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UNIVERSITY OF COLORADO

SCHOOL OF MEDICINEDEPARTMENT OF PATHOLOGY

RESIDENCY TRAINING PROGRAM

MICROBIOLOGY

VII: Curved Gram-Negative Bacilli

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

Joel Mortensen, Ph.D.

Director, Clinical Microbiology

St. Christophers Hospital for Children

Philadelphia, Pennsylvania

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Case History

A 9-day-old black female with a 2-day history of frequent bowelmovements, irritability, and decreased appetite was evaluated inthe hospital. The stool was described as yellow-green andseedy.

The infant was observed for 12 hours. During this period shetook formula without difficulty and had two normal appearingstools.

A stool specimen was submitted to the microbiology laboratoryfor routine culture. It was inoculated to routine enteric isolationculture media, including a campy-blood plate which was

incubated at 42o C under CO2 enriched atmospheric conditions.

Infant Diarrhea

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After 24-hours incubation,beta-hemolytic colonieswere observed on theblood agar plate. Thecytochrome oxidase test

was positive.

The appearance of thegram stain obtained fromone of the colonies isshown in the photograph.Note the distinctlycomma-shaped, gram-negative bacilli.

Infant Diarrhea

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Illustrated is the growth of theisolate on the surface of TCBSagar.

Infant Diarrhea

QUESTIONSWhat does the yellow pigmentindicate?

What is the most probablespecies identification?

How can this be confirmed?

ANSWERS

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The mother was contacted to determine the source of the infection.The family consisted of the mother, three older siblings, and thegrandmother, 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 atefrequently during and after her pregnancy. Two adults from thePhiladelphia area also had V. cholerae infection, and both hadfrequented the same seafood store. Cross contamination of the infantduring delivery was probable but not proven.

Infant Diarrhea

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DIAGNOSIS: INFANT DIARRHEA

Etiologic Agent: Vibrio cholerae, 01

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Abbreviated Identification ofVibrio cholerae

Smooth to creamy, gray-white, raised, non-hemolytic

colonies, 0.5- 2.0 mm after 24 hours at 35o 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

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Vibrio cholerae is a gram-negative bacterium existing in twogroups01 and non-01. Serogroup 01 causes epidemic disease,

with the El Tor biotype being 7 times more common than theClassic strain. Non-01 strains are not epidemic, and cause onlyoccasional diarrhea and sepsis.

Both 01 and non-01 strains may be recovered from shellfish in theUnited States, particularly from oysters, but also from crabs. Non-

01 strains are part of the normal free-living bacterial flora in marineand brackish waters. Illness caused by these organisms tends tooccur in the summer and fall when bacterial proliferation in coastalareas is greater. Consuming undercooked seafood is the mostcommon source of infection for humans.

Even in areas where Vibrio species are not endemic, clinicians andmicrobiology laboratory personnel should be aware of theseorganisms and their potential virulence. Treatment with eithertetracyclines or TM/SMX has been recommended.

Vibrio Cholerae: Recapitulation

NEXT CASE

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UNIVERSITY OF COLORADO

SCHOOL OF MEDICINEDEPARTMENT OF PATHOLOGY

RESIDENCY TRAINING PROGRAM

MICROBIOLOGY

VII: Oxidase-Positive Fermenters

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

Presenter: Robert Fader, Ph.D.Director Microbiology/Virology Laboratories

Spectrum Health

Grand Rapids, Michigan

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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

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Illustrated in the photograph

is the surface of a sheep

blood agar plate after 24-

hours incubation at 35o C,

on which are growingcolonies from the isolate in

this case.

The colonies appear gray,

shiny, smooth, and opaque,

with slightly convex centers.Hemolysis is absent.

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Illustrated in the photograph are a plate of MacConkey agar (left) andHektoen 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 ofShigella species was made.

Acute Diarrhea

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The reactions of the isolate in thiscase 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

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Illustrated in thisphotograph is

the bioMerieux

NE identification

strip. The

positive and

negative

reactions are

observed on the

worksheet. The

Biotype code

3160744 keyedout to:

Plesiomonasshigelloides The right side of this photograph has been cut off.

The full biotype number is: 3160744.

Acute Diarrhea

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DIAGNOSIS: ACUTE DIARRHEA

Etiologic Agent: Plesiomonas shigelloides

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Plesiomonas shigelloides

Abbreviated Identification ofPlesiomonas shigelloides

Smooth to creamy, gray-white, raised, non-hemolytic

colonies, 0.5- 1.0 mm after 24 hours at 35oC 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 +

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LABORATORY IDENTIFICATION

Plesiomonas shigelloides is a straight, rounded, short, motilegram-negative rod with each cell fitted with a single polar

flagellum. Colonies are non-hemolytic on blood agar, an importantdifferential characteristic from Aeromonas sp.

The optimum temperature of growth is 30o C. Plesiomonasspecies can be further separated from Aeromonas by being DNAsenegative, producing acid from inositol, but not from mannitol, and

being ornithine decarboxylase positive.

Plesiomonas species are also negative in the Voges-Proskauertest, while most Aeromonas species are positive, excluding somestrains ofA. caviae that are negative.

Plesiomonas shigelloides:Recapitulation

Pl h ll d

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Plesiomonas shigelloides:Recapitulation

Clinical Manifestations Plesiomonas species, the name derived from a Greek wordmeaning neighbor, are closely related to Aeromonas species.

Plesiomonas sp are ubiquitous in surface waters and soil, andcommonly 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 asmild 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

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The yellow pigment indicates that this isolate iscapable of producing acid from sucrose, a key

reaction for the presumptive identification ofVibriocholerae.

This presumptive identification may be confirmedby using either an automated or a commercial kitidentification system. In this case, the API 20E

biocode number and conventional biochemicaltests confirmed the identification ofVibriocholerae.

The identification can be further confirmed bydetermining the serovar. In this case, the isolate

was serologically identified as a 01 strain ofVibriocholerae, agglutinating with Ogawa antisera.

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The strong positive reaction for

the indole spot test does not

necessarily rule out Shigellaspecies as most strains ofS.sonnei are also indole positive.

However, the positive oxidase

reaction, as illustrated in this

photograph, serves to rule outShigella species. It is commonpractice 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.