specimens directly, (without culture for the identification of Legionella spp.) are 99% specific and 85% sensitive.

Question 38

38. Gastric biopsy material was obtained from a 35-year-old male. A suspected case of H. pylori could presumptively be identified and 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

Answer 38

A. Urea broth test and polymerase chain reaction

Tissue specimens are immersed in urea broth or placed in urea agar. A positive test indicates the presence of urease produced by H. pylori. Another method is the urea breath test, which is less invasive.

Question 39

39. 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

Answer 39

B. AACEK

AACEK (formerly HACEK) denotes the following slow-growing gram-negative bacilli causing subacute bacterial endocarditis: Aggregatibacter aphrophilus;
Aggregatibacter actinomycetemcomitans; Cardiobacterium hominis; Eikenella corrodens; and Kingella kingae.

Question 40

40. A 20-year-old 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 agar, sheep blood agar, and MTM agar. Gram staining showed small, pleomorphic gram-negative rods. What is the most likely presumptive identification?

A. Neisseria gonorrheae
B. Haemophilus ducreyi
C. Haemophilus influenzae
D. Moraxella spp.

Answer 40

B. Haemophilus ducreyi

H. ducreyi may take up to 7 days to grow on chocolate agar (requiring X factor for growth) and preferably at 33°C with high humidity. Testing for chancroid disease requires specialized media

Question 41

41. A 7-year-old female became ill after eating a chicken sandwich from a fast-food restaurant. 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 42°C and 37°C incubation). Which test(s) differentiate C. jejuni subsp. doylei from C. jejuni subsp. jejuni because both grow at 37°C and 42°C?

A. Urease
B. Hippurate hydrolysis
C. Cephalothin and nalidixic acid antibiotic disks
D. Growth at 25°C

Answer 41

C. Cephalothin and nalidixic acid antibiotic disks

To separate C. jejuni subsp. jejuni from C. jejuni subsp. doylei (both are positive for hippurate hydrolysis and grow at 37°C and 42°C), it is best to use cephalothin (30-µg disk) and nalidixic acid (30-µg disk) antibiotic testing. C. jejuni subsp. jejuni are resistant to cephalothin and susceptible to nalidixic acid. C. jejuni subsp. doylei are susceptible to both disks.

Question 42

42. A suspected B. pertussis diagnosis relies on symptoms and 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. Complement fixation
D. Enzyme immunoassay

Answer 42

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

B. pertussis produces a virulence factor, pertussis toxin (PT). The organism attaches to the respiratory ciliated epithelial cells of the upper respiratory tract, causing coughing and irritation. The recommended method for identification of the toxin in
serum is by ELISA using paired samples of acute and convalescent sera. PCR on nasopharyngeal swabs as well as DFA tests are available. MALDI-TOF MS can also be used successfully for identification.