| The transmission of antibiotic resistance in farming industry is a global problem that harm to public health,of which pig production is the main source of antibiotic resistance.For a long term,the domestic pig production consume mass of antibiotics in production,causes spreading of antibiotic resistance from the livestock production system to agricultural production system,and may invade food chain to threat public health.Therefore,it is necessary to research the pollution features and distribution of antibiotics and resistance genes aiming at the waste treatment and reuse process in large-scale pig farm,and evaluate the risk of farming environment antibiotic resistance spreading.Based on the investigation of 22 large-scale pig farms and the surrounding environment all over the country,this study made a systematic analysis on 1)distribution features and pollution status of antibiotics,resistance genes and potential resistance bacteria in manure,slurry and farmland with manure application,together with the effect of environmental factors(antibiotics and physico-chemical properties)on the spreading of antibiotic resistance in 13 pig farms;2)evaluation of potential spreading risk and distribution characteristics of antibiotics and resistance genes in groundwater environments around 9 typical pig farms.The main results are as follows:(1)Based on investigation and sample collection in 13 typical pig-breeding monitoring sites,LC-MS/MS was used to reveal the content level and contamination characteristics of 18 commonly used veterinary antibiotics such as tetracycline,sulfonamide,quinolones and macrolides in manure,slurry and farmland with manure application.The results showed that the distribution of antibiotics in different regions was distinct,but tetracycline,sulfanilamide and quinolone were most widely used in pig production,of which tetracycline,oxytetracycline,doxycycline,sulfadiazine,sulfamethoxazole,sulfanilamide oxygen pyrimidine,ofloxacin,norfloxacin were the highest residue levels in animal waste and soil.Conventional slurry treatment has limited ability to degrade veterinary antibiotics,with various and high concentrations of antibiotics still remained in the external drainage after treatment,posing risk of contamination of soil and water environment.The antibiotics contained in soils fertilized with manure were significantly lower than that of manure in contents,and showed certain similarities in composition.In contrast,there was no antibiotic residue detected in the forest soil outside the feedlot area.(2)qRT-PCR assay was used to quantify the environmental abundance of 17 common resistance genes,and reveal the effects of environmental factors on the distribution of resistance genes.The results showed that the samples from manure,slurry and soil contained a large number of resistance genes,and the rate was detected as 100% in addition to the three genes of ermB,qnrA and qnrB genes.The slurry treatment process cannot effectively remove the resistance genes,manure land application may increase resistance genes and there is a risk of antibiotic resistance spreading.There was no significant correlation between abundance changes of resistance genes in animal waste,sewage,and soil,and antibiotic concentration levels.Some resistance genes are still of high abundance without antibiotic induction.Tetracycline,Quinolone,Zn,and TP have a common selection effect on feces and soil resistance genes such as tetO,tetW,ermB and mefA/E.The results of quantification and distribution characteristics of resistance genes imply that even if reduced or stopped the use of antibiotics in the aquaculture industry,there is still the risk of antibiotic resistance spread.(3)High-throughput sequencing was used to analyze the 16 S rRNA gene diversity of bacteria,revealing the community and composition of potential resistance bacteria in manure,slurry and soils,together with the effects of environmental factors on their distribution and succession.Scarcity partial least-squares regression results showed that 421 OTUs were associated with relative abundance of resistance genes in manure,slurry and soil samples collected from different pig farms,of which 68 OTUs had a relative abundance of ? 0.01% as a major potential risk resistance bacteria.These potential resistance bacteria mainly belong to Actinobacteria,Firmicutes,and Proteobacteria at phylum level,but show abundant diversity and divergence at genus level.The antibiotic concentrations showed significant effects on the composition and distribution of resistant bacteria in the manure,slurry and soil samples,but the physicao-chemical properties only showed significant effects on the composition and distribution of resistant bacteria in the manure and soil samples.(4)Aiming at the risk of the proliferation of antibiotic residues in slurry of large-scale pig farm to groundwater environment,9 large-scale pig farms in North China were selected to study the contamination characteristics of sulfonamide,quinolones and tetracycline veterinary antibiotic residues in the groundwater around these farms,and the correlation between antibiotic contents and resistance gene copy numbers.The results of antibiotic residue detection found that 14 antibiotics were detected in 18 groundwater samples from the tested pig farms and surrounding villages and towns,including tylosin,chlortetracycline,oxytetracycline,enrofloxacin,ciprofloxacin and norfloxacin and so on,and the content varies from 0.22 to 222.3 ?g/L.By using PCA sorting and hierarchical clustering analysis,it is initially judged that veterinary antibiotics can infiltrate into the groundwater from the pig farm and further diffuse into the groundwater environment of the surrounding villages and towns,which caused the risk of public health.A variety of resistance genes can be detected in groundwater,and there is a clear linear relation between the copy numbers of qnrA,tetG,tetM and tetO and antibiotic concentration among them. |
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