Transmission Mechanism Of Plasmid-mediated Colistin Resistance Gene Mcr-1 In Salmonella Enteric Among Pigs At Slaughter,China

mcr-1,the first plasmid-mediated colistin-resistance gene,can mediate polymyxin resistance.It can be transferred horizontally among strains isolated from diverse source via plasmids and mobile genetic elements,threating public health security.This investigation put an insight into the transmission mechanism of mcr-1 gene in Salmonella strains among pigs in China.In this study,a total of 142 Salmonella strains were isolated from caecum samples collected from pigs at 5 slaughters in China from July 2013 to May 2014.PCR amplification and sequencing were performed to screen for mcr-1 gene.The results showed that 21 carried mcr-1 gene,the positive percentage of mcr-1 was 14.8%.Among them,19 were Salmonella Typhimrium,another 2 belongs to Salmonella Heidelberg and Salmonella London respectively.Agar dilution methods or broth microdilution method were conducted to test MICs of these 21 mcr-1-positive Salmonella isolates for 13 antimicrobials.MICs of colistin were 1~2 mg/L for these isolates.Over 80% of strains were also resistant to ampicillin,streptomycin,florfenicol,tetracycline,sulfamethoxazole/trimethoprim and gentamicin;76.2% showed reduced susceptibility to ciprofloxacin.The plasmid-mediated resistance florfenicol resistant gene floR and olaquindox and ciprofloxacin resistant gene oqxAB were determined by PCR and sequencing.A total of 20 and 19 isolates had the floR and oqxAB genes,respectively.The clonal relationship of mcr-1-positive isolates was determined by pulsed-field gel electrophoresis(PFGE)using the Xbal I restriction enzyme.The sequence types(STs)of these isolates were also determined by multilocus sequence typing(MLST).The data showed that most of the isolates had indistinguishable PFGE patterns and were clonally related,with serovar Typhimurium ST34(n = 19)being dominant.Determination of the transferability of mcr-1gene was performed by conjugation and transformation.However,the mcr-1 gene failed to be transferred to the recipient E.coli C600 by conjugation.We finally obtained the transformants with plasmids pHNSH138,pHNSH36 and pHNZ319 S by chemical transformation.PCR was performed to detect other resistant genes among them.The genes floR and oqxAB were identified.The genetic context surrounding the mcr-1 gene was investigated by PCR mapping and sequencing.Insertion sequence ISApl1 was only detected in the upstream of mcr-1 gene in these isolates.S1 pulsed-field gel electrophoresis(S1-PFGE)and Southern blotting were performed on 3 transformants and 21 original bacteria which carried mcr-1 gene.The mcr-1 genes were all located on a ~180 kb plasmid,except for that in strains SH33(~110 kb).We used Hiseq Technology to sequence plasmid DNA purified from transformants of Salmonella Heidelberg SH36 and Salmonella London Z4P319 S and genomic DNA extracted from the original isolate(Salmonella Typhimurium SH138,which was selected as a representative ST34 strain).The result showed that mcr-1 was located on derivatives of IncHI2-like plasmids,which also harbored IncF-type backbone.In multidrug resistant region,oqxAB,floR,sul1,cmlA and aadA2 were found.In summary,clonal spread of mcr-1-positive isolates as well as horizontal transmission of mobile elements,such as plasmids and insertion sequences,were the reason of the dissemination of the mcr-1 gene.Moreover,other resistance genes,such as floR and oqxAB,were always co-transferred with mcr-1 via IncHI2-F4:A-B5 plasmids,leading to the successful flow of mcr-1-producing Salmonella under the frequent usage of certain antimicrobials in animal sectors and clinic.