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Infect Immun, June 1998, p. 2570-2575, Vol. 66, No. 6
INSERM U.13, Hôpital Bichat-Claude
Bernard, Paris, France
Received 3 December 1997/Returned for modification 5 January
1998/Accepted 27 March 1998
The role of Enterococcus faecalis in polymicrobial
peritonitis is still debated. Virulence factors expressed in some
enterococcal strains might be involved in the pathogenicity of these
organisms. To clarify their role, three of these virulence factors
(cytolysin, gelatinase, and aggregation substance) were studied in six
isogenic strains of E. faecalis expressing various
combinations of these factors. Since the pathogenic effects of
enterococci are only moderate, the expression of their virulence might
vary from one animal species to another and from one type of infection
to another. Therefore, we evaluated these effects in two animal models,
i.e., a systemic infection in mice in which we assessed the virulence of the strains in 50% lethal dose studies and a model of
compartmentalized infection in rats in which the microbiologic and
inflammatory effects of the strains were evaluated in monomicrobial or
polymicrobial infection. In mice, significant differences were observed
in the cumulative survival curves depending on the virulence factors (P < 0.0001 [log rank test]). In rats,
monomicrobial infection induced only mild changes. In polymicrobial
peritonitis, the virulence factors mainly increased the inflammatory
response while the changes observed in the microbiologic response were
minimal. The combination of two virulence factors did not significantly
increase the severity of infection either in the mice model or the
polymicrobial rat model. These data argue for species and model
dependence of the role of the virulence factors studied here and
suggest that other important factors may be involved in the
pathogenicity of enterococci.
Many issues remain unsolved
regarding the pathogenic role of enterococci in the course of
intraabdominal infection. From the previous reports, it appears that
the enterococcal infections are rarely monomicrobial in nature,
especially in surgical patients, suggesting the role of bacterial
synergy (30, 31). Among the factors that might be involved,
the virulence factors could be of interest but have been minimally
studied (23, 25). Plasmids and conjugative transposons,
which play a key role in the acquisition of drug resistance, may also
carry these virulence traits (6), but their clinical
relevance remains unclear.
Three virulence factors of Enterococcus faecalis might have
the potential to increase the severity of intraabdominal sepsis. The
plasmid-encoded cytolysin-bacteriocin (Cly) is the best studied of
these factors. In vitro, this factor induces lysis of
erythrocytes, polymorphonuclear neutrophils, and macrophages and could
lead to a reduction in phagocytosis (29). Aggregation
substance (Agg), which is closely linked to mating response to
enterococcal sex pheromones (7), mediates adhesion of the
bacteria to host cells, such as intestinal epithelial cells
(34), renal tubular cells (27), and heart
endothelial cells (17), and could be involved in the
persistence of the organisms within the host tissues and fluids.
Gelatinase (Gel) is a non-plasmid-encoded potential virulence factor.
Although Gel strains of E. faecalis have been mainly studied in dental diseases (16), the properties given by this
extracellular metalloendopeptidase (hydrolyzis of collagen, gelatin,
and small peptides [18]) could increase bacterial
dissemination. The expression of these virulence factors might vary
from one animal species to another and from one type of infection to
another, especially because the pathogenic role of E. faecalis seems to be moderate (36, 40, 41) and
expressed only in polymicrobial infections (3, 10, 28, 30).
To address these issues, we evaluated the effects of six isogenic
strains of E. faecalis containing various combinations of virulence factors in two animal models, i.e., a model of systemic infection in mice in which we assessed the virulence of enterococcal strains in 50% lethal dose studies and a model of compartmentalized infection in rats in which the microbiologic and inflammatory effects
of the strains were evaluated in monomicrobial or polymicrobial infection in combination with Escherichia coli and
Bacteroides fragilis, two species frequently associated in
clinical samples.
Microorganisms.
Six derivatives of E. faecalis
OG1 were obtained from the University of Texas Medical School (Houston,
Tex.) and the University of Michigan (Ann Arbor, Mich.). The four OG1x
derivatives used in the present study are isogenic strains produced by
mutagenesis with nitrosoguanidine from strain OG10 (14, 39).
