| Linezolid is the last line and highly effective against infections of clinically important Gram-positive pathogens exemplified by VRE and MRSA;however,in recent years,linezolid-resistant enterococci(LRE)increased constantly.The resistance mechanism of bacteria to linezolid mainly includes the mutation of V domain of 23S rRNA,and the multidrug resistance genes cfr,optrA,etc.The latest research found that poxtA is the newly discovered resistance gene,which can mediate resistance to phenicols,oxazolidinones(hereinafter referred to as PhO antibiotics)and tetracyclines,but its prevalence in swine-derived enterococci in China is unclear.This study analyzed the resistant phenotypes of poxtA-positive swine-derived E.faecium isolated from Jiangsu province and the distribution of related resistant genes.PFGE typing,S1-PFGE and southern hybridization were carried out in order to figure out the homology relationships among strains.Conjugation as well as whole genome sequencing were used to clarify the genetic environment of the gene poxtA and the specific horizontal transfer mechanism among strains.The main research contents are as follows:1.Isolation and identification,resistance analysis and detection of PhO resistance genes of poxtA-positive E.faeciumIn this part,the anal swabs collected from 13 pig farms in Jiangsu were isolated and identified,and the new oxazolidinone resistance gene poxtA was detected.A total of 68 isolates of poxtA-positive E.faecium were isolated.The antimicrobial sensitivities of 12 antibacterials and the distribution of PhO resistance genes were performed on these strains.The results showed that the resistance rates of 68 isolates to florfenicol,tetracycline and erythromycin were the highest with rates of 100%,100%and 100%.The resistance rates to other antibacterials from high to low were ampicillin(86.76%),streptomycin(72.06%),marbofloxacin(72.06%),gentamicin(69.12%),penicillin(55.88%),rifampicin(32.35%),linezolid(27.94%),fosfomycin(19.12%),respectively.All strains are susceptible to vancomycin.Further multi-drug rsistance analysis of the strains revealed that all poxtA-positive isolates were multiple drug resistant strains,mainly resistant to 7 kinds of drugs.One strain was resistant to all antibacterials except vancomycin.PCR results showed that the fexB gene was the most prevalent among the 68 strains with the positive rate of 79.41%.The positive rates of genes optrA,floR and fexA were 17.65%,13.24%,and 13.24%.The genes cfr,cfr(B),and cfr(C)were not present in any strains.This result indicates that has been widely spread in different swine farms in Jiangsu province.2.Molecular typing of poxtA-positive E.faecium and gene localization researchIn order to better understand the prevalence and transmission characteristics of poxtA-positive strains,PFGE typing was carried on 68 poxtA-positive E.faecium,and 13 linezolid-resistant strains were subjected to Sl-PFGE and Southern hybridization and MLST.The results showed that 3 out of 68 strains were failed for typing.The other 65 strains revealed 61 PFGE bands and 7 clonal groups.There was no dominant PFGE band pattern and had a diverse distribution.Among the 7 clone groups,10 strains from AK swine farm and 5 strains from HC swine farm were the two dominant clone groups.13 linezolid-resistant E.faecium from 6 pig farms had different ST types,and the number and size of their plasmids were different.In all 13 strains,the probe had hybridized to plasmids.This result indicates that E.faecium carrying poxtA has a wide range of sources,and the plasmid-carrying characteristics of the gene poxtA suggest that it has the ability to spread horizontally among different strains.3.Study on the horizontal transmission mechanism of the gene poxtATo further explore the transmission mechanism of the gene among strains,conjugation,S1-PFGE,southern hybridization,and whole genome sequencing were applied to clarify the genetic context of poxtA and the instability of plasmids harboring poxtA.The adaptive costs of two transconjugants were also analyzed.The results showed that the two strains of E.faecium were able to conjugate into JH2-2 with a frequency of 0.87×10-8 and 0.03×10-7.Compared with JH2-2,two transconjugants,named JH2-2/pC25-1 and JH2-2/pC27-2,showed moderately increased resistance to florfenicol and linezolid.The two plasmids carrying poxtA were named pC25-1 and pC27-2.Genetic environment analysis showed that these two plasmids belong to the broad-host-range plasmids of the Inc 18 family,and both contain multiple resistance genes.There are two IS1216E elements upstream and downstream of the gene.poxtA can be excised with an IS to form a circular intermediate IS1216E-poxtA,leaving a single copy of IS on the plasmid.The instability of four adjacent IS1216E elements was demonstrated by a series of PCR and inverse PCR.The results indicated that the poxtA gene and other resistance genes may be disseminated among different Gram-positive bacteria via IS1216-mediated recombination events.Phylogenetic analysis showed that the two strains belong to ST29,and were related to various hospital-related pathogens,with a potential risk of zoonotic infection.The adaptive cost indicated that only WZ27-2/JH2-2 had a certain adaptive cost.In summary,the poxtA-positive E.faecium showed different degrees of resistance to all drugs other than vancomycin,which provides a guidance for the optimal use of antibacterial in swine farms.PFGE typing indicated that there are no dominant PFGE bands among poxtA-positive isolates.This indicated that there is no obvious dominant population and correlation among the strains.Genetic context analysis showed that at the presence of conjugative plasmids,the poxtA gene and other resistance genes may be disseminated among different Gram-positive bacteria via IS 1216-mediated recombination events. |
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