Micro 8.2 Enterobacteriaceae

Question 1

1. Biochemically, Enterobacteriaceae species are gram-negative rods that:

A. Ferment glucose, reduce nitrate to nitrite, and are oxidase negative
B. Ferment glucose, produce indophenol oxidase, and form gas
C. Ferment lactose and reduce nitrite to nitrogen gas
D. Ferment lactose and produce indophenol oxidase

Answer 1

A. Ferment glucose, reduce nitrate to nitrite, and are oxidase negative

The family Enterobacteriaceae consists of more than 100 species and represents the most commonly encountered isolates in clinical specimens. All Enterobacteriaceae species ferment glucose, are nonsporulating, and are oxidase negative (except for Plesiomonas shigelloides, recently added to the family but which is oxidase positive). Most Enterobacteriaceae species are motile, but the genera Shigella and Klebsiella are not.

Question 2

2. The ortho-nitrophenyl-β-galactopyranoside (ONPG) test is most useful when differentiating:

A. Salmonella spp. from Pseudomonas spp.
B. Shigella spp. from some strains of Escherichia coli
C. Klebsiella spp. from Enterobacter spp.
D. Proteus vulgaris from Salmonella spp.

Answer 2

B. Shigella spp. from some strains of Escherichia coli

The ONPG test detects β-galactosidase activity and is most useful in distinguishing late lactose fermenters from lactose nonfermenters. Some strains of E. coli are slow lactosefermenters and may be confused with Shigella spp., which do not ferment lactose. E. coli are ONPG positive, whereas Shigella spp. are ONPG negative.

Question 3

3. The Voges-Proskauer (VP) test detects which end product of glucose fermentation?

A. Acetoin
B. Nitrite
C. Acetic acid
D. Hydrogen sulfide

Answer 3

A. Acetoin

Acetoin or carbinol, an end product of glucose fermentation, is converted to diacetyl after the addition of the VP reagents (β-naphthol and 40% potassium hydroxide [KOH]). Diacetyl is seen as a red- to-pink–colored complex.

Question 4

4. At which pH does the methyl red (MR) test become positive?

A. 7.0
B. 6.5
C. 6.0
D. 4.5

Answer 4

D. 4.5

Both MR and VP tests detect acid production from the fermentation of glucose. However, a positive MR test result denotes a more complete catabolism of glucose to highly acidic end products, such as formate and acetate, than occurs with organisms that are VP positive only (e.g., Klebsiella pneumoniae).

Question 5

5. A positive Simmons citrate test is seen as a:

A. Blue color in the medium after 24 hours of incubation at 35°C
B. Red color in the medium after 18 hours of incubation at 35°C
C. Yellow color in the medium after 24 hours of incubation at 35°C
D. Green color in the medium after 18 hours of incubation at 35°C

Answer 5

A. Blue color in the medium after 24 hours of incubation at 35°C

The Simmons citrate test determines if an organism can utilize citrate as the sole source of carbon. The medium turns blue, indicating the presence of alkaline products, such as carbonate. Tubes are incubated a minimum of 24 hours at 35°C with a loose cap before reading.

Question 6

6. In the test for urease production, ammonia reacts to form which product?

A. Ammonium citrate
B. Ammonium carbonate
C. Ammonium oxalate
D. Ammonium nitrate

Answer 6

B. Ammonium carbonate

The test for urease production is based on the ability of the colonies to hydrolyze urea in Stuart broth or Christensen agar to form CO2 and ammonia. These products form ammonium carbonate, resulting in alkalinization. This turns the pH indicator (phenol red) pink at pH 8.0.

Question 7

7. Which of the following reagents is added to detect the production of indole?

A. p-Dimethylaminobenzaldehyde
B. Bromcresol purple
C. Methyl red
D. Cytochrome oxidase

Answer 7

A. p-Dimethylaminobenzaldehyde

The indole test detects the conversion of tryptophan (present in the media) to indole by the enzyme tryptophanase. Indole is detected by the reaction with the aldehyde group of p-dimethylaminobenzaldehyde (the active reagent in Kovac and Ehrlich reagents) in acid, forming a red complex.

Question 8

8. Decarboxylation of the amino acids lysine, ornithine, and arginine results in the formation of:

A. Ammonia
B. Urea
C. Carbonate
D. Amines

Answer 8

D. Amines

Specific decarboxylases split dibasic amino acids (lysine, arginine, and ornithine), forming alkaline amines. These products turn the pH indicators in the medium (cresol red and bromcresol purple) from yellow to purple.

