| Pathogenic E.coli is one of the common pathogens that endanger healthy of livestock and poultry,which can lead to calf diarrhea and dairy cow mastitis and other diseases.Because of its virulence factors and serotypes are complex and diverse,and different in every area,there is no ideal immune vaccine,so the antibiotics are the main measures to prevent and control disease.However,due to irregular use of antibiotics in recent years,the antibiotic resistance of E.coli clinical isolates increased rapidly,which had brought serious challenges to the disease prevention and control of animals.This study collected 112 samples from large-scale dairy farms in some areas of China from 2015 to 2016,and 84 strains of pathogenic E.coli were obtained by identification medium,housekeeping gene phoA PCR detection and animal pathogenic test.Using PCR to detect 16 kinds of virulence genes and 20 kinds of drug resistance genes PCR,the results showed that,there were 60 strains of E.coli could be detected the virulence genes with the rate of 71.43%,of which the F41 was the most prevalent genes,accounting for 31.67%of the positive strains,followed by CS31A(28.33%),F17(26.67%)and irp2(18.33%)while LT and hlyA only 1.67%.The Multiple virulence genes combinations were mainly K99+F41,in addition,the distribution of virulence genes were different in each area.A total of 84 strains of pathogenic E.coli isolates were determined by KB disk diffusion method to 20 kinds of antibiotic sensitive and resistance.The results showed that amikacin,polymyxin B and aztreonam were relatively sensitive,the sensitive rate were 66.67%,60.71%and 60.71%respectively.In contrast,amoxicillin,ampicillin and oxytetracycline were extremely resistance,the resistance rate were 91.67%,89.29%and 88.10%,respectively.Based on the result,all isolates showed 16 kinds of drug resistance spectrum,the drug resistant and resistant spectrum was differences,that of isolates from different areas.All isolates showed 15 kinds of resistant phenotype,among them,amoxicillin-cephalexin-trimethoprim-streptomycin-doxycycline-oxytetracycline-ciprofloxacin was the dominant drug resistant phenotype.In this study,the minimal inhibitory concentration(MIC)of ceftiofur and trimethoprim alone was determined against 8 pathogenic E.coli isolated from cattle by broth dilution method,the combined MIC by checkerboard method,and then calculated fractional inhibitory concentration(FIC).The mutant prevention concentration(MPC)both alone and combined was determined by the method of agar dilution method,and then the value of selectivety index(SI)(MPC/MIC)would be acquired.The results showed that the MICs of ceftiofur and trimethoprim were 0.5-4?g/mL,0.5-256 ?g/mL,respectively.And the FIC index of the combination were 0.25-1,indicating that the two drugs were synergistic or additive effect.The MPC of ceftiofur alone against 8 strains of pathogenic E.coli were 64-512 ?g/mL and SI were 64-256,while the MPC of Ceftiofur were 16-32 ?g/mL and SI decreased to 8-64 when combined with trimethoprim.The MPC Trimethoprim of alone was 512 ?g/mL and SI were 1-4,after administration with cefetaxime,the MPC and SI did not change.It indicated that ceftiofur combined with trimethoprim could not change the MPC of trimethoprim alone,but could lower the MPC of ceftiofur alone against pathogenic E.coli from cattle and reduce the resistant mutant selection window.This study detected the virulence genes and drug resistance of pathogenic E.coli isolated from cattle,the epidemic situation of virulence genes and drug resistance status were preliminarily revealed.Which have a great significance to develop a new type of vaccine based on virulence gene and the rational application of antimicrobial agents in clinical practice.By discussing the effect of ceftiofur alone or in combination with trimethoprim on MPC of pathogenic E.coli isolated from cattle,which provides a experimental evidence for narrowing resistance mutant selection window. |
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