Phenotypic and Molecular Detection of CTX-M-β-Lactamases Produced by Escherichia coli and Klebsiella spp

Abstract

Organisms producing CTX-M-␤-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-␤-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 ␤-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-␤-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.Resistance to the expanded-spectrum cephalosporins can occur in Escherichia coli and Klebsiella species via the production of extended-spectrum ␤-lactamases (ESBLs) that are capable of hydrolyzing the oxyiminocephalosporins and monobactams (11). Recently, a family of ESBLs which preferentially hydrolyze cefotaxime (CTX), the CTX-M-␤-lactamases, have been recognized and reported in the literature with increasing frequency (3). This resistance mechanism is widespread throughout the world, with reports of clinical isolates producing these ␤-lactamases from Europe, Africa, Asia, South America, and most recently North America (3, 27).CTX-M-␤-lactamases are not closely related to TEM or SHV ESBLs (7) but share high amino acid identity with chromosomal ␤-lactamases from Kluyvera georgiana (34), Kluyvera cryocrescens (17) and Kluyvera ascorbata (24). In fact, the CTX-M-5 enzyme is identical to the chromosomal gene of K. ascorbata (3). According to a recent review and new data within GenBank, CTX-M-␤-lactamases can be divided into five groups based on their amino acid sequence identities (3). Group I includes CTX-M-1, -3, -10 to -12, -15 (UOE-1), -22, -23, -28, -29, and -30. Group II includes CTX-M-2, -4 to -7, and -20 and Toho-1. Group III includes CTX-M-8. Group IV includes CTX-M-9, -13, -14, -16 to -19, -21, and -27 and Toho-2. Finally group V includes CTX-M-25 and -26. The members of these groups exhibit Ͼ94% amino acid identity within the group and Յ90% amino acid identity between groups (3).The laboratory detection of organisms producing CTX-M-␤-lactamases is not well defined. The guidelines published by the National Committee for Clinical Labor...

Study of CTX-M Type of Extended Spectrum β-Lactamase among Nosocomial Isolates of Escherichia coli and Klebsiella pneumoniae in South India

“…The entire isolates positive for bla CTX-M gene by PCR were subjected to the CTX-M group specific PCR [14]. Template DNA, positive control and visualization of the PCR products were same as above.…”

Identification of nasal colonization with β-lactamase-producing enterobacteriaceae in patients, health care workers and students in Madagascar

“…Therefore, commercial hyplex ESBL ID PCR (amPLEX, Giessen, Germany) targeting bla CTX-M as the most frequent ESBL resistance mechanism in Madagascar [4] was applied to the swabs of a subset of 251 samples in Hamburg according to the manufacturer's instructions. As well as bla CTX-M [6], the hyplex ESBL kit also targets β-lactamases of the bla TEM -and bla SHV -types [5] as well as the bla OXA-1 carbapenemase in a consensus approach, though without specifi city for the expression of an ESBL phenotype. Positive signals of the bla CTX-M -PCR were correlated with cultural results on Brilliance ESBL agar, and positive signals of the β-lactamase consensus PCR, with the general growth of Enterobacteriaceae on MacConkey II agar.…”

“…In particular, extended-spectrum β-lactamase (ESBL)-positive Enterobacteriaceae are known to be prevalent in Madagascan hospital patients [1][2][3][4], including populations at particular risks such as newborns [1]. ESBL expression causes increased resistance to penicillins and cephalosporins, driven by a variety of molecular mechanisms [5,6]. Among known mechanisms, bla CTX-M expression (CTX = resistance to the WHO-listed antibiotic drug ceftriaxone, M = Munich, Germany, as the site of first description) is the most prevalent one in Madagascar, being detected in three out of four Madagascan ESBL-positive Enterobacteriaceae [4].…”

Rapid and simple detection of bla CTX−M genes by multiplex PCR assay

“…Those methods which make possible the differentiation of different gene sequences to establish the epidemiology of these new ESBLs are particularly needed. PCR has been applied successfully to characterize bla CTX-M genes, but detection of all members from five groups required multiple PCRs with group-specific primers (Canton et al, 2002;Chanawong et al, 2002;Pitout et al, 2004) or consensus primers which only amplify few bla CTX-M alleles Saladin et al, 2002). Currently, sequencing is the only method for definitive identification of bla CTX-M genes, which is labourintensive, time-consuming and expensive.…”