Resistance And Transmission Mechanism Of Linezolid Insensitive Enterococci

Objective:Linezolid is considered as a last resort drug in treatment of severe infections caused by multidrug-resistant gram-positive pathogens,such as vancomycin-resistant Enterococcus spp.(VRE),methicillin-resistant Staphylococcus spp.and Streptococcus pneumoniae.Although most gram-positive bacteria are remain sensitive to linezoline,drug-resistant Enterococci have been reported worldwide.Our study aims to understand the detection rate and distribution characteristics of Linezolid-nonsusceptible Enterococcus(LNSE),analyze the molecular typing of LNSE,and explore the mian resistance mechanism and molecular transmission way of LNSE,so as to provide theoretical basis for the accurate prevention and control of LNSE hospital infection.Methods:1.Detection rate and distribution characteristics of the LNSERetrospective analysis antimicrobial susceptibility testing results of clinically isolated Enterococcus bacteria from a tertiary hospital from 2018 to 2019.Antimicrobial susceptibility testing was conducted by micro-broth dilution methods to confirm the resistance to linezolid of LNSE detected by the instrument method.The WHONET 5.0 software was used to statistically analyze the detection rate of LNSE,according to the standards recommended by the Clinical and Laboratory Standards Institute(CLSI;2019).2.Analysis of the homology of LNSE and the characteristic peak of protein map Multiple locus sequence typing(MLST)and MALDI-TOF biotype mass spectrometer were used to analyze the homology of the LNSE strain.The MALDI-TOF biotype was used to analyze the specific mass peaks of the protein fingerprint of the LRE strain.3.Mechanism of LRE resistancePCR was used to detect 23S rRNA,rplC,rplD,rplV,optrA,poxtA,cfr,cfr(B)linezolid resistance genes,and gene sequencing was used to verify the amplified PCR products.4.Molecular mechanism of resistance tTransmissionPlasmid transformation experiments are used to determine whether the drug-resistant gene is spread among different strains mediated by the plasmid,which leads to the acquired drug resistance of the recipient bacteria.Results:1.From 2018 to 2019,a total of 522 strains of Enterococcus were isolated and identified in our hospital.A total of 44 strains of LNSE strains(all Enterococcus faecalis)were detected,with a detection rate of 8.4%(44/522),Among them,the detection rate of Linezolid-mediated Enterococcus faecalis(LIEfs)was 3.1%(16/522),the detection rate of Linezolid-resistant Enterococcus faecalis(LREfs)was 5.4%(28/522).The 28 LRE strains had a minimum inhibitory concentration(The Minimal Inhibition Concentration,MIC)for linezolid,which was 8-16 mg/L,and all showed low levels of resistance.44 LNSE strains are mainly distributed in departments of urology,oncology,geriatrics and hepatobiliary surgery,account for 43%(19/44),9%(4/44),7%(3/44),and7%(3/44),respectively.LNSE were main isolated from the urine,aspirate,secretions and drainage,account for 52%(23/44),18%(8/44),14%(6/44),and 5%(2/44),respectively.2.Homology analysis of LRE44 LNSE strains can be divided into 25 different ST types,with ST 16 as the main type,accounting for 20.5%(9/44),ST480,ST689,ST995,ST985 each accounting for 4.5%(2/44),the rest are of different ST types.In the homology analysis of LNSE strains,the coincidence rate between the cluster analysis results of MALDI-TOF biotype mass spectrometry MSP software and the MLST results was 66.7%.A characteristic peak appeared at 9747M/Z of the protein fingerprint of the LRE strain.This peak did not appear in Linezolid-mediated Enterococcus(LIE)and Linezolid-susceptible Enterococcus(LSE).And the standard strain ATCC29212.3.The resistance genes of 44 LNSE strains.33 LNSE strains were found to be positive for the resistance gene,which can be divided into two types:single resistance gene and double resistance gene.Single resistance gene:7 strains were positive for the optrA gene,and 10 strains had mutations in the rplD gene encoding L4 ribosomal protein(C348T,A144G).Double drug resistance gene:1 LRE strains carrying both optrA and cfr genes and 1 LRE strains carrying both optrA and poxtA genes,1 strain with optrA gene positive and 23sRNA point mutation(G2601C),4 strains were optrA gene positive and rplC gene mutation(C291T,C369T,T107A),9 strains were optrA gene positive and rplD gene mutation.There were 11 LNSE strains without any resistance genes.4.Enterococcus faecalis EF29 and EF02 successfully transferred the cfr and optrA genes to the recipient bacteria OG1RF through transformation experiments,and the linezolid MIC values of the corresponding transformants of EF29 and EF02 were higher than those of the recipient bacteria(8-16 times).Conclusions:1.The detection rate of LNSE strains in our hospital is 8.4%,mainly Enterococcus faecalis,mainly distributed in the Department of Urology and Oncology,with a high detection rate.2.The main linezolid resistance mechanism of LRE strains is to carry the optrA gene,which can be used as a marker for resistance screening.cfr gene,poxtA gene and 23sRNA gene mutations are involved in resistance,but they are not the main resistance mechanism.New drug-resistant gene phenotypes(optrA+/cfr+,optrA+/poxtA+,optrA+/23sRNA+)appeared.3.Epidemiological investigations show that most LRE cases are sporadic cases.Some strains have clonal propagation.The prevalence of LRE strains in urology has both ST 16 type and ST480 type strains as the main clonal transmission,and plasmids carrying the optrA gene mediate the horizontal transmission of drug resistance genes among different strains,leading to the prevalence of polyclonal strains.4.There are new types of MLST strains(ST975,ST976,ST978-ST981,ST984,ST985,ST987,ST990,ST991,ST995,ST 1003),and the diversity of resistant strains has increased.5.After the plasmid carrying the optrA gene and cfr gene is transferred into the recipient bacteria,the sensitivity of the recipient strain to linezolid can be reduced.