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Antimicrobial Agents and Chemotherapy, June 2001, p. 1788-1793, Vol. 45, No. 6
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1788-1793.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Inhibition of Group A Streptococcus Infection
by Carboxyfullerene
Nina
Tsao,1
Tien-Yau
Luh,2
Chen-Kung
Chou,3
Jiunn-Jong
Wu,4
Yee-Shin
Lin,1 and
Huan-Yao
Lei1,*
Department of Microbiology and
Immunology1 and Department of Medical
Technology,4 College of Medicine, National
Cheng Kung University, Tainan, and Department of Chemistry,
National Taiwan University,2 and
Department of Medical Research, Veterans General
Hospital,3 Taipei, Taiwan
Received 24 October 2000/Returned for modification 29 December
2000/Accepted 2 March 2001
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ABSTRACT |
The effect of a water-soluble trimalonic acid derivative of
fullerene, carboxyfullerene, against Streptococcus
pyogenes infection was tested. Pretreatment with
carboxyfullerene was able to protect mice from S.
pyogenes infection in an air pouch model. S.
pyogenes-induced death and skin injury were inhibited dose
dependently by carboxyfullerene. Administration of carboxyfullerene via
the peritoneum and air pouch at 3 h post-S.
pyogenes infection was able to protect 33% of mice from death.
Surveys of exudates of the air pouch of carboxyfullerene-treated mice
revealed that survival of infiltrating neutrophils was prolonged and
that the bacteria were eliminated as a result of enhanced bactericidal
activity of the neutrophils. Furthermore, carboxyfullerene was able to
directly inhibit in vitro growth of S. pyogenes. These data suggest that carboxyfullerene can be considered an antimicrobial agent for group A streptococcus infection.
| |
INTRODUCTION |
The incidence of infection
with the group A streptococcus (GAS) Streptococcus pyogenes
has increased worldwide and is still associated with a high mortality
rate, especially when GAS infection induces necrotizing fasciitis and
streptococcal toxic shock syndrome (1, 3, 11, 14, 15,
21). Certain virulence factors involved in the pathogenesis of
GAS infection have been reported. These include cell surface molecules
such as M protein, opacity factor, the hyaluronic acid capsule, C5a
peptidase, and the streptococcal inhibitor of complement, as well as
secreted products such as pyogenic exotoxins, cysteine proteinase,
streptolysins O and S, hyaluronidase, streptokinase, and other enzymes
(3, 12, 15). Empirical therapy for GAS infection includes
antibiotics, aggressive surgery, and intravenous administration of
immunoglobulin (21, 22).
Buckminsterfullerenes (fullerene [C60])
have attracted much attention since their discovery and
large-scale synthesis. Fullerene is characterized as a "radical
sponge" because of its unique cage structure, which allows it to
interact effectively with free radicals (7). However,
native C60 is soluble only in organic solvents and so cannot be applied to medical therapy. A water-soluble trimalonic acid derivative of fullerene (carboxyfullerene
[C63(COOH)6]) has been
synthesized and has been found to be an effective neuroprotective antioxidant both in vitro and in vivo (2, 9).
Carboxyfullerene is a powerful free radical scavenger and can protect
cells from apoptosis in various systems (4, 5). In
previous studies, we found that carboxyfullerene was able to inhibit
the development of Escherichia coli-induced meningitis
(18) by modulating the activity of neutrophils
(16). In the present study, the effect of carboxyfullerene
on gram-positive bacterial infections was evaluated. Using an air pouch
GAS infection model, which mimics localized fasciitis (8),
we found that carboxyfullerene was able to inhibit GAS infection by
both modulating the bactericidal activity of infiltrating neutrophils
and directly inhibiting the growth of S. pyogenes.
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MATERIALS AND METHODS |
Mice.
C57BL/6 (B6) mice were purchased from The Jackson
Laboratory, Bar Harbor, Maine, or from Charles River Japan, Inc.
