Integrated Detection of Extended-Spectrum-Beta-Lactam Resistance by DNA Microarray-Based Genotyping of TEM, SHV, and CTX-M Genes

Abstract

Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to betalactam antibiotics in Gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in Gram-negative bacteria by simultaneously genotyping bla TEM , bla SHV , and bla CTX-M . The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla CTX-M (76%), bla SHV (22%), or both (2%), whereas no ESBL variant of bla TEM was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

“…The sequences of our bla TEM1 PCR products were identical to that of bla TEM1 (FJ668715.1) previously reported by Leinberger et al in K. pneumoniae from Germany [15]. Our bla TEM116 sequences were identical to that isolated from K. pneumoniae and K. oxytoca in Australia (AY265882.1).…”

Antimicrobial Resistance, Class 1 Integrons and Extended-Spectrum .BETA.-Lactamases in Escherichia coli Clinical Isolates from Patients in South Thailand

“…ESBL enzymes including TEM, SHV and CTX-M are mostly found in clinical isolates. 32) Pongpech et al 23) found that bla TEM and bla CTX-M were predominant among ESBL producing E. coli. In Argentina, CTX-M-2 is the most frequently found ESBL among clinical isolates in Argentinean hospitals.…”

Evaluation of a Commercial Microarray System for Detection of SHV-, TEM-, CTX-M-, and KPC-Type β-Lactamase Genes in Gram-Negative Isolates

“…Microarray technologies are promising genotyping systems that possess a high multiplexing capacity and can be used for detecting different ␤-lactamase genes that are present in a single strain (8,10,23). This ability can assist clinicians in directing antimicrobial therapy.…”