| The purpose of this study was to elucidate the biofilm formation and drug resistance of Escherichia coli from calves in Hohhot,Inner Mongolia,and to explore the effects and their mechanisms of berberine and thymol alone and in combination with antibiotics on biofilm formation,so as to provide theoretical basis for the treatment of calf diarrhea caused by biofilm-positive Escherichia coli.In the study,biochemical identification and molecular biological identification methods were used to isolate and identify E.coli from diseased materials.The biofilm formation ability of the isolated strains was determined by modified crystal violet staining.The drug resistance spectrum of the isolated strains was determined by microdilution method.PCR method was used to detect the efflux pump gene of the isolated strains.qRT-PCR method was used to detect the expression of efflux pump gene acr B in biofilm positive strains in biofilm state and planktonic state.The minimum inhibitory concentration(MIC)of the two drugs was determined by micro checkerboard dilution method.The effect of drugs on the adhesion value(B)of isolated strains was determined by crystal violet staining.XTT reagent was used to detect the effect of drugs on the biofilm growth curve of isolated strains.The effect of drugs on the biofilm formation of isolated strains was observed by laser confocal microscopy.The effects of drugs on the expression of biofilm formation related genes luxS,csgD,fliA and efflux pump formation related genes acrA,acrB,tolC and marA were detected by qRT-PCR.The results showed that 92 strains of E.coli were isolated from 208 calf samples,with an isolation rate of 44.23%.The number of strains with strong,medium,weak and no biofilm formation ability in 52 strains of calf diarrhea Escherichia coli were 13,9,13 and 17,respectively.The 52 isolates were sensitive to meropenem,minocycline,amikacin,and cefoxitin,and the sensitivity rates were 100%,69.2%,88.5%,and 86.5%,respectively.The resistance rates to tetracycline,doxycycline,ampicillin,trimethoprim,sulfadiazine,cefalotin,cefotaxime,and cefepime were 98%,71.2%,84.6%,90.4%,78.8%,67.3%,67.3%,and 48.1%,respectively.The number of strains resistant to three or more drugs was 48,including 33 biofilm-positive strains.Fifty of 52 isolates carried 7 efflux pump genes.Three strains with strong biofilm formation ability,large number of drug resistance and carrying efflux pump gene acr B had significantly or extremely significantly higher expression of this gene in biofilm state than in planktonic state.Berberine and thymol showed synergistic effects when combined with minocycline,respectively.Berberine alone or in combination with minocycline could significantly reduce the adhesion value(B)of biofilm-positive strains.The MIC group of thymol combined with minocycline could significantly reduce the adhesion value(B)of biofilm-positive strains,while minocycline alone had no significant effect on the adhesion value(B)of biofilm.At the same time,64,32,16 μg/m L CCCP could also significantly reduce the adhesion value(B)of strains.The MIC,1 / 2 MIC group of berberine and thymol alone,the MIC group of the two drugs combined with minocycline,and 64,32,16μg/m L CCCP could inhibit the growth and formation of biofilm in a concentration-dependent manner.qRT-PCR results showed that the MIC,1 / 2 MIC group of berberine and thymol alone,the MIC group of the two drugs combined with minocycline,and 64,32,16 μg/m L CCCP could significantly or extremely significantly down-regulate the expression of biofilm formation-related genes and efflux pump formation-related genes.In summary,the prevalence of biofilm-positive Escherichia coli from calves in Hohhot,Inner Mongolia is relatively high,and the multi-drug resistance is more serious.Berberine and thymol alone and in combination with minocycline can inhibit the synthesis of multidrug efflux pumps by down-regulating the expression of biofilm formation-related genes and efflux pump formation-related genes,thereby inhibiting the formation of biofilm. |
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