Characterization of cephalosporinases from Bacteroides fragilis, Bacteroides thetaiotaomicron and Bacteroides vulgatus.

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Abstract
The susceptibility of 80 Bacteroides fragilis group strains isolated from clinical specimens to β-lactam antibiotics was investigated by agar dilution method. Twenty strains showed high resistance to the antibiotics. The resistance level of the isolates to cephaloridine was related to the amount of β-lactamase activity (cephalosporinase; CSase) produced. B. fragilis GN11477, B. thetaiotaomicron GN11478 and B. vulgatus GN11479 were selected from among the CSase producing strains, and the enzymes were purified about 300-fold by affinity chromatography employed ampicillin as ligand bound to activated CH Sepharose 4B. The enzyme preparations gave a single protein band on polyacrylamide gel electrophoresis. The molecular weights of the three enzymes were estimated to be approximately 32, 000 and their isoelectric points were 5.2, 4.9 and 4.5, respectively. The optimal pH and the optimal temperature of the enzymes were 7.2 and 37°C, respectively. The enzyme activities were inhibited by iodine, some divalent ions, p-chloromercuribenzoate, clavulanic acid, cephamycin derivatives and cloxacillin. The enzymes showed hydrolytic activity against cephaloridine, cephalothin, cefazolin, cefuroxime and also newly introduced cephalosporins such as cefotaxime, cefoperazone and cefinenoxime. Each mouse antisera obtained against the purified enzymes showed cross-reactions with its each enzyme and others in neutralization test.