(Atsugi, Japan). They were maintained on standard laboratory chow and
water ad libitum in our animal center. The animals were raised and
cared for in accordance with guidelines established by the National Science Council of the Republic of China. Eight- to 12-week-old female
mice were used in all experiments.
Carboxyfullerene.
Two regioisomers of water-soluble
carboxylic acid C60 derivatives with
C3 or
D3 symmetry were synthesized as
described previously (2). Both
C63(COOH)6
(C3) and
C63(COOH)6
(D3) are effective free radical
scavengers. In this study, we used
C63(COOH)6
(C3) dissolved in phosphate-buffered
saline (PBS) (2 mg/ml).
Bacteria.
S. pyogenes A-20 (type M1, T1; opacity
factor negative) was isolated from the blood of a patient with
necrotizing fasciitis at the National Cheng Kung University Hospital.
S. pyogenes NZ-131 (type M49, T14) was a gift from D. R. Martin, New Zealand Communicable Disease Center, Porirua. Genotyping
of A-20 revealed the presence of speA, speB, and
speC, whereas NZ-131 had just speB
(8). S. pyogenes was cultured in tryptic soy
broth containing 0.5% yeast extract (TSBY) (Difco Laboratories,
Detroit, Mich.) for 12 h at 37°C and then subcultured in fresh
broth (1:50, vol/vol) for another 3 h. The concentration of
bacteria was determined with a spectrophotometer (Beckman Instruments,
Somerset, N.J.), with an optical density at 600 nm of 1 being equal to
108 CFU/ml (20).
Air pouch model of infection.
Mice were anesthetized by
ether inhalation and then injected subcutaneously with 1 ml of air for
three consecutive days to form an air pouch. Two days later, 0.1 ml of
bacterial suspension containing 1 × 109
S. pyogenes A-20 cells or 2 × 109 S. pyogenes NZ-131 cells was
inoculated into the air pouch (8). The 100% lethal doses
(LD100) of S. pyogenes A-20 and NZ-131
by air pouch injection in B6 mice are 1 × 109 cells and 2 × 109
cells, respectively. The animals were observed every day for a total of
5 days. In carboxyfullerene inhibition experiments, the mice were given
an air pouch injection of carboxyfullerene immediately post-S.
pyogenes injection or as late as 3 h post-S. pyogenes injection. In some experiments, carboxyfullerene was given via both air pouch and intraperitoneal injections. Survival curves were determined. Tissues around the air pouch were
excised 24 h after bacterial inoculation, fixed in 10%
formaldehyde, and embedded in paraffin. The 5-µm-thick tissues were
sliced and stained with hematoxylin and eosin. Infiltrating cells in
the air pouch were collected by injecting 1 ml of PBS into the air
pouch and aspirating the exudates by syringe with an 18-gauge needle
(8). Numbers of cells were determined with a
hemocytometer, and cell viability was determined by eosin Y exclusion.
Bacterial growth curves.
S. pyogenes A-20 was
cultured in TSBY at 37°C overnight, and then the bacterial suspension
was subcultured (1:50, vol/vol) in fresh TSBY for another 8 h. At
the time of subculture, different concentrations of carboxyfullerene
were added to the bacterial suspension, and the growth of bacteria at
different times was determined with a spectrophotometer by measuring
the absorbance at 600 nm. For exact quantification of bacteria,
bacterial suspensions collected at different times were plated on blood
agar and incubated for 24 h at 37°C. The results of one of three
experiments are reported.
Bactericidal activity of neutrophils.
Neutrophils were
purified from the blood of naïve B6 mice by Ficoll-Paque
(Amersham Pharmacia Biotech, Uppsala, Sweden) centrifugation (17). The neutrophils were resuspended
(106 in 1 ml) in 24-well plates (Falcon;
Becton-Dickinson Labware, Paramus, N.J.) and incubated for 4 h in
RPMI 1640 medium containing 10% fetal calf serum with different
concentrations of carboxyfullerene. Carboxyfullerene at 200 µg/ml is
not toxic to neutrophils in 4-h culture. The neutrophils treated with
carboxyfullerene were washed twice with PBS, and the cells were
cocultured with S. pyogenes A-20 at ratio of 20:1 (bacteria
to neutrophils) in RPMI 1640 medium for another hour. After 1 h of
incubation, the supernatant was collected by centrifugation and plated
on blood agar for quantification of bacteria.
