Phenotypic and molecular detection of CTX-M-beta-lactamases produced by Escherichia coli and Klebsiella spp

Abstract

Organisms producing CTX-M-beta-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-beta-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of Escherichia coli and Klebsiella species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum beta-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-beta-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for bla(CTX-M-1-like) genes, 95 (54%) were positive for bla(CTX-M-14-like) genes, and the remaining 56 (32%) were negative for bla(CTX-M) genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.

FIG. 1.

CTX-M PCR. Four primer sets representing specific groups of bla_CTXM genes were evaluated for specificity with DNA prepared from strains known to produce specific CTX-M β-lacatmases. The DNA templates were prepared from CF2 producing CTX-M-1, CF1 producing CTX-M-14, Rio-4 producing CTX-M-2, and Rio-3 producing CTX-M-8. H₂O represents the negative control, and L represents a 100-bp ladder (Invitrogen). Specific marker sizes in base pairs are shown on the right. Each primer pair was used in combination with both positive and negative templates. The primer groups used for each set of templates are listed below the figure and are defined in Table 2.