The Spread Mechanism And Fate Of Antibiotic Resistance Genes In Pig Waste And Their Disposal Process

The abuse of animal antibiotics has led to the serious situation of antibiotic resistance in livestock breeding environment.Antibiotic resistance genes(ARGs)and antibiotic resistant bacteria(ARB)from livestock waste can harm human health and environmental organisms through food chain contamination and farmland utilization of animal manure.In order to prevention and control of animal-derived ARGs in the environment,we selected two typical swine farms with different piggery wastewater treatment systems(PWWTSs),farm 1 and farm 2,as the target farms in this study,and carried out the following research:1)to investigate the occurrence and discharge characteristics of common ARGs in swine waste,and finding out the migration regularity of ARGs during the whole waste disposal process to assessment the impact of different waste treatments on ARGs;2)to explore the distribution characteristics of mobile genetic elements(MGEs)in swine waste and its disposal process,as well as the evolution rules of microbial community structure and composition during the whole waste disposal process,meanwhile,to elaborate the molecular diffusion mechanism of ARGs in swine waste from the perspectives of horizontal and vertical transfer.We found that sul-ARGs(sull and sul2),tet-ARGs(tetW,tetO and tetQ)qnr-ARGs(oqxB and qnrS)and erm-ARGs(emrC)were detected in all pig feces of different swine groups with a detection rate of 100%.We also found that ?-lactamase genes(bla genes;blaTEM-1,blaGES-1 blaOXA-1 and blaAmpc),which are closely related to human health,are also prevalent in swine farming environment,and it is confirmed that swine waste was confirmed to be an important repository of high-risk gene blaNDM and mcr-1.And the levels of bla gene in sow feces was significantly higher than that of piglets and fattening pigs.Furthermore,ARGs were distributed throughout the whole process of swine waste disposal process,and their change trends were as follows:ARGs increased in biological treatment unit,and significantly decreased with the sedimentation of suspended substances in sedimentation tank and filtration of ceramsite filter tank.Although of all levels of technology,the ARGs abundance has a certain amount of reduction after the treatment,there were considerable concentration of ARGs in final effluent,indicating that the current treatment technology of swine waste is not ideal for the removal of ARGsOur results also showed that MGEs(which is closely related to the spread of ARGs,like class 1 integron,class 2 integron and conjugative plasmids)were widespread in swine waste of large-scale swine farms,and there was a same rule as intl1>intl2>traA(F plasmid genetic marker gene)in both summer and winter.Importantly,the three MGEs were basically consistent with the variation trend of ARGs,and showed a significant positive correlation with them.However,they have little correlation with most dominant bacterial groups in swine waste,only treponema-2 and non-dominant bacterial groups,Rikenellaceae RC9 and Ruminococcaceae ucg-005,have a close positive correlation.In conclusion,horizontal gene transfer was the main driving factor of ARGs dissemination in swine waste,and some microbial communities have also a very important influence on the distribution of ARGs.Finally,we isolated pan-resistant Escherichia coli with blaNDM-i/blaNDM-5 from swine farm.MICs results showed that the resistance levels of blaNDM-5 positive bacteria to various antibiotics were obviously higher than that of blaNDM-1 positive strains,and we found that blaNDM-5 genes are located in a self-transfer plasmid,it can spread to antibiotic resistance to other bacteria.The sequencing results revealed that plasmid structure of pNDM5_SF01 included a complete conjugative/type/IV secretion systerm T4SS(virB--virD),which is closely related to the plasmid transfer.