The Tn917 transposon was inserted into the PAD1 plasmid,
leading to the following different phenotypes of OG1x: pAM714 with Cly
and Agg (20, 21) and pAM9058 with Agg and pAM944 with Cly
(5, 15). The two remaining strains (OG1RF and OG1SSP) are
not isogenic with the previous four strains. OG1RF is a spontaneous
mutant of OG1 with a Gel phenotype (14, 32). The OG1SSP
strain is a derivative of OG1. The Tn915 transposon was
inserted into the PCF10 plasmid, leading to the Gel and Agg phenotypes
of the OG1SSP strain (12, 13, 35). All of these strains of
E. faecalis had an intrinsic low-level resistance to
aminoglycosides, and their characteristics are listed in Table
1. The resistance phenotype, which was
encoded on the same plasmid as the virulence factor, was verified in
each experiment. Gel and Cly expression was verified in vitro before
inoculation according to the method described by Coque et al.
(8). The strains of B. fragilis (AIP5-86) and E. coli (CB1496) used in the current study have previously
been used in this model (30).
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Disparate Findings on the Role of Virulence Factors
of Enterococcus faecalis in Mouse and Rat Models of
Peritonitis
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Microbiologic characteristics of the derivatives of
E. faecalis inoculated
Animals. Female OF1 mice (Iffa Credo, L'Arbresles, France), weighing 20 to 25 g and housed 10 per cage, were used for 50% lethal dose (LD50) evaluation of the six strains of E. faecalis, which were injected intraperitoneally. Male Sprague-Dawley rats (Charles River, St-Aubin-les-Elbeufs, France), weighing 250 to 300 g and housed 5 per cage, were used for the peritonitis model. All animals had access to chow and water ad libitum throughout the experiment. All of these experiments were performed according to current European regulations.
Preparation of the microorganisms. B. fragilis was grown and diluted anaerobically in prereduced thioglycolate broth. E. coli and the six strains of E. faecalis were grown in brain-heart infusion broth. The final inoculum was made when the bacteria were in the log phase of growth. The inocula were adjusted spectrophotometrically and bacteria were diluted to give the number of microorganisms required for bacterial challenge. Purity was assessed and counts were validated for each strain immediately before inoculation (mice model) or before mixing (rat model).
LD50 in mice. The virulence of each E. faecalis strain was evaluated by inoculation of increasing concentrations of microorganisms (107 to 1011 CFU per ml) in mice. Nine groups of 10 animals were studied for each bacterium. The animals received intraperitoneally a 0.5-ml injection of the bacterial suspension. After inoculation, the animals were returned to their cages and daily mortality was recorded until day 7. Lethal dose curves were plotted, and LD50s were calculated according to the method described by Ike et al. (21).
Intraabdominal infection in rats. Semisolid agar medium was prepared by adding 2% (wt/vol) agar to the diluted broth cultures associated with barium sulfate (10% [wt/vol]). Aliquots (0.5 ml) of the final product were placed in double gelatin capsules for peritoneal implantation. Each E. faecalis strain was studied twice: once as a monomicrobial infection (108 CFU/ml, 12 animals in each group) and once as a polymicrobial infection (20 animals in each group) associated with E. coli (108 CFU/ml) and B. fragilis (109 CFU/ml).
Implantation of inoculum. The rats were anesthetized with an intramuscular injection of ketamine (30 mg/kg of body weight [Parke-Davis, Courbevoie, France]), and the gelatin capsule was inserted into the pelvic peritoneal cavity through a midline abdominal incision (40). The wound was closed with a musculoperitoneal layer and a skin layer by using interrupted nylon sutures.
Assessment of spontaneous outcome.
After implantation of the
inoculum, the animals were returned to separate cages; they were
observed and weighed daily until sacrifice. No death was observed
within 6 h of capsule implantation. In the six groups of animals
receiving a monomicrobial inoculum, sacrifice was performed at 24 h after inoculation for four animals and at day 3 for four animals. In
the groups receiving a polymicrobial inoculum, eight animals were
sacrificed at 24 h and eight animals were sacrificed at day 3. In
addition, an early sacrifice (6 h after inoculation) was planned in
both models (monomicrobial and polymicrobial) for four animals in each
group. The following pathogenicity criteria were studied: clinical
criteria (body weight and mortality), microbiological criteria
(positivity of blood cultures and bacterial counts in the peritoneal
fluid), and the inflammatory response (peritoneal concentrations of
phagocytes, tumor necrosis factor alpha [TNF-
], and interleukin-6
[IL-6] and serum concentrations of 1-acid glycoprotein).
