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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.

Extended-spectrum beta-lactamases (ESBLs) are an increasing cause of resistance to third-generation cephalosporins in Enterobacteriaceae. However, they have not been well studied in China. We investigated the prevalence, resistance, and probable gene type of ESBLs using MICs testing and polymerase chain reaction in 559 Klebsiellae pneumoniae and 427 Escherichia coli isolates collected from patients in Huashan Hospital from 1 January to 31 December 1999. The incidence of ESBL-producing strains was 51% among Klebsiellae pneumoniae (285/559) and 23.6% among Escherichia coli (101/427), most of which were collected from patients in intensive care unit and neurosurgical ward. PFGE showed that some epidemic ESBL-producing strains were present in the ICU, especially among ESBL-producing Klebsiellae pneumoniae. The major source of ESBL-producing Klebsiellae pneumoniae and Escherichia coli was sputum specimen (63.5%) and urine (64.3%), respectively. These strains were resistant to most beta-lactams (including the third-generation cephalosporins and monobactams) and non-beta-lactams (such as fluoroquinolones, aminoglycosides, tetracycline, and chloramphenicol), all or most ESBL producers were susceptible to imipenem, cefmetazole and beta-lactam/clavulanic acid. TEM was the main type of beta-lactamases and the CTX-M type of ESBLs was common in these isolates. Some ESBL-producing Escherichia coli and most ESBL-producing Klebsiellae pneumoniae produced more than one type of beta-lactamase. These data confirm that ESBL producers are common among hospital strains of Escherichia coli and Klebsiellae pneumoniae in China. It is important to monitor such strains closely and prevent their spread.