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Pryamchuk S.D.,Federal State Institution of Science State Research Center for Applied Microbiology and Biotechnology | Fursova N.K.,Federal State Institution of Science State Research Center for Applied Microbiology and Biotechnology | Anisimova V.A.,Royal Netherlands Tuberculosis Association Royal Netherlands Chemical Society | Kovalev Yu.N.,Federal State Institution of Science State Research Center for Applied Microbiology and Biotechnology | And 3 more authors.
Molecular Genetics, Microbiology and Virology | Year: 2011

An algorithm for step-by-step identification of blaCTX-M genes determining the resistance to ceph- alosporins III-IV in gram-negative bacteria is developed. The algorithm allows identification of 49 genes out of 96, which are already present in the GenBank database. The remaining 47 genes are identified in several small groups of 2 to 6 genes, excluding the bla CTX-M-14- like subgroup, which comprises 13 genes. The bla CTX-Mgenes' identification is carried out with two-step restriction fragment length polymorphism analysis (PCR-RFLP) of a 544-bp-long PCR-product. At the first stage, the blaCTX-M gene belonging to one of five subtypes (clusters), cluster 1, cluster 2, cluster 8, cluster 9, or cluster 25, is identified with the use of AluI restriction endonuclease. This stage also allows iden- tification of four genes: bla CTX-M-59 (cluster 2), bla CTX-M-63 (cluster 8), bla CTX-M-45 (cluster 9), and bla CTX-M-78 (clusters 2 and 25 hybrid). The second stage includes step-by-step identification of genes inside every cluster using a set of 26 restriction endonucleases. Thus, as a result of PCR-RFLP analysis, 23 genes of cluster 1, 11 genes of cluster 2, 4 genes of cluster 8, 9 genes of cluster 9, and 1 gene of cluster 25 are identified up to the individual type; two hybrid genes are also identified at this stage: blaCTX-M-78 (clusters 2 and 25 hybrid) and blaCTX-M-64 (clusters 1 and 9 hybrid). The above-described method is used to identify blaCTX-M genes (n = 585) in a nosocomial isolates collection of the Enterobacteriaceae family bacteria (n = 877) isolated in the Russian Federation in 2003-2007. It is shown that the majority of the genes belonged to cluster 1 (n = 543), and the majority of the genes inside this cluster were identified as bla CTX-M-15 (n = 515). Genes bla CTX-M-3(n = 25), blaCTX-M-22 (n = 1), bla CTX-M-23 (n = 1), and bla CTX-M-34 (n = 1) were identified among other types. In addition, bla CTX-M-2 (n = 1) and bla CTX-M-5 (n = 4) genes belonging to cluster 2 and bla CTX-M-9 (n = 2) and bla CTX-M-14 (n = 35) genes belonging to cluster 9 were identified. © 2011 Allerton Press, Inc.

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