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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.

SUBMITTER: Parker AC

PROVIDER: S-EPMC187887 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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

Parker A C AC Smith C J CJ

Antimicrobial agents and chemotherapy 19930501 5

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 ...[more]

PMID: 8517690

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Project description:As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.

Dataset Information

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

Publications

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

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