Roles of gyrA mutations in resistance of clinical isolates and in vitro mutants of Bacteroides fragilis to the new fluoroquinolone trovafloxacin.
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We determined whether gyrA mutations were present in fluoroquinolone-resistant laboratory mutants derived from the Bacteroides fragilis reference strain ATCC 25285 and in clinical isolates of B. fragilis. The two first-step mutants selected on ciprofloxacin (CIP) were devoid of gyrA mutations, whereas two of the three CIP-selected second-step mutants studied presented the same gyrA mutation leading to a Ser82Phe change. Unusual GyrA alterations, Asp81Asn or Ala118Val, were detected in two of the three first-step mutants selected on trovafloxacin (TRO), Mt3 and Mt1, respectively. The Ala118Val change had no effect on the susceptibility of Mt1 to CIP. No second-step mutant could be obtained with TRO as a selector. For the 12 clinical isolates studied, a Ser82Phe change in GyrA was found only in the 3 strains which showed the highest levels of TRO resistance (MIC, 4 microgram/ml). Thus, the resistance phenotypes and genotypes observed in fluoroquinolone-resistant clinical isolates of B. fragilis were similar to those found in CIP-selected laboratory mutants, whereas peculiar mutational events could be selected in vitro with TRO. (+info)
Susceptibility of clinical isolates of Bacteroides fragilis group strains to cefoxitin, cefoperazone and ticarcillin/clavulanate.
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A total of 40 strains of the B. fragilis group was isolated from clinical specimens in two hospital centers in Fortaleza from 1993 to 1997. The most frequently isolated species was Bacteroides fragilis (19 strains) and most isolates came from intra-abdominal and wound infections. The susceptibility profile was traced for cefoxitin, cefoperazone and ticarcillin-clavulanate by using the agar dilution reference method. All isolates were susceptible to ticarcillin-clavulanate (128/2 microg/ml). Resistance rates of 15 and 70% were detected to cefoxitin (64 microg/ml) and cefoperazone (64 microg/ml), respectively. Such regional results permit a better orientation in choosing this group of antibiotics for prophylaxis and therapy especially in relation to cefoxitin, which is frequently used in the hospital centers studied. (+info)
Bovine abortions associated with Bacteroides fragilis fetal infection.
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Two Santa Gertrudis cattle from a herd of 105 aborted within a 24-hour period. Bacteroides fragilis was isolated from tissues of each aborted fetus. Histopathologic lesions included placentitis and bronchopneumonia in which gram-negative, rod-shaped organisms were visible. The diagnostic workup failed to reveal other causes of abortion. Anaerobes are rarely implicated in bovine abortions, and no other report was found that described abortion in cattle due to B. fragilis. (+info)
Accumulation of norfloxacin by Bacteroides fragilis.
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The accumulation of norfloxacin by Bacteroides fragilis NCTC 9343 was determined by the modified fluorescence method. The time required to achieve a steady-state concentration (SSC) after allowing B. fragilis to accumulate norfloxacin in an aerobic or an anaerobic environment was approximately 2 min; the SSC achieved in air was 90.28 +/- 9.32 ng of norfloxacin/mg (dry weight) of cells, and that achieved anaerobically was 98.45 +/- 3.7 ng of norfloxacin/mg (dry weight) of cells. Initial rates of accumulation were determined with a range of external concentrations, as up to 8 microg/ml the concentration of norfloxacin accumulated increased proportionally to the external concentration, 12.13 ng/mg (dry weight) of cells per microg of exogenous norfloxacin per ml. At concentrations above 10 microg/ml no increase in the rate of norfloxacin accumulation was observed. From the kinetic data, a Lineweaver-Burk plot calculated a K(m) of 5.03 microg/ml and a V(max) of 25.1 ng of norfloxacin/s. With an increase in temperature of between 0 and 30 degrees C, the concentration of norfloxacin accumulated also increased proportionally at 4.722 ng of norfloxacin/mg (dry weight) of cells/ degrees C. At low concentrations of glucose (<0.2%; 11 mM), the concentration of norfloxacin accumulated was decreased. With the addition of 100 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) the mean SSC of norfloxacin was increased to 116 +/- 7.01 ng of norfloxacin/mg (dry weight) of cells; glucose had no significant effect in the presence of CCCP. Magnesium chloride (20 mM) decreased the SSC of norfloxacin to 40.5 +/- 3.76 ng of norfloxacin per mg (dry weight) of cells. These data suggest that the mechanism of accumulation of norfloxacin by B. fragilis is similar to that of aerobic bacteria and that the fluoresence procedure is suitable for use with an anaerobic bacterium. (+info)
The redox-sensitive transcriptional activator OxyR regulates the peroxide response regulon in the obligate anaerobe Bacteroides fragilis.
