A Klebsiella pneumoniae producing three kinds of class A beta-lactamases encoded by one single plasmid isolated from a patient in Huashan Hospital, Shanghai, China.

A Klebsiella pneumoniae strain was isolated from a sputum specimen of a patient in the intensive care unit in 1999 in Shanghai Huashan Hospital, China. The isolate was confirmed as an extended-spectrum beta-lactamase (ESBL) producing strain by double-disk synergy test. The results of susceptibility test showed that it was resistant to most beta-lactams (including third generation cephalosporins) and non-beta-lactam antimicrobial agents. Transconjugants were obtained at a frequency of 10(-4). A plasmid of about 60 kb was obtained from the transconjugant by plasmid extraction. Three major nitrocefin-hydrolysing bands with pIs of 5.4, 8.2 and 8.4, were shown in extracts of the transconjugant. Partial gene amplification products of bla(TEM), bla(SHV), and CTX-M-1 group gene were obtained from the isolate as well as its transconjugant. The entire bla(TEM), bla(SHV), and bla(CTX-M) in the transconjugant were amplified by PCR and the PCR products were cloned into a pHSG398 vector. Afterwards, the susceptibility of transformants and activities of beta-lactamases of transformants on antibiotics were tested. The PCR products were directly sequenced, analysed and identified as TEM-1, SHV-12, and CTX-M-3 genes. These results confirm that this strain of Klebsiella pneumoniae produces SHV-12, CTX-M-3 ESBLs and TEM-1 beta-lactamase, encoded by one single plasmid, which is responsible for the resistance of this strain to most beta-lactams.