Nonfermentative Bacilli

Question 1

What are the most appropriate screening tests to presumptively differentiate and identify the nonfermentative gram-negative bacilli (NFB) from the Enterobacteriaceae?
A. Catalase, decarboxylation of arginine, growth on blood agar
B. Motility, urease, morphology on blood agar
C. Oxidase, TSI, nitrate reduction, growth on MacConkey agar
D. Oxidase, indole, and growth on blood agar

Answer 1

C. Oxidase, TSI, nitrate reduction, growth on MacConkey agar

Note: NFB will grow on the slant of TSI or KIA but they do not acidify the butt (glucose fermentation), as do the Enterobacteriaceae. NFB can be cytochrome oxidase positive or negative, but all the Enterobacteriaceae are oxidase negative. The Enterobacteriaceae grow well on MacConkey agar and reduce nitrate to nitrite, but the NFB grow poorly or not at all and most do not reduce nitrate. Nearly 70% of the NFB recovered from clinical specimens are: Strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia

Question 2

Presumptive tests used for identification of the Pseudomonas spp. are:
A. Oxidase, oxidation-fermentation (OF) glucose (open), OF glucose (sealed), motility, pigment production
B. Growth on blood agar plate (BAP) and eosin-methylene blue (EMB) agars, lysine decarboxylation, catalase
C. Growth on MacConkey, EMB, and XLD agars and motility
D. Growth on mannitol salt agar and flagellar stain

Answer 2

A. Oxidase, oxidation-fermentation (OF) glucose (open), OF glucose (sealed), motility, pigment production

Note: The use of the OF tubes helps to determine the presumption of a nonfermentative bacillus (glucose oxidation positive and glucose fermentation negative). The positive cytochrome oxidase test and pigment production indicate a possible Pseudomonas species. Several NFB produce pigments that aid in species identification: P. aeruginosa produces yellow pyoverdins (fluorescein) and/or pyocyanin (blue aqua pigment). the characteristic grapelike odor of aminoacetophenone as well as growth at 42°C and characteristics of P. aeruginosa.

Question 3

Which tests are most appropriate to differentiate between Pseudomonas aeruginosa and Pseudomonas putida?
A. Oxidase, motility, pyoverdin
B. Oxidase, motility, lactose
C. Oxidase, ONPG, DNase
D. Mannitol, nitrate reduction, growth at 42°C

Answer 3

D. Mannitol, nitrate reduction, growth at 42°C

Note: Both organisms are oxidase positive, motile, and produce pyoverdin. Both are negative for ONPG and DNase. The differentiating tests listed above are:
P. aeruginosa (+++)
P. putida (000)

Question 4

Which test group best differentiates Acinetobacter baumannii from P. aeruginosa?
A. Oxidase, motility, NO3 reduction
B. MacConkey growth, 37°C growth, catalase
C. Blood agar growth, oxidase, catalase
D. Oxidase, TSI, MacConkey growth

Answer 4

A. Oxidase, motility, NO3 reduction

Note: Acinetobacter spp. are nonmotile rods that appear as coccobacillary forms from clinical specimens. All are oxidase negative and catalase positive. P. aeruginosa reduces NO3 to NO2, while A. baumannii does not.

Question 5

In addition to motility, which test best differentiates Acinetobacter spp. and Alcaligenes spp.?
A. TSI
B. Oxidase
C. Catalase
D. Flagellar strain

Answer 5

B. Oxidase

Note: The two genera, Acinetobacter and Alcaligenes, are very similar. Both use oxidation for the metabolism of carbohydrate, with some strains being nonsaccarolytic. Both grow well on MacConkey agar. However, Acinetobacter is nonmotile and oxidase negative. Alcaligenes is motile by peritrichous flagella and oxidase positive.

Question 6

The most noted differences between P. aeruginosa and Stenotrophomonas maltophilia are:
A. Oxidase, catalase, and TSI
B. Oxidase, catalase, and ONPG
C. Oxidase, 42°C growth, and polar tuft of flagella
D. Catalase, TSI, and pigment

Answer 6

C. Oxidase, 42°C growth, and polar tuft of flagella

Note: The two genera, Pseudomonas and Stenotrophomonas, are motile and grow well on MacConkey agar. However, P. aeruginosa is oxidase positive and grows at 42°C but is motile only by polar monotrichous flagella. S. maltophilia is oxidase negative, does not grow at 42°C, and is motile by a polar tuft or flagella.