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The peroxide response-inducible genes ahpCF, dps, and katB in the obligate anaerobe Bacteroides fragilis are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria. oxyR and dps were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions. B. fragilis OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe, Porphyromonas gingivalis. Northern blot analysis revealed that oxyR was expressed as a monocistronic 1-kb mRNA and that dps mRNA was approximately 500 bases in length. dps mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the oxyR gene [oxyR(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion of oxyR abolished the induction of ahpC and katB following treatment with hydrogen peroxide or oxygen exposure. However, dps expression was induced approximately fourfold by oxygen exposure in DeltaoxyR strains but not by hydrogen peroxide. This indicates that dps expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of DeltaoxyR mutant strains with wild-type oxyR and oxyR(Con) restored the inducible peroxide response and the constitutive response of the ahpCF, katB, and dps genes, respectively. However, overexpression of OxyR abolished the catalase activity but not katB expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of oxyR expression in the parents and in DeltaoxyR and overexpressing oxyR strains by Northern blotting and oxyR'::xylB fusions revealed that B. fragilis OxyR does not control its own expression. (+info)
In vitro and in vivo antibacterial activities of a new injectable carbapenem, S-4661, against gynaecological pathogens.
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Carbapenems are often used to treat infections in obstetrics and gynaecology because of their activity against anaerobes. S-4661, a new injectable carbapenem antibiotic, has favourable pharmacokinetic properties and is not hydrolysed by dehydropeptidase I. We investigated the in vitro and in vivo antibacterial activities of S-4661 against strains of Streptococcus agalactiae, Escherichia coli, Peptostreptococcus magnus, Bacteroides fragilis and Prevotella bivia, which are major pathogens in the fields of obstetrics and gynaecology. The MIC(50) and MIC(90) of S-4661 for these strains were 0.25 and 1 mg/L, respectively. The in vivo efficacy of S-4661 was evaluated in a rat model of intrauterine infection, namely pyometra caused by E. coli and B. fragilis. The accumulation of neutrophils in the uterus in the S-4661-treated group was less marked and the number of bacteria significantly lower than those in the untreated group. These results suggest that S-4661 may be useful for treating polymicrobial infections in the fields of obstetrics and gynaecology. (+info)
Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.
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Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli. (+info)
Bacteroides fragilis NCTC9343 produces at least three distinct capsular polysaccharides: cloning, characterization, and reassignment of polysaccharide B and C biosynthesis loci.
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Bacteroides fragilis produces a capsular polysaccharide complex (CPC) that is directly involved in its ability to induce abscesses. Two distinct capsular polysaccharides, polysaccharide A (PS A) and PS B, have been shown to be synthesized by the prototype strain for the study of abscesses, NCTC9343. Both of these polysaccharides in purified form induce abscesses in animal models. In this study, we demonstrate that the CPC of NCTC9343 is composed of at least three distinct capsular polysaccharides: PS A, PS B, and PS C. A previously described locus contains genes whose products are involved in the biosynthesis of PS C rather than PS B as was originally suggested. The actual PS B biosynthesis locus was cloned, sequenced, and found to contain 22 genes in an operon-type structure. A mutant with a large chromosomal deletion of the PS B biosynthesis locus was created so that the contribution of PS B to the formation of abscesses could be assessed in a rodent model. Although purified PS B can induce abscesses, removal of this polysaccharide does not attenuate the organism's ability to induce abscesses. (+info)