2. Case History A 9-day-old black female with a 2-day history of frequent bowel movements, irritability, and decreased appetite was evaluated in the hospital. The stool was described as “yellow-green” and “seedy”. The infant was observed for 12 hours. During this period she took formula without difficulty and had two normal appearing stools. A stool specimen was submitted to the microbiology laboratory for routine culture. It was inoculated to routine enteric isolation culture media, including a campy-blood plate which was incubated at 42 o C under CO 2 enriched atmospheric conditions. Infant Diarrhea

3. After 24-hours incubation, beta-hemolytic colonies were observed on the blood agar plate. The cytochrome oxidase test was positive. The appearance of the gram stain obtained from one of the colonies is shown in the photograph. Note the distinctly comma-shaped, gram- negative bacilli. Infant Diarrhea

4. Illustrated is the growth of the isolate on the surface of TCBS agar. Infant Diarrhea QUESTIONS What does the yellow pigment indicate? What is the most probable species identification? How can this be confirmed? ANSWERS

5. The mother was contacted to determine the source of the infection. The family consisted of the mother, three older siblings, and the grandmother, all residing together in inner city Philadelphia. There had been no travel outside the city, and all other family members were well. The mother’s favorite food was crab (photograph), which she ate frequently during and after her pregnancy. Two adults from the Philadelphia area also had V. cholerae infection, and both had frequented the same seafood store. Cross contamination of the infant during delivery was probable but not proven. Infant Diarrhea

6. DIAGNOSIS: INFANT DIARRHEA Etiologic Agent: Vibrio cholerae, 01

7. Typing: Vibrio cholerae, 01 (Ogawa) Abbreviated Identification of Vibrio cholerae Smooth to creamy, gray-white, raised, non-hemolytic colonies, 0.5- 2.0 mm after 24 hours at 35 o C incubation Ornithine+ Distinctly curved, short, gram-negative bacilli on Gram stain Arginine (-)Lysine + Cytochrome oxidase + 1% NaCl not required for growth (non-halophilic) Susceptible to vibriostatic compound 0/129 Yellow colonies on TCBS Agar Establish Species Identification

8. Vibrio cholerae is a gram-negative bacterium existing in two groups—01 and non-01. Serogroup 01 causes epidemic disease, with the “El Tor” biotype being 7 times more common than the “Classic” strain. Non-01 strains are not epidemic, and cause only occasional diarrhea and sepsis. Both 01 and non-01 strains may be recovered from shellfish in the United States, particularly from oysters, but also from crabs. Non- 01 strains are part of the normal free-living bacterial flora in marine and brackish waters. Illness caused by these organisms tends to occur in the summer and fall when bacterial proliferation in coastal areas is greater. Consuming undercooked seafood is the most common source of infection for humans. Even in areas where Vibrio species are not endemic, clinicians and microbiology laboratory personnel should be aware of these organisms and their potential virulence. Treatment with either tetracyclines or TM/SMX has been recommended. Vibrio Cholerae: Recapitulation NEXT CASE

10. A 40-year old white female was accompanied by her husband on a mission trip in Viet Nam. After being “in country” for 16 days, careful to avoid drinking local water and locally prepared food, the patient experienced abdominal cramping and watery diarrhea. The diarrhea was refractory to all over-the-counter anti- diarrheal medications. It persisted during the remainder of the trip, on the plane trip home, and for 3 days after return to the United States. A stool specimen was submitted and an organism was recovered in pure culture. The diarrhea began to abate after a 3-day course of ciprofloxacin. Acute Diarrhea

11. Illustrated in the photograph is the surface of a sheep blood agar plate after 24- hours incubation at 35 o C, on which are growing colonies from the isolate in this case. The colonies appear gray, shiny, smooth, and opaque, with slightly convex centers. Hemolysis is absent. Acute Diarrhea

12. Illustrated in the photograph are a plate of MacConkey agar (left) and Hektoen enteric agar (right) on which are growing the isolate from this case. The colonies appear lactose negative (dark blue in this photo- graph) on MacConkey, and have a pale gray-green color on Hektoen agar. A presumptive identification of Shigella species was made. Acute Diarrhea

13. The reactions of the isolate in this case when inoculated to Triple Iron agar slant is illustrated in the tube to the left. The yellow-deep indicates glucose fermentation; the red slant indicates a non-lactose fermenter. The lack of a black color in the media contained in the tube to the right indicates a negative esculin reaction. Shigella species still has not been ruled out. Acute Diarrhea What spot test should be performed? ANSWER

14. Illustrated in this photograph is the bioMerieux NE identification strip. The positive and negative reactions are observed on the worksheet. The Biotype code 3160744 keyed out to: Plesiomonas shigelloides The right side of this photograph has been cut off. The full biotype number is: 3160744. Acute Diarrhea

15. DIAGNOSIS: ACUTE DIARRHEA Etiologic Agent: Plesiomonas shigelloides

16. Plesiomonas shigelloides Abbreviated Identification of Plesiomonas shigelloides Smooth to creamy, gray-white, raised, non-hemolytic colonies, 0.5- 1.0 mm after 24 hours at 35 o C incubation Ornithine + Short, gram-negative bacilli on Gram stain Arginine +Lysine + Cytochrome oxidase + Growth of non-lactose fermenting colonies on MacConkey agar Esculin hydrolysis negative Acid from mannitol ( - )Acid from inositol +

17. LABORATORY IDENTIFICATION Plesiomonas shigelloides is a straight, rounded, short, motile gram-negative rod with each cell fitted with a single polar flagellum. Colonies are non-hemolytic on blood agar, an important differential characteristic from Aeromonas sp. The optimum temperature of growth is 30 o C. Plesiomonas species can be further separated from Aeromonas by being DNAse negative, producing acid from inositol, but not from mannitol, and being ornithine decarboxylase positive. Plesiomonas species are also negative in the Voges-Proskauer test, while most Aeromonas species are positive, excluding some strains of A. caviae that are negative. Plesiomonas shigelloides: Recapitulation

18. Plesiomonas shigelloides: Recapitulation Clinical Manifestations Plesiomonas species, the name derived from a Greek word meaning “neighbor”, are closely related to Aeromonas species. Plesiomonas sp are ubiquitous in surface waters and soil, and commonly infects cold-blooded animals such as frogs, snakes, turtles, and lizards. Humans become infected primarily through ingestion of contaminated or unwashed food. A carrier state in Thailand has been reported as high as 55% of the population. Infections in the U.S. are more prevalent in warm weather months. Shellfish-related outbreaks have been reported in Japan. Plesiomonas gastroenteritis in humans usually presents as mild watery diarrhea. Patients who are immunosuppressed or who have GI malignancies, may develop a severe colitis or cholera-like illness, related to production of an endotoxin. BACK

19. The yellow pigment indicates that this isolate is capable of producing acid from sucrose, a key reaction for the presumptive identification of Vibrio cholerae. This presumptive identification may be confirmed by using either an automated or a commercial kit identification system. In this case, the API 20E biocode number and conventional biochemical tests confirmed the identification of Vibrio cholerae. The identification can be further confirmed by determining the serovar. In this case, the isolate was serologically identified as a 01 strain of Vibrio cholerae, agglutinating with Ogawa antisera.

20. The strong positive reaction for the indole spot test does not necessarily rule out Shigella species as most strains of S. sonnei are also indole positive. However, the positive oxidase reaction, as illustrated in this photograph, serves to rule out Shigella species. It is common practice to perform a spot oxidase test on all non-lactose fermenters recovered from stool specimens to rule out oxidase- positive fermenters. Biochemical characterization was necessary to establish the final identification.