| Antimicrobial resistance(AMR)is a major concern worldwide.China is the largest pig-rearing and pork consumption country in the world.The swine-breeding industry in China is known to harbor a significant amount of antimicrobial-resistant bacteria,which not only poses a threat to the safety of pork but also to public health.However,large-scale studies on AMR in the whole China pig livestock remain scarce.It is essential to understand whether resistant E.coli strains from pigs can be transmitted to humans through the pork supply chain,the prevalence of antimicrobial resistance in each node of the supply chain,and the most prevalent resistance genes.To address these issues,E.coli strains were isolated from pig farms in 31 provinces in mainland China,and the resistance phenotypes of these strains were determined for 28 commonly used antimicrobials.Whole genome sequencing analysis was performed on isolates with public health and safety implications.The data on resistance genes,serotypes,plasmid types,and genotypes of the isolates were preliminarily analyzed.Additionally,samples were collected from slaughterhouses and terminal markets in Henan province,where severe resistance phenotypes had been detected.Then,the distribution,transmission and prevalence of resistant E.coli in the local pork supply nodes were clarified superficially.A detailed analysis of special resistant strains in the pork supply chain was also carried out,including an investigation of the genetic environment and plasmid types of mcr-1 and bla NDM-1 resistance genes.The main contents of this project are as follows:1.Samples collection and E.coli identification from pig farms in 31 provincesBetween 2018 and 2019,In each of the 31 provinces of mainland China,pig farms were randomly selected to collect pig biological samples included: rectal sponges and diarrheal feces;environmental samples included: floor swabs,nipple drinker swabs,trough swabs,vehicle swabs,feed,water,fecal slurry,etc.,2710 samples were collected totally.A total of 1871 E.coli strains were isolated and identified by a series of experimental methods such as Mac Conkey agar,eosin-methylene blue agar,PCR amplification of E.coli 16 sr DNA and simple biochemical tests.1165 strains were isolated from pig biological samples and 706 strains were from environmental samples.2.Phenotypes of 28 antimicrobials for 1871 E.coli strainsThe phenotypes of 28 antimicrobials were determined for 1871 E.coli strains using the Phoenix 100 automated microbiology system.The results showed that the resistance rates to tetracycline,chloramphenicol,moxifloxacin,and compound sulfamethoxazole were more than 80%.Additionally,E.coli strains in pig farms that resistant to carbapenems,polymyxin,and tigecycline were detected.More than 90% of the isolates were resistant to three or more tested antimicrobial agents.The degree of AMR in E.coli strains varied from region to region across the 31 provinces studied.The resistance rates were relatively high in Henan province,while they were low in Tibet.All the strains isolated from Ningxia and Beijing were resistant to three or more tested antibiotics.3.Whole genome sequencing analysis of 515 resistant E coli isolatesA total of 515 isolates resistant to carbapenem,polymyxin,tigecycline and producing ESBLs were selected for whole genome sequencing.The bioinformatic analysis results showed: the main O-serogroups were O9 a,O101,O8;and sequence types were ST10,ST101,ST48.This approach led to the detection of 109 ARGs.Of particular note was the identification of four ESBL genes(bla CTXM,bla TEM,bla OXA,and bla SHV),seven fluoroquinolone-resistance genes(aac(6’)-Ib-cr,oqx A,oqx B,qep A,qnr B,qnr D,and qnr S),three carbapenem-resistance genes(bla NDM-1,bla NDM-5,and bla NDM-7),and two colistinresistance genes(mcr-1 and mcr-3).491 isolates were found to harbor intl1 gene.More than half of the isolates(61.71%,n = 303)possessed a single intl1 gene.A total of 53 groups of plasmid replicons were detected.A Col RNAI-type plasmid was present in most of the isolates(n = 272),after which Inc FIB(n = 266)and Inc X1(n = 236)were the most common.Some E.coli harbored both mcr and bla NDM genes,then generated the complete genome sequences of an isolate through Oxford Nanopore Sequencing(ONT)and the complete genome