Sacrifice.
Animals were killed with chloroform. Blood
samples were obtained by aseptic percutaneous transthoracic cardiac
puncture for qualitative blood culture on days 1 and 3 and for
measurements of 1-acid glycoprotein concentrations in serum on day
3. After injection of 10 ml of cold phosphate-buffered saline (PBS)
intraperitoneally, a midline laparotomy was performed and peritoneal
fluid samples were recovered from all regions of the peritoneal cavity
for bacterial and cell counts (days 1 and 3). Peritoneal fluid cytokine
concentrations were specifically evaluated at 6 h after bacterial
challenge. A dilution factor taking into account the fluid present in
the peritoneal cavity prior to the injection of the 10 ml of PBS was applied in all calculations according to a technique previously described (26, 31). Blood cultures were inoculated
immediately after collection (NR-7A; Becton-Dickinson,
Le-Pont-de-Claix, France) and analyzed daily until day 5 for
identification. Serial dilutions of the peritoneal fluid were made, and
0.1 ml of each dilution was spread on agar plates for colony counts.
The limit of detection for each microbiological test was <1
log10 CFU/ml. In cases of values below this threshold, the
results were listed as 
1 log10 CFU/ml. In the statistical
analysis, these results were treated as 1 log10 CFU/ml.
Plates were incubated under appropriate conditions (aerobic and
anaerobic) for 2 to 5 days. The selective medium used for detection of
B. fragilis was Columbia agar base (Bio-mérieux, Charbonnières-les-Bains, France) with 5% sheep blood containing 75 µg of kanamycin, 7.5 µg of vancomycin, and 4 µg of pefloxacin per ml. The selective media used for aerobic culture were Drigalski agar (Diagnostics Pasteur, Marnes-la Coquette, France) and
bile-esculine-azide agar (Diagnostics Pasteur). Mueller-Hinton agar
(Diagnostics Pasteur) was used for E. faecalis counts in the
monomicrobial model.
Peritoneal cell counts. Total cell counts (polymorphonuclear neutrophils and macrophages) were made on an aliquot of the original peritoneal fluid by using a Malassez counting chamber.
Cytokine assay.
Four 1- ml samples of the original
peritoneal fluid recovered from all of the regions of the peritoneal
cavity were centrifuged (300 × g for 15 min) and then
divided into 200-µl aliquots and stored at 
80°C until assaying.
The samples were assayed in duplicate. TNF- activity was measured
with a Factor-Test-X rat TNF- enzyme-linked immunosorbent assay kit
(Genzyme Diagnostics, Cambridge, Mass.) according to a previously
described technique (37). The sensitivity of the test, which
was defined as the lowest concentration of standard which shows greater
absorbance than the mean absorbance of the 0-pg/ml sample ± 2 standard deviations (SD), was 10 pg/ml. IL-6 activity was measured by
means of a bioassay with the murine hybridoma cell line B9 according to
a previously described technique (1). The limit of detection
in peritoneal fluid was 0.2 ng/ml. The specificity of the response for
IL-6 was assessed by using polyclonal rabbit antimurine IL-6 antibodies
(Genzyme, Brussels, Belgium).
1-acid glycoprotein assay.
Blood samples were obtained by
cardiac puncture and transferred to sterile glass tubes. After
coagulation, the sera were collected and centrifuged at 500 × g for 5 min. The supernatant was divided into four aliquots
and stored at 20°C until the assay. Radial immunodiffusion was used
for the assay which involved agar containing 3% (wt/vol) immune serum
and 3% (wt/vol) polyethylene glycol 6000 (Fluka, Malakov, France). The
values of 1-acid glycoprotein were determined with a monoclonal
rabbit anti-rat 1-acid glycoprotein antibody (4). A value
of less than 200 mg/liter was considered normal. The limit of detection
was 25 mg/liter.
Statistical analysis.
