UNIVERSITY OF COLORADO SCHOOL OF MEDICINE

DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM

VII: Curved Gram-Negative Bacilli

Case Study 7a: Infant Diarrhea
Adapted from the CACMLE Teleconference first presented on Sept. 23 1998 by:

MICROBIOLOGY

Joel Mortensen, Ph.D. Director, Clinical Microbiology St. Christophers Hospital for Children Philadelphia, Pennsylvania

Infant Diarrhea
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 42o C under CO2 enriched atmospheric conditions.

Infant Diarrhea
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, gramnegative bacilli.

Infant Diarrhea
Illustrated is the growth of the isolate on the surface of TCBS agar.

QUESTIONS
What does the yellow pigment indicate? What is the most probable species identification?

How can this be confirmed?

ANSWERS

Infant Diarrhea

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 mothers 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.

DIAGNOSIS: INFANT DIARRHEA Etiologic Agent: Vibrio cholerae, 01

Abbreviated Identification of Vibrio cholerae

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

Typing: Vibrio cholerae, 01 (Ogawa)

Vibrio Cholerae: Recapitulation

Vibrio cholerae is a gram-negative bacterium existing in two groups01 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. Non01 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.

NEXT CASE

UNIVERSITY OF COLORADO SCHOOL OF MEDICINE

DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM

VII: Oxidase-Positive Fermenters

Case Study 7b: Acute Diarrhea
Adapted from the CACMLE Teleconference first presented on May 26, 1999 by:

MICROBIOLOGY

Presenter: Robert Fader, Ph.D.

Director Microbiology/Virology Laboratories Spectrum Health Grand Rapids, Michigan

Acute Diarrhea
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 antidiarrheal 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
Illustrated in the photograph is the surface of a sheep blood agar plate after 24hours incubation at 35o 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

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 photograph) on MacConkey, and have a pale gray-green color on Hektoen agar. A presumptive identification of Shigella species was made.

Acute Diarrhea
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. What spot test should be performed?

ANSWER

Acute Diarrhea
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.

DIAGNOSIS: ACUTE DIARRHEA Etiologic Agent: 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 35oC incubation Short, gram-negative bacilli on Gram stain Growth of non-lactose fermenting colonies on MacConkey agar Cytochrome oxidase + Esculin hydrolysis negative

Lysine +

Ornithine +

Arginine + Acid from inositol +

Acid from mannitol ( - )

Plesiomonas shigelloides

Plesiomonas shigelloides:
Recapitulation
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 30o 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
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

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.

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 oxidasepositive fermenters. Biochemical characterization was necessary to establish the final identification.