Multidrug Resistance Mediated By IncHI2-IS26-Class 1 Integrons In Salmonella Typhimurium

Salmonella Typhimurium is an important serovar among all nontyphoidal Salmonella.With the overuse of antimicrobials in humans and livestock,the antimicrobial resistant Salmonella has become more and more prevalent.Acquired antimicrobial resistance genes and conjugative plasmids are major reasons for the resistance dissemination.The gene cassettes can be transferred through the insertion squences(IS)and integrons transposition.Class 1 integrons,harboring the DNA elements with Int I1 integrase,can capture exogenous antimicrobial resistance genes.IS26 which often locates on plasmids mediates the transfer and the expression of neighboring resistance genes through self-movable transposons.It might be speculated that the IS26-class 1 integron might greatly promote the spread of antimicrobial resistance.The main contents and results of this study are shown as follows:1.Among 255 S.Typhimurium strains,sixty-two were positive for Inc HI2 plasmids including forty-two clinical isolates(42/180,23.3%)and twenty food isolates(20/63,31.7%).Sixty-one isolates carried IS26-int I1 except the clinical isolates SJTUF13368,and the prevalence of IS26-int I1 in Inc HI2 positive isolates was up to 98.4%(61/62).2.The antimicrobial susceptibility of sixty-one triple-positive isolates was tested by agar dilution method suggested by the Clinical and Laboratory Standards Institute(CLSI).All isolates(100.0%)were resistant to sulfisoxazole.Fifty-five(90.2%)were resistant to trimethoprim/ sulfamethoxazole and ampicillin,respectively.The resistance rate to tetracycline,nalidixic acid,chloramphenicol,streptomycin,kanamycin,amoxicillin/clavulanic acid,gentamycin,ciprofloxacin,ceftiofur,ceftriaxone and cefoxitin was 75.4%,73.8%,63.9%,60.7%,47.5%,44.3%,42.6%,39.3%,8.2%,8.2% and 1.64%,respectively.All sixty-one isolates were susceptible to amikacin,cephalothin and azithromycin.No significant difference(P>0.05)was found in resistance rates towards kanamycin,gentamycin,streptomycin,chloramphenicol,ciprofloxacin,nalidixic acid,tetracycline,trimethoprim/sulfamethoxazole,sulfisoxazole,ampicillin and amoxicillin/clavulanic among forty-one clinical isolates and twenty food isolates.3.The conjugation experiments were performed with sixty-one triplepositive isolates as donors and E.coli C600(Rifampicin resistant)as recipient.Twenty-four transconjugants were obtained by liquid mating method and eleven transconjugants were obtained by filter mating method.All transconjugants carried IS26 except SJTUF11216-TC(97.1%).Seventeen(48.6%)transconjugants carried int I1 gene including twelve from clinic and five from food.4.PCR based replicon typing method was used to identify plasmid types of thirty-five transconjugants.Fourteen transconjugants only carried Inc HI2 plasmids.SJTUF10405-TC carried both Inc HI2 and Inc I1 plasmids,and SJTUF13383-TC carried both Inc HI2 and Inc A/C plasmids.The remaining nineteen transconjugants may carry untypable plasmids.The multidrug resistance phenotypes were also transferred via the plasmid conjugation.The transfer rate of sulfisoxazole,ceftiofur and ceftriaxone resistance phenotype was the highest(100.0%),followed by resistance phenotype of nalidixic acid(96.7%),amoxicillin/clavulanic(75%),ampicillin(48.9%),trimethoprim/sulfamethoxazole(46.7%),chloramphenicol(43.5%),kanamycin(40%),gentamycin(40%),ciprofloxacin(36.4%),streptomycin(22.7%)and tetracycline(19.2%).5.The S1-PFGE method was chosen to determine the size of plasmid.Only one type plasmid existed in each of the fifteen transconjugants,and the majority transconjugants' plasmids were around 200.0 kb.Surprisingly,the plasmid size of SJTUF11216-TC was about 70.0 kb,which was possible Inc I1 plasmid.6.The 15 plasmid-harboring confirmed transconjugants' donors were genome sequenced with the next-generation sequencing method.The complete nucleotide sequences were uploaded to the MLST database and the Comprehensive Antibiotic Resistance Database to analyse their sequence type and antimicrobial resistance gene composition.The antimicrobial resistance genes with more than 90% similarity were listed including aminoglycosides resistance genes(aac(6')-Iaa,aac(6')-Ib3,aac(3)-IV,aph(4)-Ia,aph(6)-Id,aph(3')-Ia,aad A1,aad A2),quinolone resistance genes(qac H,oqx A,oqx B),tetracycline resistance genes(tet A,tet R),rifampicin resistance genes(arr-3),chloramphenicol resistance genes(flo R,cml A6,cat B3),sulfonamides resistance genes(sul1,sul2,sul3),trimethoprim resistance genes(dfr A12)and ?-lactam resistance genes(bla TEM-1,bla OXA-1,bla CTX-M-14).ST34 was the predominant ST type in all isolates and no significant difference(P>0.05)was found between the antimicrobial resistance gene category numbers.Two gene cassette arrays of class 1 integron were identified through BLASTN.This study investigated the prevalence of mobile genetic elements in antimicrobial resistant S.Typhimurium isolates.The antimicrobial resistance genes and ST type were also analysed based on the whole genome sequencing.It might reveal the spread and transferability of antimicrobial resistant S.Typhimurium mediated by Inc HI2-IS26-class 1 Integron and play a critical role in tracking and monitoring the antimicrobial resistant pathogen.