| The incidence of colibacillosis in calves has increased,with the development of the dairy and beef cattle industry in Inner Mongolia.Whether the persistent,repeated,and refractory infection of calf colibacillosis is related to the formation of Escherichia coli biofilm? Whether E.coli biofilm positive strains are sensitive to antibiotics commonly used in veterinary clinics? what is the effect of sensitive drugs on the formation of E.coli biofilm? And whether the formation of E.coli biofilm can be affected by regulating the expression of genes related to biofilm formation? So in this study,the formation of biofilm and drug resistance of E.coli isolated from calves in Inner Mongolia were clarified.The effects of the sensitive drug on the regulation of lux S,mot A,fli A,pfs,and csg D expression that related to E.coli biofilm formation were studied.In this study,biochemical identification and molecular biology methods were used to identify E.coli.The biofilm forming ability of E.coli isolates was determined by the improved crystal violet assay method.The minimum inhibitory concentrations(MICs)of the strains to 20 antibiotics were detected by the micro-dilution method in vitro,and the sensitive antibiotics were determined.The strains with strong biofilm forming ability and multi-drug resistance were selected for follow-up study.The adhesion value and the anti-biofilm effect of antimicrobial agents on E.coli were tested quantified in96-well plates by crystal violet(CV)assay for determining biofilm formation ability;Biofilm growth curve were evaluated using XTT assays;biofilm was cultured in 24-well plates and cell climbing plate,and production of biofilms and the disruption of the antimicrobial agents on biofilm was analyzed using laser confocal inverted microscope.Furthermore,the regulation of m RNA expression of lux S,mot A,fli A,pfs,and csg D genes by different concentrations of antimicrobials agent were determined by the QRT-PCR method.51 strains of E.coli were isolated from 104 samples of calf disease.The results showed that the E.coli isolates were divided into four groups with strong,moderate,weak,and absent biofilm-forming ability accounting for25.5%(13/51),17.6%(9/51),25.5%(13/51),and 31.4%(16/51),respectively.The results showed that the isolates were resistant to trimethoprim(90.2%),sulfadiazine(78.4%),tetracycline(98.0%),doxycycline(70.6%),ampicillin(84.3%),cefalotin(68.6%)and cefotaxime(68.6%).The isolates were sensitive to cefoxitin(9.8%)and meropenem(0%).Cefoxitin was selected for the subsequent study.92.2%(47/51)of the isolates were multi-drug resistant strains(resistant to 3 categories or above),94.3%(33/35)of the biofilm-positive strains were multi-drug resistant strains,and 51.4%(18/35)resistant to 6 antibiotic categories.Since the biofilm-positive strains are sensitive to cefoxitin,cefoxitin was selected for subsequent experiments.The7 isolates with strong biofilm positive and the most complicated drug-resistant phenotypes were selected as the subsequent adhesion value(B)test strains.The results of the biofilm adhesion value(B)indicated that cefoxitin showed stronger inhibition of biofilm adhesion value.Since isolate38 has the highest adhesion value and the strongest film-forming ability,follow-up studies focused on the effect of cefoxitin on the biofilm of isolate38.The results indicated that cefoxitin showed stronger inhibition of growth rate,and had serious damage to the biofilm production.QPCR results showed that cefoxitin significantly decreased the m RNA expression of lux S,mot A,fli A,pfs,and csg D,which were formed by biofilm at 1/2 MIC and MIC.Cefoxitin may inhibit the formation of biofilm by downregulating the above positive regulatory gene expression.In conclusion,the prevalence rate of E.coli biofilm-positive strains from calves was 68.6% in Inner Mongolia;51 clinical isolates are highly multi-drug resistant,and the isolates were sensitive to meropenem and cefoxitin;cefoxitin may regulate the formation of biofilms by inhibiting luxS,motA,fliA,pfs and csgD genes expression. |
