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 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) ,-Lactamase production is the most important mechanism of resistance to P-lactam antibiotics in gram-negative bacteria, and the Bacteroides fragilis group possesses a wide array of these enzymes. In general, the organisms are moderately or highly resistant to many cephalosporins and penicillins, but a-methoxyl cephamycins such as cefoxitin and the carbapenems have been highly active against Bacteroides species. In the United States, for example, imipenem resistance rates are presently low, at about 0.2%, and cefoxitin resistance (Fxr) rates have ranged up to 16% during the past decade (37, 38). High rates of Fxr are cause for concern since Fxr strains usually encountered are crossresistant to most other P-lactams (38). In the case of strains that possess metallo-p-lactamases, the cross-resistance includes the carbapenems (8, 39).Resistance of Bacteroides species to cefoxitin may involve a number of mechanisms including altered drug permeability, alterations of penicillin binding proteins, 1-lactamase production, or a combination of mechanisms (8,10,41). Excluding the relatively rare imipenem-hydrolyzing metalloenzymes, several P-lactamases capable of cefoxitin degradation have been described. These are generally cephalosporinases with a slow rate of cefoxitin hydrolysis, and they are sensitive to inhibition by clavulanate (9, 13). Recently, Aldridge et al. (1) have found that >93% of Fxr strains were still sensitive to 3-lactam-clavulanate combinations. Thus, based on these criteria, it is possible that the slow cefoxitinhydrolyzing cephalosporinases are one of the most widely disseminated mechanisms of Fxr in the Bacteroides species. In this regard, it has been suggested that these P-lactamases are not novel enzymes but rather that the strains are Fxr * Corresponding author.because of the production of much greater than normal levels of the conventional Bacteroides enzyme (36).The occurrence of high regional Fxr rates indicates the potential for clonal dissemination and/or horizontal transfer of the resistance phenotype. The transfer of Fxr by a conjugation-like mechanism has been demonstrated; however, plasmids were not involved in the transfer (10). The Fxr phenotype was mediated by the acquisition of a new P-lactamase. The present study was initiated to define the genetic basis for transmissible Fxr in Bacteroides species.The results revealed a novel genetic locus designated cfx4, which encodes an Ambler molecular class A 13-lactamase that appears to have diverged significantly from all other class A enzymes. MATERIALS AND METHODSBacterial strains and growth. Bacteroides cells were cultured anaerobically in su...

Bacteroides: the Good, the Bad, and the Nitty-Gritty

“…The most commonly discussed Bacteroides transposons of this class include Tn4399, Tn4555, and the nonreplicating Bacteroides units. The mobilizable transposon Tn4555, for example, was first detected during studies of transmissible cefoxitin resistance in a clinical isolate of Bacteroides vulgatus (182).…”

“…This enzyme is inhibited by the most commonly used ␤-lactamase inhibitors (sulbactam, clavulanic acid, and tazobactam), and agents containing these inhibitors are active against strains that produce the ␤-lactamase enzyme. The cefoxitin resistance gene, cfxA (182), has been shown to be distantly related to the B. fragilis endogenous cepA (219).…”

Resistance to -Lactam Antibiotics in Bacteroides Spp.

“…Rogers et al [64] characterised the cephalosporinase gene cepA from B. fragilis that exhibits high specific activity and showed that these typical P-lactamases form a distinct subgroup of Ambler molecular class A [65]. Similarly, a clavulanate-sensitive cephalosporinase produced by B. vulgatus, which was capable of slow degradation of cefoxitin, appears to belong to a new class-A homology group [66]. Analysis of P-lactamase from B. uniformis revealed a species-specific Bush group 2e enzyme that belongs to Ambler class A [67].…”

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

“…Mobilizable transposon Tn4555 from Bacteroides vulgatus encodes CfxA, a clinically significant broad spectrum β-lactamase conferring high-level resistance to cefoxitin and other β-lactams (Parker and Smith, 1993). The cfxA gene has been found in 80-100% of cefoxitin resistant, imipenem sensitive Bacteroides isolates (Mastrantonio et al, 1996;Parker and Smith, 1993) and more recently the gene was isolated from pathogens found in the oral cavity, Prevotella and Capnocytophaga (Iwahara et al, 2006;Jolivet-Gougeon et al, 2004).…”

“…The cfxA gene has been found in 80-100% of cefoxitin resistant, imipenem sensitive Bacteroides isolates (Mastrantonio et al, 1996;Parker and Smith, 1993) and more recently the gene was isolated from pathogens found in the oral cavity, Prevotella and Capnocytophaga (Iwahara et al, 2006;Jolivet-Gougeon et al, 2004). The cfxA gene in these oral isolates often is associated with the Tn4555 mobilization genes suggesting that this MTn or related elements are important in the spread of β-lactam resistance.…”