Results are expressed as means ± SD. The LD50s in mice were compared by a Kaplan-Meier
analysis by using a log rank test. Continuous parameters were compared
by an analysis of variance analysis, followed (in the case of
significance) by limited comparisons between the control group without
virulence factor (E. faecalis OG1X) and the other groups by
using Fisher's least significant procedures. A chi square test was
used for quantitative data. A P value of 0.05 was
considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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LD50s in mice. E. faecalis was classified into three groups according to the LD50 (Table 2). The first group, which was characterized by the lowest LD50, included E. faecalis OG1X(pAM 714) and OG1X(pAM 944). An intermediate LD50 was evidenced for E. faecalis OG1RF. Finally, the highest LD50s were observed with E. faecalis OG1X, OG1X(pAM 9058), and OG1SSP(pCF10). With a Kaplan-Meier model, a statistically significant difference was observed within the groups of animals in the delay before death (Fig. 1). Mice inoculated with E. faecalis OG1X(pAM 714), OG1X(pAM 944), and OG1RF strains had a significantly higher and earlier mortality than those receiving E. faecalis OG1X, OG1X(pAM 9058), and OG1SSP(pCF10), in which a low and delayed mortality was observed.
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Peritonitis model in rats. (i) Effect of inoculum on survival. No mortality was observed in the rat peritonitis model.
(ii) Effect of E. faecalis strains on body weight. In the polymicrobial model, maximal loss of weight occurred in most groups on day 1, except for the animals receiving the OG1X(pAM 9058) and OG1RF strains, in which maximal weight loss was reported on day 2 (Fig. 2). Progressive recovery was observed afterward. The greatest weight loss was observed in animals receiving the OG1X strain, while the least weight loss was seen with the OG1SSP(pCF10) strain. In the monomicrobial model, there was no difference in the variations of weight within the groups (data not shown). When comparing polymicrobial and monomicrobial models, a significant difference was observed in weight variations in the 3 days of the study (P < 0.01 in every case).
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, P < 0.01 compared with changes in the animals receiving the OG1SSP(pCF 10)
strain.
(iii) Effect of E. faecalis strains on blood cultures. All animals had positive blood cultures at day 1. A significant decrease in the frequency of positive blood cultures was noted between days 1 and 3 (Table 3). However, the different strains of E. faecalis did not modify the frequency of bacteremia of the other organisms. The frequency of bacteremia due to E. faecalis was more marked in the polymicrobial model than in the monomicrobial one; 75 of 90 rats (80%) had E. faecalis-positive blood cultures in the polymicrobial model versus 26 of 48 rats (55%) in the monomicrobial model (P < 0.01). In the monomicrobial model, the different strains of E. faecalis had similar frequencies of positive culture (data not shown).
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(iv) Effect of E. faecalis strains on peritoneal cultures. The bacterial titers within the peritoneal cavity between days 1 and 3 are displayed in Table 4. In the monomicrobial model, no difference was observed on day 1 or 3 or between days 1 and 3 in the different groups of animals. The peritoneal concentrations of E. faecalis were significantly higher on day 1 in the polymicrobial model than in the monomicrobial model in animals receiving E. faecalis OG1X, OG1X(pAM 9058), OG1X(pAM 714), and OG1SSP(pCF10) (data not shown). On day 3, only the animals inoculated with E. faecalis OG1X(pAM 9058) had significantly higher E. faecalis peritoneal concentrations in the polymicrobial model than in the monomicrobial model.
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(v) Effect of E. faecalis strains on peritoneal cell counts. In the polymicrobial model, an increased peritoneal cell count was observed between days 1 and 3 for all groups (Fig. 3), while an increase was noted only for the OG1SSP(pCF10) strain in the monomicrobial model. At day 1, the peritoneal cell counts were similar in each group in the polymicrobial and monomicrobial models, except for the group of animals receiving the OG1SSP(pCF10) strain, in which an increased cell count was noted in the polymicrobial model compared to the monomicrobial inoculum (P < 0.05). On day 3 in the polymicrobial model, the peritoneal cell count was significantly increased in animals receiving the OG1X(pAM 944) strain compared to the other groups (P < 0.01). Moreover, increased peritoneal cell counts were noted at day 3 in the polymicrobial model compared to the monomicrobial model in the groups receiving the OG1X(pAM 9058), OG1X(pAM 944), and OG1SSP(pCF10) strains (P < 0.05, P < 0.01, and P < 0.05, respectively).
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,
P < 0.001 between days 1 and 3.
(vi) Effect of E. faecalis strains on cytokine
levels.
