Purification and characterization of a new beta-lactamase from Bacteroides uniformis

Abstract

A beta-lactam-resistant Bacteroides uniformis strain was isolated from a clinical specimen. The strain produced large amounts of ␤-lactamase and was resistant to penicillins and cephalosporins. The specific activity of the unpurified ␤-lactamase was 4.8 U/mg of protein with nitrocefin as the substrate. The enzyme was purified 188-fold by Q-Sepharose, Sephacryl S-300, and Mono Q column passages. Kinetic parameters of the enzyme were determined by a micromethod performed in microtiter plates. ␤-Lactamase was inhibited by cefoxitin and imipenem and hydrolyzed cephalosporins more rapidly than penicillins. The molecular weight was determined by sodium dodecyl sulfate-gradient gel electrophoresis to be 32,500, and the isoelectric point was 4.5.Resistance to antimicrobial agents in Bacteroides spp. is mediated by several mechanisms. The most important mechanism of resistance to beta-lactam antibiotics to date is ␤-lactamase production. ␤-Lactamases are produced by a majority of resistant isolates of the Bacteroides fragilis group as well as by other anaerobes (17). At least four types of ␤-lactamases have been described for members of the B. fragilis group, but the most common type is a constitutively produced, chromosomally encoded cephalosporinase that has no activity against cefoxitin and imipenem. Recent studies have shown that at least 90% of all B. fragilis group strains produce ␤-lactamases and that 25% of the strains produce high levels of ␤-lactamase (7). Members of the B. fragilis group of organisms are the anaerobic bacterial pathogens most frequently recovered from humans. Bacteroides uniformis is not often found in anaerobic infections. It accounts for less than 5% of the B. fragilis group species found in clinical specimens (11). The aim of this study was to purify and characterize a ␤-lactamase produced by B. uniformis by a microtechnique for determination of kinetic parameters and enzyme inhibition. MATERIALS AND METHODSBacterial strain. B. uniformis B 371 was isolated from a wound in a kidney transplant patient at the Huddinge University Hospital, Stockholm, Sweden. The strain was identified by the procedures of Holdeman et al. (14).Antimicrobial agents. The following agents were tested: ampicillin and benzylpenicillin (Astra, Södertälje, Sweden), cefoxitin and imipenem (Merck Sharp & Dohme, West Point, Pa.), cephalothin (Eli Lilly and Co., Indianapolis, Ind.), piperacillin (Lederle, Pearl River, N.Y.), and cephaloridine and nitrocefin (Glaxo Pharmaceuticals, Greenford, United Kingdom).Antimicrobial susceptibility tests. MICs were determined by the agar dilution method on paper disc method agar (AB Biodisk, Solna, Sweden) with the addition of 5% defibrinated horse blood. The inoculum was approximately 10 6 CFU/ml and was applied by a Steers replicator and incubated for 48 h at 37ЊC in anaerobic jars (GasPak; BBL Microbiology Systems, Cockeysville, Md.).Medium. The medium used was prereduced proteose peptone-yeast extract medium prepared as described previously (20).Production of ␤-lactamase. B. uniform...

Resistance to -Lactam Antibiotics in Bacteroides Spp.

“…However, an unusual P-lactamase from B. unformis strain B 371, which appears to belong to group 2e, has been reported. This P-lactamase showed a substrate profile that resembles enzymes of Ambler class C, except for its inhibition by clavulanic acid [68]; class C enzymes have not been encountered previously in Bacteroides SPP.…”

Protection of Salmonella by ampicillin-resistant Escherichia coli in the presence of otherwise lethal drug concentrations

“…(b) Measurement of b-lactamase activity, b-galacosidase activity and ampicillin concentrations Beta-lactamase activity was measured using a spectrophotometric assay (Hedberg et al 1995) with nitrocefin (Glaxo Research, Oxoid, UK) as the chromogenic cephalosporin substrate. The colour change from yellow to red when nitrocefin was cleaved by b-lactamase was monitored over a 30 min period at 495 nm.…”

Cloning and Biochemical Characterization of a Class A β-Lactamase from Prevotella intermedia

“…Several studies have reported increasing resistance to antibiotics in gram-negative anaerobes, especially to ␤-lactam antibiotics, mostly by the production of ␤-lactamases (3,4,8,13,14). Only a few genes encoding ␤-lactamases have been cloned and sequenced in members of the family Bacteroidaceae (Bacteroides fragilis, Bacteroides vulgatus, and Bacteroides uniformis) but not in Prevotella and Porphyromonas species (12,16,18,20). A preliminary work concluded that P. intermedia and Prevotella buccae were the predominant ␤-lactamase-producing species among anaerobic gram-negative rods isolated from periodontal pockets, with 35 and 42% of these, respectively, ␤-lactamase-positive strains (9).…”

“…Repeated isoelectric focusing experiments with crude extracts were necessary to visualize a discrete reactive band with a pI of 5.4. The pI reported for Bacteroides species was 5.8, but CfxA2 differed from CfxA by a glutamic acid (acid) replacing a lysine (base) in position 272 (12,16). The hydrolysis of ␤-lactams was monitored at 37°C in sodium phosphate buffer (0.05 M; pH 7.0) with 20 g of ␤-lactamase in a 500-l reaction mixture.…”

“…For cloning experiments, chromosomal DNA from P. intermedia NI-1187 was obtained with conventional phenol-chloroform extraction methods, restricted with EcoRI, ligated in pZErO-2-Kan, and transferred by electroporation in Escherichia coli Top 10 (InvitroGen, Leek, The Netherlands) (12,16,20). A bank of approximately 10 6 recombinant clones was obtained on kanamycin selective plates and yielded about 10 colonies of ␤-lactamase-producing E. coli transformants on ampicillin selective plates (40 g/ml).…”