Genetic and biochemical analysis of a novel Ambler class A β-Lactamase responsible for cefoxitin resistance in Bacteroides species.

Abstract

A clinical isolate of Bacteroides vulgatus was resistant to tetracycline, clindamycin, ampicillin, cephaloridine, cefoxitin, and other 13-lactam antibiotics except imipenem. 13-Lactam resistance was mediated by a membraneassociated, clavulanate-sensitive cephalosporinase capable of degrading cephalosporins and penicillins. Cefoxitin also was degraded but at a slow rate. The cefoxitin resistance (Fxr) determinant was cloned from B. vulgatus genomic libraries that were prepared in Escherichia coli and then mated with Bacteroidesfragilis for the identification of Fxr strains. Analysis of B. fragilis strains with the cloned Fxr determinant revealed the presence of a new 13-lactamase protein with the physical and enzymatic properties of the 13-lactamase found in the original B. vulgatus isolate. The 13-lactamase gene (cftA) was subcloned on a 2.2-kb DraI-HindIII fragment, and the nucleotide sequence was determined. These results showed that cfxA encoded a protein of 321 amino acids and 35,375 molecular weight. Mutant strains in which the cfxA structural gene was disrupted by insertional inactivation lost both Fxr and 13-lactamase activity. Comparison of CfxA with other f3-lactamases showed a relationship with the active-site serine 13-lactamases in the Ambler molecular class A, although CfxA had apparently diverged significantly. This was exemplified by the substitution in CfxA at 13 of 25 amino acid residues previously identified as being invariant in class A 13-lactamases. These results suggest that CfxA may represent a new class A homology group which diverged very early. Originally published Antimicrobial Agents and Chemotherapy, Vol. 37, No. 5, May 1993