Study On The Detection Of Drug Resistance Genes And FloR Gene Transmission In The Environment Of Duck Farms In Zhanjiang Area

With the continuous expansion of breeding scale,there are more and more types of animal diseases,antibiotics were more and more widely used in the treatment of diseases,and even serious abuses have occurred,resulting in the infectiousness of bacteria and the occurrence of multiple expected phenomena,reduce the effectiveness of treatment.The presence of drug-resistant genes was a key factor in the development of resistance in bacteria.Animal-derived bacteria develop drug resistance,and the drug-resistant genes it carries would be transmitted through the food chain and then passed on to humans,which would have a major inpact on human health.Salmonella was one of the most important zoonotic pathogens,and antibiotics were used for its control.Flufenicol,an amido alcohol antibiotic,was specifically used in veterinary medicine and was widely used in the prevention and treatment of animal diseases.The selective pressure exerted by long-term improper use of flufenicol in duck farms will cause its drug resistance to continue to increase.In order to understand the effects of flufenicol residues in the environment on environmental safety,the indoor florfenicol stress soil model was established,and the susceptibility test of soil bacteria to antibacterial drugs was carried out by double dilution method of broth and paper diffusion method;PCR method was used to investigate the drug-resistant genes in duck farm environment in Zhanjiang,and Salmonella was isolated from duck farm environment by microbial isolation and identification method.Sensitivity of isolated strains to 22 antibiotics by double dilution method of broth;the spread of salmonella florfenicol resistance gene floR was analyzed by PCR amplification,conjugation test,and electrical conversion test.The main findings are as follows:1.Effect of flufenicol stress on soil bacterial resistanceFlufenicol was added to the soil at a concentration of 0 mg/kg,0.1 mg/kg,1 mg/kg,10mg/kg,100 mg/kg.Soil samples were collected on the 14 d,28 d,and 56 d after adding the drug.The susceptibility test of isolated soil dominant bacteria was carried out by double dilution method of broth,and susceptibility test of antibiotics was carried out by paper diffusion method.The results showed that the bacterial drug resistances were increased with the increasing concentration of florfenicol in soil.The susceptibilities of florfenicol-sensitive bacteria to 9 of 18 antimicrobial agents were significantly higher than that of florfenicol resistant bacteria?P<0.01?.2.Detection of drug-resistant genes in duck farm environment and analysis of drug resistance of Salmonella in duck farm areaExtract total DNA from duck feces,soil,and water.The PCR was used to detect the distribution of 25 common resistance genes such as floR,bla _CTX-M,aad A1,Sul1,tet M,ere A and oqx A in the duck farm environment.The results showed that 25 drug resistance genes were detected in water,24 were detected in soil,23 were detected in feces.Among them,the detection rate of aad A1 gene in soil reached 100%,13 drug resistance genes with detection rate higher than 80%,and 5 species with detection rate less than 50%;the detection rate of floR gene and aad A1 gene in water was 100%,12 drug resistance genes with detection rate higher than 80%,and 8 drug-resistant genes with detection rate less than 50%.Besides,there were 9 kind of drug-resistant genes that detection rate reached100%in feces,9 kinds of drug-resistant genes with detection rate higher than 80%,and 1kind of genes with detection rate less than 50%.In addition,5 or more drug resistance genes were detected in 140 samples of feces,soil and water.Isolation,identification of Salmonella and the minimum inhibitory concentration?MIC?of the isolated strains on 22drugs including ceftriaxone,florfenicol and ciprofloxacin etc were determinated.The results showed that 92 strains of Salmonella isolated and identified had a resistance rate of up to 100%to 9 drugs such as amoxicillin,lincomycin,oxytetracycline and so on.All isolated Salmonella can tolerate at least 9 drugs,and 55.43%?51/92?of the strains can tolerate more than 16 drugs,and 4.35%?4/92?of the strains was resistant to up to 21 drugs.Conclusion:Drug resistance genes were ubiquitous in the duck farm environment,and the phenomenon of multiple genes coexisting at the same time was serious.The drug resistance genes detected in feces,soil and water from the same farm were similar.Salmonella isolated from duck farms was more resistant,and all of them exhibit multi-drug resistance,at least 9 antibiotics.3.The spread of Salmonella resistance gene floRSalmonella carrying florfenicol resistance gene floR isolated from the duck farm environment was used as the donor bacteria,and E.coli C600 was used as the receptor bacteria for the conjugation test.The PCR method was used to detect the florfenicol resistance gene floR in the zygote,and the plasmid of floR positive bacteria was extracted by the kit.The method of electro-transformation and PCR were used to detect the spread of the floR gene.The results showed that 3 strains of Salmonella carrying floR were combined with E.coli C600 to obtain 3 strains of zygote with a zygosity rate of 100%?3/3?;3 strains of positive zygote carrying floR were used as donor bacteria,and E.coli DH5?was used as the receptor bacteria to conduct the electric transformation test,one strain of Salmonella was successfully transformed.The obtained transformants and zygotes were confirmed by PCR detection.The PCR amplification product sequence of the positive transformants was same as the floR gene sequence.Conclusion:Salmonella florfenicol resistance gene floR can be spread horizontally through plasmid-mediated.