Novel genetic environment of the plasmid-mediated KPC-3 gene detected in Escherichia coli and Citrobacter freundii isolates from China

The imipenem and meropenem-resistant strains Citrobacter freundii HS70 and Escherichia coli HS510 were isolated from patients in Shanghai, China. By isoelectric focusing, PCR amplification and sequencing, these strains were each found to produce four β-lactamases: TEM-1, KPC-3, SHV-7 and CTX-M-14. A conjugation experiment and plasmid restriction digestion revealed that the blaKPC-3 gene was located on the same plasmid in both isolates. Bidirectional primer walking sequencing showed that the nucleotide sequence surrounding the 3.8 kb blaKPC-3 contained a 671-bp insertion similar to that previously characterized in China. The insertion was located between the promoter and the coding region of the blaKPC-3 gene. Susceptibility testing performed on recombinant strains carrying the blaKPC-3 gene with or without the insertion revealed that minimum inhibitory concentrations of imipenem, meropenem, cefepime, and cefotaxime for E. coli EMU-KPC3 (without insertion) were four times higher than that of E. coli EKPC3 (with insertion). The 671 bp insertion reduced blaKPC-3 expression significantly. Taken together, these results suggest that KPC-3-producing C. freundii and E. coli have begun to emerge in our hospital.