The animals inoculated with E. faecalis
OG1X(pAM 714) and OG1RF had higher concentrations of TNF- in the
peritoneal fluid than those receiving E. faecalis OG1X
(P < 0.05, P < 0.05) and OG1X(pAM
944) (P < 0.05, P < 0.05) (Fig.
4). The animals receiving E. faecalis OG1RF had higher peritoneal concentrations of IL-6 than
those inoculated with E. faecalis OG1X (P < 0.01), OG1X(pAM 944) (P < 0.001), OG1X(pAM 9058)
(P < 0.05), and OG1SSP(pCF10) (P < 0.05). Moreover, animals given E. faecalis OG1X(pAM 714) had
higher concentrations of IL-6 in the peritoneal fluid than those
receiving E. faecalis OG1X(pAM 944) (P < 0.05).
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(vii) Effect of E. faecalis strains on 1-acid
glycoprotein concentrations.
The concentrations of 1-acid
glycoprotein in plasma were significantly higher in animals receiving a
polymicrobial inoculum than in those receiving E. faecalis
alone (P < 0.01), except for animals inoculated with
E. faecalis OG1X(pAM 9058) and OG1X(pAM 714) (Fig.
5). In the polymicrobial model, only the
animals given E. faecalis OG1SSP(pCF10) had higher 1-acid
glycoprotein concentrations than the other groups (P < 0.05). There was no difference within groups in the concentrations of
1-acid glycoprotein in plasma in the monomicrobial model.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The literature on the pathogenic role of E. faecalis strains is somewhat unclear. Their potential pathogenic effects are quite subtle and are not evidenced in nondiscriminative models of infection. In addition, the results of an experimental model of enterococcal infection seem to differ from one model to another, as illustrated by our results.
The direct injection of microorganisms within the peritoneum represents a model of systemic sepsis induced within a very short period of time (9, 33). This model allows a good approach to identify quickly the most pathogenic strains. However, this experimental design may not resemble the development of infection in patients, which commonly occurs over days. Therefore, the information provided by the peritoneal implantation of a septic capsule is of interest. This model, which represents a compartmentalized sepsis with prolonged infectious and inflammatory responses, allows study over a prolonged period of time of the relationships between the host and the offending organisms and among the various organisms inoculated. Taking these features into consideration, we chose these two models in order to study different aspects of the role of virulence factors of enterococci.
Several experimental investigations, mainly performed with rats, have demonstrated the synergistic role of enterococci (2, 11, 28, 30). In these studies, the enterococcal strains did not express any known virulence factor. Nevertheless, increased bacteremia (10, 30) or increased mortality (2, 10) was reported when enterococcus was part of the inoculum. Our current results confirm these observations of bacterial synergy. Moreover, we demonstrated that the pathogenicity of E. faecalis in rats is minimal when inoculated alone, an issue frequently suggested in clinical studies but rarely assessed. All of the parameters that we tested were in agreement in demonstrating that polymicrobial infection and monomicrobial infection significantly differed. On the other hand, there was divergence between the parameters according to the strain tested in the rat polymicrobial model.
Our observations with the mouse model poorly predicted the effects of enterococci in rats. To the best of our knowledge, the virulence of a microorganism is rarely assessed in two different models in the same study, raising difficulties in the interpretation of the results of previous studies. The rat model has been largely used to evaluate the mechanisms of bacterial synergy (2, 11, 28, 30), and the information obtained seems to be clinically relevant (40, 41). Since we have previously demonstrated that enterococcal pathogenicity is expressed in a dose-dependent fashion (30, 31), the results reported with the rat model could be related to an insufficient concentration of enterococci in the inoculum. In addition, virulence factors might not be expressed in rats or might not be important in the model. Only few studies assessing the virulence of enterococci are available, and these are always performed with monomicrobial infections in mouse and rabbit models (5, 21, 24). It is not possible from the data available to determine whether virulence factors are expressed in intraabdominal infections or at what level and whether this expression is similar in poly- and monomicrobial infections.
