bla(VIM-2) cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital

Abstract

We investigated the phenotypic and genetic properties of metallo-beta-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 beta-lactamase, a class B enzyme previously found only in P. aeruginosa isolates from Europe. VIM-2-producing isolates of Pseudomonas putida were also detected. Resistance was transferable from both these species to P. aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing P. aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P. putida isolates were diverse. Sequencing showed that the bla(VIM-2) genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a P. aeruginosa isolate had bla(VIM) located downstream of a variant of aacA4. bla(VIM) also lay in a class 1 integron in a representative P. putida strain, but the organization of this integron was different from that sequenced from the P. aeruginosa strain. In conclusion, the metallo-beta-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance.

FIG. 1.

Schematic presentation of PFGE patterns of XbaI-digested genomic DNA of bla_VIM-2-positive isolates of P. aeruginosa. M in lane 1 is a size marker (a chromosome of Saccharomyces cerevisiae). The numbers of isolates with serotypes O:11, O:12, and OPA are shown under each PFGE profile, and total numbers are in parentheses. OPA, O polyagglutination; NT, serotype not tested.

FIG. 2.

Schematic structure (not to scale) of integrons in isolates of P. aeruginosa YMC 95/1/704 and P. putida YMC 97/8/322, which had approximate sizes of 5.6 and 3 kb, respectively. The ORFs are boxed. The names of unknown orfs “i,” “ii,” and “iii” are arbitrary. Arrows indicate their transcriptional orientation. orf “ii” had a reversed orientation. Primers used for sequencing are shown under the integron structures (see text for primer sequences).