Correlation Analysis On Multiple Drug Resistance Mechanism Of Escherichia Coli From Different Sources In Livestiock Farms

Long-term exposure to antimicrobial drug can lead to drug resistance in bacteria,and with the widespread use and abuse of antimicrobial drugs,the emergence of multidrug-resistant Escherichia coli（E.coli）has not only brought a heavy burden to the global economy,but also seriously threatened the health of both animals and humans.The resistance mechanism of multiple drug-resistant E.coli is very complex,and has rapidly evolved under the drug selection pressure.In addition,the drug resistance genes can spread widely with the participation of environmental vectors.Therefore,it is of great significance to understand the drug resistance mechanism and analyze its correlation of multidrug-resistant E.coli from different sources in animal husbandry,and guide the clinical application of antimicrobial agents.In this paper,correlation of multi-drug resistance mechanism in E.coli from different sources of some pig and chicken farms around Chongqing was researched,so as to understand the evolving mechanism of multi-drug resistance and provide reference for rational use of antimicrobial agents.1.Drug resistance rate and associated genes detectionThe resistance rates of 108 tested E.coli strains isolated from 11 pig farms around Chongqing in 2007-2008 and 2 chicken farms in Rongchang,Chongqing and Lu zhou,Sichuan in 2016 and 2020 to 19 kinds of antibiotics were tested by disk diffusion method and broth dilution method,respectively.After that,according to drug resistance spectrum,39 typical multidrug-resistant E.coli strains were selected to detected the positive rate of 17 horizontally transferable resistance genes by PCR,and the correlation between drug resistance phenotype and drug resistance genotype was analyzed.Results:93 of 108 tested strains were multi-resistant strains,and the percentage is 86.11%.The main resistance spectrum is 4 kinds and 5 kinds,which accounted for41.67%,and the most widely resistant spectrum was 12 kinds of 1 strain.Among the 19 tested antibacterial drugs,the drug resistance rates of 4 drugs as SXT（92.59%）,ampicillin（86.11%）,tetracycline（84.26%）,and oxytetracycline（64.81%）were higher than 50%,and all the tested stains had no resistant to cefepime.The drug resistance rates of cotrimoxazole（all>88%）,tetracycline（all>80%）and cefazolin（all>17%）were similar in stains isolated form different pig and chicken farms.However,the drug resistance rates of pig isolates were significantly higher than that of chicken isolates for other drugs.In all 39 typical multidrug-resistant strains,floR（71.79%）,bla_TEM（61.54%）,qnrS（51.28%）and sul2（51.28%）had higher detection rates,and 6 other drug-resistant associated genes including tetO,tetX,bla_SHV,aac（3）-Iva,fexA,and fexB were undetected.Correlation analysis on drug resistance phenotype and drug resistance gene showed that bla_CTX-M had higher correlation with resistance of cefazolin,cefuroxime and ceftazidime,and mcr-1 had higher correlation with resistance of polymyxin B and polymyxin E,both of which reached more than 80%.2.MLST typing of 39 tested typical multidrug-resistant E.coli strainsAccording to the MLST typing method of E.coli provided by MLST database,the seven housekeeping genes as adk,fumC,gyrB,icdA,mdh,purA and recA were PCR and sequenced,and cluster analysis was performed for 39 typical multidrug resistant E.coli strains,and the genetic evolution relationship between the strains was analyzed.Then,according to the common phylogenetic group identification method of E.coli,chuA,yjaA and TSPE4.c2 were tested by multiplex PCR,and the E.coli populations were determined by the gene bands displayed on the electrophoretic map.Finally,the correlation analysis of phylogenetic group,ST type and drug resistance was carried out.Results:There were 29 ST types in 39 tested strains,among which 3 were newly discovered and named ST12677,ST12678,ST12679.Among which,12 ST types belonged to 6 clonal groups,and the