The Identification And Functional Analysis Of Novel Tn916-like Conjugative Transposons In Enterococci From Humans,Pigs And Poultry

With the widely use in veterinary clinical with antibiotics,the drug resistance of bacteria is becoming more and more important,especially there is great difficulty in clinic of some drug-resistant genes horizontal or vertical transmission.And enterococcus have been considered stores of many kinds of drug-resistant genes,which has found spread a variety of resistance genes by enterococcus(erythromycin resistance genes erm B,tetracycline resistance genes tet M,gentamicin resistant gene aac-aph D and so on).Tetracycline is now commonly used as broad-spectrum antibiotics,whose resista nce of mechanism is more serious and complex,such as major facilitator superfamilyactive efflux genes tet(L),tet(K)and the ribosome protective protein tet(M),tet(O),etc.Tet(M)is the most major and common gene among these,the main reason is that tet(M)can be transferred with the transposon Tn916/Tn1545 family between gram positi ve and negative bacteria.Enterococcus play an important role in spreading of tetracyc line resistance Once acquire tetracycline resistance gene,so the study of enterococcus resistance transmission mechanism is particularly important.In this study,we isolated 413 enterococci strains from poultry,swine and human at first,then amplified the tetracycline resistance gene tet(M)and tet(M)-int in this process.After PCR,there were some products were different from Tn916,their fragment were longer.So we selected 2 strains to do the whole genome sequencing.The result showed that the 2 strains had tetracycline resistant gene tet(M)-tet(L)and chloramphenicol resistance gene cat.By overlapping PCR,bacterial conjugation testing and cyclic intermediate amplification to analyze the epidemiology and transposon function analysis.It shows that,in different sources of Enterococci,many strains had the chain of tet(M)-tet(L)and tet(M)-tet(L)-cat,but some of these connected with transposase int.By overlapping PCR,we obtained two Tn916-like transposons,which were 21.9Kb and 25.3Kb,then we named Tn6258 and Tn6259.They were detected in enterococci from poultry,swine and human,the rates were 11.5%,7.3%,7.2% and 6.4%,0.09%,2%.Tn6258 and Tn6259 can also conjugative transfer in enterococci from poultry,swine and huaman,Except Tn6259 in Enterococci from poultry.Then we design reverse primer to amplify cyclic intermediates for Tn6258 and Tn6259.Conjugative experment and cyclic tests show that two transposons can transfer.They can transfer resistance genes between the species and others.In summary,in different sources of enterococci we discovered the new Tn916-like conjugative transposon-Tn6258 and Tn6259.Compared with Tn916,Tn6258 integrated tetracycline resistant gene tet(L);while the Tn6259 not only integrates the tetracycline resistance gene tet(L),also integrated the chloramphenicol resistance gene cat.Further experiments showed two transposons can horizontal transfer the resistance gene,It increased the difficulty of the treatment and provide some theoretical basis for resistance rapidly spread.