| Enterococcus is an important gram-positive opportunistic pathogen that can be widely found in soil,plants,human and animal gastrointestinal tract.At the same time,Enterococcus is considered to be a reservoir of antibiotic resistance genes that can accelerate the spread of antibiotic resistance genes.Teatment failures caused by multidrug-resistant bacteria in breeding industry not only influence the economic benefits,but also pose a great threat to human health.optr A gene was recently discovered which can mediate both amide alcohols(animal specific)and oxazolidinones(medical)resistance among bacteria.Since it was first discovered in2015,it has been reported in many species of bacteria obtained from worldwide clinical medicine,farmed animals,slaughterhouses,retail meat products.In this study,optr A-positive enterococci from different sources in a large-scale pig farm were taken as the research object to explore.In this study,flies,spiders,sewage,drinking water,fecal samples of pigs,birds and breeders were collected from a large-scale pig farm,and then enterococci were isolated and identified.The horizontal transmission characteristics and transmission mechanism of optr A gene and its mobile genetic elements in the local ecological environment under the condition of large-scale pig breeding model were studied by experimental methods of PCR detection,antibiotic susceptibility test,whole genome sequencing,genotypic antibiotic resistance analysis,MLST typing,core-genome single nucleotide polymorphism analysis,conjugation experiments,S1-PFGE and Southern blot.137 strains of Enterococcus faecalis,1 strain of Enterococcus faecium,14 strains of Enterococcus casselifavus,3 strains of Enterococcus gallinarum,1 strain of Enterococcus hirae and 2 strains of Enterococcus avium were isolated in this study.optr A gene detection showed that there were 125 strains of optr A-positive enterococci,including 106 strains of E.faecalis、1 strain of E.faecium、14 strains of E.casselifavus、4 strains of E.gallinarum and 1 strain of E.aviam.The susceptibility of optr A-positive enterococci to 10 antimicrobial agents was determined by broth dilution method.The results showed that all optr A-positive enterococci were sensitive to tigecycline and vancomycin and showed different levels of resistance to other antibiotics.The result of whole genome sequencing of optr A-positive enterococci shows that antibiotic-resistance phenotype of enterococci was consistent with the antibiotic resistance genes carried by optr A-positive enterococci,and one type of antibiotic-resistance pattern was corresponding to multiple antibiotic resistance genotypes.The results of multilocus sequence typing(MLST)of optr A-positive E.faecalis and E.faecium showed that 18 known ST types and 10 new ST types were obtained.ST691 was the most prevalent ST type,and its sources of hosts was also the most abundant.The genetic relationship analysis of optr A-positive E.faecalis showed that some strains from pig,water,bird and spider hosts of sources had similar genetic relationship,suggesting that optr A-positive E.faecalis could spread among different hosts in this farm,while the genetic relationship of optr A-positive E.faecalis from the same source were complex,indicating that optr A gene might be transferred horizontally among these strains.Genetic environment analysis of optr A gene showed that Tn6674/Tn6674-like(71/106)was the most prevalent type of optr A genetic environment.E.faecalis carrying Tn6674/Tn6674-like belongs to multiple ST types,presenting complex genetic relationship and they were distributed in all sources.Different ST types of E.faecalis obtained from different sources which carrying Tn6674/Tn6674-like were selected as donor strains for conjugation and transfermation experiments and the results showed that the range of transfer frequencies was7.3×10-8-3.5×10-4.16 S r RNA sequencing,MLST typing,overlapping PCR and antibiotic susceptibility tests were performed to verify the obtained transconjugants.The results showed that Tn6674/Tn6674-like in these strains were transferable.The wild strains and corresponding transconjugants were conducted by S1-PFGE,Southern bolt and the third generation whole genome sequencing,which confirmed that Tn6674/Tn6674-like were located on the chromosome and was horizontally transferred to the chromosome of the recipient strains by homologous recombination.In conclusion,optr A-positive enterococci were found widely distributed in this large-scale pig farm,and the condition of multiple-antibiotic resistance in optr A-positive enterococci was serious.MLST typing and core-genomes single nucleotide polymorphism analysis found that optr A-positive E.faecalis may be transmitted among different hosts in this pig farm,and environmental media such as water(drinking water,sewage),insect vectors(spiders)and birds may play an important role in the transmission of optr A gene in this pig farms.The molecular mechanism of horizontal transfer of Tn6674/Tn6674-like was revealed by optr A genetic environment analysis and transferability exploration of Tn6674/Tn6674-like. |
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