Question 9

9. Lysine iron agar (LIA) showing a purple slant and a blackened butt indicates:

A. Escherichia coli
B. Citrobacter spp.
C. Salmonella spp.
D. Proteus spp.

Answer 9

C. Salmonella spp.

LIA is used as an aid for the identification of Salmonella species. It contains phenylalanine, lysine, glucose, thiosulfate, ferric ammonium citrate, and bromcresol purple. Salmonella spp. produce H2S from thiosulfate. This reduces ferric ammonium citrate, forming ferrous sulfate and causing the butt to blacken. Salmonella also decarboxylate lysine to produce alkaline amines, giving the slant its purple color and differentiating it from Citrobacter spp., which are lysine decarboxylase negative.

Question 10

10. Putrescine is an alkaline amine product of which bacterial enzyme?

A. Arginine decarboxylase
B. Phenylalanine deaminase
C. Ornithine decarboxylase
D. Lysine decarboxylase

Answer 10

C. Ornithine decarboxylase

Putrescine is the amine product of the decarboxylation of ornithine.

Question 11

11. Which genera are positive for phenylalanine deaminase?

A. Enterobacter, Escherichia, and Salmonella
B. Morganella, Providencia, and Proteus
C. Klebsiella and Enterobacter
D. Proteus, Escherichia, and Shigella

Answer 11

B. Morganella, Providencia, and Proteus

Phenylalanine deaminase oxidatively deaminates phenylalanine, forming phenylpyruvic acid. When a solution of ferric chloride is added, the iron reacts with phenylpyruvic acid, forming a green-colored complex. Phenylalanine deaminase is found in the genera Morganella, Providencia, and Proteus and is an excellent test to determine if an organism belongs to this group. Rarely, isolates of Enterobacter may be phenylalanine deaminase positive as well.

Question 12

12. Kligler iron agar (KIA) differs from triple-sugar iron agar (TSI) in the:

A. Ratio of lactose to glucose
B. Ability to detect H2S production
C. Use of sucrose in the medium
D. Color reaction denoting production of acid

Answer 12

C. Use of sucrose in the medium

Both KIA and TSI contain 10-fold more lactose than glucose, peptone, and phenol red to detect acid production (turns yellow) and sodium thiosulfate and ferrous ammonium sulfate to detect H2S production. However, TSI contains sucrose, and KIA does not. Organisms fermenting either sucrose or lactose will turn the slant of the agar tube yellow. Therefore, some organisms (e.g., many species of Cedecea, Citrobacter, Edwardsiella, and Serratia) will produce a yellow slant on TSI but a red slant on KIA.

Question 13

13. The malonate test is most useful in differentiating which members of the Enterobacteriaceae family?

A. Shigella
B. Proteus
C. Salmonella subgroups 2, 3 (the former Arizona)
D. Serratia

Answer 13

C. Salmonella subgroups 2, 3 (the former Arizona)

The malonate test determines whether an organism can utilize sodium malonate as the sole source of carbon. Malonate is broken down, forming alkaline metabolites that raise the pH of the broth above 7.6. This causes bromthymol blue to turn from green to deep blue (Prussian blue). E. coli, Shigella, and most Salmonella are malonate negative, whereas Enterobacter and Salmonella (formerly Arizona) subgroups 2, 3a, and 3b are positive. Proteus, Providencia, Serratia, and Yersinia are also malonate negative.

Question 14

14. Which genera of the Enterobacteriaceae family are known to cause diarrhea and are considered enteric pathogens?

A. Enterobacter, Klebsiella, Providencia, and Proteus
B. Escherichia, Salmonella, Shigella, and Yersinia
C. Pseudomonas, Moraxella, Acinetobacter, and Aeromonas
D. Enterobacter, Citrobacter, and Morganella

Answer 14

B. Escherichia, Salmonella, Shigella, and Yersinia

Escherichia, Salmonella, Shigella, and Yersinia are responsible for the majority of enteric diarrhea cases attributable to the Enterobacteriaceae family.

Question 15

15. An isolate of E. coli recovered from the stool of a patient with severe bloody diarrhea should be tested for which sugar before sending it to a reference laboratory for serotyping?

A. Sorbitol (fermentation)
B. Mannitol (oxidation)
C. Raffinose (fermentation)
D. Sucrose (fermentation)

Answer 15

A. Sorbitol (fermentation)

An isolate of E. coli (shiga-like producing toxin of E. coli [STEC]) recovered from a stool culture in hemorrhagic colitis can be definitely identified only by serotyping. The isolate is identified as E. coli by the usual biochemical reactions. The strain of E. coli responsible for hemorrhagic colitis is O157:H7 and is usually negative for sorbitol fermentation. Colonies of this strain of E. coli appear colorless on MacConkey agar with sorbitol added.

