The Effects Of Layer Manure Composting On Tetracycline Resistance Genes Under Doxycycline Stress

Antibiotic resistance genes（ARGs）have been defined as novel environmental pollutants with a risk of transmitting antibiotic resistance.Livestock and poultry manure is an important repository for veterinary antibiotics and ARGs.Concerning the changes in ARGs in livestock manure compost under the stress of veterinary antibiotics is particularly important for environmental health and ecological safety.Therefore,in this study,tetracycline ARGs were used as the research object,and three different concentrations of doxycycline were added to the laying hen compost to investigate the tetracycline ARGs and mobile genetic elements（MGEs）of laying hens composted under doxycycline stress.Impact,providing a reference for reducing and eliminating the risk of contamination of tetracycline-based ARGs.Using sawdust as a conditioning agent,a dynamic box composting test device was used to carry out the composting test of laying hens.In this experiment,doxycycline was absent in the blank control group,and 5,10 and 20 mg/kg of doxycycline were added to the treatment group,with 3 replicates in each group;during the 25-day experimental period,the ARGs（tetA,tetC,tetG,tetH,tetO,tetM,tetQ,tetT,tetW and tetX）and MGEs（int1 and int2）,doxycycline concentration and compost physicochemical and microbial indicators were determined.The main results obtained in this paper are as follows:（1）The relative abundance of total ARGs decreased significantly by 28.75%（P<0.05）,and the relative abundance of tetC,tetH,tetO,tetM,tetQ,tetT and tetW decreased significantly by 36.77%～99.31%.（P<0.05）,the relative abundance of tet A,tetG and tetX increased significantly by 1.07～20.13 times（P<0.05）.The relative abundance of total MGEs was significantly increased by 13.23 times（P<0.05）,and the int1 was significantly increased by 15.59 times（P<0.05）,while int2 had no significant change（P>0.05）.The total ARGs of tetracyclines were significantly positively correlated with MGEs（r=0.57,P<0.01）,and there was a significant positive correlation between efflux pumps and inactivated enzymes ARGs and MGEs（r=0.72～0.87,P<0.01）.However,ribosome-protected protein ARGs were significantly negatively correlated with MGEs（r=0.37,P<0.01）.（2）At the end of composting,the degradation rates of doxycycline in the experimental groups with initial concentrations of 5,10 and 20 mg/kg were 64.05%,65.30%and 65.22%,respectively;the concentration of doxycycline in compost was tetH,tetO,tetM,tetQ,tetT and tetW showed a very significant positive correlation（r=0.56～0.89,P<0.01）,and the low concentration of doxycycline was more correlated（r=0.75～0.89,P<0.01）.（3）The diversity of bacterial community structure in composting under doxycycline stress was significantly decreased（P<0.05）,and the activity of invertase was significantly decreased（P<0.05）.The dominant flora in composting,Firmicutes,was significantly positively correlated with tetH,tetT and tetW（r=0.37-0.69,P<0.01）,Bacteroidetes and Proteobacteria.tetA,tetC,tetG,tetX,int1 and int2 showed a significant positive correlation（r=0.44～0.89,P<0.01）;Bacteroides,Anaerococcus and tetH,tetO,tetM,tetQ,tetT and tetW showed a very significant positive correlation（r=0.58～0.89,P<0.01）.（4）The temperature was extremely significantly correlated with other ARGs except tetO,tetM and tetQ（P<0.01）;H₂O content,EC,TN,TOC and NH⁴⁺-N were extremely significantly positively correlated with tetH,tetO,tetM,tetQ,tetT and tetW（P<0.01）;the C/N was extremely significantly positively correlated with tetX and int1（P<0.01）.The most significant physical and chemical indicators affected the change of ARGs and MGEs in composting were temperature,H₂O content and NH⁴⁺-N.In summary,layer manure composting can significantly reduce the absolute abundance and relative abundance of most ARGs,but the relative abundance of MGEs will increase significantly;composting can significantly affect the microbial community structure under doxycycline stress,and thus affected the abundance of ARGs and MGEs.