title: Oligonucleotide microarrays with horseradish peroxidase-based detection for the identification of extended-spectrum β-lactamases.
abstract: Production of extended-spectrum β-lactamases (ESBLs) is the one of most widespread and clinically significant mechanism of Enterobacteriaceae resistance towards modern β-lactam antibiotics. There are known 400 ESBLs, with the majority represented by the enzymes of TEM, SHV and CTX-M families. Oligonucleotide microarrays with colorimetric detection have been developed for the purposes of determination of ESBLs and inhibitor-resistant β-lactamases using horseradish peroxidase (HRP). Specific oligonucleotide probes have been designed for the identification of β-lactamase family and important SNPs responsible for the broadening of substrate specificity and tolerance to inhibitors. Multiplex PCR has been developed for simultaneous amplification and labeling of full-size genes of TEM-, SHV- and CTX-M-type β-lactamases with biotin. The labeled target DNA is then hybridized with specific oligonucleotide probes immobilized on a porous membrane support. After hybridization, biotin-labeled DNA duplexes are stained with the streptavidin-HRP conjugate detected colorimetrically. Design of oligonucleotide probes and optimization of hybridization conditions ensure the specificity of all control ESBLs identification. The newly developed method has been successfully used to identify bla(TEM), bla(SHV) and bla(CTX-M) genes in 90 clinical isolates of Enterobacteriaceae: 70% were found to carry bla(TEM), 50% bla(SHV), 50% bla(CTX-M); with the following distribution of CTX-M subclusters: 68% bla(CTX-M-1), 4% bla(CTX-M-2), and 14% bla(CTX-M-9). No ESBL of TEM-type and IRT phenotype assigned to TEM- or SHV-type β-lactamases had been detected; 24.6% of clinical samples show two types of ESBLs simultaneously. A mixture of CTX-M-1-like and SHV-5-like genes was the most abundant combination detected. Membrane microarray technique with colorimetric detection provides both high specificity and effectiveness of screening for ESBL- and IRT-producing samples.
authors.affiliation: Dept. Chem. Enzymology, Chemistry Faculty, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia. mrubtsova@gmail.com
authors.forename: Mayya Yu
authors.initials: MY
authors.lastname: Rubtsova
authors.forename: Maria M
authors.initials: MM
authors.lastname: Ulyashova
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Edelstein
authors.forename: Alexey M
authors.initials: AM
authors.lastname: Egorov
mesh_terms: D001483:Base Sequence; D003124:Colorimetry; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000188:drug therapy / Q000382:microbiology; D005798:Genes, Bacterial; D006735:Horseradish Peroxidase; D006801:Humans; D008826:Microbial Sensitivity Tests; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D001618:beta-Lactamases / Q000145:classification / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D006735:Horseradish Peroxidase; D001618:beta-Lactamases