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Journal of Clinical Microbiology, July 1998, p. 2138-2139, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Bacillus thuringiensis subsp.
konkukian (Serotype H34) Superinfection: Case Report and
Experimental Evidence of Pathogenicity in Immunosuppressed
Mice
Eric
Hernandez,1,*
Francoise
Ramisse,2
Jean-Pierre
Ducoureau,3
Thierry
Cruel,1 and
Jean-Didier
Cavallo3
Laboratoire de
Biologie1 and
Service de
Réanimation,3 Hôpital des
Armées Begin, 94160 Saint Mandé, and
Centre
d'Etude du Bouchet BP3, 91710 Vert-le-Petit,2
France
Received 15 January 1998/Returned for modification 29 January
1998/Accepted 13 April 1998
 |
ABSTRACT |
We present a case of severe war wounds infected by Bacillus
thuringiensis serotype H34 and describe the experimental protocol used to demonstrate its ability to infect mice after cutaneous inoculation. This case is interesting because B. thuringiensis is considered to be a contaminant in laboratories
and receives inadequate attention.
| |
TEXT |
Bacillus species are
widely distributed in nature, and finding a Bacillus sp. in
medical practice, aside from B. anthracis and B. cereus, is often considered clinically irrelevant. B. thuringiensis is closely related to B. cereus and is
used extensively around the world as a pesticide in forestry and
agriculture (1). The two species can be differentiated only
by the production of the plasmid-encoded delta endotoxin, which is
pathogenic for larvae of Lepidoptera. As this characteristic
is not consistent, many consider B. thuringiensis to be a
variant of B. cereus.
Infection in humans is unusual and, except for reports on infection of
the gastrointestinal tract or laboratory contamination, there is only
one clinical report of infection by this microorganism (2).
We describe a case of severe war wounds infected by B. thuringiensis serotype H34 (4) and present experimental
evidence for the pathogenicity of this strain in a mouse model of
cutaneous infection.
Case report.
The patient was a healthy 28-year-old French
soldier severely wounded in March of 1995 by a land mine explosion in
former Yugoslavia. When he was admitted to the emergency room of the French military hospital in Sarajevo, Yugoslavia, the patient presented
with hemorrhagic shock, a pulmonary blast, shrapnel lesions in the left
leg, and multiple fractures in the left knee. Immediate
antibioprophylaxis, given on the battlefield, included penicillin G
(5 × 106 U/24 h) associated with metronidazole (1.5 g/24 h). After immediate surgery, the patient was evacuated within
24 h to the Begin Army Hospital (Paris, France). Upon admission,
he was found to have an oral temperature of 38.7°C, a pulse of 100 beats/min, blood pressure of 130/90 mm Hg, and a respiratory rate of 24 breaths/min. His leukocyte count was 10,200 cells/mm3. He
was immunocompetent and a nonsmoker and had no history of illness.
Physical examination revealed abscesses on the left thigh and knee, and
biopsy specimens of the wounds were obtained surgically. Microscopic
examination showed large gram-positive rods with endospores. After
routine culture, the colonies on blood agar medium were shown to be
large (5 mm), beta-hemolytic, flat, white, and rough. Catalase was
positive, and oxidase was negative. The bacterium was mobile.
Biochemical tests performed with API 50-CHB and 20-E (Biomerieux,
S. A., Lyon, France) identified the strain as B. cereus, disagreeing with the results obtained by the Vitek system (Biomerieux Vitek, St. Louis, Mo.), which identified the strain as
B. thuringiensis. The results of the biochemical reactions are presented in Table 1.
As this finding was unusual, the strain was sent to M. Lecadet
and her colleagues at the World Health Organization collaborating center for entomopathogens (Unité des Bactéries
Entomopathogènes, Institut Pasteur, Paris, France), who
identified the strain as B. thuringiensis subsp.
konkukian (serotype H34).
The presence of crystal in sporulated culture, which is characteristic
of
B. thuringiensis, was recorded by direct examination
of a
fresh preparation under a phase-contrast microscope or after
specific
coloration of proteins with Coomassie brilliant blue
solution.
The standard characteristics according to the method described by
Sneath (
5) were determined mainly by the API
Bacillus (API-20E and API-50CH) system.
H serotyping based on flagellar antigens was performed according to the
agglutination method described by de Barjac (
3)
with
specific antisera for the 79 known
B. thuringiensis
serovars.
