Study Of Metallo-beta-lactamase Producing Status In Clinical Isolates Of Gram-negtive Bacteria In Hunan

Objective (1) To survey the distribution and drug resistance of carbapenem-non-susceptible Acinetobacter baumannii and Pseudomonas aeruginosa in xiangya hospital.(2) To investigate epidemiological characteristics of metallo-beta-lactamase(MBL) in Gram-negative bacteria in Hunan.Methods (1) Non-duplicate clinical isolates of carbapenem-non-susceptible Acinetobacter baumannii and Pseudomonas aeruginosa in xiangya hospital from January1,2011to June30,2011were collected. Identification of isolates was carried out by automatic microorganism analytical system Vitek-2. Antimicrobial susceptibility was tested by Kirby-Bauer method and the data were analyzed by WHONET5.4software.(2) Totally482non-duplicate clinical isolates from15hospitals in Hunan during January1,2011to June30,2011were collected. EDTA-synergy test and combination disk diffusion were used for phenotypic screening of MBL. And the positive strains were further confirmed by E-test MBL strips.(3) PCR were preformed for detection of MBL genes including IMP, VIM-2, SIM, SPM, GIM and NDM-1respectively, in strains that had a positive phenotype of MBL. The positive products of PCR were sequenced and performed BLAST assay.(4) Conjugation test was used to detect if MBL gene can transfer through plasmids. ERIC-PCR was used for DNA typing and homology analysis in both IMP and VIM-2gene positive strains.(5) Genomic DNA from a strain of NDM-1producing K.pneumoniae was used as template for PCR amplification of the whole length NDM-1gene. Recombinant plasmid pET-28a (+)-NDM-1were constructed.Results (1) Of the193carbapenem non-susceptible A. baumannii isolated in Xiangya hospital, the resistance rates to15of19antimicrobial agents were higher than80%. Resistance rates of piperacillin, piperacillin/tazobactam and gentamicin were the highest (100%), while resistance rates to cefoperazone/sulbactam(39.4%) and minocycline (44.6%) were the lowest. The39carbapenem non-susceptible P. aeruginosa only showed higher sensitivity to polymyxin B(100%) and amikacin(41.0%), and the resistance rates of them to other antimicrobial agents were all higher than50.0%. Especially to cefotaxime the resistance rate was100%.13pan-resistant strains (only sensitive to colistin) were found including11strains of P. aeruginosa and2strains of A. baumannii.(2) Among482multidrug-resistant Gram-negative bacteria,25had a positive phenotype of MBL and23were identified of at least one MBL gene positive by PCR and sequencing. Among them,13strains of P. aeruginosa carries IMP-9MBL gene,1strains of P. aeruginosa carries IMP-1,6strains of P. aeruginosa carries VIM-2,1strain of P. putida carries both IMP-9and VIM-2, another P. putida carries VIM-2and1 strain of K.pneumoniae carries both NDM-1and IMP-4genes. There were no SIM, SPM and GIM gene positive strains.(3) The blaNDM-1gene-harboring plasmid was successfully transferred to E.coli J53. ERIC-PCR showed that there were multiple genotypes among14IMP-producing strains, while6VIM-2-producing strains belonged to the same genotype.(4) Confirmed by PCR, double-enzyme digested assessment and sequencing, recombinant plasmid pET-28a (+)-NDM-1was constructed successfully.Conclusions (1) Multidrug resistance of carbapenem-non-susceptible Acinetobacter baumannii and Pseudomonas aeruginosa was serious in xiangya hospital.(2) The major MBL-producing strains in Hunan were P.aeruginosa. IMP-9and VIM-2were two major MBL genes.(3) This study first identified NDM-1gene in K.pneumoniae in Hunan.(4)Recombinant plasmid pET-28a(+)-NDM-1was constructed successfully.