Priamchuk S D, Fursova N K, Anisimova V A, Kovalev Iu N, Abaev I V, Kuzhel'naia E N, Svetoch E A
Mol Gen Mikrobiol Virusol. 2011(4):7-13.
The algorithm of the identification of the bla(CTX-M) genes coding CTX-M-type beta-lactamases providing resistance to cephalosporins III-IV was developed. This algorithm provides identification of 49 genes of 96 genes presented in the GenBank database so far. Remaining 47 genes can be identified as consisting of small sub-groups composed of 2-6 genes with the exception of sub-group of the bla(CTX-M-14)-like genes composed of 13 genes. The identification of the bla(CTX-M) genes is based on two-step restriction fragment length polymorphism analysis of 544 bp PCR-product (PCR-RFLP). In the first step, determination of subtype (cluster) of the bla(CTX-M) gene occurred using the restriction nuclease Alu I: cluster 1, -2, -8, -9 or -25. Moreover, four genes can be identified just at this step: bla(CTX-M)-59, (cluster 2); bla(CTX-M-63) (cluster 8), bla(CTX-M-45) (cluster 9), and bla(CTX-M-78) (hybrid gene between cluster 2 and cluster 25). At the second step gene identification goes on inside of each cluster separately using a set of 26 restriction nucleases. As a result of the PCR-RFLP-analysis, 23 bla(CTX-M) genes can be identified at the cluster 1, 11 genes--at the cluster 2, 4 genes--at the cluster 8, 9 genes--at the cluster 9, 1 gene--at the cluster 25, and 2 hybrid genes: bla(CTX-M-78) (between clusters 2 and 25), and bla(CTX-M-64) (between clusters 1 and 9). The described algorithm was used for identification of the blac(CTX-M) genes (n = 585) detected in Enterobacteriaceae nosocomial isolates (n = 877), collected from Russial hospitals in 2003-2007. It was shown that major genes belonged to cluster 1 (n = 543), namely--bla(CTX-M-15) gene (n = 515), bla(CTX-M-3) (n = 25), bla(CTX-M-22) (n = 1), bla(CTX-M-23) (n = 1), and bla(CTM-34) (n = 1). Moreover, the genes atributed to cluster 2 were identified: bla(CTX-M-2) (n = 1), and bla(CTX-M-5) (n = 4); and genes belonged to cluster 9: bla(CTX-M-9) (n = 2), and bla(CTX-M-14) (n = 35).
开发了用于鉴定编码对第三代至第四代头孢菌素耐药的CTX-M型β-内酰胺酶的bla(CTX-M)基因的算法。该算法可鉴定出GenBank数据库中目前已有的96个基因中的49个基因。其余47个基因可被鉴定为由2至6个基因组成的小亚组，但由13个基因组成的bla(CTX-M-14)样基因亚组除外。bla(CTX-M)基因的鉴定基于对544 bp PCR产物的两步限制性片段长度多态性分析（PCR-RFLP）。第一步，使用限制性核酸酶Alu I确定bla(CTX-M)基因的亚型（簇）：簇1、-2、-8、-9或-25。此外，仅在这一步就可以鉴定出4个基因：bla(CTX-M)-59（簇2）；bla(CTX-M-63)（簇8），bla(CTX-M-45)（簇9），以及bla(CTX-M-78)（簇2和簇25之间的杂交基因）。第二步，使用一组26种限制性核酸酶在每个簇内分别进行基因鉴定。PCR-RFLP分析的结果是，在簇1中可鉴定出23个bla(CTX-M)基因，在簇2中可鉴定出11个基因，在簇8中可鉴定出4个基因，在簇9中可鉴定出9个基因，在簇25中可鉴定出1个基因，以及2个杂交基因：bla(CTX-M-78)（簇2和簇25之间）和bla(CTX-M-64)（簇1和簇9之间）。所描述的算法用于鉴定2003年至2007年从俄罗斯医院收集的肠杆菌科医院分离株（n = 877）中检测到的bla(CTX-M)基因（n = 585）。结果表明，主要基因属于簇1（n = 543），即bla(CTX-M-15)基因（n = 515）、bla(CTX-M-3)（n = 25）、bla(CTX-M-22)（n = 1）、bla(CTX-M-23)（n = 1）和bla(CTM-34)（n = 1）。此外，还鉴定出了属于簇2的基因：bla(CTX-M-2)（n = 1）和bla(CTX-M-5)（n = 4）；以及属于簇9的基因：bla(CTX-M-9)（n = 2）和bla(CTX-M-14)（n = 35）。
