Identification And Function Analysis Of The Novel Transposon In Enterococci

In recent years,with the frequent use of antibiotics,the emergence of multidrug resistant enterococcus has increased gradually,now enterococcus has become one of the main pathogens of nosocomial infection.However,the transposon plays an important role in the formation of multi-resistant enterococcus.Transposon is a kind of mobile genetic elements,witch can confer antibiotic resistance genes and virulence genes,resulting in the antibiotic resistance and virulence of the pathogen transfer and disseminate rapidly,then that will accelerate the adaptive evolution of pathogenic bacteria.So researching the horizontal transfer of antibiotic resistance genes mediated by transposon in enterococcus has important clinical significance.In this study,86 enterococcus strains separated from pigs and humans were used for detecting the bacitracin zinc resistant gene bcrB,tetracycline resistance gene tet(M),and we also used the technology of plasmid conjugation and electricity transformation,southern hybridization,whole genome sequencing etc,to discuss the relationships of bacitracin zinc resistant gene bcrB and tetracycline resistance gene tet(M)with transposons.The results of detection of bcrB showed that it can exist in pig and human enterococci strains,and the prence of it in pig and human is 47.4%(27/57)and 13.8%(4/29),respectively.Compared the prence of bcrB in pigs with that in humans,the former was much higher than the latter,this may be largely because bacitracin,especially licensed as the feed additive in swine industy,was more widely used compared with the human medicine practice.Conjugation experiments showed that bcrB can transfer and the transfer frequencies of it in human and pig are(4.5 ± 0.3)×10-3 and(6.0 ± 0.5)×10-5,respectively.Multilocus sequence typing(MLST)following the protocols(http://www.mlst.net/)indicated that there was no genetic similarity between the bcrB-carrying strains derived from human and pigs,with an exception of E.faecalis ST116,which was shared by both human(H7)and swine(P33)strain,so the problem of cross-species transmission in pig and human enterococci shoud be concern.The genetic envirment of bcrB revealed that it located on a novel composite transposon which contains repeat suqences at both ends and target site duplications squences is TTAGGAA.The position of bcrB shows that it located on a plasmid of 56.5 Kb,which contained two multi-antibiotic resistant gene clusters : erm(B)-aadE-spw-lsa(E)-lnu(B)and aad E-sat4-aphA-3.The gene cluster erm(B)-aad E-spw-lsa(E)-lnu(B)seemed to originate from enterococcal strains and had been acquired by methicillin-resistant S.aureus(MRSA)/methicillin-susceptible S.aureus(MSSA)CC9 and CC398 several times on different occasions in Europe and Asia,while the other gene cluster aadE-sat4-aphA-3 was firstly detected in E.faecium and ever had been present in staphylococcal strains and even in Gram-negative organism like Campylobacter coli,highlighting the potential and importance of E.faecium acting as donors of antimicrobial resistance genes for other pathogens.The detection rate of etracycline resistance gene tet(M)is 21%(12/57).Conjugation experiments showed that the gene of tet(M)could transfer,and the transfer frequencies is 3.5×10-5.The product that amplificated from tet(M)to tndx is 5.2kb larger than anticipation,and further Whole genome sequencing obtained a novel conjugative transposon,named Tn6247.Sequence analysis revealed that a 5.2kb fragment containing the tet(L)-pre/mob-repU-IS1216E gene was integrated,but a 2.6 Kb fragment containing the group ? intron was deleted in Tn6247,as compared with Tn5397.Tn6247 completely inserted into a plasmid-associated fic gene by creating the direct target duplications of 10 bp(5'-GAAGGAAATG-3')in both ends.In conclusion,a novel conjugative transposon,designed Tn6247,and a novel Zinc bacitracin resistance composite transposon which located on a mult-drug resistance plasmid were found in this study.The identification and analysis of novel transposon and mult-drug resistance plasmid will provide some theoretical explanations and basises for quickly appearring of mult-drug resistance enterococcus.