Harr 7.2 - Microbiology - Enterobacteriaceae

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 by catheterization also showed gram-negative rods, 100,000 col/mL. Given the following results, which is the most likely organism? A. Proteus vulgaris B. Salmonella typhi C. Yersinia enterocolitica D. E. coli

E. coli Typically, the IMViC reactions for the organisms listed are: E. coli (++00) S. typhi (0+00) Y. enterocolitica (V+00) P. vulgaris (++00) Note: indole reaction is variable for Y. enterocolitica

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 a hamburger lunch 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

E. 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.

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

E. coli O157:H7, Salmonella spp., Shigella spp. Preliminary serological grouping of the Salmonella spp. and Shigella spp. should be performed, since reliable commercial polyvalent antisera are available. Sorbitol-negative (MacConkey agar with sorbitol) colonies of E. coli should be tested using commercially available antisera for somatic "O" antigen 157 and flagellar "H" antigen 7. However, Yersinia pestis isolates should be sent to a public health laboratory for testing, since clinical laboratories generally do not have the typing sera available.

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

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, Yersinia pestis.

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

4.5 Both MR and VP tests detect acid production from the fermentation of glucose. However, a positive MR test 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).

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

A. E. 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 Klebsiella pneumonia and E. coli. ESBL strains are detected by demonstrating their resistance to β-lactam antibiotics.

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

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

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

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.

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

Aminoglycosides, trimethoprim-sulfamethoxazole, third-generation cephalosporins The drugs of choice for the Enterobacteriaceae 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.

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

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.

A positive Simmons citrate test is seen as 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.

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

C. freundii strains C. freundii and Salmonella spp. are H2S positive and indole, VP, and phenylalanine deaminase negative. Biochemical characteristics that help to differentiate C. freundii from Salmonella include lactose fermentation (50% of C. freundii are lactose positive, whereas 100% of Salmonella are lactose negative) and urease production (70% of Citrobacter are positive and greater than 99% of Salmonella are negative).

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

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. Young children may also exhibit bloody stools when infected with Campylobacter.

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

Citrobacter freundii Biochemical differentiation is essential because Citrobacter isolates may give a false-positive agglutination test with Salmonella grouping sera. C. freundii strains, like Salmonella spp., are usually H2S producers and may be confused with Salmonella spp. unless the proper biochemical tests are utilized. C. freundii and Salmonella spp. are adonitol, indole, and malonate negative. However, C. freundii is KCN positive, whereas Salmonella spp. are KCN negative.

Which of the following organisms are generally positive for β- galactosidase?

E. coli Enterobacteriaceae are grouped according to their ability to ferment lactose, a β-galactoside. Salmonella, Shigella, Proteus, Providencia, and Morganella are usually lactose nonfermenters.

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

E. coli Serological confirmation of Shigella isolates is based upon 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.

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

E. 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.

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

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

Biochemically, the Enterobacteriaceae are gram-negative rods that:

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 ferment glucose and are oxidase negative and nonsporulating. Most Enterobacteriaceae are motile, but the genera Shigella and Klebsiella are not.

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

Hydrogen sulfide (H2S) production, sucrose fermentation, indole, and VP

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

Indole K. oxytoca and K. pneumoniae are almost identical biochemically except for the ability to produce indole. Both organisms are usually positive for urease, sucrose, and citrate. However, K. oxytoca is indole positive and K. pneumoniae is indole negative.

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

Indole, oxidase, MacConkey, and blood agar plates All Enterobacteriaceae are oxidase negative. Because E. coli and Proteus spp. comprise a majority of the organisms recovered from clinical specimens, they can be initially identified through rapid testing without additional overnight testing. E. coli display a positive indole test, and the colonial morphology on MacConkey agar is distinctive, showing flat, pink (lactose-positive) colonies with a ring of bile precipitation. Proteus spp. swarm on blood agar and are indole negative.

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? (Results on picture) Antibiotic susceptibility: resistant to carbenicillin and ampicillin

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 for Klebsiella.

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

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

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

MacConkey agar Cefsulodin-irgasan-novobiocin (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; 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.

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:

Morganella spp. Morganella are biochemically similar to Proteus spp., both being lactose negative, motile, and positive for phenylalanine deaminase and urease. However, Morganella can be differentiated from Proteus spp. based upon H2S, indole, ornithine decarboxylase, and xylose fermentation. Ewingella spp. are usually positive (70%) for lactose fermentation, whereas the other three genera are lactose negative.

Which genera are positive for phenylalanine deaminase?

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.

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

Motility, urease, and phenylalanine deaminase (Morganella and Proteus spp. are motile, produce urease, and deaminate phenylalanine.)

