Multilocus Sequence Typing And Drug Resistant Mechanism Study Of Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, but due to the wide use of antimicrobials in recent years, its drug resistance has become a domestic and even a global problem, which brings great challenge to clinicians of infectious diseases. Carbapenems are taken as the last resort of treatment for multidrug resistant Gram-negative bacteria. While, the resistance of Carbapenems in Pseudomonas aeruginosa is increasing incessantly, which would render the clinicians into the situation of no drug. In order to give some reference to clinicians on rational use of Carbapenems and to establish a scientific drug resistance prevention strategy, this study analyzed the epidemiological genetic background of Carbapenem-nonsusceptible Pseudomonas aeruginosa in China by molecular typing method, and further investigated their drug resistant mechanisms.This study collected2818Pseudomonas aeruginosa isolates from22provinces and65hospitals in China from Jan.2010to Dec.2010, and tested their in vitro susceptibility to16regularly used antimicrobials by disk diffusion method. According to the susceptibility results,896Carbapenem-or Ceftazidime-nonsusceptible isolates were selected and proceeded to MLST (Multilocus Sequence Typing) analysis. DNA sequence assembling software and website were used to distribute allele type and sequence type (ST), and BioNumerics software were used to analyze the genetic diversity. In vitro antimicrobial susceptibility tests displayed that the resistance rate of imipenem (IPM), meropenem (MEM) and ceftazidime (CAZ) were23.1%,18.1%and15.3%respectively, while that of the other main β-lactam antimicrobials were among15%to25%. MLST results revealed that in896Pseudomonas aeruginosa isolates,632isolates belonged to116STs,201made up104new found STs, and the other63constituted34novel STs by mutations in one or several alleles. The primary STs were ST244, ST277, ST235and ST274, and so on. ST categories had significant correlation with bacterial number, which suggested the variety and dispersal of STs. Minimum spanning tree created from BioNumerics software also indicated that those isolates were from diverse clones.Carbapenem resistance mechanism study of627imipenem-or meropenem-nonsusceptible Pseudomonas aeruginosa isolates was conducted. All the isolates were carried on β-lactamases gene screen by PCR, after which PFGE (Pulsed Field Gel Electrophoresis) homology analysis and S1-PFGE and Southern blot hybridization were conducted for P-lactamases gene positioning. Four different imipenem or meropenem resistant phenotype isolates, i.e.137IPM-R, MEM-I/S,40IPM-I/S, MEM-R,118IPM-R, MEM-R and75IPM-S, MEM-S, mutation type of oprD gene was detected by PCR and sequencing the whole length of oprD gene. A total of35different resistant phenotype isolates were selected for efflux pumps gene expression test, i.e.9IPM-R, MEM-I/S,9IPM-I/S, MEM-R,9IPM-R, MEM-R and8IPM-S, MEM-S, qPCR (Quantitive Real-Time PCR) was used to measure the relative expression level of RND family efflux pumps. Finally the random transposon mutagenesis method was applied to investigate the potential new resistance mechanism of Carbapenem. p-lactamases screen revealed that only29of627isolates contained carbapenemase, the primary of which were metallo-β-lactamases IMP-1, IMP-25and VIM-2. S1-PFGE and Southern blot located them on plasmids. In the imipenem or meropenem resistant isolates, the oprD gene had different type of mutations, in which the amino acid mutation was the main type. qPCR reavealed that efflux pumps were only overexpressed2.8%of35imipenem or meropenem resistant isolates.Consequently, Carbapenem nonsusceptible Pseudomonas aeruginosa in China dispalyed a genetic diversity in whole, and no major clones were spreading. The primary Carbapenem resistant mechanism was the oprD gene mutation, and the carbapenemase harboring or the efflux pumps overexpression were not the main reason.