Study On CTX-M Type ESBLs In Escherichia Coli And Klebsiella Pneumoniae

The production of extended-spectrum β -lactamases (ESBLs) is the most common mechanism of resistance to the third generation cephalosporins and monobactaoms for enterobacterium. ESBLs that is a large species of β -lactamases can hydrolyze the third generation cephalosporins and monobactams. ESBLs are mainly produced by Klebsiella pneumoniae and Escherichia coli. The genotype of ESBLs producing strains differs in different countries areas and hospitals because of the different use of antibacterial agents. In order to clarify the predominant ESBLs genotype in Zhejiang Province, We investigated the genotypes distribution of CTX-M type ESBLs in ESBLs producing Escherichia coli and Klebsiella pneumoniae in Zhejiang province. 119 clinical strains of Escherichia coli and Klebsiella pneumoniae confirmed to produce ESBLs were conserved in our laboratory, and the ESBLs genotypes were detected by polymerase chain reaction (PCR). The primers of CTX-M-1, CTX-M-2, CTX-M-8 and CTX-M-9 groups were designed. The genotypes of p-lactamases were confirmed by gene cloning and sequencing of the PCR products. The novel CTX-M-29 was inserted into pET28 and expressed inEscherichia coli BL21 (DE3). The transformants were detected to confirm if they produced ESBLs. The pIs of the expressed protein were detected by isoelectric focusing (IEF).110 strains (92.44%) produced CTX-M type ESBLs, including CTX-M-14 (82 strains), CTX-M-24 (12 strains), CTX-M-22 (7 strains), CTX-M-9 (2 strains), CTX-M-27 (1 strain), CTX-M-28 (1 strain) and CTX-M-29 (1 strain). 4 strains produced CTX-M-14 and CTX-M-22. The former studies showed that 8 strains produced SHV type ESBLs, including SHV-12 (7 strains) and SHV-5 (1 strain). None of the strains produced TEM type ESBLs. 5 strains were negative by PCR according to the former results of study on TEM-types SHV-type and CTX-M-type ESBLs. 6 strains produced two kinds of ESBLs and 1 strain procuced three kinds of ESBLs.A new CTX-M type p-lactamase was detected and named to be CTX-M-29 (GenBank accession NO AY267213). After prokaryotic expression, the transformant was confirmed to be ESBLs producing strain and the pi of the expressed protein was about 9.0.In brief, CTX-M type is the most common genotype in plasmid-mediated ESBLs produced by Escherichia coli and Klebsiella pneumoniae in Zhejiang province; CTX-M-29 is a novel ESBLs and the GenBank accession number is AY267213. It is thefirst time to report CTX-M-27 and CTX-M-28 in China.