| As the scale of urban sewage treatment in China increases,the amount of residual sludge also increases year by year.However,due to the long-term “heavy water and light sludge”,the situation of sludge treatment and disposal is very severe.Antibiotic-resistant genes(ARGs)are new biological pollutants in the environment.The accumulation of ARGs in sewage in sludge has little effect on their removal.Therefore,controlling and reducing the spread and pollution of ARGs during the process of sludge resource utilization has become an urgent problem to be solved.Vermicomposting is a resource utilization technology that uses the synergistic effect of earthworms and microorganisms to decompose and transform difficult-to-degrade organic matter in sludge.It has low cost,simple operation,sustainable treatment of sludge,and vermicompost is rich in plant-available nutrients and abundant agricultural beneficial bacteria.It has a high market value.Previous studies have shown that vermicomposting has a selective reduction effect on ARGs,but insufficient understanding of the spread of ARGs in sludge vermicomposting is the main reason for the difficulty in controlling ARGs.The earthworm intestine is a suitable microenvironment for the potential spread of ARGs.However,few people have studied earthworm intestinal digestion.Therefore,it is very important to study the impact of earthworm intestinal digestion on ARGs in sludge.This study takes the earthworm intestine as the research object and uses RP4 plasmids to use metagenomic sequencing and fluorescence quantitative PCR and other molecular biology techniques to measure the ARGs and mobile genetic elements(MGEs)before and after earthworm digestion and in various sections of the earthworm intestine.Analyze the distribution and abundance changes of ARGs and MGEs and RP4 plasmids to clarify the impact of earthworm intestinal digestion on horizontal gene transfer(HGT)of ARGs.The results show:(1)Chromosomes and plasmids mediate the vertical and HGT of ARGs in sludge,respectively,resulting in lower reduction of ARGs during vermicomposting.In this study,the effects of vermicomposting on vertical and HGT of ARGs in sludge were investigated by detecting the abundance changes of ARGs and MGEs on chromosomes and plasmids during vermicomposting.For better comparison,the control without earthworm was set up in parallel.The results showed that the sludge vermicomposting for first 10 days was the peak of ARGs transfer,displaying the significant(P<0.05)increase of ARGs abundance on plasmids and chromosomes,except for tet M.The abundances of erm F,erm B,tet X,and sul1 on plasmids significantly increased by 1.02-fold,1.97-fold,2.43-fold,and 0.75-fold in the vermicomposting compared with the control(P<0.05),while only erm B on chromosomes significantly increased in the vermicomposting compared with the control(P<0.05).Compared with the control,the selected MGEs abundance of intⅠ1 on plasmids significantly enriched by 1.63-fold in the vermicomposting,while its abundance on chromosomes was diametrically opposite,its abundance in the control was larger than vermicomposting.In the10-20 d of composting,the abundance of MGEs and total ARGs in chromosomes and plasmids decreased in both treatments,with a faster decrease in the vermicomposting.In addition,the MGEs had a significant positive correlation(P<0.05)with erm F,erm B,and sul2 on plastids,while no significant correlation among MGEs and all ARGs on chromosomes was recorded during vermicomposting.The redundancy analysis revealed that the changes of ARGs were related to the MGEs and environmental changes during vermicomposting,and the environmental factors had a stronger effect on ARGs and MGEs on plasmids than those on chromosomes.(2)After previous studies determined the impact of earthworm digestion on the HGT of ARGs,an earthworm digestion experiment was conducted.Five days after earthworm digestion,metagenomics was performed on the earthworm digestion products and the earthworm intestine.The results show that among the top ten abundant ARGs,compared with the control,8 decreased and 2 increased,indicating that earthworms selectively reduced ARGs during composting.The abundance of multidrug-resistant genes accounted for30.3-46.1% in each treatment,but the reduction of multidrug-resistant genes by earthworm digestion was not ideal.Compared with the control,the types of plasmids,integrases and insertion sequences in vermicompost increased by 28.6%,14.1%,and 35.6%,respectively,and their concentrations increased by 36.8%,396.3%,and 62.5%,respectively.Among them,the concentration of integrase in vermicompost increased nearly 4-fold compared to the control.The main source of integrase is pathogenic bacteria,among which Salmonella increased by 212-fold and Klebsiella increased by 485-fold.The results show that after earthworm intestinal digestion,there is still a risk of HGT of ARGs in vermicompost,and human pathogenic bacteria integrate ARGs more frequently.For microorganisms,metagenomic sequencing results show that the abundance of various actinomycete genera in the earthworm intestine is very high,further proving the ability of the earthworm intestine to degrade organic matter.Through functional annotation of the KEGG PATHWAY database,it was found that the relative abundance of the top 10 functional genes in vermicompost increased by 9.86% compared to the control,of which carbohydrate metabolism increased by9.57%.The results show that earthworm intestinal digestion can increase microbial metabolic activity in the vermicomposting and accelerate the decomposition of organic matter in sludge.In each experimental treatment,27-30% of ARGs hosts are Proteobacteria bacteria,and efflux pumps are the main drug resistance mechanism for bacteria in vermicomposting.(3)By adding Escherichia coli carrying the RP4 plasmids for earthworm digestion experiments,the results show that compared with the control,the conductivity of the experimental treatment increased significantly by 12.81%(P<0.05),ammonia nitrogen decreased by 12.15%(P<0.05),and nitrate nitrogen increased by 2.76-fold(P<0.05),indicating that after adding Escherichia coli,the ability of vermicomposting to decompose organic matter during the digestion stage increased significantly and significantly promoted the progress of nitrification.The abundance of bacteria and eukaryotic microorganisms in the earthworm gizzard is extremely high.After being mechanically ground by the gizzard,the abundance in the stomach and hindgut drops sharply.After adding Escherichia coli carrying RP4 plasmids,the abundance of aph A gene in each section of earthworm intestine increased.In the hindgut,the aph A gene in the experimental treatment was significantly increased(P<0.05)by 4.89-fold and 4.21-fold compared to the original earthworm and control,respectively,indicating that the earthworm hindgut is an important place for HGT of ARGs.Most of the HGT of RP4 plasmids occurs between bacteria rather than eukaryotic microorganisms.Compared with the control,the abundance of tnp A-04 gene in gizzard,stomach and hindgut increased by 28.57%,23.60% and 4-fold(P<0.05),respectively.Finally,after 5 days of digestion,compared with the control,both ARGs and MGEs in earthworm digestion products increased,indicating that RP4 plasmids increased the risk of HGT of ARGs in earthworm digestion products.In summary,the earthworm digestion stage during vermicomposting is the peak period for the transfer of ARGs,and the HGT mediated by plasmids carrying ARGs is the main way for the spread of ARGs in vermicomposting.In the earthworm intestine,Proteobacteria bacteria are the largest potential hosts of ARGs,and efflux pumps are the main drug resistance mechanism for bacteria in vermicomposting.The earthworm hindgut is the main gathering area for RP4 plasmids and an important place for HGT of ARGs.Finally,adding RP4 plasmids increases the risk of HGT of ARGs in earthworm digestion products. |
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