Study On Genotype Of CTX-M-type Extended-spectrum Beta-lactamases By Denaturing High-performance Liquid Chromatography

Objective To evaluate the accuracy of denaturing high-performance liquid chromatography (DHPLC) by comparing with sequencing and investigate the genotype distribution of CTX-M-type extended-spectrumβ-lactamase(ESBLs) by DHPLC in three affiliated hospitals of Central South University.Methods A total of 171 multidrug resistant Enterobacteriaceae isolates were collected from three affiliated hospitals of Central South University. 142 ESBLs-producing isolates were confirmed by phenotypic confirmatory tests as recommended by the CLSI(2006) .The blaCTX-M genes of standard strains and clinical ESBLs-producing isolates were amplified by multiplex PCR followed by DHPLC and genotype determination. Meanwhile, 25 selected isolates were amplified by specific PCR and sequenced to assess the accuracy of DHPLC method for the genotype of CTX-M- type ESBLs.Results 142 ESBLs-producing isolates were confirmed by phenotypic confirmatory tests from 171 multidrug resistant Enterobacteriaceae isolates. 109 isolates carried blaCTX-M gene among 142 ESBLs-producing isolates,and the rate was 76.8%( n = 109 out of 142). The 109 clinical isolates included 52 from group 1, 57 belonging to group 9.The proportion of the Escherichia coli carring blaCTX-M gene was the highest, followed by Klebsiella pneumoniae and Enterobacter cloacae.The 109 clinical isolates were found present four different CTX-M genotypes by DHPLC, including CTX-M-3(33 isolates), CTX-M-15(19 isolates), CTX-M-14(52 isolates)and CTX-M-9(5 isolates). Among the 25 selected isolates,which were amplified by specific PCR,14 belong to CTX-M-1 group and 11 belong to CTX-M-9 group. The genotype of the 25 isolates were CTX-M-15 ,CTX-M-3, CTX-M-14 and CTX-M-82,which were confirmed by sequencing. The DHPLC typing results of 25 isolates suggested that 24 isolates were verified uniformly by the sequencing results, but one CTX-M-15 producing isolate typed by DHPLC has been shown CTX-M-82 by sequencing.Conclusions (1) DHPLC uses the special DNA chromatographic column to genotype the sample by comparing with the standard chromatograph peak chart,operated under the partly denatured temperature condition.It is a fast and powerful tool for genotyping of the blaCTX-M genes with accurate, rapid and economic advantages. (2) Genotyping of the blaCTX-M genes were conducted by DHPLC in three affiliated hospitals of Central South University for the first time.Four different subtypes of CTX-M-type ESBLs were detected,which are CTX-M-3, CTX-M-15,CTX-M-9,and CTX-M-14.Among them, CTX-M-14 was the main subtype. (3) This is the first report of a novel CTX-M-type ESBLs in the word,named CTX-M-82. (4) Many species of Enterobacteriaceae isolates were found present CTX-M ESBLs in the three affiliated hospitals of Central South University and Escherichia coli, Klebsiella pneumoniae and Enterobacter cloaca were the most common CTX-M ESBLs-producing species.