Question 7

A nonfermenter recovered from an eye wound is oxidase positive, motile with polar monotrichous flagella, and grows at 42°C. Colonies are dry, wrinkled or smooth, buff to light brown in color, and are difficult to remove from the agar. In which DNA homology group should this organism be placed?
A. Pseudomonas stutzeri
B. Pseudomonas fluorescens
C. Pseudomonas alcaligenes
D. Pseudomonas diminuta

Answer 7

A. Pseudomonas stutzeri

Note: P. stutzeri produces dry, wrinkled colonies that are tough and adhere to the media as well as smooth colonies. B. pseudomallei produces similar colony types but is distinguished by biochemical tests and susceptibility to the polymyxins. The colonies of P. stutzeri are buff to light brown because of the relatively high concentration of cytochromes.

Question 7

Which Pseudomonas is usually associated with a lung infection related to cystic fibrosis?
A. P. fluorescens
B. P. aeruginosa
C. P. putida
D. Burkholderia pseudomallei

Answer 7

B. P. aeruginosa

Note: P. aeruginosa is often recovered from the respiratory secretions of cystic fibrosis patients. If the patient is chronically infected with the mucoid strain of P. aeruginosa, the biochemical identification is very difficult. The mucoid strain results from production of large amounts of alginate, a polysaccharide that surrounds the cell.

Question 7

Which organism is associated with immunodeficiency syndromes and melioidosis (a glanders-like disease in Southeast Asia and northern Australia)?
A. Pseudomonas aeruginosa
B. Pseudomonas stutzeri
C. Pseudomonas putida
D. Burkholderia pseudomallei

Answer 7

D. Burkholderia pseudomallei

Note: B. pseudomallei produces wrinkled colonies resembling P. stutzeri. Infections are usually asymptomatic and can be diagnosed only by serological methods. The organism exists in soil and water in an area of latitude 20 degrees north and south of the equator (mainly in Thailand and Vietnam). Thousands of U.S. military personnel were infected with these bacteria during the 1960s and 1970s. The disease may reactivate many years after exposure and has been called the “Vietnamese time bomb.”

Question 7

Which biochemical tests are needed to differentiate Burkholderia cepacia from S. maltophilia?
A. Pigment on blood agar, oxidase, DNase
B. Pigment on MacConkey agar, flagellar stain, motility
C. Glucose, maltose, lysine decarboxylase
D. TSI, motility, oxidase

Answer 7

A. Pigment on blood agar, oxidase, DNase

Note: Both organisms produce yellowish pigment and have polar tuft flagella, but the oxidase and DNase tests are differential.

B. cepacia: Green-yellow pigment on BAP, oxidase +, DNase Neg, Motility +, Glucose OF (open) +, Maltose OF (open) +, Lysine decarboxylase +

S. maltophilia: Lavender-green pigment on BAP, oxidase Neg, DNase +, Motility +, Glucose OF (open) +, Maltose OF (open) +, Lysine decarboxylase +

Question 8

The following results were obtained from a pure culture of gram-negative rods recovered from the pulmonary secretions of a 10-year-old cystic fibrosis patient with pneumonia:
Oxidase = +, Motility = +, Glucose OF (open) = +, Gelatin hydrolysis = +, Pigment = Red (nonfluorescent), Growth at 42°C = +, Arginine dihydrolase = +, Flagella = + (polar, monotrichous)
A. Burkholderia pseudomallei
B. Pseudomonas stutzeri
C. Burkholderia cepacia
D. Pseudomonas aeruginosa

Answer 8

D. Pseudomonas aeruginosa

Note: The oxidase test and red pigment (pyorubin), as well as growth at 42°C, distinguish P. aeruginosa from the other pseudomonads listed, particularly B. cepacia, which is also associated with cystic fibrosis.

Question 9

Alcaligenes faecalis (formerly A. odorans) is distinguished from Bordetella bronchiseptica with which test?
A. Urease (rapid)
B. Oxidase
C. Growth on MacConkey agar
D. Motility

Answer 9

A. Urease (rapid)

Note: Alcaligenes and Bordetella are genera belonging to the Alcaligenaceae family. The two organisms are very similar biochemically, but B. bronchiseptica is urease positive. Both organisms are oxidase positive, grow on MacConkey agar, and are motile by peritrichous flagella, B. bronchiseptica grows well on MacConkey agar but other species of Bordetella are fastidious gram-negative rods.

Question 10

Chryseobacterium spp. are easily distinguished from Acinetobacter spp. by which of the following two tests?
A. Oxidase, growth on MacConkey agar
B. Oxidase and OF (glucose)
C. TSI and urea hydrolysis
D. TSI and VP

Answer 10

A. Oxidase, growth on MacConkey agar

Note: Chryseobacterium spp. and Acinetobacter spp. often produce a yellow pigment on blood or chocolate agar and are nonmotile. Acinetobacter spp. are oxidase negative, grow on MacConkey agar, and are coccobacillary on the Gram stain smear. In contrast, Chryseobacterium spp. are oxidase positive, do not grow on MacConkey agar, and are typically rod shaped. Chryseobacterium meningosepticum is highly pathogenic for premature infants.

