| Streptococcus suis is a zoonotic pathogen,which not only causes huge economic losses to the pig industry,but also threatens human health and even life.Current studies have confirmed that S.suis is a reservoir of antimicrobial resistance genes,and its resistance-related mobile genetic elements such as integrative and conjugative element(ICE),plasmids,and prophages play key roles in the horizontal spread of its resistance genes.In this study,the methods of isolation and identification of S.suis,whole genome sequencing,bioinformatics analysis,MLST typing,natural transformation test,electrotransformation test,competition test and other methods were used to analyze the antimicrobial resistance phenotype of S.suis,identify antimicrobial resistance genes and the mobile genetic elements related to antimicrobial resistance and the analysis of the mechanism of horizontal transmission provide a theoretical basis for the prevention and control of antimicrobial resistance and the rational use of antimicrobials.From 2006 to 2018,131 clinical isolates of S.suis in five provinces including Guangdong,Henan,Shandong,Shanxi,and Hebei were identified and their genus were sequenced.Bioinformatics analysis showed that 131 strains of S.suis carried a total of 28 antimicrobial resistance genes,of which the tetracycline resistance gene tet had the highest carrying rate of 100%(131/131),followed by the macrolide resistance gene erm,the carrier rate was 80.9%(106/131).The results of antimicrobial resistance phenotype analysis showed that the highest resistance rate was 100%(131/131)to tetracycline,followed by 96.9%(127/131)to erythromycin.The results of differential analysis of antimicrobial resistance genes carried by different ST types showed that131 S.suis strains were divided into 55 genotypes,of which 48 strains of S.suis carried erm(B)and tet(O)genotypes.Distributed in all ST types,while carrying antimicrobial resistance genes ant,aph(3’)-III,mef(A),msr(D),tet(40),erm(B),tet(O)genotypes There were 19 strains of S.suis,which were concentrated in ST7 type.The results of serotype analysis showed that 109 strains could be classified into 18 serotypes,and 22 strains were unknown serotypes.Type 2 accounted for the highest proportion at 27.5%,followed by type 3(10.7%)and type 9(9.2%).At the same time,this study also found a novel ICESsu SC128 carrying the tigecycline resistance gene tet(M)variant,as well as two mobile genetic elements ICESsu SC117 and plasmid p SC262 containing the macrolide resistance gene erm(T).ICESsu SC128 is located on the chromosome of S.suis SC128,with a size of36.097 kb,and contains both tet(L)and tet(M)genes,which have cyclization activity and can be inserted into the recipient bacterium rpl L locus by horizontal transfer.Through gene cloning and functional analysis,it was found that ICESsu SC128-derived tet(M)contributed to tigecycline resistance,but not tet(L).Compared with the reference sequence,33 amino acid positions of Tet(M)from ICESsuSC128 were mutated.The ICESsu SC117 carrying the erm(T)gene is located on the chromosome of S.suis SC117,with a size of 64.013 kb.It has cyclization activity and can be inserted into the rpl L locus of the recipient strain by horizontal transfer,giving the recipient bacteria erythromycin and lincomycin and clindamycin resistance phenotype.Analysis of the upstream regulatory region of erm(T)gene showed that erm(T)from ICESsu SC117 was constitutively expressed.It is speculated that it may lead to the fitness cost of ICESsu SC117.The plasmid p SC262 carrying the erm(T)gene is 5.125 kb in size and contains the drug resistance gene erm(T).Using S.suis P1/7,human Streptococcus pneumoniae D39 and Staphylococcus aureus RN4220 as the recipient bacteria,through the transformation test,all successfully obtained p SC262 positive transformants,and all have erythromycin,lincomycin and clindamycin resistance phenotype.Analysis of the upstream regulatory region of erm(T)gene showed that erm(T)from plasmid p SC262 was constitutively expressed.It is speculated that this may result in the fitness cost of plasmid p SC262.The results of growth curves and competition experiments showed that ICESsu SC117 had an adaptive cost in S.suis,and plasmid p SC262 had an adaptive cost in both S.suis and human S.pneumoniae.In conclusion,this study analyzed the antimicrobial-resistant phenotypes and genotypes of 131 S.suis strains from five provinces in my country through phenotypic determination and whole-genome sequencing,and found that tetracyclines and macrolides were generally resistant,it may be related to the mobile genetic elements it carries.The study further identified three novel mobile genetic elements ICESsu SC128,ICESsu SC117 and plasmid p SC262,and confirmed that they can all be transferred horizontally.The erm(T)gene located on ICESsu SC117 and plasmid p SC262 is constitutively expressed,resulting in the fitness cost of ICESsu SC117 and plasmid p SC262,revealing that the erm(T)gene fails to occur in S.suis and S.pneumoniae important reason for the general popularity.This study provides a theoretical basis for suppressing the interactive transmission of mobile genetic elements carrying tetracycline and macrolide resistance genes in animal and human streptococci. |
