Isolation Of Escherichia Coli From Diarrhea Calves And Establishment Of Multiplex PCR For Diarrheagenic Escherichia Coli

The intestinal microbial system of calves has not been stably established and is susceptible to bacterial infection.The main manifestation is diarrhea of calves,which will cause serious harm to the growth and development of calves and the performance of production.Diarrheagenic Escherichia coli（DEC）,one of the main pathogenic bacteria causing diarrhea of calves,is an important zoonotic proto-microbe,which seriously threatens the public health safety in the area.In recent years,the misuse of antibiotics has enhanced the resistance of DEC,further increasing the difficulty of prevention and treatment of the disease.In view of the above situation,this study investigated and collected samples from 5 large-scale dairy farms in Guanzhong area of Shaanxi Province,isolated and identified pathogenic bacteria in the collected samples,established 5 kinds of DEC multiplex PCR detection methods,and detected the DEC isolates and their drug resistance.The results as follow:1.126 diarrhea samples of calves were collected from 5 large-scale dairy farms in Guanzhong area of Shaanxi Province,and 323 strains of 20 kinds of bacteria were isolated and identified from the samples.The three bacteria with the highest detection rate were Escherichia coli,84.13%,Enterococcus faecalis,19.84%and Klebsiella pneumoniae,18.25%.2.We have established a multiplex PCR assay that can detect 5 kinds of DEC.We based on the National Food Microbiology Test Standard,GB 4789.6-2016,which gave11 virulence genes of 5 types of DEC,and with uidA gene as positive reference.The enterogenous Escherichia coli（EAEC）,enterohemorrhagic Escherichia coli（EHEC）and toxic Escherichia coli（ETEC）,enteroinvasive Escherichia coli（EIEC）and enteropathogenic Escherichia coli（EPEC）can be detected in two systems.System A was reaction identification of EAEC and EHEC and the best reaction system for:final concentrations of uid A,pic,agg R,ast A,stx1 and stx2 primers are 0.2μmol/L,0.3μmol/L,0.4μmol/L,0.5μmol/L,0.4μmol/L,0.4μmol/L respectively,2×Accurate Taq Master Mix 10μL,0.5μL bacterial solution template,and add dd H₂O to 20μL.The optimal amplification conditions were:predenaturation at 94℃for 10 min and 30 s;30cycles of denaturation at 98℃for 10 s,annealing at 59℃for 30 s,extension at 72℃for 1 min and 30 s;final extension at 72℃for 2 min.System B was reaction identification of EAEC and EHEC and the best reaction system for:final concentrations of inv E,esc V,bfp B,lt,stp and sth primers are 0.3μmol/L,0.4μmol/L,0.3μmol/L,0.3μmol/L,0.4μmol/L,0.4μmol/L respectively,2×Accurate Taq Master Mix 10μL,0.5μL bacterial solution template,and add dd H₂O to 20μL.The optimal amplification conditions were:predenaturation at 94℃for 10 min and 30 s;30 cycles of denaturation at 98℃for 10 s,annealing at 59℃for 30 s,and extension at 72℃for 1 min.Final extension at 72℃for 2 min.3.The multiplex PCR assay was tested for specificity and sensitivity,and results showed that the established multiplex PCR method was not have specific reaction against 6 common intestinal bacteria namely Salmonella,Cloakobacter pyogenes,Proteus,Klebsiella pneumoniae,Enterococcus faecalis and Staphylococcus aureus;The detection limits of ETEC,EIEC,EPEC,EAEC and EHEC are 1.09×10³ CFU/m L,2.43×10³ CFU/m L,5.71×10³ CFU/m L,7.98×10³ CFU/m L and 1.27×10³ CFU/m L.4.The multiplex PCR assay was used to detect 183 strains of Escherichia coli isolated from clinical samples,and 17 strains of diarrheagenic EScherichia coli were obtained,including 6 strains of EAEC,4 strains of ETEC,4 strains of EPEC,2 strains of EHEC and 1 strain of EIEC.5.Drug sensitivity test results showed that 17 DEC strains were resistant to ampicillin,amoxicillin,gentamicin,enrofloxacin and tetracycline,and were sensitive to cefoxitin,ceftazidime,meropenem,amicacin and polymyxin.