Question 16

16. Care must be taken when identifying biochemical isolates of Shigella because serological cross reactions occur with:

A. Escherichia coli
B. Salmonella spp.
C. Pseudomonas spp.
D. Proteus spp.

Answer 16

A. Escherichia coli

Serological confirmation of Shigella isolates is based on O antigen typing. If a suspected Shigella spp. is serologically typed with polyvalent sera before it has been
correctly identified biochemically, a false-positive confirmation may occur with an isolate that is E. coli (i.e., anaerogenic, non–gas-producing, lactose-negative or -
delayed, and nonmotile strains). These strains were formerly known as the Alkalescens-Dispar serotype.

Question 17

17. Which species of Shigella is most commonly associated with diarrheal disease in the United States?

A. Shigella dysenteriae
B. Shigella flexneri
C. Shigella boydii
D. Shigella sonnei

Answer 17

D. Shigella sonnei

The Shigella spp. are lactose nonfermenters that, for the most part, are biochemically inert and are classified into serogroups A, B, C, and D as a result of their biochemical similarity. S. sonnei is the species most often isolated from diarrhea cases in the United States. It is more active biochemically than the other species because of ornithine decarboxylase and β-galactosidase activity. These enzymes, found in most strains of S. sonnei, distinguish it from other Shigella species.

Question 18

18. Which of the following tests best differentiates Shigella species from E. coli?

A. H2S, VP, citrate, and urease
B. Lactose, indole, ONPG, and motility
C. Hydrogen sulfide, MR, citrate, and urease
D. Gas, citrate, and VP

Answer 18

B. Lactose, indole, ONPG, and motility

E. coli, when positive for lactose, indole, and ONPG, are usually motile. Shigella species do not ferment lactose or produce indole, lack β-galactosidase, and are nonmotile.

Question 19

19. Which genera of Enterobacteriaceae are usually nonmotile at 36°C?

A. Shigella, Klebsiella, and Yersinia
B. Escherichia, Edwardsiella, and Enterobacter
C. Proteus, Providencia, and Salmonella
D. Serratia, Morganella, and Hafnia

Answer 19

A. Shigella, Klebsiella, and Yersinia

Shigella spp. and Klebsiella spp. are, for the most part, nonmotile. Yersinia can be motile at 22°C but is nonmotile at 36°C. Other members of Enterobacteriaceae that have been isolated from human specimens and are usually nonmotile include Leminorella, Rahnella, and Tatumella (considered unusual enteric pathogens)

Question 20

20. Fever, abdominal cramping, watery stools, and fluid and electrolyte loss preceded by bloody stools 2 to 3 days before is characteristic of shigellosis but may also result from infection with:

A. Campylobacter spp.
B. Salmonella spp.
C. Proteus spp.
D. Yersinia spp.

Answer 20

A. Campylobacter spp.

Shigella spp. and Campylobacter spp. are both causes of diarrhea, abdominal pain, fever, and sometimes vomiting. Blood is present in the stools of patients infected with Shigella as a result of invasion and penetration of the bowel by the organisms. Young children may also exhibit bloody stools when infected with Campylobacter.

Question 21

21. Cold enrichment of feces (incubation at 4°C) in phosphate-buffered saline prior to subculture onto enteric media enhances the recovery of:

A. Enterotoxigenic Escherichia coli
B. Salmonella paratyphi
C. Hafnia alvei
D. Yersinia enterocolitica

Answer 21

D. Yersinia enterocolitica

Cold enrichment is especially useful when specimens contain large numbers of normal flora that are sensitive to prolonged exposure to near-freezing temperature. In addition to Yersinia, the technique has been used to enhance recovery of Listeria monocytogenes from specimens containing other bacteria.

Question 22

22. Which group of tests, along with colonial morphology on primary media, aids most in the rapid identification of Enterobacteriaceae?

A. MR and VP, urease, and blood agar plate
B. Phenylalanine deaminase, urease, and CDC agar plate
C. Bacitracin, β-lactamase, and MacConkey agar plate
D. Indole, oxidase, MacConkey, and blood agar plates

Answer 22

D. Indole, oxidase, MacConkey, and blood agar plates

Question 23

23. A routine, complete stool culture procedure should include media for the isolation of E. coli O157:H7 as well as:

A. Salmonella, Shigella, Yersinia, Campylobacter, and Staphylococcus aureus
B. Vibrio cholerae, Brucella, and Yersinia spp.
C. Staphylococcus aureus, group B streptococci, and group D streptococci
D. Clostridium difficile, Clostridium perfringens, and Yersinia spp.

