Distribution Of ICE In Macrolide-resistant Streptococcus Suis Strains And Its Horizontal Transfer Mechanism

Streptococcus suis is an important zoonotic pathogen causing huge economic problems in the pig industry.It shows multiple antibiotics resistance,especially for macrolide and tetracycline antibiotics.In recent years,the horizontal transfer of antibiotic resistant genes mediated by integrative and conjugative elements(ICEs)has been paid more and more attention in the Streptococcus,but few studies have been studied in Streptococcus suis.In this paper,we test the resistance and molecular epidemiology of 109 strains of Streptococcus suis isolated in 2016,and analyze the distribution of ICEs in clinical isolates of Streptococcus suis.The core genes were classified and the junction sites of ICEs were detected and anlysised.The integrative form and circular form of ICEs were also detected by PCR by different primer groups.The horizontal spread of ICEs in Streptococcus suis was studied by conjugation experiments.Finally,we explored the effects of different selection pressures on ICE transfer in order to gain a better understanding of the mechanism by which ICE mediates the spread of antibiotic resistance.1.Resistance of Streptococcus suis to macrolide and tetracycline antibioticsThe broth microdilution method was used to detect the antimicrobial sensitivity of five antibiotics against 109 S.suis strains,which were isolated from pig farms in Jiangsu Province.The results showed that the resistant rates of erythromycin,tilmicin,clindamycin,lincomycin and tetracycline were 90.83%,90.83%,97.25%,99.08%and 92.66%,respectively.The results showed that the resistance of Streptococcus suis was prevalent and showed high levels of drug resistance.PCR was used to detect the common macrolides[erm(A),erm(B),erm(C),mef(A),and msr(D)]and tetracyclines[tet(O)and let((M)]resistance genes in S.suis clinical isolates.99%of the macrolide resistant isolates harbour erm(B),the strains showed MLSB resistance phenotype,which is the main way of the resistance of macrolide.Among them,45%carried mef(A),and 45%carried msr(D).In tetracycline resistant strains,88%carried tet(O),18.8%carried tet(M),and 8.9%carried both tet(O)and tet(M).Nine kinds of resistant genotypes were obtained in S.suis isolates,with high frequency of erm(B)+tet(O)(66/109).Streptococcus suis is highly resistant to macrolides and tetracyclines,which may be caused by the widespread use of clinical antibiotics.2.Identification of ICE and its distribution and transfer in Streptococcus suisThe distribution of ICEs in clinical isolates of S.suis was further studied.PCR was used for the detection of the core genes of ICEs,including Type ? secretion system(T4SS)gene virB4,integrase gene int and Relaxase gene.We classified core genes and detect the junction site of ICEs.The integrative form and circular form of ICEs were also detected.The results showed that 84.4%of S.suis isolates harbour virB4 gene.52.3%of the strains haboured serine family integrase gene intser,37.6%of the strains haboured tyrosine family integrase gene intTyr,20.2%of the strains containing both intser and intTyr.Only 7.3%of the strains were detected for Relaxase.Tyrosine family integrase specifically recognized a 15bp att sequence at the 3'-end of rpIL,which is TTATTTAAGAGTAAC form 5' to 3'.S.suis strains HB1011 and LP081102 were used as donors and S.suis strain P1/7RF as the recipent,and the conjugants were obtained by conjugation experiments,indicating that ICEs could be transferred horizontally in Streptococcus suis.The above results show that ICE is widely distributed in Streptococcus suis,and also shows that ICE can spread among Streptococcus suis.3.Effects of selective stress on the cyclization rate of ICE in Streptococcus suisIn order to gain a depth understanding of the mechanism of resistant gene transmission mediated by ICE,this chapter explores the effects of different selection pressures on the excison of ICE.The regulation of ICE excision was investigated under different conditions(exponential growth or stationary phase,erythromycin treatment,DNA damage by exposition to mitomycin C and Hydrogen peroxide H2O2 treatment).Quantitative PCR was firstly performed to measure the amounts of cicular form of ICEs,the cyclization rate was calculated as the copy number of attP sites per hyd copy(chromosomal locus).The results showed that the cyclization rate of ICESsuYSJ4 and ICESsuHB 1011 had a significant increase in the stationary phase compared with the exponential growth phase(P<0.01).Erythromycin and mitomycin C treatment increased the cyclization rate of ICESsuYSJ4,but there was no significant difference(P>0.05).H2O2 treatment could significantly increase the cyclization rate of ICESsuYSJ4(P<0.05).The results indicated that ICE of Streptococcus suis could only be a small part of the excision and cyclization,and were more likely to be cut down from the genome in the stationary phase than in the exponential growth phase.The unfavorable living environment could promote the excision.Erythromycin,DNA damage agents and oxidative stress can promote the excision and cyclization of ICEs carried by Streptococcus suis.In summary,the resistance of Streptococcus suis to macrolides and tetracycline antimicrobial agents is quite serious.erm(B)and tet(O),which performed drug resistance,were located in the integrated and engaging elements(ICEs).ICEs are widely distributed among Streptococcus suis and can spread among Streptococcus suis.Normally,most of ICEs integrate in the genome,only a small partcan excision pontaneously.Nevertheless,antibiotics associated with ICE,DNA damage agents and oxidative stress can promote the excision and cyclization of ICEs.