In Vitro Susceptibility Test Of Vancomycin Combined With Other Drug For MRSA And Detecition Of SCCmec Genotyping

ObjectiveMethicillin-resistant Staphylococcus aureus（MRSA）is a common pathogen in infection.Vancomycin,as the first choice drug for MRSA infection,has been widely used in clinical practice,resulting in a high degree of drift in the minimum inhibitory concen-tration（MIC）.Therefore,a drug-combined strategy was employed to reduce vancomycin use,narrow the mutation selection window（MSW）,and reduce the production of resistant strains.Polymerase chain reaction（PCR）is recognized as the"Gold Standard"of detection of MRSA.SCCmec typing of MRSA by PCR can be used to explore the main types of MRSA in this region,providing the basis and countermeasures for clinical treatment of MRSA infection and prediction of the prevalence of resistant strains.Materials and MethodsFrom January 2018 to November 2018,83 strains of MRSA（excluding the strains repeatedly isolated from the same patient）isolated from various types of samples submitted for examination in the outpatient department and inpatients of Linyi People’s Hospital were collected.1.The MIC values of standard bacterial ATCC 43300 against MRSA and 83 clinically isolated MRSA strains were determined by broth microdilution and checkerboard dilution for vancomycin and piperacillin/tazobactam alone and in combination,and the partial inhibitory concentration index（FICI）was calculated.The time-kill curve of standard bacterial ATCC 43300 of MRSA was plotted based on colony counts at different drug concentrations and time points.2.The anti-drug mutation concentration（MPC）of standard bacterial ATCC 43300 and83 clinically isolated MRSA strains of vancomycin and piperacillin/tazobactam alone and in combination was determined by agar dilution method,and the selectivity index（SI）was calculated.3.The mec A gene,fem A gene and SCCmec genotype of 83 clinically isolated MRSA strains were studied by PCR amplification and gel electrophoresis,and the PCR amplification products of 2 positive strains of SCCmec genotype were randomly selected for sequencing.Then,BLAST was compared with gene sequence published in Gene Bank database（http://www.ncbi.nlm.nih.gov/BLAST）.Results1.For the MRSA standard strain ATCC 43300,MIC values of vancomycin and piperacillin/tazobactam alone respectively were 0.5μg·ml^-1 and 128/16μg·ml^-1;MIC values when combined were 0.125+32/4μg·ml^-1.The MPC values respectively were16μg·ml^-1 and 4096/512μg·ml^-1;The MPC values when combined were 2+521/64μg·ml^-1.After combined treatment,the MIC value of each antibiotic was significantly lower than that of MPC strain,and both MSW_VAN and MSW_TZP were narrowed,the SI_VANand SI_TZPwere reduced by 2 times,the FICI value was 0.5,showing synergistic antibacterial effect.The time-kill curve experiment showed that when the drug concentration used alone exceeded the MIC value,the two drugs had certain antibacterial activity.But when the drug was used in combination,the drug with 1/2×MIC showed some antibacterial activity,and the antibacterial effect was significantly higher than that when the drug was used alone（P<0.01）.2.For 83 strains of clinical isolates of MRSA,the MIC values of vancomycin and piperacillin/tazobactam were in the range of MIC_VAN(0.25～2μg·ml^-1)and MIC_TZP(8/1～512/64μg·ml^-1)respectively.MIC values in combination ranged from MIC_VAN(0.0625～0.5μg·ml^-1)and MIC_TZP(4/0.5～256/32μg·ml^-1).The MPC values of MPC_VAN(4～64μg·ml^-1)and MPC_TZP(128/16～8192/1024μg·ml^-1).The MPC values in combination were in the range of:MPC_VAN(0.5～8μg·ml^-1)and MPC_TZP(16/2～1024/128μg·ml^-1).After combined use,the MSW_VAN and MSW_TZP were narrowed,the SI_VAN and SI_TZP were were significantly reduced（P<0.01）.3.For 83 strains of clinical isolates of MRSA were tested by PCR.The results showed that the positive rate of mec A gene and fem A gene in MRSA was 100%（83/83）.Among 83MRSA isolates,the positive rate of SCCmec II was 13.25%（11/83）.The detection rate of SCCmec III type was 9.64%（8/83）;The positive rate of SCCmec IVa was 72.29%（60/83）.The detection rate of untyped strains was 4.82%（4/83）,and it could be found that the majority of MRSA infections in this region were SCCmec IVa type CA-MRSA.Conclusions1.In vitro antibacterial activity of vancomycin in combination with piperacillin/tazobactam against MRSA,mainly manifested as synergistic effect.2.The MIC,MPC and SI value of vancomycin combined with piperacillin/tazobactam on MRSA were significantly decreased and the MSW was narrowed,which could effectively control the production of resistant mutant strains.3.The current infection of MRSA in this region is mainly the infection of CA-MRSA type SCCmecIVa.