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

OBJECTIVE

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

METHODS

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.

RESULTS

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.

CONCLUSION

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.