Investigation On Drug Resistance Of Salmonella Isolates From Food Animals And Characteristics Of The Molecular Transmission Of 16S RRNA Methylases Genes

Salmonella is the important foodborne pathogenic bacteria and its antimicrobial resistance poses a major threat to human health.In this study,we investigated the prevalence and drug resistance of Salmonella isolates from food animals in several regions of seven China's provinces,and further explored the characterization of molecular transmission of16S rRNA methylase?16S-RMTase?genes among the multidrug-resistant S.Indiana isolates.Our study would provide the basic data for the risk assessment of drug resistance and the theoretical basis for reducing drug resistance in bacteria.A total of 2163 samples were collected in Guangdong,Jingsu,Jingxi,Fujian,Guangxi,Zhejiang and Hainan Province druing 2016 to 2017.A total of 310 Salmonella strains were obtained through isolation and identification,and the isolation rate was 14.3%.The highest isolation rate of Salmonella strains was from duck samples,and it was 26.3%?85/323?.There was no significant difference in the isolation rates of Salmonella strain among pig,chicken and goose samples.297 of 310 Salmonella isolates were successfully serotyped,and 19serotypes were identified,including the five predominant serotypes S.Typhimurium,S.Derby,S.Meleagridis,S.Indiana and S.Rissen.Antimicrobial susceptibility test of the 310 Salmonella isolates were performed by the agar dilution method.More than 60%of the 310 isolates showed resistance to six drug including cefotaxime and ciprofloxacin.while,less than 5%of these isolates showed resistance to amikacin,colistin and fosfomycin.The resistance rates of different serotypes of Salmonellas varied greatly.S.Indiana isolates were presented to be the most serious multiple drug resistance and resistant to more than eight classes of antibiotics accounted for 96.4%?27/28?.Furthermore,42%?42/100?of S.Typhimurium was resistant to more than eight classes of antibiotics.There was no significant difference in other serotypes of multiple drug resistance.The occurrence of ESBLs,PMQR and 16S rRNA methylase gene among Salmonellas were determined by PCR amplification and sequencing.For the ESBLs genes,the resistance rates of bla_CTX-M-positive isolates were 12.9%,and the dominant subtype was bla_CTX-M-55?32strains?,followed by bla_CTX-M-27?8 strains?and bla_CTX-M-14?2 strains?,and no other subtypes were found.In addition,bla_CTX-M-55 and bla_CTX-M-27 were detected in 8 isolates,simultaneously.The detection rates of bla _TEM-1 and bla _OXA-1 were 26.5%and 35.5%,respectively.For the PMQR genes,aac?6'?-Ib-cr has the highest detection rate,35.2%,followed by oqxAB,with the detection rate of 35.2%.The other PMQR genes was detected in several isolates.Ten isolates carried qepA genes,and qnrS,qnrB and qnrD genes were detected in two isolates.Thirteen isolates carrying 16S-RMTase genes were detected,and 12and 1 carried rmtB and armA genes,respectively.All of these isoaltes belonged to S.Indiana,and concurrently conferred high-level resistance to cefotaxime,amikacin and ciprofloxacin.PFGE and MLST were carried out to analyze the genetic relatedness among 28 S.Indiana isolates.All the isolates were ST17 and divided into 13 PFGE profiles.13 S.Indiana isolates carrying rmtB/armA were divided into 7 PFGE profiles.10 isolates carrying rmtB gene were divided into 4 PFGE profiles,with a high similarity.These 10 isolates have a relatively low similarity of PFGE profiles to that of the remained 18 S.Indiana isolates,indicating clonal spread of rmtB-positive isolates in local areas.The 13 isolates carrying rmtB/armA were conducted with conjugation experiment.The results showed that 10 of 12 isolates carrying rmtB were successfully transferred by conjugation.I-Ceu I-PFGE and Southern blotting showed that the rmtB and armA genes were located on chromosome among the remained 3 isolates with failing to conjugation.For the ten transconjugants,qepA and bla_CTX-M-27 were co-transferred with rmtB in eight strains.In another two strains,qepA was co-transferred with rmtB.S1-PFGE and Southern blotting showed that these ten transconjugants harbored only one plasmid,with sizes of¹00 kb?n=2?and¹30 kb?n=8?,respectively.Replicon typing was conducted in rmtB-positive transconjugants and the results showed that all of rmtB-carrying plasmids belonged to F2:A1:B1.All rmtB-carrying plasmids carried pemKI,hok-sok,vagCD and ccdAB addiction systems.RFLP analysis showed four EcoRI digestion profiles,with a high degree of similarity Southern blotting hybridization was performed on EcoRI digestion fragments showed that rmtB,qepA and bla_CTX-M-27 genes were almost located on¹0 kb,⁴.3 kb and⁶.5 kb DNA bands,repectively.The four representative rmtB plasmids were selected and sequenced.The sizes of the pGDD3,pGDD5,pGDD16,and pGDD21 plasmids were 127734 bp,100935 bp,129455 bp,and 130794 bp,respectively,containing¹10-¹40 open reading frames.These four plasmids contained a typical IncF?-type backbone structures,and are highly similar to the F2:A-:B-plasmid pHN3A11 carrying rmtB and qepA,with the major difference being between the bla_TEMEM and ycgA genes.Each of the four rmtB positive plasmids contained a multidrug resistance region,and the multidrug resistance region on plasmid pGDD5 was¹2kb,including IS26-?intI1-?dfr-qepA-ISCR3-groEL/intI1-?rmtB-?Tn3(bla _TEM-1).The multiple drug resistance on another three rmtB-carrying plasmids are almost identical,with size of 14.5 kb,and it contained resistance genes such as qepA,rmtB and bla_CTX-M-27 as well as complete or truncated inserts or transposons.This is a new discovery in this research.In summary,the multidrug resistance of Salmonella isolates from food animal is serious,especially S.Indiana.The ESBLs,PMQR and 16S rRNA methylase genes were found with different prevalence in Salmonella.Further,this study revealed the emergence of S.Indiana isolates that were concurrently resistant to amikacin,cefotaxime and ciprofloxacin from waterfowl husbandry in Guangdong province,China.This was due to the clonal expansion and horizontal transmission of F2:A1:B1 plasmids involved in the spread of rmtB,qepA,and/or bla_CTX-M-27 in S.Indiana isolates.This is worrisome that an epidemic plasmid is involved in co-spread of rmtB,qepA and bla_CTX-M-27 in salmonella in China.Future studies are necessary to monitor the resistance in salmonella,especially multidrug-resistant S.Indiana,so as to better understand its potential threat to public health.