Study Of Metallo-beta-lactamase And Corresponding Integron In Pseudomonas Aeruginosa

Objective Investigate and analyze occurrence of metallo-beta-lactamase and corresponding integron in clinical isolates of Pseudomonas aeruginosa for exploring methods to supervise these resistance status.Methods 1) Pseudomonas aeruginosa strains isolated from Renmin Hospital of Wuhan University during Jan 2002 to Jun 2004 were collected and identified by VITEK 32 system. The minimal inhibitory concentrations (MICs) of them to 10 antibiotics, including Imipenem, Piperacillin, Cefotaxime, Ceftazidime, Cefepime, Aztreonam, Ciprofloxacin, Gentamycin, Amikacin and Chloromycetine were performed by agar dilution method. 2) Clinical isolates with reduced susceptibility to Imipenem and/or Ceftazidime were subjected to PCR (Polymerase Chain Reaction) analyses with primers specifically target to bla_VIM and bla_IMP genes for detecting metallo-beta-lactamase genes maybe harbored. 3) The inhibitor-potentiated disk diffusion test using substrate-inhibitor combination (Imipenem and EDTA, Cefatazimide and thiolactic acid) was established and the results were evaluated with those of PCR. 4) Integron corresponding to metallo-beta-lactamase were detected and analyzed by PCR-mapping and subsequential DNA sequencing approach.Results 1) Among 567 isolates collected, 102 (18.0%) of them resisted to Imipenem, while 143 (25.2%) for Cefatazimide. 2) 11 isolates get positive result with bla_VIM primers in PCR, and one of them was confirmed being bla_VIM-2 by DNA sequencing. 3) Compared with PCR, Imipenem-EDTA disk diffusion test showed high sensitivity (90.9%) and high specificity (99.3%) when adopted cut-off value of 7.0mm and with EDTA of 0.5μmol/disk. 4) Of 102 Imipenem resistant isolates, 42 (41.7%) carried class I integron. While for the metallo-beta-lactamase producer, all 11 isolates did. PCR-mapping showed that integrons is highly correlated with metalla-beta-lactamase genes. PCR-mapping products of one isolate named IRPA2 were subjected to DNA sequencing. The result gave a novel integron structure. Besides bla_VIM-2, it also harboreda new aacA4 allele, which encoding aminoglycoside 6'-N-acetyltransferase, conferring resistance to aminoglycoside agents. 5) Isolates producing metallo-beta-lacatamase possed wide resistance profile. They resisted to most beta-lactams and even Ciprofloxacin, Gentamycin, and Chloromycetine. Only were they still sensitive to Aztreonam, Amikacin and Cefepime.Conclusion Pseudomonas aeruginosa isolates produce metallo-beta-lacatamase with high rate (10.8%). It is right time to effective monitoring and control. And Imipenem-EDTA disk diffusion test is a convenient and credible method for doing that. While integron-gene cassette system is strongly correlated with metal lo-beta-lactamase, detection of integron with PCR-mapping and other strategies could give useful epidemic information, and it also provides possibility to detect other antibiotics resistant gene at one time.