Study On The Distribution And Dissemination Mechanism Of Integrative And Conjugation Element In Streptococcus Suis

Antibiotics was typically used for treatment of bacterial infections in livestock and poultry,increasing resistance of bacterial to commonly used antimicrobials and generating threat to public health.Streptococcus suis was a multidrug-resistant zoonotic pathogen that causes meningitis,septicemia,and arthritis in humans and swine,and was one of the major pathogens that led to substantial economic losses in the swine industry.Streptococcus suis(S.suis)was an important antimicrobial resistance reservoir promoting the spread of resistance genes to various streptococci.The dissemination of resistance genes was associated with different mobile genetic elements(MGEs).Integrative and conjugative elements(ICEs)are a diverse group of mobile genetic elements(MGEs)primarily existing in a host chromosome and retaining the ability to excise and transfer by conjugation.ICEs have been shown to act as vital vectors for the dissemination and transfer of genes related with antimicrobial resistance and virulence.In addition,some ICEs were reported to be capable of mobilizing other types of MGEs,such as non-conjugative plasmids,via the formation of a cointegrate molecule.With the development of high-throughput sequencing,various types of ICEs have been identified in both Gram-negative and Gram-positive bacteria.Among them,the ICESa2603-family,one of the largest streptococcal ICE families,has been identified in various Streptococcus species of different origins,but comparative research of ICEs is still absent.In this study,202 clinical S.suis strains,isolated from 6 provinces including Hunan,Hubei,Tianjin,Henan,Xinjiang,and Heilongjiang,were used to investigate detection and dissemination mechanism of ICEs through antimicrobial susceptibility testing,Conjugation assays,and sequencing of whole genome DNA and next generation.Antimicrobial susceptibility testing showed that all strains were sensitive to vancomycin and linezolid,but showed highly rate of resistance to erythromycin,tetracycline and gentamicin(69%)and a relative less rate of resistance to florfenicol(23%).The analysis of the draft genomes of 202 S.suis isolates showed that the positive rate of putative ICEs and IMEs(integrative and mobilizable elements)was 54%(109/202),38.6%(78/202)and a total positive rate of 92.6%,respectively.It indicated that those MGEs were had been prevalent in S.suis.The result showed that high levels of the resistance of tetracycline and erythromycin.Conjugation assays were performed using E.faecalis JH2-2(Rif^r)and S.suis BAA(Rif^r)as the recipients.In mating experiments,27 S.suis strains carrying ICEs as donors showed that the ICEs could transfer from S.suis into the recipient E.faecalis JH2-2 and S.suis BAA at frequencies ranged from 1.4x 10^-6and 8.8 x 10^-2.Multilocus sequence typing(MLST)was used to investigate the molecular prevalence of ICE-positive isolates in this study.MLST typing of 27 ICE-positive isolates presented 16different number of MLST and exhibited significant diversity.The analysis of complete sequences of 27ICEs showed that these 27 ICEs can be classified into ICESa2603 family and ICESa2603-like family according to the recombinase of tyrosine or serine.In this study,this is the first report of seven new integrative sites(omd A?ara C?gdh A,mal X,DUF3329,MTH1,and car B)located on ICESa2603 family and ICESa2603-like family ICEs,which showed that ICEs have a widely range of integrated site in chromosome.In this work,we also identified a novel optr A+fex A+erm(A)+spc-carrying TU and a novel plasmid p5303 carrying cfr(D)gene as well as an erm(B)+optr A-carrying UCS.we report the detection of IS1216E-mediated cointegrate formation between TU,plasmid,and ICE.The cointegrate formation with an ICE enabled plasmid and TU transfer via conjugation and indirectly leads to the horizontal spread of important resistance genes.This work enriched the theory that the ICEs distribution,transmission,and conjugation,which provides a scientific basis for transfer and spread of non-conjugative elements,and put a theoretical basis for curbing the spread of drug resistance in Streptococcus suis.