Question 11

A gram-negative coccobacillus was recovered on chocolate agar from the CSF of an immunosuppressed patient. The organism was nonmotile and positive for indophenol oxidase but failed to grow on MacConkey agar. The organism was highly susceptible to penicillin. The most probable identification is:
A. Acinetobacter spp.
B. Pseudomonas aeruginosa
C. Pseudomonas stutzeri
D. Moraxella lacunata

Answer 11

D. Moraxella lacunata

Moraxella spp. are oxidase positive and nonmotile, which distinguishes them from Acinetobacter spp. and most Pseudomonas spp. Moraxella spp. are highly sensitive to penicillin, but Acinetobacter spp. and Pseudomonas spp. are penicillin resistant. M. lacunata is implicated in infections involving immunosuppressed patients.

Question 12

Cetrimide agar is used as a selective isolation agar for which organism?
A. Acinetobacter spp.
B. Pseudomonas aeruginosa
C. Moraxella spp.
D. Stenotrophomonas maltophilia

Answer 12

B. Pseudomonas aeruginosa

Cetrimide (acetyl trimethyl ammonium bromide) agar is used for the isolation and identification of P. aeruginosa. With the exception of P. fluorescens, the other pseudomonads are inhibited along with related nonfermentative bacteria.

Question 13

A specimen from a 15-year-old female burn patient was cultured after debridement, and the following results were obtained:
Oxidase = +, Catalase = +, Ornithine decarboxylase = Neg, Arginine dihydrolase = +, Penicillin = Resistant, Colistin (Polymixin B) = Susceptible, Lysine decarboxylase = Neg, Motility = +, Glucose = + for oxidation (open tube), Maltose = Neg for oxidation (open tube), Aminoglycosides = Susceptible
These results indicate which of the following organisms?
A. Acinetobacter baumannii
B. Moraxella lacunata
C. Pseudomonas aeruginosa
D. Acinetobacter lwoffii

Answer 13

C. Pseudomonas aeruginosa

Note: P. aeruginosa is a cause of a significant number of burn wound infections; these organisms can exist in distilled water and under chlorinated water. Acinetobacter spp. are oxidase negative and Moraxella spp. are highly susceptible to penicillin, ruling them out as possible causes.

Question 14

A yellow pigment-producing organism that is oxidase positive, nonmotile, and does not grow on MacConkey agar is:
A. Acinetobacter baumannii
B. Acinetobacter lwoffii
C. Burkholderia cepacia
D. Chryseobacterium meningosepticum

Answer 14

D. Chryseobacterium meningosepticum

Note: All species of Acinetobacter are oxidase negative and grow on MacConkey agar. Chryseobacterium spp. produce yellow pigment (like Acinetobacter) but are oxidase positive and do not grow on MacConkey agar. B. cepacia also produces a yellow pigment but is motile.

Question 15

Which reagent(s) is (are) used to develop the red color indicative of a positive reaction in the nitrate reduction test?
A. Sulfanilic acid and alpha-naphthylamine
B. Ehrlich’s and Kovac’s reagents
C. o-Nitrophenyl-B-D-galactopyranoside
D. Kovac’s reagent

Answer 15

A. Sulfanilic acid and alpha-naphthylamine

Note: In the nitrate test, nitrites formed by bacterial reduction of nitrates will diazotize sulfanilic acid. The diazonium compound complexes with alpha-naphthylamine, forming a red product. Media containing nitrates are used for the identification of nonfermenters. When testing nonfermenters, it is wise to confirm a negative reaction using zinc dust. The diazonium compound detects nitrite only, and the organism may have reduced the nitrates to nitrogen, ammonia, nitrous oxide, or hydroxylamine. Zinc ions reduce residual nitrates in the media to nitrites. A red color produced after addition of zinc indicates the presence of residual nitrates, confirming a true negative reaction. If a red or pink color does not occur after adding zinc, then the organism reduced the nitrate to a product other than nitrite, and the test is considered positive.

Question 16

A culture from an intra-abdominal abscess produced orange-tan colonies on blood agar that gave the following results:
Oxidase = +, KIA = Alk/Alk (H2S)+, DNase = +, Growth at 42°C = Neg, Nitrate reduction = +, Motility = + (single polar flagellum), Ornithine decarboxylase = +
the most likely identification is:
A. Shewanella putrefaciens
B. Acinetobacter spp.
C. Pseudomonas aeruginosa
D. Chryseobacterium spp.

