Research Of Vancomycin Resistant Enterococcus Faecium In Tracing And Clinical Application

Enterococcus faecium is one of the most important pathogens of nosocomial infection,which can cause multiple parts of human body infection,including urinary tract infections,blood flow system infections,endocarditis,intra-abdominal and pelvic infections,skin and skin structure infections and rarely central nervous system infections.The Enterococcus faecium harbour intrinsic resistance to cephalosporins,clindamycin,trimethoprim-sulfamethoxazole and low-level resistance to aminoglycoside antimicrobial agents and acquired resistance to glycopeptide antimicrobial agents,so that clinical treatment is very difficult to the multiple resistance Enterococcus faecium infection selected antimicrobial agents are extremely limited.The trend of vancomycin resistant Enterococcus faecium(VREfm)is increasing every year and the situation is not optimistic.The study was based on the drug resistant phenotype,genotype,resistance transposon structure,multilocus sequence typing and whole genome sequence of VREfm to study its genetic background and related drug-resistance mechanism which are great significance to the prevention and control spread of the bacterium.Objective: To investigate the drug resistance characteristics of 43 strains of VREfm isolated from the clinical specimens of affiliated hospital of southwest medical university and hospital in Chengdu from January 2015 to July 2017,and to provide evidence for clinically rational use of antimicrobial agents;To analyze the drug resistance gene,resistance transposon structure and multilocus sequence typing of 43 strains of VREfm to study genetic background.To analyze the basic characteristics of SY1,LY19,LY22 three strains of VREfm,and to study their related drug resistance mechanism.Methods:(1)43 strains of VREfm were Collected from January 2015 to July 2017 in affiliated hospital of southwest medical university and Chengdu in clinical isolates.Re-identification of strains using MALDI-TOF MS and 13 kinds of antimicrobial drug resistance were detected by VITEK2-Compact.The MIC value of vancomycin and teicoplanin was detected by broth microdilution method,and the drug resistance phenotype was determined.(2)Detection of vancomycin-resistant genes van A,vanB and vanD in all strains by PCR and sequencing techniques.The resistance transposon structure analysis through primers overlapping PCR method and the analysis of all strains Sequence type(ST)by using the Multilocus sequence typing(MLST).(3)Through the second generation high throughput sequencing whole genome sequencing of SY1?LY19?LY22 of VREfm,mosaic,assembly,gene prediction and gene annotation are done,resistance genes and orthologous gene are comparative analysis in tracing.Results:(1)43 strains of VREfm to penicillin,ampicillin,ciprofloxacin,levofloxacin,erythromycin and clindamycin drug resistance rate were 100%,the linezolid,tigecycline,quinupristin-dalfopristin,streptomycin high level,tetracycline,gentamicin high level,nitrofurantoin resistance rates were 100%,100%,97.7%,88.4%,83.7%,44.2%,4.7% respectively.The MIC value of vancomycin and Teicoplanin were higher(VAN?64 ?g/ml,TEC?32 ?g/ml).(2)the test results of drug resistance gene showed that 43 strains of VREfm were vanA genotypes.The Tn1546 structure were divided into five types,of which the type B was the highest proportion up to 79.1%(34/43),the remaining type C was 9.3%(4/43),the type D was 7.0%(3/43),type A was 2.3%(1/43),and the type E was 2.3%(1/43),and IS1216 V reverse insertion sequence in Type B and C.MLST results showed seven types and was ST17,ST78,ST203,ST363,ST555,ST1392,ST1394,respectively.ST17 accounted for 74.4%(32/43),ST78 accounted for 14%(6/43),and the others were 1 strain for each ST.seven STs belonged to CC17,ST1392 and ST1394 were newly discovered ST.(3)sequencing results showed that the SY1 genome contained 2435331 bp bases,and G+C bases accounted for 38.5%.a total of 2909 coding genes,Among them,24 drug-resistant genes,257 unique gene family numbers;LY19 genome contained 2567058 bp bases,and G+C bases accounted for 38.3%.A total of 3135 coding genes,Among them,27 drug-resistant genes,330 unique gene family numbers;LY22 genome contained 2516184 bp bases,and G+C bases accounted for 38.4%.A total of 3078 coding genes,among them,29 drug-resistant genes,323 unique gene family numbers.LY19 contains tetO ribosomal protection protein gene,aad(6)aminoglycoside nucleotidyltransferase gene and AAC(6')-Ie-APH(2'')-Ia aminoglycoside bifunctional enzyme gene,LY22 contains tetK efflux pump gene,AAC(6')-Ie-APH(2'')-Ia aminoglycoside bifunctional enzyme gene.Conclusions:(1)43 strains of VREfm are multiple resistant strains and belong to vanA phenotype.linezolid,tigecycline,quinupristin-dalfopristin are the first choice for the treatment of VREfm.(2)the 43 strains of VREfm were vanA gene type,and the IS1216 V reverse insertion sequence located in some resistance transposon structure.all isolates belonged to CC17,ST17 and ST78 are the main ST.(3)tetO and tetK are the genes that caused tetracycline resistance,AAC(6')-Ie-APH(2'')-Ia and aad(6)are the genes that caused gentamicin high level and streptomycin high level resistance,respectively.