Distribution Of Antibiotic Residues And Antibiotic Resistance Genes In Fecal Contamination And Surrounding Environment Of Typical Pig Farms In Sichuan Province

In recent years,antibiotics have been widely used in China ’s pig breeding industry.Only a small part of them can be metabolized,absorbed and degraded when they enter animals.Most of the antibiotics are discharged in the form of raw drugs through feces and wastewater,and finally enter the ecological environment.Within the scope of the country ’s pig breeding,Sichuan Province is a major pig breeding province,so it is inevitable to use a large amount of antibiotics in the process of pig breeding.Moreover,antibiotics can enter the environment through a large amount of excrement and sewage discharge,and carry out a high concentration of residues.antibiotic resistance genes(ARGs)are likely to cause the increase of antibiotic resistance genes,and will cause harm to human life and health.Therefore,it is very important to study antibiotic residues and ARGs pollution in typical pig farms and their surrounding environment.In this study,17 typical pig farms and their surrounding environment in Sichuan Province were selected as the research objects.Based on solid phase extraction-high performance liquid chromatography-tandem mass spectrometry(SPE-UPLC-MS /MS),26 veterinary antibiotics in seven categories including sulfonamides,quinolones,macrolides,tetracyclines,β-lactams,chloramphenicols,and lincosamides were used as test antibiotics to systematically analyze the antibiotic residues in pig manure,soil,and water.The drug sensitivity of Enterobacteriaceae isolated from the environment was analyzed by micro-broth method,and the distribution of drug resistance genes in Enterobacteriaceae was systematically analyzed by drug resistance gene detection.Based on the metagenomic sequencing method,the microbial community structure characteristics and community function annotation in the samples were analyzed.The main findings are as follows:(1)An SPE-UPLC-MS/MS method for the detection of antibiotic residues was developed based on the investigation of 17 typical pig breeding monitoring sites in Sichuan Province.The results showed that a total of 23 antibiotics were detected in the sampling points of typical pig farms,and the concentration of antibiotics ranged from0.019 μg/kg to 36744.670 μg/kg.Antibiotic content in pig feces samples ranged from2.359 μg/kg to 36616.382 μg/kg.The antibiotic content in the soil surrounding the pig farm ranged from 0.159 μg/kg to 128.288 μg/kg.The antibiotic content in the surrounding water body of pig farm ranged from 0.008μg /L to 114.321μg /L.Tetracycline and quinolone antibiotics were the highest among all the verified antibiotics,especially in fecal samples,which were 91932.037 μg/kg and 8054.112μg/kg,respectively.The detection rate of chloramphenicol antibiotics in fecal,soil and water samples was extremely low,ranging from 0.025 μg/kg to 2.359 μg/kg,among which soil samples were not detected.(2)The gradient dilution coated plate method was used to screen the enterobacter strains of fecal,soil and water samples,and a total of 85 enterobacter strains were screened and isolated.According to the detected antibiotic residue data,18 representative enterobacter strains were selected,and then the microbroth method was used to analyze their drug sensitivity.The results showed that,Most antibiotics contain resistance to the situation.Forty-five ARGs were detected and analyzed by common PCR.The results showed that the detection rates of tetracycline,quinolones,chloramphenicol and β-lactam reached 73.3%,80%,70% and 90%,respectively,and all the samples could be detected: Three tetracycline resistance genes(tet B,tet C,tet M),two quinolone resistance genes(qnr D,qnr S),two chloramphenicol resistance genes(flo R,cml A4),and two β-lactam resistance genes(SHV,CTX-M).(3)Microbial community structure and drug resistance gene characteristics in pig feces,soil and water samples were analyzed based on metagenomic sequencing.The results showed that the microbial population at the phylum classification level was relatively simple,and the main microbial groups in feces,soil and water samples were Firmicutes,Bacteroidetes and Proteobacteria.In feces,soil and water samples,ARGs sul1 、 sul2 、 mdt B 、 tet W/N/W 、 APH3-IIIa 、 tet44 、 ANT6-Ia were the main pollution characteristics in typical pig farm environment.