Oligonucleotide microarrays with horseradish peroxidase-based detection for the identification of extended-spectrum β-lactamases.

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.

Rubtsova MY ，Ulyashova MM ，Edelstein MV ，Egorov AM ... -  《-》

Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis.

Cohen Stuart J ，Dierikx C ，Al Naiemi N ，Karczmarek A ，Van Hoek AH ，Vos P ，Fluit AC ，Scharringa J ，Duim B ，Mevius D ，Leverstein-Van Hall MA ... -  《-》

High diversity of extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae from Portugal.

Machado E ，Coque TM ，Cantón R ，Novais A ，Sousa JC ，Baquero F ，Peixe L ，Portuguese Resistance Study Group ... -  《JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY》

Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae.

Dallenne C ，Da Costa A ，Decré D ，Favier C ，Arlet G ... -  《-》

Distribution of bla(TEM), bla(SHV), bla(CTX-M) genes among clinical isolates of Klebsiella pneumoniae at Labbafinejad Hospital, Tehran, Iran.

Feizabadi MM ，Delfani S ，Raji N ，Majnooni A ，Aligholi M ，Shahcheraghi F ，Parvin M ，Yadegarinia D ... -  《-》