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

Negative DNase DNase is not produced by Cedecea spp. but is produced (along with proteinases) by Serratia spp. Other key differential tests include lipase (positive for Cedecea, negative for Serratia) and gelatin hydrolysis (negative for Cedecea, positive for Serratia).

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

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, which helps to differentiate it from other Enterobacteriaceae.

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

O, Vi, and H antigens The Kaufmann-White schema groups the 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. There are over 2,200 serotypes of Salmonella.

Putrescine is an alkaline amine product of which bacterial enzyme?

Ornithine decarboxylase (Putrescine is the amine product of the decarboxylation of ornithine.)

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?

Pathogenic E. coli can be lactose positive or lactose negative Possible pathogenic strains of E. coli should be picked from MacConkey agar and subcultured onto MacConkey agar with sorbitol. After subculture, these strains can be serotyped or sent to a reference laboratory. Most E. coli normal flora ferment D-sorbitol and appear pink to red on MacConkey-sorbitol agar. The E. coli strain O157:H7 causes the enteric disease hemorrhagic colitis. It ferments D-sorbitol slowly or not at all and appears as colorless colonies on MacConkey-sorbitol agar.

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

Proteus spp. Proteus spp. are urease positive as are approximately 70% of Y. enterocolitica isolates. Both organisms are lactose negative and motile. However, Yersinia is motile at 22°C and not at 35°C (demonstrated using motility media).

Three blood cultures taken from a 30-year-old cancer patient receiving chemotherapy and admitted with a urinary tract infection grew lactose-negative, motile, gram-negative rods prior to antibiotic therapy. Given the following biochemical reactions, which is the most likely organism?

Proteus vulgaris Although P. mirabilis is more frequently recovered from patients with urinary tract infections, 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.

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

S. 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 owing to ornithine decarboxylase and β-galactosidase activity. These enzymes, found in most strains of S. sonnei, distinguish it from other Shigella species.

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

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 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.

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

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.

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

Salmonella, Shigella, Yersinia, Campylobacter, and Staphylococcus aureus V. cholerae and C. difficile are usually not included in a routine stool culture. If Vibrio spp. are suspected, a special request should be included. Although MacConkey agar will support the growth of Vibrio spp., normal enteric flora overgrow and occlude these organisms. C. difficile culture requires special media (CCFA) that inhibit other anaerobic flora and facultative anaerobic flora and should be requested specifically if symptoms warrant. MacConkey agar with sorbitol will allow the E. coli O157:H7 to be recovered. Yersinia spp. can be detected on a regular MacConkey agar plate.

A leg-wound culture from a hospitalized 70-year-old diabetic man grew motile, lactose-negative colonies on MacConkey agar. Given the following biochemical reactions at 24 hours, what is the most probable organism?

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.

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) tested negative for oxidase, indole, urease, motility, and H2S The most likely identification is:

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.

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

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 lactose fermenters and may be confused with Shigella spp., which do not ferment lactose. E. coli are ONPG positive while Shigella spp. are ONPG negative.

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

Y. 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.

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 Which tests are most appropriate for identification of this enteric pathogen?

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

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

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.

Enterobacter sakazakii can best be differentiated from Enterobacter cloacae by which of the following characteristics?

Yellow pigmentation and negative sorbitol fermentation E. sakazakii is called a yellow-pigmented E. cloacae and is best differentiated from E. cloacae by sorbitol fermentation (95% positive for E. cloacae and 0% for E. sakazakii). In addition, E. cloacae is usually positive for urease and malonate (65% and 75%, respectively) and E. sakazakii is usually negative (1% and < 20%, respectively). Both species are usually motile and arginine dihydrolase positive.

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 (cefsulodin-irgasan-novobiocin) displayed "bulls-eye" colonies after 48 hours 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?

Yersinia enterocolitica Most members of the Enterobacteriaceae family produce detectable growth on MacConkey agar within 24 hours. Yersinia enterocolita produces non-lactose-fermenting colonies on MacConkey agar, salmon-colored colonies on Hektoen agar, and yellow or colorless colonies on XLD agar. If Yersinia enterocolitica is suspected, specialized agar (CIN) is employed. The typical bulls-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, since Yersinia spp. are oxidase negative and Aeromonas spp. are oxidase positive.

Following a 2-week camping trip to the Southwest (US), 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, resulting in growth of gram-negative rods resembling "closed safety pins." The organism grew on MacConkey's agar showing non-lactose-fermenting colonies. Testing demonstrated a nonmotile rod that was biochemically inert. What is the most likely identification?

Yersinia pestis Yersinia 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.

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

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's and Ehrlich's reagents) in acid, forming a red complex.

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

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 the Enterobacteriaceae that have been isolated from human specimens and are usually nonmotile include Leminorella, Rahnella, and Tatumella.

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?

Sorbitol (fermentation) An isolate of E. coli 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.