Detection Of Metallo-β-lactamse Genes In Multidrug-resistant Pseudomonas Aeruginosa And Its Resistance Mechanisms Against Polymyxin B

Objectives:To analyze the resistant profile against polymyxin B and other antibiotics and to detect the common metallo-β-lactamse genes in Pseudomonas aeruginosa isolated from six hospitals of zhejiang province from Jan,2006 to March, 2010; To investigate the resistant mechanism among multiple-drug resistant Pseudomonas aeruginosa strains against polymyxin B.Methods:Pseudomonas aeruginosa isolates were collected from six hospitals of zhejiang province. Antibiotic susceptibilities were determined by agar dilution method and E-test. Genes of the common metallo-β-lactamse were detected by specific PCR. Polymoxin B resistant Pseudomonas aeruginosa strains were created from by in vitro induction using step-wise increasing of polymoxin B concentration and selection process. The sequence of pmrAB abd PhoPQ of the clinically isolated strains and the induced polymoxin B resistant strains were studied by using PCR amplification and sequencing of the relevant genes. Results:1. Among the 9 antibiotics tested, the resistant rates of 437 Pseudomonas aeruginosa isolates to meropenem, ciprofloxacin and cefoperazone/sulbactam were higher than 30.0%, while the susceptible rate to polymyxin B was 93.4%;2. One of 169 strains was positive for IMP-4 gene, but none for VIM genes.3. Three polymoxin B resistant strains were induced and screened from 6 clinically isolated multi-drug resistant strains. MICs against polymoxin B of the induced strains arose from 1,1, 1μg/ml to 128,128,64μg/ml respectively, while the MICs of other antibiotics did not change.4. PCR amplification and DNA sequencing of the two-component system proteins PmrB and PhoQ genes identified mutations of G to A and T to C in pmrB gene, and a single mutation of A to T in phoQ in the induced strain NO.B919d comparing to its parent strain of NO.B919.Conclusions:1. While most clinically isolated Pseudomonas aeruginosa strains are multidrug-resistant against a wide range of antibiotics, they are still susceptible to polymyxin B.2. Among tested Pseudomonas aeruginosa strains, positive rate of metallo-β-lactamse genes is only 0.59%(1/169), of which the gene type is IMP-4.3. The resistant strains against polymix B can be induced in vitro. The resistance mechanism of the induced strains may be related with point mutation in pmrB and phoQ genes and as wellas a partial deletion of phoQ gene.