Miscellaneous and Fastidious Gram-Negative Rods

Question 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

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

Question 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 = +
Indole = +
VP = Neg
Urease = ±
TCBS agar = Green colonies
Arginine decarboxylase = Neg
KIA = Alk/Acid
Lactose = Neg
String test = Neg

A. Vibrio cholerae
B. Vibrio parahaemolyticus
C. Shigella spp.
D. Salmonella spp.

Answer 2

B

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. 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

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

Catalase = +
Motility = +
Growth at 42°C = +
Grape odor = Neg
Oxidase = +
Hippurate hydrolysis = +
Nalidixic acid = Susceptible
Pigment = Neg
Cephalothin = Resistant

The most likely identification is:
A. Pseudomonas aeruginosa
B. Campylobacter jejuni
C. Campylobacter fetus
D. Pseudomonas putida

Answer 3

B

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 will not grow at 42°C but will grow at 25°C and 37°C.

Question 4

Which atmospheric condition is needed to recover Campylobacter spp. from specimens inoculated onto a Campy-selective agar at 35°C–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

Campylobacter spp. are best recovered in a micro-aerophilic 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 48–72 hours before reporting no growth.

Question 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

Helicobacter 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 mucus-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.

Test H. pylori C. jejuni
Nitrate
reduction Neg +

Hippurate
hydrolysis Neg +

Urease + Neg

Cephalothin
sensitivity Sensitive Resistant

Nalidixic acid
sensitivity Resistant Sensitive

Question 6

Which of the following tests should be done first in order to differentiate Aeromonas spp. from the Enterobacteriaceae?
A. Urease
B. OF glucose
C. Oxidase
D. Catalase

Answer 6

C

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 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

Which is the best rapid test to differentiate Plesiomonas shigelloides from a Shigella species on selective enteric agar?
A. Oxidase
B. Indole
C. TSI
D. Urease

Answer 7

A

P. shigelloides is a lactose nonfermenter 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.

Question 8

Which are the best two tests to differentiate A. hydrophilia from P. shigelloides?
A. Oxidase and motility
B. DNase and VP
C. Indole and lysine decarboxylase
D. Growth on MacConkey and blood agar

Answer 8

B

Both of these bacteria cause diarrhea, grow well on enteric agar, and may be confused with Enterobacteriaceae. Both organisms are positive for oxidase, motility, indole, and lysine decarboxylase. The following reactions are differential:

Test A. hydrophilia P. shigelloides
β-Hemolysis + Neg
on sheep
blood agar

DNase + Neg

VP + Neg

Question 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, bacteremia, as well as wound and dental infections in humans?
A. Actinobacillus
B. Pseudomonas
C. Campylobacter
D. Vibrio

Answer 9

A

Actinobacillus spp. (formerly 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 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

Which of the following tests may be used to differentiate Cardiobacterium hominis from Actinobacillus spp.?
A. Gram stain
B. Indole
C. Anaerobic incubation
D. Oxidase

Answer 10

B

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

Question 11

A mixture of slender gram-negative rods and coccobacilli with rounded ends was recovered from blood cultures following a patient’s root canal surgery. Given the following results after 48 hours, what is the most likely organism?

Catalase = Neg
Urease = Neg
Oxidase = +
Indole = Neg
Growth on blood and chocolate agar = + (with pitting of agar)
Growth on MacConkey agar = Neg

Ornithine decarboxylase = +
Lysine decarboxylase = +
X and V requirement = Neg Carbohydrates = Neg (no acid produced)

A. Eikenella corrodens
B. Actinobacillus spp.
C. Cardiobacterium hominis
D. Proteus spp.

Answer 11

A

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 a pitting of the agar where colonies are located. The smell of bleach may be apparent when the plates are uncovered for examination. Actinobacillus spp. and C. hominis both utilize several carbohydrates, and Proteus spp. are oxidase negative.

Question 12

Kingella kingae can best be differentiated from Eikenella corrodens using which medium?
A. Sheep blood agar
B. Chocolate agar
C. MacConkey agar
D. XLD agar

Answer 12

A

Both Kingella 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

Kingella kingae is usually associated with which type of infection?
A. Middle ear
B. Endocarditis
C. Meningitis
D. Urogenital

Answer 13

B

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

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 = +
Growth on MacConkey agar = Neg
Catalase = +
Motility = Neg

A. Actinobacillus spp.
B. Eikenella spp.
C. Capnocytophaga spp.
D. Pseudomonas spp.

Answer 14

C

Capnocytophaga 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.