Answer 23

A. Salmonella, Shigella, Yersinia, Campylobacter, and Staphylococcus aureus

Question 24

24. Which group of tests best identifies the Morganella and Proteus genera?

A. Motility, urease, and phenylalanine deaminase
B. Malonate, glucose fermentation, and deoxyribonuclease (DNase)
C. Indole, oxidase, MR, and VP
D. Indole, citrate, and urease

Answer 24

A. Motility, urease, and phenylalanine deaminase

Question 25

25. Which group of tests best differentiates Enterobacter aerogenes from Edwardsiella tarda?

A. Motility, citrate, and urease
B. H2S production, sucrose fermentation, indole, and VP
C. Lysine decarboxylase, urease, and arginine dihydrolase
D. Motility, H2S production, and DNase

Answer 25

B. H2S production, sucrose fermentation, indole, and VP

Question 26

26. Cronobacter sakazakii (formerly Enterobacter sakazakii) can best be differentiated from Enterobacter cloacae by which of the following characteristics?

A. Yellow pigmentation and negative sorbitol fermentation
B. Pink pigmentation and positive arginine dihydrolase
C. Yellow pigmentation and positive urease
D. H2S production on TSI

Answer 26

A. Yellow pigmentation and negative sorbitol fermentation

Question 27

27. Members of the genus Cedecea are best differentiated from Serratia spp. by which test result?

A. Positive motility
B. Positive urease
C. Positive phenylalanine deaminase
D. Negative DNase

Answer 27

D. Negative DNase

Question 28

28. Which of the following organisms is often confused with the Salmonella species biochemically and on plated media?

A. Escherichia coli
B. Citrobacter freundii
C. Enterobacter cloacae
D. Shigella dysenteriae

Answer 28

B. Citrobacter freundii

Question 29

29. A gram-negative rod is recovered from a catheterized urine sample from a nursing home patient. The lactose-negative isolate tested positive for indole, urease, ornithine decarboxylase, and phenylalanine deaminase and negative for H2S. The most probable identification is:

A. Edwardsiella spp.
B. Morganella spp.
C. Ewingella spp.
D. Shigella spp.

Answer 29

B. Morganella spp.

Question 30

30. Which single test best separates Klebsiella oxytoca from K. pneumoniae?

A. Urease
B. Sucrose
C. Citrate
D. Indole

Answer 30

D. Indole

Question 31

31. Which of the following organisms, found in normal fecal flora, may be mistaken biochemically for the genus Yersinia?

A. Klebsiella spp.
B. Proteus spp.
C. Escherichia coli
D. Enterobacter spp.

Answer 31

B. Proteus spp.

Question 32

32. Why might it be necessary for both pink (lactose-positive) and colorless (lactose-negative) colonies from an initial stool culture on MacConkey agar to be subcultured and tested further for possible pathogens?

A. Most Shigella strains are lactose positive
B. Most Salmonella strains are maltose negative
C. Most Proteus spp. are lactose negative
D. Pathogenic Escherichia coli can be lactose positive or lactose negative

Answer 32

D. Pathogenic Escherichia coli can be lactose positive or lactose negative

Question 33

33. Which agar that is used for routine stool cultures is the medium of choice for the isolation of Yersinia strains from stool specimens?

A. Salmonella–Shigella agar
B. Hektoen enteric agar
C. MacConkey agar
D. CNA agar

Answer 33

C. MacConkey agar

CIN medium is the best agar for the isolation of Yersinia strains because it inhibits growth of other coliforms, but it is not used routinely in clinical laboratories. Yersinia spp. grow well on MacConkey agar incubated at 37°C, but the colonies are much smaller than the other Enterobacteriaceae species; therefore, 25°C is the temperature recommended for isolation. Some serotypes of Yersinia may be inhibited on more selective media, such as Salmonella–Shigella or Hektoen. CNA agar inhibits the growth of gram-negative bacteria.

Question 34

34. Which organism is sometimes mistaken for Salmonella and will agglutinate in Salmonella polyvalent antiserum?

A. Citrobacter freundii strains
B. Proteus mirabilis strains
C. Shigella sonnei strains
D. Escherichia coli

Answer 34

A. Citrobacter freundii strains

Question 35

35. A bloody stool cultured from a 26-year-old woman after 3 days of severe diarrhea showed the following results at 48 hours after being plated on the following media:
    MacConkey agar: Little normal flora with many non–lactose-fermenting colonies
    Hektoen enteric agar: Many blue-green colonies
    Campylobacter blood agar and C. difficile agar: No growth
    Clear colonies (from MacConkey agar): Negative for oxidase, indole, urease, motility, and H2S
    The most likely identification is:

A. Shigella spp.
B. Salmonella spp.
C. Proteus spp.
D. Escherichia coli

Answer 35

A. Shigella spp.

Shigella is the most likely organism biochemically. E. coli are usually indole and motility positive, and Proteus are motility and urease positive. Most Salmonella are H2S positive. Shigella and Campylobacter cause bloody diarrhea because they invade the epithelial cells of the large bowel; however, Campylobacter spp. do not grow on MacConkey agar, and they are oxidase positive.