In the mouse model, the highest mortality was observed for animals given the strain with the cytolysin factor. This is in agreement with the results obtained by Ike et al. (21), who reported that inoculation of a Cly-producing strain of E. faecalis caused an increased mortality and a 90% decrease in LD50 compared to a control strain. Moreover, in previous studies performed with models of endophthalmitis (24, 38) or endocarditis, disease severity was markedly increased after inoculation of an enterococcal strain producing Cly (5). In clinical practice, the Cly phenotype, although difficult to detect, seems to be common, as reported by Ike et al. (22), who tested 97 clinical isolates of E. faecalis and showed that 60% were hemolytic. The data reported by Huycke et al. also suggested the pathogenicity of Cly in cases of enterococcal bacteremia in which a fivefold increased risk of death was observed in patients infected with an E. faecalis strain demonstrating a Cly phenotype (19).
In our models of the mouse and rat, Agg did not seem to exert a significant virulence. In the mouse model, the mortality level induced by strains mating Agg and no Cly was low and delayed. The effects of adherence and persistent infection, which were linked to the properties of Agg (17, 27, 34), were not observed in our rat model, suggesting that Agg has only a minor role to play in the severity of peritoneal infection. Agg is the most difficult factor to assess in vivo, since there is no clinical marker of its activity. Only a molecular approach could demonstrate its activation, as previously performed by other authors (8).
Gelatinase is probably the least-studied virulence factor. In the mouse model, this factor induced an increased mortality compared to the control strain OG1X, as well as a 68% decrease of the LD50. In the rat model, the Gel strain seemed to be the least pathogenic, with only moderate weight loss and a mild peritoneal cellular reaction. The pathogenicity of Gel strains of E. faecalis in intraabdominal infections is purely hypothetical and has never been demonstrated. However, since a relationship between Gel and gentamicin resistance has been reported in one study (8), therapeutic difficulties could be expected in the presence of this factor.
The effect obtained with the OG1X strain (control strain) in the polymicrobial model is unclear. Significant and prolonged weight loss combined with an increased frequency of positive blood cultures was noted in animals receiving this strain. Reversion of this mutant to a wild expression of gelatinase might be involved, although this strain acts differently from the OG1RF strain. A lower level of interaction of the other OG1 derivatives with E. coli and B. fragilis might also be involved. In this setting, the gelatinase may damage the virulence factors of these organisms. Even though the cytolysin demonstrates bacteriocin activities that might be deleterious to other bacteria, such properties have not been reported with gelatinase. On the other hand, OG1X was generated with nitrosoguanidine, a technique which could induce a mutation in other genes.
The combination of two virulence factors (Cly and Agg or Agg and Gel)
gave results different from those reported with a single factor. A
combination of Cly and Agg generated the lowest LD50 in the
mouse model. These results suggest a possible interaction between
Cly and Agg, the mechanism of which remains to be elucidated. Similar
synergy has been previously reported with the combination of Cly and
Agg, which caused an increased level of mortality in a rabbit model of
endocarditis (5). In contrast, the combination of Agg and
Gel did not seem to result in increased pathogenicity. In the rat
model, the effects of the combinations were minimal, but the
inflammatory response was increased, as assessed by the concentrations
of 1-acid glycoprotein in plasma.
In conclusion, our study confirms the previously reported mechanisms of bacterial synergy between enterococci and other organisms. However, the marked discrepancy in results obtained from mice and rats renders any extrapolation to clinical practice difficult. The use of one virulence factor only minimally influenced the course of the disease. In contrast, the combination of two factors (Cly and Agg) seemed to be responsible for the more severe peritoneal infection which resulted in mice. In view of our results, other components shared by these strains may have a greater influence on the pathogenicity of enterococci. A good candidate could be the bacterial wall and, more specifically, some of its constituents, such as peptidoglycan or lipoteichoic acid, as has previously been suggested with experimental peritonitis (31).
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ACKNOWLEDGMENTS |
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We thank B. Murray, Texas University, Houston, and D. B. Clewell, Michigan University, Ann Arbor, for providing the strains of
E. faecalis; and C. Poüs, Faculté de Pharmacie
de Chatenay-Malabry, for performing the 1-acid glycoprotein assay.
This work was supported by a grant from the French Foundation for Medical Research.
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FOOTNOTES |
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* Corresponding author. Present address: Service d'Anesthésie C, Groupe Hospitalier Sud, Ave. René Laënnec, Salouel, 80054 Amiens Cedex 1, France. Phone: (33) 3-22-45-59-55. Fax: (33) 3-22-45-53-40. E-mail: pmontrav{at}planete.net.
Editor: E. I. Tuomanen
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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