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RESULTS |
Inhibition of GAS infection by carboxyfullerene.
In the air
pouch model of GAS infection, inoculation of 109
S. pyogenes A-20 cells (the LD100 for
mice at 48 h) causes hair loss, suppuration, and bleeding on the
skin around the air pouch at 24 h (8); the mice die
at 48 h. The effect of carboxyfullerene on GAS-induced mortality
was evaluated. As shown in Fig.
1A, carboxyfullerene pretreatment inhibited S. pyogenes A-20-induced death dose
dependently. Injection of 10 mg of carboxyfullerene/kg of body weight
resulted in 25% survival (2 of 8 mice), 20 mg/kg resulted in 63%
survival (5 of 8), 30 mg/kg resulted in 75% survival (6 of 8), and 40 mg/kg completely protected mice from S. pyogenes
A-20-induced death (Fig. 1A). In control mice, the structure of the
epidermis, dermis, and subcutaneous fat on the skin around the air
pouch was destroyed, but in carboxyfullerene-treated mice, the skin
remained intact (Fig. 2). Thus,
carboxyfullerene can inhibit S. pyogenes A-20-induced death
as well as skin injury.
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FIG. 1.
Inhibition of GAS infection by carboxyfullerene. (A)
Carboxyfullerene inhibits S. pyogenes A-20-induced death
in B6 mice dose dependently. Groups of four B6 mice were inoculated via
the air pouch with 109 S. pyogenes A-20
cells per mouse. Various doses of carboxyfullerene were administrated
via air pouch immediately after injection of S. pyogenes
A-20. The mice were monitored for death every day for a total of 4 days. A total of eight mice were pooled in two experiments.
Carboxyfullerene alone at 40 mg per kg has no effect on mice.
(B) Therapeutic effect of carboxyfullerene against S.
pyogenes A-20 infection in B6 mice. Groups of four B6 mice were
inoculated via the air pouch with 109 S. pyogenes A-20
cells per mouse. Carboxyfullerene (40 mg per kg per mouse) was
administrated via air pouch at 3 or 6 h postinfection. In one
group, carboxyfullerene was administrated via both air pouch and
intraperitoneal injection (40 mg per kg per route) at 3 h after
infection. The mice were monitored for death every day for a total of 4 days. A total of eight mice were pooled in two experiments. (C)
Therapeutic effect of carboxyfullerene against S.
pyogenes NZ-131 infection in B6 mice. Groups of six B6 mice
were inoculated via the air pouch with 2 × 109
S. pyogenes NZ-131 cells per mouse. In the preventive
experiment, carboxyfullerene (40 mg per kg per mouse) was administrated
immediately after inoculation of S. pyogenes NZ-131 via
air pouch or intraperitoneal injection. In the therapeutic experiments,
carboxyfullerene (40 mg per kg per mouse) was administrated at 3 h
postinfection via air pouch or combined air pouch and intraperitoneal
injection. The mice were monitored for death every day for 5 days
(n = 6).
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FIG. 2.
Inhibition of GAS-induced skin damage by
carboxyfullerene. Groups of four B6 mice were inoculated via the air
pouch with 109 S. pyogenes A-20 cells per
mouse. Carboxyfullerene (40 mg/kg) was administrated immediately after
injection of S. pyogenes A-20, and the mice were
sacrificed at 24 h postinfection. The skin tissue around the air
pouch was excised and fixed in 10% formaldehyde, as described in
Materials and Methods. Five-micrometer-thick sections were stained with
hematoxylin and eosin. (A) PBS-inoculated control. The arrow
indicates intact epidermis. (B) S. pyogenes A-20
treatment. (C) S. pyogenes A-20 and carboxyfullerene
treatment. The top arrow indicates intact epidermis; the bottom arrow
indicates infiltrating neutrophils. Magnification, ×100.