Question 36

36. Which one of the following organisms (are) is generally positive for β-glycosidase (utilizes lactose)?

A. Salmonella spp.
B. Shigella spp.
C. Proteus spp.
D. Escherichia coli

Answer 36

D. Escherichia coli

Enterobacteriaceae species are grouped according to their ability to ferment lactose, a β-galactoside. Salmonella, Shigella, Proteus, Providencia, and Morganella are usually lactose nonfermenters. Others—including certain strains of E. coli, S. sonnei, H. alvei, Serratia marcescens, and some Yersinia—appear to be lactose nonfermenters because they lack the permease enzyme that actively transports lactose across the cell membrane. However, true lactose nonfermenters do not possess β-galactosidase. The test for β-galactosidase uses the substrate o-nitrophenyl-β-galactopyranoside. At an alkaline pH, β-galactosidase hydrolyses the substrate, forming o-nitrophenol, which turns the medium yellow.

Question 37

37. In the Kauffmann-White schema, the combined antigens used for serological identification of the Salmonella spp. are:

A. O antigens
B. H antigens
C. Vi and H antigens
D. O, Vi, and H antigens

Answer 37

D. O, Vi, and H antigens

The Kaufmann-White schema groups Salmonellae on the basis of the somatic O (heat-stable) antigens and subdivides them into serotypes based on their flagellar H (heat-labile) antigens. The Vi (or K) antigen is a capsular polysaccharide that may be removed by heating. In the United States, commercially available slide agglutination tests utilize polyvalent antisera (A, B, C-1, C-2, D, E, and Vi) to group the Salmonella spp. because 95% of isolated organisms belong to groups A through E with the Vi antiserum identifying the capsular or “K” antigen of Salmonella serotype typhi. Antiserum is also used against the flagella (“H”) antigens. If the Vi antigen is positive and the O antigen is negative, the isolate must be heated in boiling water for 15 minutes to remove the capsule and retested with the antisera. There are over 2,200 serotypes of Salmonella.

Question 38

38. The drugs of choice for treatment of infections with Enterobacteriaceae are:

A. Aminoglycosides, sulfamethoxazole-trimethoprim, third-generation cephalosporins
B. Ampicillin and nalidixic acid
C. Streptomycin and isoniazid (isonicotinylhydrazide [INH])
D. Chloramphenicol, ampicillin, and colistin

Answer 38

A. Aminoglycosides, sulfamethoxazole-trimethoprim, third-generation cephalosporins

The drugs of choice for the Enterobacteriaceae species vary, and several genera display patterns of resistance that aid in their identification. K. pneumoniae and Citrobacter diversus are resistant to ampicillin and carbenicillin; most Enterobacter spp. and Hafnia are resistant to ampicillin and cephalothin. Proteus, Morganella, and Serratia are resistant to colistin. Providencia and Serratia are resistant to multiple drugs. Several genera are resistant to chloramphenicol and most are resistant to penicillin.

Question 39

39. The Shiga-like toxin (verotoxin) is produced mainly by which Enterobacteriaceae species?

A. Klebsiella pneumoniae
B. Escherichia coli
C. Salmonella typhimurium
D. Enterobacter cloacae

Answer 39

B. Escherichia coli

Strains of E. coli that produce one or both of the Shiga-like toxins (SLT I and SLT II) can cause bloody diarrhea (hemorrhagic colitis). In the United States, E. coli strain O157:H7 is the serotype most often associated with hemorrhagic colitis.

Question 40

40. Infections caused by Y. pestis are rare in the United States. Those cases that do occur are most frequently located in which region?

A. New Mexico, Arizona, and California
B. Alaska, Oregon, and Utah
C. North and South Carolina and Virginia
D. Ohio, Michigan, and Indiana

Answer 40

A. New Mexico, Arizona, and California

Approximately 15 cases of Y. pestis infection are confirmed in the United States annually. Most originate in the Southwest. It is necessary to be aware of this regional occurrence because untreated cases are associated with a mortality rate of approximately 60%. Y. pestis is not fastidious and grows well on blood agar. It is inactive biochemically, and this helps differentiate it from the other Enterobacteriaceae species. Y. pestis is the only member of the Enterobacteriaceae family that infects humans via an insect vector (animal transmission by a flea bite).

