| The unscientific and irrational use of antimicrobial drugs in dairy farms has led to the emergence of more serious drug resistance in Escherichia coli.Therefore,it is important to carry out epidemiological investigation and study of pathogenic Escherichia coli in calf diarrhea and analyze the distribution of virulence genes and drug resistance genes.In this study,cases of calf diarrhea in cattle farms around the Hohhot area were studied,and Escherichia coli was isolated and cultured using molecular biology and biochemical identification,and the distribution of virulence and drug resistance genes of the isolated strains was analyzed.On this basis,whole-genome sequencing was performed to target the screened E12 strain with high pathogenicity and drug resistance.Meanwhile,a network pharmacology approach was used to study the mechanism of action of Gegenqinlian Decoction for the treatment of calf diarrhea caused by atypical enteropathogenic Escherichia coli.It laid a theoretical foundation for elucidating the epidemiological trend of Escherichia coli in calf diarrhea in Hohhot area and guiding the rational use of veterinary clinical drugs.The details of the study and the results were as followed:1.The isolated strains were identified by biochemical and molecular biological methods,and then their drug resistance and pathogenicity were determined by drug-sensitive paper diffusion method and mouse pathogenicity test,and then the germline subgroups and strain sequence type were determined by polymerase chain reaction.The results showed that the isolated strains were all consistent with the biochemical characteristics of Escherichia coli,21 strains of Escherichia coli were isolated,and the isolation rate was 60%.The isolates were identified by polymerase chain reaction using the Escherichia coli specific genes uida and 16SrRNA,and the isolates were consistent with the positive control.Germline grouping of the isolates revealed that 19%of the isolates were from group A,29%from group B1,9%from group B2 and 43%from group D.Multilvcus sequence typing analysis of the isolates revealed that the isolates were classified into 15 ST types.The results of drug sensitivity tests showed the resistance levels of the isolates to 20 antimicrobial drugs,and the isolates were more than 50%resistant to penicillin,ampicillin and cephalosporins,and extremely sensitive to meropenem.The rates of resistance to tetracycline and doxycycline were 33%and 29%,respectively.Resistance rates to ciprofloxacin,levofloxaciu and enrofloxacin were 48%,33%and 33%,respectively.The rates of resistance to amikacin,kanamycin and gentamicin were 19%,24%and 38%,respectively.The resistance rates to cotrimoxazole and erythromycin were 48%and 15%,respectively.The resistance rates to florfeuicol,chloramphenicol and polymyxin B were 29%,33%and 5%,respectively.Nine pathogenic strains of Escherichia coli with calve diarrhea were isolated by mouse pathogenicity test.2.Polymerase chain reaction was used to determine the distribution of virulence genes in the isolates,and the results showed that the detection rates of virulence genes for the adhesion class were eaeA(76%),fimC(95%),fimA(62%),IuxS(95%),ompA(52%)and flu(24%).The detection rates for iron transporter protein like virulence genes were fyuA(14%),iroN(33%),iutA(19%),irp5(9.5%),Iss(9.5%),and iucD(9.5%).The detection rates for toxin-like virulence genes were F17(9.5%),hlyF(14%),ropS(33%),phoA(90%)and Ecs3703(57%).3.Polymerase chain reaction was used to determine the distribution of resistance genes in the isolates,and the results showed that the detection rates of tetracycline resistance genes in isolated strains were tetA(19%),tetB(29%)and tetD(14%),respectively.The detection rates for fluoroquinolone resistance genes were gyrA(76%),gyrB(95%),parC(95%),parE(100%),qnrD(43%)and qnrS(9.5%),respectively.The detection rates for β-lactam resistance genes were blaCTX-M(29β),blaTEM(29%)and blaSHV(9.5%),respectively.The detection rate of chloramphenicol resistance genes was floR(24%).The detection rate for sulfa resistance genes was sul2(33%).The detection rates for aminoglycoside resistance genes were aac(3’)-Ⅱa(29%),aacC(33%),aadAI(24%),and strA-B(57%).4.The whole gene map of highly pathogenic Escherichia coli E12 was obtained by whole gene sequencing,and its genomic features were analyzed.The virulence genes of the sequenced strains E12 were compared by the virulence factors of pathogenic bacteria(VFDB)database,and the sequenced strains contained a total of 366 virulence genes.The antibiotic resistance genes(ARDB)and antibiotic resistance(CARD)databases were used to identify the resistance genes of the sequenced strains E12,and a total of 127 resistance genes in 4 categories were found to be included in chromosomes and plasmids,and the resistance phenotypes and genotypes were basically consistent.5.A network model of drug-active ingredient-gene target for the treatment of calf diarrhea caused by atypical enteropathogenic Escherichia coli with Gegenqinlian Decoction was constructed by protein interaction network analysis in network pharmacology,STRING database and Cytoscape software.The results showed that Gegenqinlian Decoction contained 53 active chemical components,among which formononetin,coptisine,epiberberine,quercetin,β-sitosterol,naringenin,kaempferol and baicalin were the main components.The core targets between the drug targets of Gegenqinlian Decoction and the three genetic targets of atypical enteropathogenic Escherichia coli acting on cattle and calf diarrhea genetic targets were PTGS2,IL1B,CXCL8,AKT1,IL6,VEGFA,CASP8,CASP3,and TNF as known by Venny’s analysis.The intersection targets were analyzed by GO gene annotation and KEGG pathway enrichment analysis mainly involved in inflammatory response,positive regulation of RNA polymerase Ⅱ and Ⅲ promoter transcription,HIF-1 signaling pathway,inflammatory bowel disease,IgA-producing intestinal immune network,VEGF signaling pathway and MAPK signaling pathway to complete the regulation of calve diarrhea and fever,which revealed the mechanism of pharmacological effect of Gegenqinlian Decoction on calve diarrhea caused by atypical enteropathogenic Escherichia coli. |
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