| The widespread use of antibiotics in veterinary has led to many cross-resistance genes such as optrA,poxt A and lsa(E).Enterococcus has a wide range of mobile genetic elements such as insertion sequences,transposons,and plasmids,and its ability of quickly acquiring resistant genes has been making Enterococcus becoming a "repository" and "transit station" for resistant genes.Moreover,the transfer of resistant genes from Enterococcus to other Gram-positive pathogens,such as Staphylococcus.spp and Streptococcus.spp,posing a serious threat to the health of human and animal.In this study,91 optrA/poxt A/lsa(E)positive Enterococcus were isolated and identified from farms and slaughterhouses in Nanyang,Xuchang,Zhoukou,Xinmi and other regions of Henan Province in 2017-2019.The epidemic plasmids in optrA/poxt A/lsa(E)positive Enterococcus and transmission characteristics of mobile genetic elements(insertion sequence,transposon,plasmid),involving in the horizontal transmission of resistance genes,were identified by the whole genome sequencing,plasmid replicon typing,resistance gene environment analysis,MLST,antimicrobial susceptibility testing,conjugation assay,S1 PFGE and Southern blot hybridization,electrotransformation assay,in vitro induction of linezolid,and real-time q PCR.Antimicrobial susceptibility testing results showed 91 Enterococcus were only sensitive to tigecycline,and resistant to other 11 kinds of antibiotics with different degrees.The antimicrobic resistance genotypes of 91 Enterococcus were basically consistent with the resistance phenotypes carrying in Enterococcus.The genetic environment of optrA,poxt A and lsa(E)genes showed different characteristics in 91 Enterococcus,optrA gene exhibiting a diversity of characteristics,the lsa(E)gene located on the aad E-spw-lsa(E)-lnu(B)resistance gene cluster,and the poxt A gene flanking by IS1216 E.The replicon typing showed that the replicons related to pheromone-responsive conjugative plasmid were widespread in E.faecalis(58/62),while the pLG1-like related replicon was widespread in E.faecium(20/20).In E.faecalis,pheromone-responsive conjugative plasmids could promote the horizontal transfer of optrA gene located on themselves,poxt A gene on the mobilizable plasmids,and lsa(E)gene island on the chromosome.The optrA carrying pheromone-responsive conjugative plasmid in E.faecalis could transfer intraspecies with a frequency of 1.1×10-2 。 Besides,with the help of pheromone-responsive conjugative plasmids,poxt A carrying mobilizable plasmid could not only transfer intraspecies but also intergeneric.While the intraspecific transfer occurring,poxt A carrying mobilizable plasmid were fused into the optrA pheromone-responsive conjugative in the same strain,forming a new optrA-poxt A-co-carrying plasmid through homologous sequence mediated homologous recombination.While the intergeneric transfer occurring,poxt A-(confirmed as poxt A2 by sequencing)and cfr(D)-co-carrying Inc18 plasmid could transfer between Enterococcus and Staphylococcus.Moreover,pheromone-responsive conjugative plasmid is indispensable in the intraspecific horizontal transfer of lsa(E)gene island located on chromosome by chromosome and chromosome homologous recombination.In E.faecium,pLG1-like plasmids promoted the horizontal transfer of optrA/lsa(E)located on themselves,optrA/poxt A located on the mobilizable plasmids,and optrA gene island located on the chromosome.optrA/lsa(E)carrying plasmids could occur intra-and interspecific transfer,with the help of the pLG1-like plasmids,the intra-and interspecific transfer of optrA/poxt A carrying mobilizable plasmids could occur and the optrA gene island located on chromosomes could transfer intraspecific by homologous recombination of chromosomes.Besides,the pLG1-like plasmid is indispensable in promoting the horizontal transfer of optrA/poxt A mobilizable plasmids and chromosome optrA gene islands.Moreover,the pLG1-like plasmids were fused into chromosome while occurring interspecific transfer through homologous recombination mediated by homologous sequences.In E.faecium,the poxt A mobilizable plasmid could formed by transposon Tn6657(IS1216E at both ends)in Staphylococcus inserted into plasmid p ISMMS_VER4_p6 in E.faecium.Besides,the plasmid fusion of poxt A plasmid and pLG1-like plasmid were occurred in the process of transfer,which was mediated by the transposition of IS1216 E,and the poxt A carrying plasmid could format double copy poxt A carrying plasmids by IS1216 E translocation.Moreover,the poxt A multi-copy carrying plasmids could form by IS1216 E mediated translocation in vitro exposure to linezolid.To sum up,firstly,this study found pheromone-responsive conjugative plasmid and pLG1-like plasmid were prevalent in optrA/poxt A/lsa(E)positive E.faecalis and E.faecium,respectively.Two types plasmids played an important role in the horizontal transmission of optrA,poxt A and lsa(E)genes in Enterococcus.Secondly,the role of transposition and translocation mediated by insertion sequence IS1216 E in promoting the diversification of poxt A carrying plasmid and the formation of multiple copies of poxt A gene in E.faecium was analyzed,which was confirmed that IS1216 E accelerated the horizontal transmission of poxt A gene in Enterococcus.In brief,this study clarified the transmission characteristics of optrA,poxt A and lsa(E)genes,and emphasized the role of mobile genetic elements in the horizontal transmission of resistance genes occurring intraspecific,interspecific,and intergeneric in Enterococcus,enriching the theory of bacterial resistance transmission. |
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