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The therapeutic effect of carboxyfullerene after GAS infection was
examined next. As shown in Fig.
1B, the mice died within
48 h of
injection of 10
9 S. pyogenes A-20 cells.
Administration of 40 mg of carboxyfullerene/kg
in the air pouch at 3 or
6 h postinjection only delayed the death
of the mice; the mice
still died by 72 h. When a combination of
air pouch and
intraperitoneal administration of 40 mg of carboxyfullerene/kg
at
3 h postinfection was used, there was a marginal effect, as
12.5%
of the mice survived. To repeat this experiment, we used
another
isolate,
S. pyogenes NZ-131, which is less virulent than
S. pyogenes A-20. The LD
100 of NZ-131
is 2 × 10
9 cells at 72 h via air pouch
inoculation. Pretreatment with carboxyfullerene
protected the mice from
S. pyogenes NZ-131-induced death in a
dose-dependent manner
(data not shown); 40 mg of carboxyfullerene/kg
completely protected
mice from
S. pyogenes NZ-131-induced death
(Fig.
1C).
Intraperitoneal injection of 40 mg of carboxyfullerene/kg
protected
only 50% of mice from death; thus, intraperitoneal injection
of
carboxyfullerene is less effective than local injection in
the air
pouch at inhibiting
S. pyogenes-induced death.
Administration
of 40 mg/kg in the air pouch at 3 h postinfection
protected 17%
of the mice from death. When combined air pouch and
peritoneal
injection at a total dose of 80 mg/kg was used, the
protective
effect increased to 33%. These results suggest that
carboxyfullerene
has a partial therapeutic effect on GAS infection. Its
inhibitory
effect is influenced by the dose of carboxyfullerene and the
virulence
of the
S. pyogenes isolate
used.
Carboxyfullerene not only prolongs the survival of infiltrating
neutrophils but also accelerates the clearance of bacteria.
The
working mechanism of carboxyfullerene-mediated inhibition was
investigated next. Histological examination showed that neutrophils had
infiltrated the subcutaneous area after carboxyfullerene treatment
(Fig. 2C). Therefore, the infiltrating neutrophils in the exudates were
collected from the air pouches, and their total numbers and viability
were determined. It was found that neutrophil infiltration of the air
pouches began at 3 h, reached a maximum at 6 h, and then
declined from 9 h to 24 h after inoculation of S. pyogenes A-20. In carboxyfullerene-treated mice, the kinetics of
neutrophil infiltration were slightly slower than in untreated controls
but the total numbers of infiltrating cells did not differ between the
two groups (Fig. 3A). However, the
viability of the infiltrating neutrophils dropped significantly in
untreated mice: at 6 h post-S. pyogenes A-20 injection,
most of the infiltrating cells (>90%) were dead. In contrast, the
viability of neutrophils in carboxyfullerene-treated mice remained at
over 85% (Fig. 3B). Further examination of remnant bacteria in the
exudates revealed that the bacteria were cleared quickly: no bacteria
(<100 CFU/ml) were found in the exudates by 6 h after
carboxyfullerene treatment. In contrast, bacteria continued to grow in
the air pouches of untreated mice (Fig.
4). To avoid the carryover effects of
remnant carboxyfullerene in the exudates, the exudates collected at
different times were diluted at least 100× with PBS, and the lower
limit of detection was 100 CFU/ml. The bactericidal activity of
neutrophils enhanced by carboxyfullerene was further demonstrated.
Peripheral neutrophils from naïve mice were isolated and
incubated for 4 h with different concentrations (5 to 200 µg/ml)
of carboxyfullerene and then washed with PBS to remove the
carboxyfullerene, after which the bactericidal activity of neutrophils
was determined. With no carboxyfullerene treatment, S. pyogenes A-20 was present at (3.2 ± 1.1) × 108 CFU/ml; with carboxyfullerene at 5, 50, and
200 µg/ml, numbers of bacteria were (3.8 ± 2.1) × 108, (1.1 ± 1.0) × 108, and (8.5 ± 2.1) × 106 CFU/ml, respectively. These results
suggest that carboxyfullerene not only prolongs neutrophil survival but
also enhances their bactericidal activity.