sequencing results showed that the two genes were located on two independent plasmids,and the plasmid backbone had high homology with the published human source AMR plasmids.There were ISs(insertion sequences)upstream or downstream of these resistance genes.Genome sequencing of two highly tigecycline-resistant isolates revealed that tet(X4)was carried by an Inc X1 plasmid in both isolates.Plasmid conjugation experiments revealed that most of the bla NDM-carrying,mcr-carrying,and tet(X4)-carrying plasmids were conjugative and conferred phenotypes of carbapenem,colistin,and high tigecycline resistance in their bacterial recipients.Single nucleotide polymorphism(SNP)analyses revealed that a large proportion of the pig farm isolates showed high genetic similarity(228/515,as few as three SNPs)to the human isolates.Among the 287 human commensal isolates,45 STs were also shared by the 515 drug-resistant isolates from pig farms.4.Epidemiological characteristics of E.coli AMR in Henan pork supply nodesA total of 408 E.coli strains were isolated from 924 samples collected from pig farms,slaughterhouses,and retail markets in Henan province.The AMR of E.coli strains from different nodes varied significantly.The resistance rate of E.coli from pig farms was significantly higher than that from slaughterhouses and terminal markets.There was no significant difference in resistance rates between isolates from abattoirs and markets.Approximately 93.19%,66.43% and 67.53% of the isolates from farms,slaughterhouses,and retail markets were resistant to three or more antibiotic classes,respectively.Farms had the highest proportion of multidrug-resistant strains,with no significant difference between slaughterhouses and retail markets.The ST10 and ST101 sequence types were commonly identified among the isolates from farms,slaughterhouses,and retail markets,and mcr-1 and bla NDM-1 genes were found in these two STs.Two bla NDM-1 positive strains harbored sul2,sul3,flo R,tet A,bla TEM,and bla CTX-M simultaneously.Meanwhile,the mcr-1 positive isolate additionally carried sul3,flo R,bla TEM,and cfr ARGs.Virulence factor-encoding genes such as fim H,fim C,ast A,tra T,tsh,omp T,sit A,iro N,and iut A were detected among these two STs strains.The detection rates of fim C,tra T,tsh,sit A,iro N,and iut A genes were higher in slaughterhouses than those in farms and retail markets.5.Genetic environment analysis of carbapenem and polymyxin resistant strains8 carbapenem-resistant and colistin-resistant isolates were discovered in the pork supply node in Henan province.PCR amplification result showed all of them carried mcr-1 and bla NDM-1 genes.8 E.coli strains were almost resistant to antibiotics tested except tigecycline.The whole genome sequences revealed the 8 E.coli bla NDM-1 genes were located on putative Inc FII-type plasmids;7 E.coli mcr-1 genes were carried by putative Inc X4-type plasmids.The other one was not predicted any putative mcr-1-carrying plasmids.However,the mcr-1 was found on the chromosome though third sequencing technology analysis,and it was located between an IS30-like element ISApl1 and a PAP2-like encoding gene.These three genes consisted of an “ISApl1-mcr-1-orf” segment.The bla NDM-1-carrying plasmid contained 109 open reading frames.The MDR region of this plasmid comprised two antimicrobial resistance gene cassettes,including a 9.9-kb cassette harboring the aph(3’)-VI and bla NDM-1 genes and a 5.3-kb cassette harboring the bla TEM and rmt B genes.The 9.9-kb cassette was flanked by ISAba14 and ISVsa3,while the 5.3-kb cassette was flanked by two IS26 elements.Overall,this study provides a preliminary analysis of the current levels of antimicrobial resistance in E.coli from domestic pig sources in China and the epidemiology of resistant E.coli within the pork supply chains in certain regions.It not only has clinical implications for pig health and production,but also contributes to promoting public health and safety related to both human and animal health.In addition,this study provides valuable data for the development of predictive models for antimicrobial resistance risk,as well as for the development of new and emerging industries like big data analytics and intelligent agriculture. |
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