In brief, an early exponentially growing culture is
inoculated
on the agar surface of a small, special tube filled with
soft
nutrient agar. If mobile, the bacteria migrate in 24 h. The
cells
obtained by this method are then tested for agglutination with
various antisera.
Antimicrobial susceptibility was tested by E test and disk diffusion.
Interpretive criteria for
Staphylococci were used because
there are no National Committee for Clinical Laboratory Standards
guidelines for interpreting the results of susceptibility tests
on
Bacillus spp. Results (MICs in micrograms per milliliter and
zone diameters in millimeters) showed resistance to penicillin
G (MIC,
32; diameter, 8) and ampicillin (MIC, 16; diameter, 12)
and a
paradoxical susceptibility to piperacillin (MIC, 1; diameter,
27). The
presence of
-lactamase was indicated by the Cefinase
test
(Biomerieux), performed in liquid medium. The strain was
also
susceptible to imipenem (MIC, 0.047; diameter, 34 mm), vancomycin
(MIC,
2; diameter, 18), gentamicin (MIC, 0.5; diameter, 24) and
ciprofloxacin
(MIC, 0.25; diameter, 31).
The patient recovered after 15 days of intensive care, multiple
surgical cleansings, and an antibiotic treatment of ciprofloxacin
associated with gentamicin that was administered for 10 days.
The isolation of
B. thuringiensis from wound abscesses
raised the question of whether this bacterium was the causative agent
of the infection or simply a contaminant, leading us to investigate
the
potential pathogenicity of this strain in mammals with a mouse
model of
cutaneous infection. After depilation of a 2-cm
2 area of
their skin, six groups of 5-week-old female BALB/c mice
were infected
by an application of a bacterial suspension containing,
respectively,
10
5, 10
6, or 10
7 CFU per mouse. For
each inoculum, 4 days before the experiment,
one group of mice was
immunosuppressed by intravenous injection
of cyclophosphamide (200 mg/kg of body weight); a control group
was not immunosuppressed.
Cutaneous inflammatory lesions occurred
in all animals when the
10
7 CFU inoculum was used. Lesions healed spontaneously
after 48
h in nonimmunosuppressed mice but increased in the other
group.
Two days after the infection, the mice were sacrificed, and the
inner side of the skin (4 cm
2) was aseptically removed and
grown on a blood agar medium.
B. thuringiensis was recovered
only in the samples from the immunosuppressed
mice. The bacterial count
was correlated with the quantity of
the cutaneous bacterial inoculum.
Bacteriological examination
of tissue stained by Gram stain and
periodic acid-Schiff stain
revealed that
B. thuringiensis
was associated with tissue necrosis
and polymorphonuclear infiltrates
(Fig.
1).
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FIG. 1.
Gram stain of tissue revealing association of B. thuringiensis with tissue necrosis and polymorphonuclear
infiltrates. Abbreviations: b, bacteria; f, fat; m, muscle; n, deep
necrosis. Magnification, ×400.
|
|
The animal model used demonstrates the ability of
B. thuringiensis subsp.
konkukian (serotype H34) to
produce infection and
myonecrosis in the absence of an effective immune
response. Likewise,
the clinical data suggest that disease may also be
produced in
patients with tissue devitalization caused by massive
tissue destruction.
Like
B. cereus, this bacterium forms
spores which are normally
found in the soil. Presumably, soil and dust
particles projected
by the land mine explosion were the source of the
organism in
this unusual case of infection. This reported episode of
soft-tissue
infection and necrosis by
B. thuringiensis is
reminiscent of
B. cereus infection after severe trauma
(
6).
| |
ACKNOWLEDGMENTS |
We thank Alan Tallmon, minister of the French Reformed Church, for
his help in the preparation of the manuscript. We also thank Marguerite
Lecadet (Unité des Bactéries Entomopathogènes, Institut Pasteur, Paris, France) for her valuable help in identifying B. thuringiensis subsp. konkukian.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Biologie, Hôpital des Armées Begin, 69 Avenue de Paris,
94160 Saint Mandé, France. Phone: 1-43-98-50-00. Fax:
1-41-46-63-21. E-mail: hnz.eric{at}mailexcite.com.
| |
REFERENCES |
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Journal of Clinical Microbiology, July 1998, p. 2138-2139, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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