Genetic and biochemical analysis of a novel Ambler class A beta-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 beta-lactam antibiotics except imipenem. beta-Lactam resistance was mediated by a membrane-associated, 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 Bacteroides fragilis for the identification of Fxr strains. Analysis of B. fragilis strains with the cloned Fxr determinant revealed the presence of a new beta-lactamase protein with the physical and enzymatic properties of the beta-lactamase found in the original B. vulgatus isolate. The beta-lactamase gene (cfxA) 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 beta-lactamase activity. Comparison of CfxA with other beta-lactamases showed a relationship with the active-site serine beta-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 beta-lactamases. These results suggest that CfxA may represent a new class A homology group which diverged very early.