Effect Of Water Extracts Of Gallnut On Drug Resistance Gene Expression Of Escherichia Coli Isolated From Chicken Houses

Escherichia coli(E.coli)is one of the most common zoonotic pathogens.Poultry can excrete E.coli in their faeces,and it can spread in the air and constantly accumulate,which causes higher concentration of E.coli in the air environment and pollutes the chicken house.E.coli can enter the body again with the breathing of chicken and cause the outbreak of colibacillosis in chicken.Meanwhile,with the long-term use of antibiotics in veterinary clinical,the antimicrobial resistance has become widespread amongst E.coli,indicating difficulties in the treatment of E.coli diseases,a rise in the economic loss of poultry industry,and a risk in the health of the chickens and farm staff.However,there is little information about the drug resistant E.coli isolated from the air of the chicken farm.The aim of the present study was to determine the antimicrobial resistance patterns of E.coli isolated from the air of the chicken house and the phylogenetic analysis of 16 S rRNA methylation resistance gene rmtB,and to investigate the effect of water extract of Gallnut on drug resistance gene expression in E.coli isolates,which provides more detailed information of antimicrobial resistance patterns and experimental basis for Chinese traditional medicine to improve the drug resistance in E.coli isolates.Methods: The natural sedimentation was used to collect E.coli in the air of large-scale chicken houses.The E.coli isolates were identified by morphological characteristics,biochemical characteristics and 16 S rDNA gene amplification and characterized by antimicrobial susceptibility testing and antibiotic resistance genes using standard disk diffusion tests and PCR,respectively.The genetic evolution analysis of 16 S rRNA methylase gene rmtB was based on the NCBI GeneBlast.Minimal Inhibitory Concentration(MIC)was measured by microdilution method to evaluate the antibacterial effect of water extracts of TCM to E.coli.After treatment with 1/2 MIC,1/4 MIC,1/8 MIC concentration of water extract of Gallnut,the total RNA was prepared and then the drug resistance gene expression was detected by Real-time PCR(RT-PCR).Results:(1)A total of 63 isolates were identified as E.coli from the air environment in the chicken houses.Antibiotic resistance profiles showed that the drug resistance rate of E.coli isolates to the Oxacillin and Penicillin both reach to 93.65%,and 92.06% of E.coli come into being drug resistance to Gentamicin.Multiple drug resistance phenomenon is very common and the rate of multiple resistant reach to 98.41%.(2)The PCR results showed that each E.coli isolate harbored at least one drug resistance gene of quinolone and sulfonamide.Among the seventeen resistance genes,qnrS(100%)was the most common genotype identified.Most of the isolates contained multi drug resistant genes,which carried at least 4 drug resistant genes and up to 15 drug resistant genes.Out of 63 isolates,33.33% E.coli strains carried 13 drug resistant genes.(3)The nucleotide sequence and phylogenetic tree analysis of E.coli strains showed that the genetic variation in all isolated samples is low,while the the genetic variation between isolated samples and isolates in NCBI database is high.(4)Among the 11 kinds of TCM,the Gallnut and Radix Scutellariae exhibited stronger antibacterial activity than Fructus forsythia,Sophora flavescens,Lonicera japonica,Coptis chinensis and Euphorbia humifusa.Howerer,Arnebia euchroma and Radix isatidis demonstrated no antimicrobial effect.(5)E.coli was incubated with the water extract of Gallnut in different concentrations what were 1/2 MIC,1/4 MIC and 1/8 MIC.RT-PCR results showed that all different concentrations of the water extract of Gallnut can reduce the gene expression level of 16 S rRNA methylation resistance gene armA and rmtB of E.coli from the chicken houses.Conclusion: The drug resistance and multiple resistance of E.coli obtained from the air environment of chicken house was widespread and serious.Moreover,most of E.coli isolates contained durg-resistant genes.Gallnut extract decreased the mRNA expression of resistance genes in E.coli.The water extract of Gallnut can reduce the mRNA expression level of drug resistance genes in E.coli of the air of chicken house.