Question 41

41. A leg culture from a nursing home patient grew gram-negative rods on MacConkey agar as pink to dark pink oxidase-negative colonies. Given the following results, which is the most likely organism?
    TSI = A/A
    Indole = Neg
    MR = Neg
    VP = +
    Citrate = +
    H2S = Neg
    Urease = +
    Motility = Neg
    Antibiotic susceptibility: Resistant to carbenicillin and ampicillin

A. Serratia marcescens
B. Proteus vulgaris
C. Enterobacter cloacae
D. Klebsiella pneumoniae

Answer 41

D. Klebsiella pneumoniae

K. pneumoniae and E. cloacae display similar IMViC (indole, MR, VP, and citrate) reactions (00++) and TSI results. However, approximately 65% of E. cloacae strains are urease positive compared with 98% of those of K. pneumoniae. Enterobacter spp. are motile, and Klebsiella are nonmotile. The antibiotic pattern of resistance to carbenicillin and ampicillin is characteristic of the nonmotile Klebsiella spp.

Question 42

42. Four blood cultures were taken over a 24-hour period from a 20-year-old woman with severe diarrhea. The cultures grew motile (room temperature), gram-negative rods. A urine specimen obtained via catheterization also showed gram-negative rods (100,000 col/mL). Given the following results, which is the most likely organism?
    TSI = A/A gas
    Indole = +
    VP = Neg
    MR = +
    H2S = Neg
    Citrate = Neg
    Urease = Neg
    Lysine decarboxylase = +
    (PD) Phenylalanine deaminase = Neg

A. Proteus vulgaris
B. Salmonella typhi
C. Yersinia enterocolitica
D. Escherichia coli

Answer 42

D. Escherichia coli

Question 43

43. A stool culture from a 30-year-old man suffering from bloody mucoid diarrhea gave the following results on differential enteric media:
    MacConkey agar = clear colonies
    XLD agar = clear colonies
    Hektoen agar = green colonies
    Salmonella–Shigella agar = small, clear colonies
    Which tests are most appropriate for identification of this enteric pathogen?

A. TSI, motility, indole, urease, Shigella typing with polyvalent sera
B. TSI, motility, indole, lysine, Salmonella typing with polyvalent sera
C. TSI, indole, MR, VP, citrate
D. TSI, indole, MR, and urease

Answer 43

A. TSI, motility, indole, urease, Shigella typing with polyvalent sera

The most likely organism is a species of Shigella. Typically, Salmonella spp. produce H2S-positive colonies that display black centers on the differential media (except on MacConkey agar). The biochemical tests listed are necessary to differentiate Shigella from E. coli because some E. coli strains cross-react with Shigella-typing sera. Shigella spp. are one of the most common causes of bacterial diarrhea; group D (S. sonnei) and group B (S. flexneri) are the species most often isolated.

Question 44

44. A leg-wound culture from a hospitalized 70-year-old man with diabetes grew motile, lactose-negative colonies on MacConkey agar. Given the following biochemical reactions at 24 hours, what is the most probable organism?
    H2S (TSI) = Neg
    Indole = Neg
    MR = Neg
    VP = +
    DNase = +
    Citrate = +
    Urease = Neg
    (PD) Phenylalanine deaminase = Neg
    Ornithine and lysine decarboxylase = +
    Arginine decarboxylase = Neg
    Gelatin hydrolysis = +

A. Proteus vulgaris
B. Serratia marcescens
C. Proteus mirabilis
D. Enterobacter cloacae

Answer 44

B. Serratia marcescens

S. marcescens has been implicated in numerous nosocomial infections and is recognized as an important pathogen with invasive properties. Gelatin hydrolysis and DNase are positive for both the Proteus spp. and Serratia, but the negative urease and phenylalanine deaminase are differential. E. cloacae does not produce DNase, gelatinase, or lysine decarboxylase but will display red-pigmented colonies on MacConkey agar.

Question 45

45. Three blood culture specimens taken from a 30-year-old patient with cancer receiving chemotherapy and admitted with a urinary tract infection (UTI) grew lactose-negative, motile, gram-negative rods prior to antibiotic therapy. Given the following biochemical reactions, which is the most likely organism?
    H2S (TSI) = +
    Indole = +
    MR = +
    VP = Neg
    Citrate = Neg
    Urease = +
    DNase = +
    (PD) Phenylalanine deaminase = +
    Gelatin hydrolysis = +
    Ornithine decarboxylase = Neg

A. Proteus vulgaris
B. Proteus mirabilis
C. Serratia marcescens
D. Klebsiella pneumoniae

Answer 45

A. Proteus vulgaris

Although P. mirabilis is more frequently recovered from patients with UTIs, P. vulgaris is commonly recovered from immunosuppressed patients. P. mirabilis is indole negative and ornithine decarboxylase positive but otherwise is very similar to P. vulgaris.

