[Identification and expression of blaCTX-M-14 and blaCTX-M-24]

To identify the ESBL gene and the prevalence in Escherichia coli and Klebsiella pneumoniae strain isolated from Huashan Hospital, Shanghai.

Isolates were confirmed as an ESBL producing strain by double-disk synergy test and NCCLS Confirmatory Test. Antibiotic susceptibilities were determined by standard agar dilution procedure on Mueller-Hinton agar. To determine whether the resistance was transferable, the conjugation experiment was performed; plasmids were isolated from clinical isolates and transcojugants. The partial bla(gene) of ESBL producing isolates and their transcojugants were detected by PCR using universal primers for TEM, SHV, CTX-M-1group, Toho-1group, CTX-M-13group respectively. The entire bla(CTX-M-13) group were amplified by PCR using the primers outside the Open Reading Frame (ORF) of CTX-M-13group beta-lactamases; the PCR products of entire bla(CTX-M-13)group were cloned into vector and the recombinant plasmids were transformed into the recipient strain for expression; the PCR products were also directly sequenced and analyzed; the clinical isolates of ESBL producers were detected by PFGE.

ESBL producers were resistant to most beta-lactams and non-beta-lactams. Most transconjugants were obtained at frequency of 10(-4) approximately 10(-5) and resistance to non-beta-lactams was cotransferred with the ESBL activity to the transconjugant. A plasmid of about > 23.1 kb was obtained from each tansconjugant by plasmid extraction. Partial gene amplification products of CTX-M-13 group gene were obtained from isolates and their transconjugants. The bla(CTX-M-13)group from 4 transconjugants were identified as bla(CTX-M-14), and other six were bla(CTX-M-24); those ESBLs were mediated by plasmids (> 23.1 kb); the transformants producing CTX-M-14 or CTX-M-24 were resistant to most beta-lactams, which were much more resistant to cefotaxime than to ceftazidine; PFGE patterns of those isolates were different.

clinical isolate of Escherichia coli and Klebsiella pneumoniae isolated from Huashan Hospital, Shanhai produced CTX-M-14 or CTX-M-24, which caused the isolate resistant to most beta-lactams; no clone spread in those isolates was found.