Metallo-β-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme

A total of 209 clinical isolates of Pseudomonas (193 Pseudomonas aeruginosa, 10 P. putida, 4 P. stutzeri, and 2 P. fluorescens isolates) with reduced susceptibilities to imipenem and/or ceftazidime were subjected to PCR assays with primers specific for bla_IMP-1, bla_IMP-2, bla_VIM-1, and bla_VIM-2 and sequence analysis to identify the metallo-β-lactamases (MBLs) prevalent among these organisms in Taiwan; and 21 isolates gave positive results. Five isolates including two P. putida and three P. stutzeri isolates were found to carry bla_IMP-1, and six isolates including five P. putida and one P. stutzeri isolates harbored bla_VIM-2. The remaining 10 isolates were P. aeruginosa, and all were found to carry a novel variant of bla_VIM-2, designated bla_VIM-3. There are only two nucleotide differences between bla_VIM-2 and bla_VIM-3, leading to two amino acid alterations. Our findings indicate that VIM-2 and its variant have become the most prevalent metalloenzymes in Pseudomonas in Taiwan. Southern hybridization with the bla_VIM-2-, bla_VIM-3-, and bla_IMP-1 -specific probes revealed that only two VIM-2-producing P. putida isolates appeared to carry the MBL gene on plasmids. Pulsed-field gel electrophoresis showed that six VIM-3-producing P. aeruginosa isolates and two IMP-1-producing P. stutzeri isolates were genetically related, suggesting that the spread of these MBL genes in Taiwan could be due to clonal dissemination as well as genetic exchange between different clones.