| Enterobacteriaceae are widely present in the gastrointestinal tract of humans and animals as well as in the daily environment,and are a common conditional pathogenic bacteria.With the abuse of antibiotics,these bacteria have developed different degrees of resistance to commonly used antibacterial drugs,which not only restrict the development of animal husbandry,but also seriously threaten the public health safety of human beings.Studies have shown that the detection rate and drug resistance rate of Enterobacteriaceae in the clinic has increased year after year,with the most serious drug resistance rate of E.coli.The high drug resistance rate of E.coli has made it a repository of drug resistance genes and plays an important role in the horizontal transmission of drug resistance genes.To this end,this study intends to isolate and identify animal-derived E.coli in samples sent from farms in some areas of Jilin Province,monitor drug susceptibility and analyze drug resistance genes,the results of which can lay the foundation for the scientific and rational use of veterinary clinical drugs in Jilin Province,and also provide a theoretical basis for the study of the enterobacterial drug resistance gene environment.The main research contents are as follows.1.Isolation and identification of E.coli from different animal sources and drug susceptibility analysisThe isolation and identification of 246 samples from 16 farms of different animal sources in some areas of Jilin province were carried out,and a total of 66 strains of E.coli were isolated by Gram staining and 16 Sr DNA sequencing analysis,with an isolation rate of 26.8%;drug susceptibility testing of the isolated strains showed that the isolated bacteria had a high resistance rate to tetracycline,flupenthixol,marbofloxacin and tetracycline.The results showed that the isolates were resistant to tetracycline,florfenicol,marbofloxacin and tetracycline at51.5%,59% and 57.6%,respectively;the isolates were resistant to enrofloxacin,azithromycin,ceftriaxone,gentamicin and polymyxin B at different degrees,the resistance rates were 45.5%,7.5%,42.4%,21.2% and 3%,respectively;some strains were multi-drug resistant,among which quadruple,quintuple and heptadruple resistance were more frequent;the isolates were only sensitive to tigecycline and imipenem;further,the isolates were sensitive to tetracycline and imipenem.Further,66 strains of E.coli were typed by pulsed field gel electrophoresis(PFGE typing),among which 14 strains could not be digested by Xbal enzyme and could not be typed for the time being.52 other strains of E.coli were divided into 30 PTs,with 36%-100%similarity between strains and clonal transmission.2.Analysis of drug resistance genes and target mutations in E.coli of different animal originsThe results showed that the isolates all carried aph A1 resistance genes,which could mediate resistance to aminoglycosides;some strains carried tet A,tet W,flo R,qnr S and other resistance genes,which could mediate resistance to tetracyclines,chloramphenicol and quinolones,respectively;the drug targets of seven fluoroquinolone-resistant strains were analyzed.The results showed that there were two different types of mutations in the isolated strains,and seven strains had mutations in gyr A(Ser83Leu/Asp87Asn)and par C(Ser80Ile)loci.It is noteworthy that multi-drug resistant strain C3 not only carries a gene cassette containing five drug resistance genes,but also mediates resistance to aminoglycosides,chloramphenicol and β-lactams,and at the same time its resistance can be transmitted horizontally by means of conjugate transfer.3.Whole genome sequencing and analysis of multi-drug resistant strain C3The whole-genome sequencing and analysis of the screened multi-drug resistant strain C3 were performed using the ONT whole-genome sequencing platform.The results showed that the whole genome of C3 strain was 5,109,751 bp long,containing two plasmids,carrying 71 resistance genes,16 gene islands and 946 virulence genes;the two plasmids were typed,in which Contig00002 was Inc FIB(98.39% similarity)or Inc FIC(95.79 similarity),and Contig00003 The plasmid Contig00003 contains a 27 kb drug resistance gene cluster and a novel mobile element ICE,which carries 9 drug resistance genes(flo R,cml A,bla CTX-M,bla OXA10,qnr S,tet A,dfr A,aad A1,arr)that can mediate The plasmid also carries three complex transposons,IS26,ISVsa3 and IS102,which carry various drug resistance genes such as bla CTX-M,bla OXA10,flo R,tet A,aph(3,)-I,etc.Among them,the complex transposons IS26 and ICE,ISVsa3 and ISVsa3 have drug resistance.The new transposon IS26 and ICE,ISVsa3,have overlapping resistance genes;the resistance genes carried by IS102 are similar to those carried by IS26 and ISVsa3;the traceability analysis of the novel mobile element ICE revealed that its main backbone originated from Escherichia coli(accession number CP050201.1),and it is presumed that it may be derived from the evolutionary transfer of ICE from other Escherichia coli. |
