Dept. Chem. Enzymology, Chemistry Faculty, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia.
Biosens Bioelectron. 2010 Dec 15;26(4):1252-60. doi: 10.1016/j.bios.2010.06.053. Epub 2010 Jul 3.
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.
超广谱β-内酰胺酶（ESBLs）的产生是肠杆菌科对现代β-内酰胺类抗生素产生耐药性的最广泛和最具临床意义的机制之一。目前已知有 400 种 ESBLs，其中大多数由 TEM、SHV 和 CTX-M 家族的酶代表。已经开发出带有比色检测的寡核苷酸微阵列，用于使用辣根过氧化物酶（HRP）测定 ESBL 和抑制剂耐药的β-内酰胺酶。已经设计了特定的寡核苷酸探针，用于鉴定β-内酰胺酶家族和负责拓宽底物特异性和对抑制剂耐受性的重要 SNP。已经开发了多重 PCR 用于用生物素同时扩增和标记 TEM-、SHV-和 CTX-M 型β-内酰胺酶的全长基因。然后，将标记的靶 DNA与固定在多孔膜载体上的特异性寡核苷酸探针杂交。杂交后，用链霉亲和素-HRP 缀合物染色生物素标记的 DNA 双链体，并用比色法检测。寡核苷酸探针的设计和杂交条件的优化确保了所有对照 ESBL 鉴定的特异性。新开发的方法已成功用于鉴定 90 株肠杆菌科临床分离株中的 bla(TEM)、bla(SHV)和 bla(CTX-M)基因：发现 70%携带 bla(TEM)，50% bla(SHV)，50% bla(CTX-M)；CTX-M 亚群的分布如下：68% bla(CTX-M-1)，4% bla(CTX-M-2)，和 14% bla(CTX-M-9)。未检测到 TEM 型和 IRT 表型的 TEM 型 ESBL；24.6%的临床样本同时显示两种类型的 ESBL。检测到最丰富的组合是 CTX-M-1 样和 SHV-5 样基因的混合物。带有比色检测的膜微阵列技术提供了筛选 ESBL 和 IRT 产生样本的高特异性和有效性。
