Isolation,identification,pathogenicity And Drug Resistance Of APEC From Some Areas Of Anhui Province

Avian pathogenic Escherichia coli(APEC)is the pathogenic agent of Escherichia coli disease in poultry.APEC serotypes are numerous,without cross protection,often resulting in mixed infection,prevention and treatment is difficult.In recent years,the abuse of antibiotics has led to the emergence of multi-drug resistant strains,which hinder the prevention and control of poultry Escherichia coli,restrict the economic development of livestock and poultry industry,and threaten the public health safety of human beings.Therefore,the pathogenicity and drug resistance of APEC in some areas of Anhui province were studied in this study,which is helpful to understand the pathogenesis and drug resistance of APEC in Anhui region,and provides medication reference for the prevention and treatment of APEC in Anhui region,which is of great significance for protecting human public health security.In this study,92 APEC isolates were isolated and identified from diseased materials in some areas of Anhui Province.The genotyping,14 virulence genes and animal pathogenicity tests were carried out to explore the relationship among them.Secondly,12 drug resistance genes were detected and drug sensitivity experiments were conducted to explore the association between drug resistance genotype and drug resistance phenotype.The specific research contents and results are as follows:1.Isolation and identification of APEC in some areas of Anhui provinceThe isolated APEC birds were collected from Feidong,Feixi,Huaibei city,Shouxian county of Huainan City,Shucheng County of Lu An city,Huaiyuan County of Bengbu City and other Anhui regions.First,fresh liver and lung were collected aseptically and inoculated in LB solid medium.Eosin-Methylene Blue Agar(EMB)solid medium was used for isolation and culture.After identification by Gram staining and biochemical test,primer of pho A gene was designed for PCR identification,and 92 strains of E.coli were isolated and identified.The specific primers of chu A,yja A and Tsp E4.C2 were used to determine the genotyping of 92 isolates by PCR.The results showed that 19 isolates belonged to group A(20.65%)and 35 isolates belonged to group B1(38.04%).There were20(21.74%)strains belonging to B2 group and 18(19.57%)strains belonging to D group.2.Study on pathogenicity of APEC in some areas of Anhui provinceAccording to the virulence gene classification,14 virulence genes were selected and specific primers were used to detect virulence genes of 92 isolated strains by PCRtechnology.The results showed that,the positive rate of 10 virulence genes including fim C,iuc D,iut A,omp A,mat,iss,tra T,ibe B,yij P and iro N was over 50%,among which omp A had the highest detection rate of 98.91%,mat 95.65% and fim C 91.30%.All 14 virulence genes were detected,and each strain could carry 3-14 virulence genes.Five isolates(A2,A46,A65,A68 and A80)carrying the maximum number of virulence genes were selected for animal pathogenicity test.The experimental animals were 7-day-old white-feathered chickens.According to the pre-test,the injection dose was 5×108CFU/ chick,and the control group was injected with equal dose of PBS.According to the death of chickens and pathological changes of necropsy,A2,A68 and A80 were highly pathogenic strains,while A46 and A65 were low pathogenic strains.3.Study on resistance of APEC in some areas of Anhui provinceTwelve commonly used antibacterial drugs were selected according to the types of drugs,and the drug sensitivity of 92 isolates was detected by disk agar diffusion method(K-B method).The results showed that 92 isolates of APEC were fully resistant to florfenicol(Chloramphenicol),Sulfisoxazole(sulfonamides)and tetracycline(tetracycline).88 strains(91.30%)were sensitive to ceftazidime(penicillins),and all strains showed different degrees of drug resistance to 14 antibiotics,with serious multiple drug resistance.The detection rate of penicillin resistance gene bla TEM,bla CTXM,colistin resistance gene mcr and quinolone resistance gene gyr B was above 90%.The detection rate of β-lactam resistance gene qnr A was the lowest 4.35%.All 12 drug resistance genes were detected.In conclusion,among 92 strains of APEC isolated and identified in some areas of Anhui in this study,group B1 was the main dominant subgroup,with high carrying rates of fim C,omp A and mat virulence genes.The drug resistance of tetracycline,multidrug resistant is serious,and multiple drug resistance is common.gyr B,mcr,bla TEM and bla CTXM were highly carried.