What Constitutes an Extended-Spectrum β-lactamase?

In a recent publication, Madinier et al. describe the cloning and characterization of a Class A β-lactamase, CfxA2, from Pervotella intermedia (3). CfxA2 differs by only a single amino acid residue from CfxA, an extended-spectrum β-lactamase in Bacteroides vulgatus CLA-341 (3). This is an important finding, as it continues to document the spread of antibiotic resistance determinants among normal flora species that colonize diverse niches fo the human host (oral and intestinal). Previously, we showed that CfxA can mediate resistance to nearly all β-.actam antibiotics (including cephamycins) but not to carbapenems or related compounds; thus, we concluded that CfxA is an extended-spectrum β-lactamase (5). Since cefoxitin is an important drug in the treatment of Bacteroides spp. infections, we felt that the ability of cfxA to mediate cefoxitin resistance was a particularly significant aspect of our studies. However, in their publication of CfxA2, Madinier et al. (3) make the claim that “high level of resistance of B. vulgatus CLA-341 towards cefoxitin should be attributed to a resistance mechanism other than CfxA production, such as porin mutation.” This claim is based on the MIC values obtained for the cloned cfxA2 gene in a heterologous host (Escherichia coli) and in B. vulgatus NI-2869. Further, kinetic experiments with purified CfxA2 were used to support their position. In making their claim, Madinier et al. ignore the considerable genetic and biochemical evidence indicating that CfxA is an extended-spectrum β-lactamase responsible for cefoxitin resistance in Bacteroides spp. Evidence supporting our position is as follows. (i) In Table 1 of our original paper (5) and in many subsequent studies we have shown that the cloned cfxA gene (containing no other intact genes) can be transferred to any cefoxitin-sensitive Bacteroides spp. strain and confer high-level cefoxitin resistance. Controls with vector alone do not become resistant to cefoxitin. In addition, the transmissible Tn4555 (Genbank accession no. {"type":"entrez-nucleotide","attrs":{"text":"U75371","term_id":"5566608","term_text":"U75371"}}U75371) containing the cfxA gene mediates cefoxitin resistance when present in single copy on the chromosome of Bacteroides species. (ii) Similar to Madinier et al. (3), we showed (Table 2 of reference 5) that the β-lactamase activity of CfxA with cefoxitin as a substrate was very low ( 91% of the cefoxitin in the media after overnight incubation (Table 1 of reference 5). In contrast, Bacteroides spp. with the vector alone could not degrade cefoxitin. This indicates that CfxA can slowly degrade cefoxitin even though the rate cannot be readily monitoerd by standard spectrophotometric assays. (iii) Experiments with cfxA in our laboratory were all performed in the natural host, Bacteroides (5), where the gene encodes a highly expressed β-lactamase as determined by specific activity of cell extracts (5). Madiner et al. (3) performed some experiements in E. coli, where β-lactamase and other resistance genes of Bacteroides origin are rarely expressed or are not correctly processed (1, 2, 4-8). Further, experiments performed in B. vulgatus NI2869 containing cfxA2 exhibited extremely low MIC values for amoxicillin (and all β-latams) and no β-lactamase specific activities were reported. These MIC values were even lower than those seen in their E. coli work, suggesting that there is a significant problem with expression of their cloned cfxA2 gene (3). Finally, the authors never directly tested cfxA in their B. vulgatus NI-2869 host, so there is no way to correct for differences conferred by strain background. These observations establish that the β-lactamase gene cfxA mediates a broad-spectrum β-lactam resistance in Bacteroides spp. that is not dependent upon other mechanisms for resistance. This brings us back to the original question: what is an extended-spectrum β-lactamase? Is it an enzyme that rapidly degrades a wide raneg of β-lactam substrates or is it an enzyme that can mediate resistance to a wide range of β-lactams? Clearly, CfxA falls in this latter group and should be considered a broad-spectrum enzyme that mediates resistance to cefoxitin and other β-lactam drugs regardless of the enzymatic rate.