Study On Dissemination Of Linezolid-resistance And Investigation Of Risk Factors Associated With Linezolid-resistant Enterococcus Faecalis

Objective:The ABC transporter OptrA mediates resistance to oxazolidinones（linezolid and tidazolamide）and amido alcohols（chloramphenicol and florfenicol）.Based on the existing research results of the subject,we speculate that the horizontal transfer of drug resistance genes may be an important reason for the high carrying rate of optrA gene,so we explore the mechanism of optrA gene transmission.Then we investigated the risk factors of linezolid-resistant Enterococcus faecalis infection in the First Affiliated Hospital of Chongqing Medical University from 2014 to 2018.Methods:1.S1-PFGE and Southern-blot were used to analyze the location of optrA of 44 strains of linezolid-resistant Enterococcus faecalis.The filter-membrane conjugation experiment was used to determine whether the resistant genes could be transferred horizontally,and the successfully conjugated strains were performed to replicon plasmid typing.2.The complete genome of the strain P10748 was extracted and sequenced using the Illumina MiSeq platform.SeqMan Pro was used to assemble sequencing fragments.Gene prediction and annotation were supplemented with GeneMarkS,RAST,ORF Finder and BLAST.The genetic environment of multiple similar plasmids was compared by SnapGene mapping.3.LRE（MIC≥8mg/L）was collected from our hospital from 2014 to2018 and case-control retrospective analysis was used to investigate the risk factors of LRE infection.Results:1.Pulse field gel electrophoresis and DNA hybridization showed that26 strains（59%）of optrA carried on the plasmid,and optrA existed on the chromosome of 18 strains（41%）.Plasmid conjugation experiments showed that among the 26 strains of antibiotic-resistant bacteria where optrA was located on the plasmid,88.5%（23/26）were successfully transferred,but none of the optrA in the chromosome（0/18）was transferred.The plasmid typing test showed that the rep₉ plasmid（pCF10 prototype,sex pheromone plasmid）predominated（18/23,78%）.2.The sequencing of the complete plasmid genome of P10748 showed that resistance genes（optrA and fexA）and multiple sex pheromone-responsive genes were present in the plasmid.In the isolates carrying optrA,three key sex pheromone response genes（prgA,prgB,and prgC）were detected.The clump assay showed that varying degrees aggregation was detected in most strains.3.A total of 1902 Enterococcus faecalis were identified in our hospital from 2014 to 2018,of which 91（4.8%）LRE isolates were collected.Transfer from external hospital,age,malignancy,tracheal intubation and previous treatment of fluoroquinolone are independent risk factors for LRE infection.Conclusions:1.Gene optrA is the most important linezolid resistance mechanism in Enterococcus faecalis.2.Linezolid resistance mediated by optrA in Enterococcus faecalis can be widely transmitted by sex pheromone plasmid transfer.3.External hospital transfer,malignancy,tracheal intubation,and antibiotics previously treated were independent risk factors for LRE infection.