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UNIVERSITY OF COLORADO
SCHOOL OF MEDICINE
DEPARTMENT OF PATHOLOGY
RESIDENCY TRAINING PROGRAM
MICROBIOLOGY
VII: Curved Gram-Negative Bacilli
Case Study 7a: Infant Diarrhea
Adapted from the CACMLE Teleconference first presented on Sept. 23 1998 by:
Joel Mortensen, Ph.D.
Director, Clinical Microbiology
St. Christopher’s 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 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.
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
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. 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
MICROBIOLOGY
VII: Oxidase-Positive Fermenters
Case Study 7b: Acute Diarrhea
Adapted 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
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 +
Acid from mannitol ( - )
Arginine +
Acid from inositol +
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.