Transfer of imipenem resistance in Bacteroides fragilis was studied. Clinical isolate B. fragilis 10-73 was highly resistant to imipenem. Imipenemresistance was transferred from 10-73 to B. fragilis strain TM4000at a frequency of 10~6/input recipient by a filter mating technique. The resistance could also be retransferred. B. fragilis 10-73 and both primary and secondary transcipients produced an imipenem-hydrolyzing metallo-/Mactamase. Acquisition of imipenemresistance correlated with the appearance of plasmid DNAwith a size (ca. 13.6kb) similar to that of the donor strain. TM4000 transformed by electroporation with purified DNAof the 13.6-kb plasmid pBFUKl produced the metallo-/Mactamase and was resistant to imipenem. Transfer was resistant to DNasetreatment and no transfer was seen with a sterile filtrate of the donor culture. It is suggested that gene transfer in B. fragilis has the properties of a conjugation system rather than those of transformation or transduction.
The Il-lactamase from Bacteroides fragilis GAI-30144 hydrolyzed imipenem, oxyiminocephalosporins, cephamycins, and penicillins. Enzyme activity was inhibited by EDTA. Zinc completely reversed inactivation of the enzyme by EDTA. The molecular mass of purified enzyme was estimated to be 33,000 daltons.Carbapenems such as imipenem were initially believed to be stable to the hydrolytic action of P-lactamases. However, some bacterial strains have been reported to be resistant to imipenem by producing 13-lactamase (2, 7, 8, 10).Bacteroides fragilis is frequently isolated from clinical specimens. While the clinical isolates generally contain P-lactamase (3), resistance to imipenem is rare in this organism. Recently, we discovered that two B. fragilis strains isolated from clinical specimens (GAI-30079, GAI-30144) were highly resistant to imipenem and investigated the biochemical properties of the ,-lactamases extracted from these organisms.The MICs were determined by an agar dilution method by using modified GAM (5) showed a high level of activity to both cephalosporins and penicillins. Furthermore, the substrates usually considered to be stable to B. fragilis P-lactamases, i.e., ceftizoxime, moxalactam, and imipenem, were significantly hydrolyzed by this enzyme. The ratios of relative Vmax to Km suggested that cephaloridine is the best substrate, followed by penicillin G, ceftizoxime, imipenem, and moxalactam, in that order. In addition, the enzyme hydrolyzed cephamycins, while aztreonam was quite stable (data not shown). These observations suggested that characteristic resistance to ceftizoxime, moxalactam, cephamycins, and imipenem is involved in P-lactamase activity. The substrate profile of the GAI-30079 enzyme was quite similar to that of the GAI-30144 enzyme. The imipenem-hydrolyzing 1-lactamase from GAI-30144 was subjected to further investigation.The ,-lactamase from GAI-30144 was not inhibited by 1 mM clavulanic acid or sulbactam. It was completely inhibited by 1 mM p-chloromercuribenzoic acid and EDTA. When the enzyme was completely inactivated by EDTA, 0.5 mM ZnCl2 was added. The inhibitory effects of EDTA were completely reversed by the addition of Zn2+ (Fig.
A study was undertaken in Japan to evaluate the susceptibility patterns of Bacteroides fragilis group species (849 strains) isolated from December 1986 through May 1991. All of the strains, which included B. fragilis (610 strains), Bacteroides thetaiotaomicron (201 strains), and Bacteroides distasonis (38 strains), were studied for susceptibility to imipenem and 16 other antimicrobial agents by an agar dilution method. Metronidazole was the most active agent; the minimal concentration for 90% inhibition (MIC90) was 0.78 micrograms/mL, and no isolate was noted to be resistant to it during the entire study period. Amongst the beta-lactam agents tested, imipenem was the most effective antimicrobial drug; the rate of resistance to this agent was only 3.3%. Cefoperazone/sulbactam (MIC90, 6.25 micrograms/mL) and cephamycins (MIC90, 25-50 micrograms/mL) were found to be more active against B. fragilis strains, whereas minocycline (MIC90, 6.25 micrograms/mL) showed better activity against B. thetaiotaomicron and B. distasonis strains. Increasing resistance to imipenem was observed in B. fragilis (2.0%-5.9%) and B. thetaiotaomicron (2.5%-6.1%) during the 4-year study period. Detailed investigation of beta-lactamases from imipenem-resistant strains demonstrated that, amongst them, six of the B. fragilis strains for which the MICs of imipenem were > or = 25 micrograms/mL were producing imipenem-hydrolyzing metallo-beta-lactamase.