Question 46

46. Three consecutive stool cultures from a 25-year-old male patient produced scant normal fecal flora on MacConkey and Hektoen agars. However, colonies on CIN agar displayed “bull’s eye” colonies after 48 hours of incubation. The patient had been suffering from enterocolitis with fever, diarrhea, and abdominal pain for 2 days. What is the most likely identification of this gram-negative rod?

A. Escherichia coli
B. Proteus mirabilis
C. Yersinia enterocolitica
D. Klebsiella pneumoniae

Answer 46

C. Yersinia enterocolitica

Most members of the Enterobacteriaceae family produce detectable growth on MacConkey agar within 24 hours. Y. enterocolita produces non–lactose-fermenting colonies on MacConkey agar, salmon-colored colonies on Hektoen agar, and yellow or colorless colonies on XLD agar. If Y. enterocolitica is suspected, specialized agar (CIN) is employed. The typical “bull’s eye” colonies, dark red with a translucent border, can be confused with Aeromonas spp. that appear similarly on CIN agar. To differentiate, an oxidase test must be performed because Yersinia spp. are oxidase negative and Aeromonas spp. are oxidase positive.

Question 47

47. A 6-year-old female patient was admitted to the hospital following 2 days of severe diarrhea. Cultures from three consecutive stool samples contained blood and mucus. Patient history revealed that she had eaten a hamburger at a fast-food restaurant 3 days earlier. Which pathogen is most likely responsible for the following results?
    Growth on:
    XLD agar = Yellow colonies
    HE agar = Yellow colonies
    Mac agar = Light pink and dark pink colonies
    Mac with sorbitol agar = Few dark pink and many colorless colonies

A. Salmonella spp.
B. Shigella spp.
C. Escherichia coli O157:H7
D. Yersinia enterocolitica

Answer 47

C. Escherichia coli O157:H7

Inflammation with bleeding of the mucosa of the large intestine (hemorrhagic colitis) is a result of an enterohemorrhagic E. coli (EHEC) infection associated with certain serotypes, such as E. coli O157:H7. The source of the E. coli infection is from ingestion of undercooked ground beef contaminated with fecal matter or drinking raw milk.

Question 48

48. Following a 2-week camping trip to the southwest United States, a 65-year-old male patient was hospitalized with a high fever and an inflammatory swelling of the axilla and groin lymph nodes. Several blood cultures were obtained, and cultures showed growth of gram-negative rods resembling “closed safety pins.” The organism grew on MacConkey agar showing non–lactose-fermenting colonies. Testing demonstrated a nonmotile rod that was biochemically inert. What is the most likely pathogen identified?

A. Yersinia pestis
B. Klebsiella pneumoniae
C. Proteus vulgaris
D. Morganella morganii

Answer 48

A. Yersinia pestis

Y. pestis is the cause of bubonic and pneumonic plague. Bubonic plague causes swelling of the groin lymph nodes (bubos), whereas pneumonic plague involves the lungs. The infection caused by bubonic plague may result in fulminant bacteremia that is usually fatal. The transmission is from rodents (rats, ground squirrels, or prairie dogs) to humans by the bite of fleas (vectors) or by ingestion of contaminated animal tissues. Pneumonic plague is acquired via the airborne route when there is close contact with other pneumonic plague victims.

Question 49

49. The majority of clinical laboratories with a microbiology department should have the capability of serotyping which pathogenic Enterobacteriaceae species?

A. Yersinia enterocolitica, Shigella spp.
B. Escherichia coli O157:H7, Salmonella spp., Shigella spp.
C. Yersinia pestis, Salmonella spp.
D. Edwardsiella spp., Salmonella spp.

Answer 49

B. Escherichia coli O157:H7, Salmonella spp., Shigella spp.

Preliminary serological grouping of the Salmonella spp. and Shigella spp. should be performed because reliable commercial polyvalent antisera are available. Sorbitolnegative (MacConkey agar with sorbitol) colonies of E. coli should be tested by using commercially available antisera for somatic “O” antigen 157 and flagellar “H” antigen 7. However, Y. pestis isolates should be sent to a public health laboratory for testing because clinical laboratories generally do not have the typing sera available.

Question 50

50. Direct spread of pneumonic plague disease occurs by which route?

A. Fecal–oral route
B. Rat bite
C. Ingestion of contaminated tissue
D. Inhalation of contaminated airborne droplets

Answer 50

D. Inhalation of contaminated airborne droplets

Bubonic plague involves an inflammatory swelling of the lymph nodes of the axilla and groin, whereas pneumonic plague is associated with an airborne route involving the lungs. Both infections are caused by the same member of the Enterobacteriaceae family—Y. pestis.