Answer 16

A. Shewanella putrefaciens

Note: S. putrefaciens produces abundant H2S on KIA or TSI. Shewanella are the only nonfermenters that produce H2S on these media.

Question 17

Chryseobacterium spp. and B. cepacia are easily differentiated by which test?
A. Motility
B. OF glucose
C. Oxidase
D. Cetrimide agar

Answer 17

A. Motility

Note: B. cepacia (93%) are weakly oxidase positive and motile. Chryseobacterium spp. are oxidase positive but are nonmotile.

Question 18

A 15-year-old female complained of a severe eye irritation after removing her soft-contact lenses. A swab of the infected right eye was obtained by an ophthalmologist, who ordered a culture and sensitivity test. The culture was plated on blood agar and MacConkey agar. At 24 hours, growth of a gram-negative rod that tested positive for cytochrome oxidase was noted. The Mueller-Hinton sensitivity plate showed a bluish-green “lawn” of growth that proved highly resistant to most of the antibiotics tested except amikacin, tobramycin, and ciprofloxacin. What is the most likely identification?
A. Burkholderia cepacia
B. Pseudomonas aeruginosa
C. Stenotrophomonas maltophilia
D. Acinetobacter baumannii

Answer 18

B. Pseudomonas aeruginosa

Note: P. aeruginosa is an opportunistic organism that is not part of the human normal flora. Contact lens solution contamination, eye injury, or contact lens eye trauma are factors that contribute to P. aeruginosa eye infections. The characteristic blue-green pigment on Mueller-Hinton agar (pyocyanin pigment) produced by P. aeruginosa and the high resistance to antibiotics aid in its identification.

Question 19

Which of the listed Pseudomonas spp. is associated with the following virulence factors: exotoxin A, endotoxins, proteolytic enzymes, antimicrobial resistance, and production of alginate?
A. P. fluorescens
B. P. putida
C. P. stutzeri
D. P. aeruginosa

Answer 19

D. P. aeruginosa

Note: P. aeruginosa is highly resistant to many antimicrobial drugs as well as being one of the most often cultured opportunistic organisms. This virulence factor allows for many nosocomial infections such as those of the urinary tract, wounds (burn patients), bacteremia, respiratory tract, and CNS.

Question 20

A 20-year-old horse groomer exhibited a “glanders-like” infection. His history indicated he had suffered several open wounds on his hands 2 weeks before the swelling of his lymph nodes. A gram-negative rod was recovered from a blood culture that grew well on blood and MacConkey agars. Most of the biochemical tests were negative, including the cytochrome oxidase test. What is the most likely identification?
A. Burkholderia mallei
B. Pseudomonas aeruginosa
C. Pseudomonas stutzeri
D. Burkholderia pseudomallei

Answer 20

A. Burkholderia mallei

Note: Burkholderia mallei is rarely transmitted to humans. It is the causative agent of glanders in mules, donkeys, and horses. It is not part of the humans skin flora and the most likely transmission to humans is through broken skin.

Question 21

A Vietnam War veteran presented with a “glander-like” infection (melioidosis). Several blood cultures produced gram-negative rods that were positive for cytochrome oxidase, oxidized glucose and xylose, and grew at 42°C. What is the most likely organism?
A. Stenotrophomonas maltophilia
B. Burkholderia pseudomallei
C. Pseudomonas aeruginosa
D. Acinetobacter spp.

Answer 21

B. Burkholderia pseudomallei

Note: Burkholderia pseudomallei infections often produce abscesses in organs (liver, spleen, lungs) as well as on the skin, in soft tissue, and in joints and bones. Vietnam War veterans especially may harbor these organisms, which are limited to tropical and subtropical environments (Southeast Asia and Australia). The organism may surface years later after surviving in a latent state within phagocytes. The surfaces of rice paddies in Northern Thailand have a high prevalence of this organism.

Question 22

Cytochrome oxidase-positive, nonfermentative gram-negative bacilli were recovered from the stool of a cystic fibrosis (CF) patient. The isolates produced wet (mucoidy) colonies on blood agar. Which identification is most likely?
A. Acinetobacter spp.
B. Pseudomonas alcaligenes
C. Pseudomonas stutzeri
D. Pseudomonas aeruginosa

Answer 22

D. Pseudomonas aeruginosa

Note: Infected CF patients usually do not escape P. aeruginosa infections completely. P. aeruginosa produces alginate that accounts for the “wet, mucoidy” appearance of colonies. This overproduction of alginate is thought to cause the inhibition of phagocytosis. The result is chronic infections in CF patients with the “wet” form of P. aeruginosa.