remaining 17 ST types were isolated.ST10,ST23and ST206 were relatively small clones.The 39 strains were mainly distributed in phylogenetic groups A,B1 and D.Most ST types of isolates from pig and chicken farms were scattered,but some ST types and clonal relationships were associated among isolates from different sources of chicken farms.There was no ST type crossover but ST clonal complex crossover between pig and chicken farm-derived strains.For example,both ST10 and ST12677 pig farm-derived strains,and ST48 and ST1638chicken farm-derived strains belonged to the ST10 clonal complex,and were resistant to cotrimoxazole,ampicillin and florfenicol.ST23 pig farm-derived strain and ST88chicken farm-derived strains belonged to ST23 clonal complex,and were resistant to cotrimoxazole,ampicillin,florfenicol,tetracycline,oxytetracycline,doxycycline,cefazolin and cefuroxime.ST206 pig farm-derived strains and ST5764 chicken farm-derived strains belonged to ST206 clonal complex,and were resistant to ampicillin,florfenicol,tetracycline,oxytetracycline and enrofloxacin.In addition,the ST155 type chicken farm air-sourced stain 2-K30.1 and water-sourced stain 2-S1.8,which both belonged to group B1,carried the same drug resistance genes,and the drug resistance spectrum were basically consistent.Similarly,two chicken farm human-sourced strains 2-R3.5 and 2-R12.8 of ST88 belonged to group A and carried basically consistent drug resistance genes and drug resistance spectrum,and two pig farm strains 29-1 and 4-5 of group A,ST12677 carried the same drug resistance genes,and the drug resistance spectrum were basically the same as each other.These results showed that there was correlation in phylogenetic classification,ST type and drug resistance between this two kinds of isolates.3.Genome sequencing analysis of 9 typical multidrug-resistant strainsBased on the above results,9 typical multidrug-resistant strains（4 strains from pig farms and 5 strains from chicken farms）were tested with the whole genome sequenced by Illumina HiSeq.NCBI Blast+annotated and analyzed gene protein sequences with KOG,COG,NR,PFAM,Swissprot and other databases.Compared with CARD antibiotic resistance Database,the related gene information leading to multiple drug Resistance in the genome was obtained,and the drug resistance gene correlation was analyzed of the 9 tested strains.Results:Diverse 105～120 antibiotic resistance genes were present in all genomes of 9 strains,including about 28 efflux pump regulatory factors,20 efflux pump protein genes and 70 drug-specific resistance genes.There were many horizontally transferred drug-specific resistance genes,including 9 kinds of aminoglycosides,6 kinds of tetracycline,4 kinds ofβ-lactam,3 kinds of sulfonamides and 3 kinds of trimethoprim,2 kinds of acyl alcohols,2 kinds of macrolides,1 kind of peptide,1 kind of fosfomycin,and 1 kind of quinolone.The 9 strains carried similar efflux pump families,which were all belonged to MFS,RND and SMR families.The drug resistance genes of 2-K30.1and 2-S1.8 strains from chicken farm and 4-5 and 29-1 strains from pig farm were identical,respectively.There were 17 horizontally transferable resistance genes in both pig and chicken sourced stains,including tet34,tetG,tetA,tetR,aadA,aadA2,mcr-1,dfrA12,mphA,qnrS1,fosA3,floR,cmlA1,bla_CMY-46,sul1,sul2,and sul3.In summary,All the 109 strains had serious multiple drug resistance and complex drug resistance types.There was a certain correlation between drug resistant phenotypes and drug resistant genotypes of 39 strains.ST10 was the common ancestor of 39isolated strains from both pig farms and chicken farms,but most ST types were scattered.Certain correlation may exist among ST types,drug resistance rate and drug resistance genes.Some clonal transmission of drug resistance between two derived strains in the inner farm was detected.All the 9 strains carried the same efflux pump family gene and similar horizontally transferable resistance genes,and their multiple drug resistance mechanisms may be correlated.