Fate Of Antibiotic Resistance Genes And Its Micro-ecological Mechanisms In The Process Of Dairy Manure Resource Utilization

A large number of antibiotic resistance genes(ARGs)existed in livestock manure,which will transfer and spread among microbial populations,and even spread to humans through the food chain to threaten human health.Dairy manure is the main kind of livestock manure,but the occurrence and the dynamics of ARGs in dairy manure and its subsequent resource utilization processes are not clear,the relevant micro-ecological mechanisms are not clear,and the transmission and dissemination risks of ARGs in dairy manure still needs to be explored.In the present study,dairy manure derived from a large-scale dairy farm in Hohhot were chosen to analyze the occurrence of ARGs,and the fate of ARGs in dairy manure and its subsequent resource utilization processes were investigated.Furthermore,the relevant micro-ecological mechanisms and the potential transmission and dissemination risks of ARGs were also explored and evaluated.The main results are as follows:(1)Based on metagenome sequencing,the diversity and abundance of ARGs in large-scale dairy manure and its treatment process were analyzed.The results showed that there was a high diversity of ARGs in dairy manure,with up to 1721 ARG subtypes,which mainly divided into several drug classes such as multi-drug,peptides,β-lactams and tetracyclines,and also divided into several resistance mechanisms including coding antibiotic efflux,antibiotic target alteration,antibiotic target protection and multiple resistance mechanism.After treatment,the ARGs subtypes increased from 1283 to 1417 and 1435,and the total abundance of ARGs increased from 150.64 reads/ng DNA to 241.98 and 166.14 reads/ng DNA.Redundancy analysis and network analysis showed that phyla Firmicutes,Bacteroidetes,Plantomycetes,Actinobacteria and Proteobacteria could explain 88.3% of the changes of ARG drug class.Genera Actinomadura,Glycomyces,Luteimonas,Saccharomonospora and Thermobifida and so on were potential hosts carrying ARGs,and their distributions in dairy manure dominated the distributions of ARGs.(2)Considering the large amount of liquid manure and tetracycline antibiotics as clinical drugs,the lab-scale anaerobic digestion of dairy manure was carried out.The dynamics of typical tetracycline ARGs(tet A,tet C,tet G,tet O,tet Q,tet T,tet W and tet X)were analyzed by fluorescence quantitative PCR(q PCR)to explore the effects of polyethylene microplastics(PE MPS)on ARGs during anaerobic digestion.The results showed that PE MPs make it difficult to reduce ARGs by anaerobic digestion,and even increased the abundance of ARGs after anaerobic digestion,especially in the presence of OTC or/and Cu.Microbial community analysis showed that PE MPs increased the abundances of some bacteria related to the hydrolysis,acidification and gas production,such as Clostridium,unclassified＿Clostridiales,unidentified＿MBA08and unidentified＿OPB54 and so on.Moreover,network analysis and redundancy analysis showed that those bacteria were potential hosts of ARGs,and PE MPs drives the change of potential hosts via changing the chemical parameters to enrich ARGs.(3)The anaerobic digested biogas slurry and biogas residue were applied to the soil as fertilizers,and the pot experiment was carried out to track the changes and distributions of typical tetracycline ARGs in the soil.The results showed that tet G,tet O,tet Q,tet T,tet W and tet X were existed in soil,which were consistent with biogas slurry and biogas residue.Compared with digested products,tet A and tet C disappeared during storage.The application of biogas slurry and biogas residue increased the abundance of ARGs in soil,which was 4.51～63.60 times higher than that in control group.However,the increase of biogas slurry application did not lead to the accumulation of ARGs in soil.Planting vegetables changed the distributions of ARGs in soil: the abundances of six ARGs in the soil planted with Brassica napus and Raphanus sativus were higher than those in control soil without vegetables,and there were significant differences in the abundance of ARGs in non-rhizosphere soil and rhizosphere soil.The main reason maybe that the microbial community diversity and bacteria composition are different among different soils,and genera Altererythrobacter,unclassified＿Microbacteriaceae,Brevundimonas,unclassified＿Bacillaceae and unclassified＿Micrococcales dominated the distributions of ARGs in soil and play an important role in the changes of ARGs.(4)ARGs in soil could be transferred to plants,and the application of dairy manure biogas slurry and biogas residue promotes that transfer.Tracking the changes of ARGs in the process of dairy manure resource utilization,it was found that ARGs were reduced,especially in the storage stage of biogas slurry and biogas residue.Mobile genetic elements(MGEs)are the vectors of ARGs’ horizontal gene transfer.In the present study,three MGEs vary greatly,and only int I1 has always existed in the process of dairy manure resource utilization and even transferred to plants,indicating that int I1 was related to the transfer of ARGs among dairy manure,soil and plants.ARGs are also positively correlated with a variety of human pathogens,including Aeromonas,Agrobacterium,Bordetella,Campylobacter,Clostridium,Corynebacterium,Enterococcus,Helicobacter and Ralstonia,suggesting that the resource utilization of dairy manure may lead to the transmission and dissemination of ARGs among pathogens,which might pose potential risks to natural environment and human beings.This study clarified for the first time that there are a large number and diversity of ARGs in dairy manure derived from a large-scale dairy farm in Hohhot,which emphasizes the severity of ARGs in dairy manure.It also found that manure application promoted the transfer of ARGs between soil and plants and had the risks of ARGs’ s transmission and dissemination,although the abundance of ARGs were reduced compared with raw dairy manure.The key species groups driving the fate of ARGs were revealed and the relationship between key species groups and environmental factors was explored.The results of this study provided a basis for understanding the transmission and dissemination of ARGs from “source” to “sink”,and helps to control or reduce the risks of ARGs caused by resource utilization of dairy manure.