A nationwide survey of the susceptibility of 433 isolates of Bacteroides fragilis and 149 isolates of Bacteroides thetaiotaomicron was conducted from December 1986 through November 1989 in Japan. These strains were collected from 16 university hospitals and one metropolitan hospital. Metronidazole was the most active drug against both species, with no resistant isolates found. The activity of imipenem and sulbactam-cefoperazone was good, with very low resistance rates determined in Bacteroides fragilis (1.4% and 1.6%, respectively) and in Bacteroides thetaiotaomicron (3.4% for both drugs), and was comparable to that of metronidazole. Cefoxitin, cefmetazole, cefotetan, cefbuperazone, latamoxef and ceftizoxime were found to be more active against Bacteroides fragilis, for which resistance rates were 3.2 to 9.5%, than against Bacteroides thetaiotaomicron, for which resistance rates were 18.1 to 21.8%. Rates of piperacillin resistance in the two species were 12.9% and 26.8%, respectively. Clindamycin was very active at a low concentration (MIC50 of 0.39 to 1.56 mg/l), but 24% and 27.5% of Bacteroides fragilis and Bacteroides thetaiotaomicron isolates, respectively, were resistant to this agent.
The accumulation of macrolide antibiotics in Bacteroides fragilis ATCC 25285 was increased in the order erythromycin, josamycin, and rokitamycin, depending on hydrophobicity. The half-times of efflux were also prolonged in the same order. Furthermore, MICs of the antibiotics were correlated with the extent of hydrophobicity. These findings suggest that the macrolide antibiotics are accumulated in B. fragilis by means of their hydrophobic properties, and the efficient accumulation of the drugs may explain the susceptibility of this gram-negative bacterium to macrolides.There are several lines of evidence that macrolides are intrinsically inactive against gram-negative bacteria (1, 7). Since the site of action of macrolides has been thought to be the ribosomal subunits located in cytoplasm (16), efficient permeation across the surface envelope is required for the drugs to be effective. It has thus been proposed that the resistance in gram-negative organisms is due to the permeability barrier of the outer membrane (7,15,18,19). However, some anaerobic gramn-negative rods such as Bacteroides spp. which carry complete outer membranes (11) are usually susceptible to lower concentrations of macrolide antibiotics (21, 23), suggesting differences in the properties of the outer membrane between gram-negative anaerobes and aerobes. Although some information on the transport of macrolide antibiotics in aerobic bacteria (7, 15) is available, little is known about antibiotic transport in anaerobic gramnegative bacteria.In view of the considerable clinical significance of the anaerobic bacteria (6), particularly the Bacteroides group, this study was undertaken to examine macrolide transport in Bacteroidesfragilis. Since we were particularly interested in relating the permeability of macrolide antibiotics to their physical properties such as hydrophobicity, the transport of three macrolides which differ greatly in their hydrophobicity was systematically assessed.B. fragilis ATCC 25285 was grown at 35°C in an anaerobic glove box containing an atmosphere of 80% N2, 10% Co2, and 10% H2. The medium was GAM (Gifu Anaerobic Medium) broth (Nissui Seiyaku Co.) containing (per liter) 10 g of Proteose Peptone, 13.5 g of serum digest, 5 g of yeast extract, 2.4 g of beef extract, 1.2 g of liver extract, 3 g of glucose, 2.5 g of potassium dihydrogen phosphate, 3 g of sodium chloride, 5 g of soluble starch, 0.3 g of L-cysteine hydrochloride, 0.3 g of sodium thioglycolate, 3 g of soy peptone, and 10 g of peptone. The medium was supplemented with 0.0005% hemin and menadione. Bacteria were harvested by centrifugation, washed, and suspended in anaerobic salt solution (ABS) containing 20 mM MOPS (morpholinepropanesulfonic acid)-Tris (pH 7.0), 155 mM NaCl, 5.3 mM KCI, 1 mM MgCl2, 1.7 mM CaCl2, and 3 mM L-cysteine, all under anaerobic conditions. Antibacterial activity (expressed as MIC) was determined by the agar dilution technique, as described previously (12 the drug concentration in octanol and water phases at 25°C spectrophotometrically.Uptake...
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