Question 15

. Smoothgraycoloniesshowingnohemolysisare recovered from an infected cat scratch on blood
and chocolate agar but fail to grow on MacConkey agar. The organisms are gram-negative pleomorphic rods that are both catalase and oxidase positive
and strongly indole positive. The most likely organism is:
A. Capnocytophaga spp.
B. Pasteurella spp.
C. Proteus spp.
D. Pseudomonas spp.

Answer 15

B

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 colonies of E. coli. Pseudomonas spp. are also catalase and oxidase positive but can be ruled out because they grow on MacConkey agar and do not produce indole.

Question 16

Which media should be used to recover Bordetella pertussis from a nasopharyngeal specimen?
A. Chocolate agar
B. Blood agar
C. MacConkey agar
D. Bordet–Gengou agar

Answer 16

D

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

Question 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

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 and is responsible for many cases of fever of unknown origin. Brucella spp. are facultative intracellular organisms and grow very slowly, usually requiring 4–6 weeks for recovery. Brucella melitensis is the most frquently recovered species.

Question 18

In addition to CO2 requirements and biochemical characteristics, Brucella 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

Question 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

F. tularensis is a fastidious gram-negative rod that is best recovered from lymph node aspirates and tissue biopsies. It is oxidase negative, nonmotile, and inert biochemically. Cysteine blood agar is the medium of choice, but F. tularensis will grow on commercially prepared chocolate agar because it contains X factor and is supplemented with a growth enrichment (IsoVitaleX) that contains cysteine. F. tularensis may not grow well on MacConkey agar.

Question 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

L. pneumophila should be recovered on buffered
CYE agar. This agar is nonselective, but can be made more selective for Legionella spp. by addition of
the antibiotics cefamandole, polymixin B, and anisomycin. Any small, glistening, convex colonies on buffered CYE agar after 2–3 days of incubation that do not grow on L-cysteine–deficient buffered CYE agar or routine nonselective media should be further tested by the direct fluorescent antibody test (DFA) for confirmation of L. pneumophila.

Question 21

Haemophilus influenzae causes ocular infections (pinkeye) and requires X and V factors in the primary medium for growth. The subspecies Haemophilus influenza (biogroup) aegyptius can further be identified and differentiated by which two tests?
A. Indole and xylose
B. Glucose and urease
C. Oxidase and catalase
D. ALA test and oxidase

Answer 21

A

H. influenzae and subspecies H. aegyptius are both glucose, urease, oxidase, and catalase positive.
H. influenzae (biotype II) is positive for both indole and xylose, whereas H. aegyptius is negative for both tests. Biotype II encompasses 40%–70% of H. influenzae strains recovered from clinical specimens. H. influenzae subspecies aegyptius is responsible for epidemics of conjunctivitis in children.

Question 22

Haemophilus species that require the V factor (NAD) are easily recovered on which primary agar plate?
A. Blood agar made with sheep red cells
B. Blood agar made with horse red cells C. Chocolate agar
D. Xylose agar

Answer 22

C

The V factor, NAD, must first be released from RBCs before it can be assimilated by Haemophilus spp. Chocolate agar is made by heating blood agar in order to lyse RBCs. The released NAD is directly available to those Haemophilus species requiring it. Chocolate agar also contains the X factor (hemin). All Haemophilus except H. ducreyi and H. aphrophilus require V factor, while X factor is required by H. influenzae, H. haemolyticus, and H. ducreyi.

Question 23

Which of the following products is responsible for satellite growth of Haemophilus spp. around colonies of Staphylococcus and Neisseria spp. on sheep blood agar?
A. NAD
B. Hemin
C. Indole
D. Oxidase

Answer 23

A

Colonies growing on sheep blood agar secreting NAD (V factor) or producing β-hemolysins (which lyse the sheep RBCs releasing NAD) allow pinpoint-size colonies of Haemophilus spp. to grow around them. 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

Which of the following plates should be used in order to identify Haemophilus haemolyticus and Haemophilus 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 agar with sheep red cells added
D. Chocolate agar and Mueller–Hinton agar with X factor added

Answer 24

B

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 on the next page summarizes the characteristics of the Haemophilus spp.

Question 25

The majority of Haemophilus influenzae infections are caused by which of the following capsular serotypes?
A. a
B. b
C. c
D. d

Answer 25

B

The majority of H. influenzae infections occur in children under 5 years old and are caused by capsular serotype b, one of six serotypes designated a through f. This strain appears to contain a virulence factor that makes it resistant to phagocytosis and intracellular killing by neutrophils. Serotyping of Haemophilus is performed by mixing colonies with agglutinating antibodies available as commercial agglutination kits.

