Effects Of Earthworm On Antibiotic Resistance Genes And Microbial Community During Copper-containing Swine Manure Vermicomposting

In recent years,the emergence of antibiotic resistance genes as emerging environmental pollutants has increasingly attracted public attention.In the livestock and poultry breeding industry,antibiotics and heavy metals are widely used to the prevention and treatment of animal diseases and the promotion of animal growth,resulting in livestock and poultry manure as an important repository for antibiotic,heavy metal and antibiotic resistance genes.These residues not only affect the indigenous microbes but also pose a great threat to public health,as the spread of faeces in farmland,soil and water.Therefore,it is of great significance to understand the effect of different concentrations of heavy metals on the relative resistance genes in livestock manure and explore the effect of vermicomposting on the reduction of resistance genes in pig manure containing copper.This research set three concentrations of copper treatment group,respectively control group(0mg/kg),low-concentration group(200mg/kg),high-concentration group(2000mg/kg),and then set up copper-containing pig manure added mature earthworm(Eisenia fetida)treatment group to construct a pig manure vermicompost model.Pig manure samples were collected on the 0th,2nd,10 th,20th,30 th,and 40 th days after modeling,and the sample metagenomic DNA was extracted.Real-time fluorescent quantitative polymerase chain reaction technique was used to detect the resistance genes associated with heavy metal copper,macrolide ARGs(ermA,ermB,ermF)and tetracycline ARGs(tetA,tetB,tetT),a copper-resistance gene pcoD,a mobile genetic elements intI2.To reveal the relative abundance of ARGs,CRGs and MGEs in swine manure under different concentrations of copper stress,and to investigate the effects of vermicompost on the abatement of antibiotic resistance genes in pig manure containing copper.On this basis,16 S rDNA gene sequencing was performed on the Illumina MiSeq sequencing platform to analyze the changes of microbial communities during the various stages of composting.The results showed that under the stress of copper,the relative abundance of ARGs in swine manure at all stages of composting was significantly changed.The addition of low concentrations of copper had a significant increase in the abundance of three macrolide resistance genes.High concentration of Cu significantly increased ermA and mid-to-late tetB abundance.At the 20 th day of composting,the tetT abundance was at two concentrations of Cu.The effects were significantly increased(P ≤ 0.05),and intI2 may play an important role in the evolution of ARGs.The results showed that under copper stress,the relative abundance of ARGs in swine manure at all stages of composting was significantly changed.The addition of low concentrations of copper had a significant increase in the abundance of three macrolide resistance genes.High concentration of Cu significantly increased the abundance of ermA and tetB in subsequent period.At the 20 th day of composting,the abundance of tetT is significantly increased under the low and high concentrations of Cu(P ≤ 0.05),and intI2 may play an important role in the evolution of ARGs.Vermicomposting can reduce the relative abundance of antibiotic resistance genes in copper-containing swine manure.Adding earthworm to swine manure in two concentrations will significantly reduce the effects of ermB,ermF,tetB,and tetT.16S rDNA sequencing was performed on the 1,2 and 40 days of the copper treatment and plutonium treatment samples of this test model.The results showed that the compost swine manure had significant effects on microbial community structure on the 2nd and 40 th day under copper stress comparing control group.From the level of the gate,on the 2nd day,the increase of Firmicutes and Actinobacteria content occupied Proteobacteria and Bacteroidetes in the pig manure.On the 40 th day,the addition of copper caused a significant drop in the proportion of Firmicutes.The effect of earthworms on the microbial communities of copper-containing swine manure was mainly due to differences in Firmicutes,and the contents in the Scorpion group were significantly higher.The content of Fibrobacteres and Saccharibacteria in the middle and late stages was the main difference with the copper treatment group.From the results of the whole experiment,the simulation of vermicomposting in coppercontaining pig manure revealed the variation pattern of relative abundance of antibiotic resistance genes in swine manure under different copper stress conditions,and the effect of earthworm on the reduction of antibiotic resistance genes in copper containing pig manure was preliminary investigated.