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FIG. 3.
Numbers (A) and viability (B) of infiltrating
neutrophils in air pouches after carboxyfullerene treatment of GAS
infection. Groups of four B6 mice were inoculated via the air pouch
with 109 S. pyogenes A-20 cells per mouse.
Carboxyfullerene was administrated via air pouch immediately after
injection of S. pyogenes A-20, and infiltrating cells
were collected at different times postinfection, as described in
Materials and Methods. Treatments:  , S. pyogenes A-20
only;  , S. pyogenes A-20 plus carboxyfullerene (40 mg/kg);  , carboxyfullerene (40 mg/kg) only.
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FIG. 4.
Bacteria in air pouches after carboxyfullerene treatment
of GAS infection. Groups of four B6 mice were inoculated via the air
pouch with 109 S. pyogenes A-20 cells per
mouse. Carboxyfullerene was administered via air pouch immediately
after injection of S. pyogenes A-20. Exudates containing
bacteria were collected, and bacteria were counted at different times
postinfection, as described in Materials and Methods. Treatments: ,
S. pyogenes A-20 only; , S. pyogenes
A-20 plus carboxyfullerene (40 mg/kg).
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Inhibition of in vitro GAS growth by carboxyfullerene.
The
direct effect of carboxyfullerene on in vitro growth of GAS was
examined. As shown in Fig. 5,
carboxyfullerene was able to inhibit the growth of S. pyogenes A-20 directly in an in vitro culture, and its inhibition
was dose dependent. Based on the above data, we conclude that
carboxyfullerene inhibits GAS infection both by enhancing the
bactericidal activity of neutrophils and by directly inhibiting growth
of S. pyogenes.
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FIG. 5.
Inhibition of in vitro growth of S.
pyogenes A-20 by carboxyfullerene. S. pyogenes
A-20 (4 × 106cells/ml) was cultured with various
concentrations of carboxyfullerene in TSBY, and bacterial growth was
determined by counting colonies on blood agar plates, as described in
Materials and Methods. One of three experiments is represented. Amount
of carboxyfullerene: , none;  , 5 µg/ml; , 50 µg/ml;  ,
200 µg/ml.
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DISCUSSION |
GAS not only causes serious diseases in humans but also is
associated with a high mortality rate, especially in streptococcal toxic shock syndrome, which kills 30 to 60% of patients in 72 to
96 h (15). Penicillin, which has been the recommended
drug for GAS infection (13), is less effective against
severe infections because of its short postantibiotic effect and
reduction of activity against stationary-phase organisms. Nowadays,
therapy for invasive GAS infection consists mainly of clindamycin,
aggressive surgery, and intravenous administration of immunoglobulins
(15). However, inappropriate use of antibiotics can lead
to the emergence of resistant bacteria. In this study, we demonstrate
that carboxyfullerene inhibits GAS infection both through direct
inhibition of bacterial growth and by enhancing the bactericidal
activity of neutrophils. These findings suggest a new way to treat GAS infection.
Fullerenes have a unique cage structure that allows them to interact
effectively with biomolecules and free radicals. These properties of
fullerenes have generated great interest in their use in biomedical
research (6, 7). It is necessary to convert hydrophobic
C60 into one of its water-soluble derivatives
before using it as a free radical scavenger or an antioxidant in
medical or therapeutic applications. A water-soluble trimalonic acid
derivative of C60, carboxyfullerene
[C63(COOH)6], has
recently been synthesized and has been reported to have protective
effects in various systems. These effects include protection of
cultured cortical neurons from excitotoxic injury in vitro, delaying of
neuronal deterioration in a transgenic model of familial amylotrophic
lateral sclerosis (2), blocking of apoptosis signaling
induced by transforming growth factor-
in human hepatoma cells
(5), and prevention of iron-induced oxidative injury in
rat brain (9). In a previous study, we found that
carboxyfullerene was able to inhibit E. coli-induced meningitis and that it did so through enhancing the host immune response rather than by direct antimicrobial activity
(18). Furthermore, we found that carboxyfullerene was able
to decrease the damage of infiltrating neutrophils at the blood-brain
barrier during E. coli-induced meningitis (16).
