Isolation Of E. Coli And Proteus Mirabilis From Calf Diarrhea Identification,Virulence Gene Detection And Drug Resistance Analysis

Calf diarrhea is a common and frequent gastrointestinal disease,which can lead to the direct death of calves and bring serious economic losses to breeding farms.Both Escherichia coli and Proteus Mirabilis can cause calf diarrhea,among which Escherichia coli is the main pathogen of calf diarrhea.With the abuse of antibiotics in aquaculture,the resistance of E.coli and Proteus Mirabilis is becoming more and more serious.Monitoring and epidemiological investigation of drug resistance and resistance genes of calf E.coli and Proteus Mirabilis are helpful to understand the prevalence and transmission mechanism of drug resistance of E.coli and Proteus Mirabilis,and provide guidance for clinical drug use.E.coli and Proteus Mirabilis were isolated and identified in 198 calf source samples from 8 dairy farms in part of Ningxia.Macconkey,Ered methblue,SS AGAR,BHI,PCR and K-B drug-sensitive disk method were used to isolate and identify the isolated strains,detect virulence genes,pathogenicity,drug resistance genes and drug resistance.The results were as follows:(1)The feces of calves with diarrhea in some areas of Ningxia were collected for isolation and identification,and 151 strains of Escherichia coli and 13 strains of Proteus mirabilis were obtained,with isolation rates of 76.26%(151/198)and 6.57%(13/198)respectively.(2)80 E.coli isolates were selected for the detection of 6 virulence factors.The detection rates of curli pili(crl)and fim H(fim H)were 68.75% and 66.25%,respectively,which were much higher than other virulence genes detected.The combination of crl+fim H virulence genes dominated,accounting for 35%.(3)The 13 strains of Proteus Mirabilis isolates were tested for 6 virulence factors,and it was found that the carrying rates of pili gene(pmf A),thermophilic pili gene(atf A),metalloproteinase gene(zap A)and urease gene(ure C)were the highest,all of which were100%.The rates of adhesion pili gene(uca A)and virulence protein gene(rsb A)in urothelial cells were 92.31%.(4)Mice were infected with Proteus mirabilis with 6 virulence genes,and all of the mice died within 24 hours.After autopsy,pericardial effusion,swelling of liver,spleen and kidney,swelling of small intestine and congestion and swelling of large intestine were found.(5)The drug susceptibility test of 80 E.coli isolates and 13 Proteus Mirabilis isolates showed that the drug resistance rates of penicillin and ampicillin were 100% and 97.5%and 100% and 100%,respectively.The drug resistance rates of tetracycline and cotrimoxazole were 93.75%,92.50%,100%,92.31%,respectively.The drug resistance rates of norfloxacin,ciprofloxacin and ofloxacin were 63.75%,52.50%,46.25% and53.84%,46.15%,30.77%,respectively.All were most sensitive to amikacin,and the resistance rate was less than 20%.(6)Eighty isolates of Escherichia coli and 13 isolates of Proteus mirmiris were selected for drug resistance gene detection.The results showed that β-lactam bla TEM type gene carrying rate was the highest,and the positive rate was above 95%.The qnr S gene carrying rate of quinolones was the highest,with positive rates of 51.25% and 46.15%,respectively.The aac(3)-IV gene carrying rate of aminoglycosides was the highest,with positive rates of 27.5% and 23.08%,respectively.The highest rate of tet A resistance gene was found in Tetracycline,with positive rates of 66.25% and 69.23%,respectively.The carrying rate of drug resistance gene of sulfamides sul2 was the highest,with positive rates of 73.75% and 84.62%,respectively.In this study,the isolation rate,prevalence of virulence genes,drug resistance and prevalence of drug resistance genes of calving E.coli and Proteus Mirabilis were obtained in some areas of Ningxia,which provided experimental basis for clinical rational drug use,prevalence of virulence genes and drug resistance genes.