Question 26

Which Haemophilus species is generally associated with endocarditis?
A. H. influenzae
B. H. ducreyi
C. H. aphrophilus
D. H. haemolyticus

Answer 26

C

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 δ-aminolevulinic acid (ALA) test. H. influenzae and H. haemolyticus are incapable of synthesizing protoporphyrin from δ-ALA and are negative for this test.

Question 27

Which Haemophilus species is difficult to isolate and recover from genital ulcers and swollen lymph nodes?
A. H. aphrophilus
B. H. ducreyi
C. H. haemolyticus
D. H. parahaemolyticus

Answer 27

B

H. ducreyi requires exogenous X factor and causes genital lesions referred to as “soft chancres.” The medium used for recovery is commercial chocolate agar or gonococcus base medium containing 1%–2% hemoglobin, 5% fetal calf serum, and 1% IsoVitaleX enrichment. The plates must be incubated in a 3%–5% CO2 environment for 2–3 days. Most specimens are recovered from heterosexuals, and outbreaks in the United States are traced to female prostitutes.

Question 28

Which of the following is a characteristic of strains of Haemophilus 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

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

Question 29

A small, gram-negative coccobacillus recovered from the CSF of a 2-year-old 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. Haemophilus aphrophilus

Answer 29

B

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. H. aphrophilus does not require either X factor or V factor for growth.

Question 30

The δ-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

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 δ-ALA test determines the ability of an organism to synthesize protoporphyrin intermediates in the biosynthetic pathway to hemin from the precursor compound δ-aminolevulinic acid. Haemophilus species that need exogenous X factor
to grow are unable to synthesize protoporphyrin from δ-ALA and are negative for the δ-ALA test. These include H. influenzae, H. haemolyticus, H. aegyptius, and H. ducreyi.

Question 31

An elderly woman who cared for several domestic cats was hospitalized with suspected cat-scratch disease (CSD). Blood cultures appeared negative, but a small, slightly curved pleomorphic gram-negative bacillus grew on BHI agar (brain, heart infusion agar with 5% horse or rabbit blood). What is the most likely identification?
A. Bartonella spp.
B. Brucella spp.
C. Kingella spp.
D. Haemophilus spp.

Answer 31

A

Bartonella spp. are difficult to grow on primary culture media. When CSD is suspected from the patient’s history, blood cultures should be smeared and
Gram stained. Bartonella spp. are biochemically inert, meaning that they are negative for oxidase, catalase, indole, and urease tests. Therefore, commercial identification systems, DNA amplification for various genes, and indirect immunofluorescence assays are used to identify these organisms.

Question 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–Genjou, and Regan–Lowe (with 10% charcoal) agars. All media grew a gram-negative coccobacillus. Carbohydrate and biochemical tests were negative. What is the most likely identification?
A. Haemophilus influenza
B. Bordetella pertussis
C. Haemophilus parainfluenzae
D. Bordetella bronchiseptica

Answer 32

B

B. pertussis, the cause of whooping cough, is highly contagious during the 5–10 day period after acquisition. The incidence of whooping cough is greater in nonimmunized individuals, and therefore, is higher in children under 1 year of age. 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.

Question 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 from the aerobic blood culture bottle only. 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

Persons handling samples suspected of containing F. tularensis must wear gloves and use a biological safety cabinet (follow biosafety Level II controls).
For cultures, biosafety Level III controls must be followed. Tularemia 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

A neonate was readmitted to the hospital with
a diagnosis of meningitis. The 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 a non–lactose fermenter. Colonies were oxidase, DNase, and gelatinase positive, and oxidized glucose and mannitol.
What is the most likely identification?
A. Haemophilus influenza
B. Chryseobacterium meningosepticum C. Stenotrophomonas maltophilia
D. Acinetobacter baumannii

Answer 34

B

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

A 46-year-old dog warden was admitted to the hospital with several puncture bite wounds encountered while wrangling with 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 stain 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

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

The HACEK group of organisms (Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella spp.) are all known for which type of infection?
A. Urinary tract
B. Endocarditis
C. Pharyngitis
D. Tonsillitis

Answer 36

B

Blood cultures growing small gram-negative rods shoud alert the microbiologist to the possiblility of infection with one of the five HACEK organisms. Although responsible for less than 5% of bacterial endocarditis overall, greater than half of endocarditis cases caused by gram-negative rods result from one of them.

Question 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 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

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. Since culture can take up to 10 days, rapid diagnosis by direct immunofluorescence and DNA amplification are preferred. Direct fluorescent antibody 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.