The bactericidal activity of neutrophils against E. coli in
vitro was also shown to be enhanced by carboxyfullerene
(unpublished data). Although the bactericidal activity of
neutrophils against E. coli and S. pyogenes has
been shown to be enhanced by carboxyfullerene, the phagocytic activity of neutrophils has not (unpublished data). Infiltrating neutrophils from carboxyfullerene-treated air pouches have greater viability than
those from untreated controls, suggesting that carboxyfullerene might
modulate neutrophil activity by enhancing the survival of activated
neutrophils. Nevertheless, our data suggest that carboxyfullerene has
dual roles: modulation of neutrophils to enhance its bactericidal activity and direct inhibition of S. pyogenes growth. In
addition to its effects on neutrophils, other in vivo effects of
carboxyfullerene administration have not been excluded; we are
currently investigating this. Since we do not have appropriate methods
of quantitating carboxyfullerene, its pharmacokinetics in vivo are not understood.
The therapeutic effect of carboxyfullerene is greater against E. coli-induced meningitis than against S. pyogenes-induced septic shock. The LD100 of
E. coli in the meningitis model was 5 × 105 cells, and E. coli in the brain
was limited until 12 h after infection, so administration of
carboxyfullerene as late as 9 h after infection still had a
protective effect (18). In contrast, in S. pyogenes-induced septic shock, the number of bacteria (1 × 109 to 2 × 109 cells)
inoculated into the air pouch is much higher than the number of
E. coli cells used in the meningitis model. S. pyogenes replicates quickly and invades the bloodstream at 6 h postinjection, and it secretes cysteine protease. Once S. pyogenes spreads systemically, it is difficult to inhibit even by
combined treatment with both air pouch and intraperitoneal injection.
When the inoculation dose of S. pyogenes is lowered from
109 to 108 cells, the
survival rate ranges from 20% in untreated mice to 80% in
carboxyfullerene-treated mice (data not shown). The highest dosage used
in this study (a total of 80 mg/kg by air pouch and intraperitoneal
injection) was far below the toxic dose. The LD50 of fullerenol in mice by intraperitoneal injection is 1,000 mg/kg (19). For therapeutic purposes, the dosage can be
increased further in future studies.
In a previous study, carboxyfullerene was unable to inhibit E. coli growth directly (18), but in this study it was
able to inhibit S. pyogenes A-20 growth. Carboxyfullerene
inhibits the growth of gram-positive, but not gram-negative, bacteria, as confirmed with several different strains of bacteria (unpublished observations). This difference is interesting and worth further investigation. We have also reported that carboxyfullerene is able to
inactivate enveloped viruses such as dengue virus and Japanese
encephalitis virus by a light-independent mechanism (10). It blocks virus replication at the attachment and penetration stages,
suggesting a direct interaction between carboxyfullerene and virions.
The inhibition of gram-positive bacteria such as S. pyogenes
is an example of direct contact between carboxyfullerene and the
gram-positive cell wall. Carboxyfullerene has not only free radical
scavenging activity that might modulate neutrophil activity but also an
antimicrobial effect on gram-positive bacteria such as S. pyogenes. This dual activity of carboxyfullerene permits this nonantibiotic compound to affect both phagocytes and the bacterium
itself during infection.
| |
ACKNOWLEDGMENTS |
This work was supported by grant NHRI-GT-EX89B717L from the
National Health Research Institute, Department of Health, Republic of China.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China. Phone: 886-6-2353535, ext. 5643. Fax: 886-6-2097825. E-mail:
hylei{at}mail.ncku.edu.tw.
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0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1788-1793.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
