Detection Of Linezolid Resistance Gene OptrA And It’s Dissemination Mechanism In Streptococcus Suis

Streptococcus suis(S.suis)is an important zoonotic pathogen.The horizontal transfer of resistance genes caused by the widespread use of antibiotics is the main reason for the drug resistance of S.suis.Linezolid is the first generation of oxazolidinone antimicrobial drugs,approved for clinical use in human medicine and known as "the last-resort antimicrobial agents for the control infections caused by MDR Gram-positive pathogens".Although it has not been approved for animal treatment,linezolid resistant strains of animal origin have appeared in many countries,which have attracted extensive attention from scholars at home and abroad.178 strains of S.suis were isolated from Henan,Jiangxi,Hunan,Hubei,Anhui,Shanxi and Shaanxi provinces as the research object,to investigate epidemiology,genetic environment and transmission mechanism of optr A in S.suis,and provide theoretical data for preventing and controlling the spread of optr A in S.suis.PCR method was used for detect the linezolid resistance gene.The sensitivity of 51 optr A positive strains to antibiotics was determined by broth dilution method.The genetic environment of 51 strains of optr A was analyzed by whole genome sequencing(WGS).The circular intermediate form of the integrative and conjugative element(ICE)was detected by reverse PCR.Conjugation experiment was used to verify ICE transferred into the recipient strain S.suis P1/7;PFGE method was used to genotype 51 optr A positive strains and analyze their genetic relatedness.The results showed that 51 of 178 isolates were positive for optr A and the positive rates was 28.7%,2 strains carried both optr A and cfr genes,the positive rates of cfr was1.1%,and other linezolid resistance genes were not detected.The drug sensitivity test results showed that the 51 optr A positive strains were all multi-drug resistant,and all the strains were resistant to 4 or more drugs.The number of strains resistant to 7 kinds of antibiotics was the highest(23 strains,accounting for 45.1%).Only 1 strain was resistant to 4 kinds of antibiotics,accounting for 2.0%.It was noteworthy that 2 strains were resistant to all 9 kinds of tested antibiotics.WGS analysis showed that optr A environment of 51 strains could be classified into12 types,I to XII,with complex and diverse genetic backgrounds.There were 18 strains,whose optr A was located in a complex transposon consisting of IS1216 insertion sequence.A putative transcriptional regulator gene ara C was lacated downstream of optr A in 5 strains.There were 5 strains,the ferredoxin was located in the upstream of optr A.In 3 strains,the optr A was located on transposon Tn6675.One strain,optr A and methicillin-resistant gene dfr G were co-located in the transposon composed of upstream ISEnfa1 and downstream IS1216.Importantly,strain 1112 S carried a optr A,what was associated with a single copy of IS6 family insertion sequence ISS1 S,and was tandem with erm(B)and tet(O)genes,which were located on the integrative and conjugative element ICESa2603 family-like ICESsu1112 S.Reverse PCR results showed that ICESsu1112 S could be excised from chromosomes and form circular intermediate.Multiple PCR results showed that the integrated,excised and looped forms of ICESsu1112 S co-existed in the genome.ICESsu1112 S could transferred into the recipient strain S.suis P1/7 with a frequency of 5.9×10-6 transconjugants per donor cell.Subsequently,the PCR method was used to investigate the distribution of ICESa2603family-like ICE in optr A-positive strains.The result showed that all 18 of 51 strains were found to carry ICESa2603 family ICE,suggesting that ICE elements play an important role in the transfer and transmission of resistance genes in S.suis.In order to understand the relationship of optr A-positive strains,51 optr A-positive strains were classified by PFGE.Clear PFGE profiles of 37(72.5%)strains were obtained,including 26 profiles,indicating that 51 optr A-positive strains from 7 provinces were mainly transmitted horizontally.In conclusion,the carrying rates of S.suis to optr A was increased in 7 provinces and optr A may be horizontal transmission mediated by mobile genetic elements(MGEs).Therefore,the study of MGEs carrying optr A transfer and transmission is crucial to prevent and control the transfer of linezolid resistance.