Methicillin-resistant Staphylococcus Aureus Phenotype And Genotype Analysis Of Resistance

Objective:This study will investigate that the mechanism of multidrug resistance of methicillin-resistant Staphylococcus aureus (MRSA) and its nature of drug resistance. The clinical-isolated MRSA will be established for finding new targets of the drug effectiveness through the phenotype and genotype. This study potentially optimizes the clinical use of antibiotics for treating MRSA infection and preventing the expansion of restraint strains.Methods:(1)152strains of Staphylococcus aureus were collected in the period of January2012to December2012from the laboratory of the First Hospital of Shanxi Medical University, the Second Hospital of Shanxi Medical University and Shanxi People’s Hospital. All the strains were identified as Staphylococcus aureus using BioMerienx VITEK-Ⅱ bacteria identification system and51of these strains were further identified as MRSA using the K-B (Kirby-Bauer) agar disk diffusion method according to the operation rules of the the United States National Clinical Laboratory Standards Institute (NCLSI) and BioMerienx VITEK-Ⅱ bacteria identification system. The resistant phenotype and the drug sensitivity test of51strains of MRSA were also measured.(2) The minimum inhibitory concentration (MIC) of vancomycin was tested by using E-test card.(3) The extracted DNA of the51MRSA strains were analysed by using sequence specific primers polymerase chain reaction (PCR) to examine the genes of mecA, mecI and mecRl.(4) The DNA from4specific and representative strains was further amplified using PCR methods and the PCR products were examined for gene sequencing.(5) Analysed the resistance gene island (SCCmec) typings of MRSA by multiplex PCR.(6) We also investigate the virulence genes of MRSA by using multiplex PCR.Results:(1) All the MRSA strains are highly resistant to the drugs of beta-lactams, and the resistance rate was up to100%. The rates of MRSA resistant to ciprofloxacin, levofloxacin, clindamycin, erythromycin, tetracycline, teicoplanin, sulfamethoxazole, fosfomycin, linezolid, rifampicinand vancomycin are8.2%,86.3%,86.3%,84.3%,82.4%,0%,15.7%,29.4%,0%,39.2%, and0%.(2) All the MRSA strains were sensitive to vancomycin.(3) The mecA, mecl and mecR1gene were100%detected in the whole experimental strains.(4) Gene sequencing shows that the homology of the experimental strains and the strains from database are overlapped more than97%.(5) The SCCmec typings of the major experimental strains are Type II and III and some strains might be the new subtypes of Type II.(6) Detection of virulence genes shows that the presence rate of femA gene, enterotoxin gene see, enterotoxin gene sed, enterotoxin gene sec, epidermal stripping gene etb and toxic shock syndrome toxin gene tst in throughout strains are100%,76.5%,47.1%,5.9%,49%,5.9%.43.1%(22/51) strains are carried two kinds of enterotoxingene,39.2%(20/51) strains are carried two kinds of toxin gene,5.9%(3/51) strains are carried three kinds of toxin gene.Conclusion:(1) The resistance to beta-lactam of the whole collected MRSA100%, but these experimental strains are sensitive to vancomycin, teicoplanin and linezolid,but to other drugs. With the drug resistant trend seriously, we need to standardize the management to the efficacy of antibiotics that can delay the process of drug resistance.The monitoring and clinical report of the MIC are required to be intensified for the common drugs including vancomycin, teicoplanin, linezolid and others, pay attention to patients with MRSA who were poor efficacy to vancomycin.(2) Through gene amplification and sequencing of mec gene, the predominant type is A in this region. Homology of the experimental strains and the norm strains was more than97%and the homology was high. The larger variant strains were not appeared.(3) Some new subtypes are discovered by using multi-PCR technology in MRSA SCCmec types of51strains. This does not only prove the local MRSA SCCmec types or subtypes, but also illustrate the trend and local MRSA multiple drug resistance status and futher provid theoretical basis for effective prevention strategies, treatment measures, controling MRSA spread and outbreaks. Furthermore, this study would provide the theoretical evidence for for the use of multiple drug resistance.(4) The detection of virulence gene shows that some local strains are highly drug-resistant and the strains which involves the latest discovered SCCmec subtypes can resist four types of antibacterial drugs in two classifications. Two toxin genes can be detected in the same strain. The resistance degree is serious and the multi-drug resistance pattern is complex.