Question 51

51. Which isolates of the Enterobacteriaceae family most commonly produce extended-spectrum β-lactamase (ESBL)?

A. Escherichia coli and Klebsiella pneumoniae
B. Yersinia enterocolitica and Yersinia pestis
C. Morganella morganii and Proteus vulgaris
D. Salmonella typhi and Shigella sonnei

Answer 51

A. Escherichia coli and Klebsiella pneumoniae

Point mutations occur in most members of the Enterobacteriaceae family that result in production of a β-lactamase that hydrolyzes broad-spectrum antibiotics, such as the cephalosporins, as well as penicillin and monobactam antibiotics. These are known as ESBL producers. The most common ESBL organisms are K. pneumoniae and E. coli. ESBL strains are detected by demonstrating their resistance to β-lactam antibiotics.

Question 52

52. Additional methods of definitive identification for the Enterobacteriaceae family include which of the following:

A. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF-MS)
B. Serodiagnosis
C. Nucleic acid–based studies
D. All of the above

Answer 52

D. All of the above

Serotyping for grouping of Salmonella spp., Shigella spp., and E.coli 0157:H7 using commercially available slide agglutination test kits is done by most clinical laboratories. Nucleic acid–based studies are usually performed in reference laboratories if the demand is minimal in a clinical laboratory. MALDI-TOF MS is used
for the identification of Enterobacteriaceae species. Microbial genomic research
showed that many mass spectral peaks represented ribosomal proteins when compared
with 16S rRNA gene sequencing and could be used to identify clinically important bacteria (Enterobacteriaceae) with the incorporation of mass spectral libraries and software. One limitation has been identified in that MALDI-TOF MS cannot differentiate between E. coli and Shigella spp. Species-level identification compared with the reference method showed optimal performance in the case of many of the Enterobacteriaceae.

Question 53

53. Which of the following Shigella spp. serotypes is the most often isolated in the United States?

A. Serotype A (Shigella dysenteriae)
B. Serotype B (Shigella flexneri)
C. Serotype C (Shigella boydii)
D. Serotype D (Shigella sonnei)

Answer 53

D. Serotype D (Shigella sonnei)

S. sonnei (Serotype D) is the most isolated species of Shigella in the United States. Humans are the only reservoirs, and of the estimated 500,000 cases of shigellosis in the United States per year, S. sonnei is responsible for 85% of the infections. It is primarily a pediatric infection, with 60% seen in children under 10 years of age.

Question 54

54. Which organism is transmitted by ingesting undercooked ground beef or raw milk resulting in inflammation and bleeding of the mucosa of the large intestine (i.e., hemorrhagic colitis) which can also lead to hemolytic uremic syndrome (HUS) ?

A. Escherichia coli (STEC)—Shiga toxin
B. Escherichia coli (ETEC)—enterotoxigenic
C. Escherichia coli (EAEC)—enteroaggregative
D. Proteus mirabilis

Answer 54

A. Escherichia coli (STEC)—Shiga toxin

E. coli (STEC) is the organism referred to as enterohemorrhagic E. coli, which produces a toxin similar to the Shiga toxin produced by S. dysenteriae. Inflammation and bleeding of the mucosa of the large intestine can lead to kidney failure and HUS. HUS is characterized by low platelet counts and hemolytic anemia, leading to transfusions and dialysis and death. The two most common serotypes are 0157:H7 and 0157:NM (nonmotile). Unlike in dysentery, the wet preps of the stools usually lack white blood cells (WBCs).

Question 55

55. This organism is transmitted by ingesting contaminated fresh water and/or shell fish, resulting in watery, dysentery-like stools and chronic diarrhea. Culture on stool media and initial testing show the following results:
    MacConkey agar = Both lactose-fermenting and non–lactose-fermenting colonies
    Sheep blood agar = Nonhemolytic, shiny, opaque, smooth, nonspreading
    Oxidase = +
    DNase = Neg
    String test = Neg
    The most likely preliminary identification is?

A. Proteus vulgaris
B. Escherichia coli
C. Salmonella spp.
D. Plesiomonas shigelloides

Answer 55

D. Plesiomonas shigelloides

P. shigelloides, now considered a new member of the Enterobacteriaceae family, causes gastrointestinal infections. Research shows that it may cross-react with Shigella grouping antisera (Group D) causing misidentification. If an oxidase test is performed first, this error can be avoided (Shigella = oxidase negative, whereas Plesiomonas = oxidase positive).