Multidrug Resistance Associated Mechanism In Clinical Isolates Of Pseudomonas Aeruginosa

Objective: To study the mechanism of multidrug resistant Pseudomonas aeruginosa.Methods: Whonet software was used to screen multidrug resistant isolates of Pseudomonas aeruginosa from clinical isolates. E-test and EDTA disc potentiation test were conducted to screen Metallo-β-lactamase positive strains, PCR was used to detect the resistant genes of MBL. Induction test for resistance of Levofloxacin and Meropenem was performed by multiple-step method in 3 strains of P. aeruginosa. The induced resistant strains and the multidrug resistant strains were measured by detecting the MICs using microdilution method and CLSI standards, the MICs were checked with and without Efflux Pump Inhibitors (Phe-Arg-β-naphthylamide). Based on the variety of MICs for selection of the positive phenotype strains, PCR was used to detect MexAB-OprM efflux pump genes. RT-PCR was used to test the levels of mRNA for oprM gene, and DNA sequencing was adopted to analyze the resistance-related mexR gene. AmpC was screened by Cefoxitin, Ceftazidime, Cefotaxime discs and these discs with APB. ESBLs producers confirmated according to the criteria of guidelines of CLSI. Imipenem and multisubstrates were used to detect the inducible AmpC β-lactamase, PCR was used to detect genes of AmpC and ESBLs. Result: 70 multidrug resistant P. aeruginosa strains were screened from 850 clinical isolates by Whonet software from October 2004 to October 2005. 52 strains of 70 P. aeruginosa were shown to produce Metallo-β-lactamase by the phenotype method. Among them, 37 and 3 strains showed specific bands of imp, vim gene, respectively, and 1 strain showed spm gene. 3 susceptible strains were resistant to Levofloxacin and Meropenem by induced test, and PCR result showed specific bands of efflux pump genes. 46 strains were positive in phenotype tests,among them, specific bands of oprM, mexB and mexR genes were deteced simultaneously from 42 strains, but, 2 strains had no efflux pump genes. 58 strains had oprM, mexB and mexR genes in 70 strains, and 10 strains possessed no efflux pump genes. The result of OD for RT-PCR products showed that 3 mutidrug resistant strains and 1 induced resistant strain increased their levels of mRNA compared with PAO1. DNA sequencing showed 2 strains were 100% homology and 1 strain had a 2bp (AT) insertion mutations in 434 position of the multidrug resistant strains. 1 indced resistant strain showed a C to T substitution at nucleotide 222 resulting in a nonsence mutation and a 1bp (A) insertion at nuclenotide 436. 1 strain and 52 strains showed ESBLs and AmpC positive phenotype. Among them, the specific band of dha (1 strain) and tern (1 strain) were detected. There were no shv, ctx genes. From the 52 AmpC positive strains of P. aeruginosa, 33 strains produced the inducible AmpC β-lactamase. The strains with inducible AmpC β-lactamase using cefotaxime, piperacillin, aztreonam, cefepime, ceftazidime, cefotaxime/clavulanate, ceftazidime/ clavulanate as substrate were 4, 22, 24, 8, 10, 2,20, respectively, inducible AmpC β-lactamase were not found in 18 strains. 57 strains showed two or more different genes from 70 multidrug resistant isolates of P. aeruginosa. Conclusion: Resiatance to microbial agents of P. aeruginosa in 301 hospital is related to genes of tern, dha, imp, vim, oprM, mexB and mexR. These genes and the active efflux pumps contribute to the multidrug resistance in P. aeruginosa.