| Objectives:Enterococcus widely exists in mammalian intestines and natural environment.Among the elderly and patients with low immune function,Enterococcus is considered to be the second to third most common hospital pathogen that endangers life.Linezolid is mainly used to treat infections caused by multi-resistant gram-positive bacteria.The emergence of linezolid resistant enterococci has weakened the efficacy of linezolid in treating VRE and MASA.Obtaining optrA gene is one of the main mechanisms leading to linezolid resistance of enterococci,which brings great difficulties to clinical anti-infection treatment.Therefore,it is urgent to carry out research on the diffusion mechanism of linezolid resistant enterococci resistance gene.In this study,the transmission mechanism of linezolid resistant Enterococcus faecalis with optrA gene positive collected from our hospital from September 2018 to July 2022 was studied,and the antibiotic resistance rate,antibiotic resistance gene carrying rate and clone transmission of linezolid resistant Enterococcus faecalis were obtained by means of MLST typing,antibiotic sensitivity test,splicing test and whole-genome sequencing.The purpose of this study is to clarify the transmission mechanism of optrA gene in linezolid resistant Enterococcus faecalis and provide theoretical and scientific basis for clinical treatment and prevention and control of antibiotic resistant bacteria.Methods:1.Non-repeated enterococci clinically isolated in the Second Affiliated Hospital of Kunming Medical University from September 2018 to July 2022 were collected,and the collected strains were frozen in a refrigerator at-80 ℃.39 strains of Enterococcus faecalis with linezolid MIC ≥ 8 μ g/m L were determined by MALDI-TOF MS and micro broth dilution method as experimental strains.2.Seven housekeeping genes of Enterococcus faecalis(aro E,gdh,gki,gyd,pst S,xpt,yqi L)were amplified by polymerase chain reaction,and the positive products were sequenced and verified.The ST typing and clonal transmission of linezolid resistant Enterococcus faecalis with optrA gene positive were analyzed by Pub MLST online database.3.Through antibiotic sensitivity test and whole-genome sequencing,we can understand the antibiotic resistance phenotype and antibiotic resistance genotype of linezolid resistant Enterococcus faecalis with positive optrA gene,and analyze the horizontal transmission mechanism and genetic environment of optrA gene through splicing experiment,so as to provide a theoretical basis for antibiotic resistance prevention and control and rational use of clinical antibiotics.Results:1.From September 2018 to July 2022,a total of 39 strains of Enterococcus faecalis with a MIC ≥ 8 μ g/m L of linezolid determined by MALDI-TOF MS and micro broth dilution method were collected as experimental strains.The sample sources include: 22 strains of urine(57%,22/39),7 strains of drainage fluid(18%,7/39),4 strains of wound secretion(10%,4/39),2 strains of pus(5%,2/39),2 strains of blood(5%,2/39),2 strains of bile(5%,2/39);Departments are mainly distributed in urology and ICU.Linezolid resistant Enterococcus faecalis showed multiple antibiotic resistance,and the antibiotic resistance rate to tetracycline,minocycline,erythromycin,clindamycin and chloramphenicol was higher,all above 80%.Low resistance rate to furantoin,penicillin,ampicillin,vancomycin and teicoplanin.2.Thirty-nine strains of linezolid resistant Enterococcus faecalis carried 18 kinds of antibiotic resistance genes,the highest rate of tetracycline-resistant gene tet(M)was 97.4%(38/39),followed by the rate of chloramphenicol-resistant gene fex A was92.3%,and the rate of optrA gene was 76.9%(30/39).The MLST typing of 30 strains of optrA gene-positive linezolid resistant Enterococcus faecalis showed that there were 21 ST types,ST16 is the ST type with the highest proportion of optrA gene positive linezolid resistant Enterococcus faecalis,accounting for 30.0%(8/30),followed by ST585,accounting for 10.0%(3/30).Homology analysis shows that there is no hospital clonal transmission.3.Ten strains of linezolid resistant Enterococcus faecalis with optrA gene positive were selected as donors and Enterococcus faecalis JH2-2 as receptors for the conjugation experiment.Only three strains were successfully conjugated,with a conjugation success rate of 30%(3/10).The transconjugants showed fourfold increases in the MICs of linezolid compared with recipient strains.The antibiotic resistance gene analysis of Enterococcus faecalis resistant to linezolid with optrA gene positive showed that the co-localization of optrA genes and fex A,erm(A)could be transferred to the recipient bacteria with the plasmid,resulting in the resistance of the recipient not only to linezolid,but also to erythromycin and chloramphenicol.Conclusions: The optrA gene is the main resistance mechanism of linezolid resistant Enterococcus faecalis,which can be transmitted at the intra-specific level by splicing.Linezolid resistant Enterococcus faecalis can carry multiple antibiotic resistance genes at the same time,which is manifested as multiple antibiotic resistance.The optrA gene can be transferred horizontally with other antibiotic resistance genes,such as fex A and erm(A),and the horizontal transfer can carry multiple antibiotic resistance genes at the same time,Causing the spread and even outbreak of antibiotic-resistant bacteria.ST16 is the main ST type of linezolid resistant Enterococcus faecalis in our hospital.Homology analysis shows that there is no clonal transmission. |
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