Effects Of Microplastics On The Microbiome And Antibiotic Resistome Of Earthworm Gut

The long-term application of plastic films and organic fertilizers in agricultural production has caused the co-pollution of microplastics(MPs)and antibiotic resistance genes(ARGs)in agricultural soils,threatening the soil ecological environment and human health.Earthworm is an important indicator organism of soil pollution,and its gut is a hotspot for the research on MPs and ARGs.However,the response rules of the microbiome and antibiotic resistome in the MPs-contaminated soil-earthworm system remain unclear.In this study,Eisenia fetida was used as the test organism,and polyvinyl chloride microspheres(PVC-MPs),the main component of farmland plastic films,were used as the target pollutant.Based on the actual residual amounts of MPs in agricultural soil,an indoor simulated soilearthworm ecosystem was constructed to investigate the residual characteristics of PVCMPs in soil and earthworm gut and to reveal their impact on the enzyme activity of soil and earthworm,and the microbial functional diversity,microbial community structure composition as well as the diversity and abundance of ARGs in the soils and earthworm guts.The main findings are listed below.(1)PVC-MPs(10 mg/kg and 100 mg/kg)had no significant effect on the survival rate of earthworms.Inoculation of earthworms reduced the amounts of PVC-MPs in the soil,and PVC-MPs could enter the gut of earthworms through ingestion.PVC-MPs exposure had no significant effect on the catalase and peroxidase activities in the earthworm-uninoculated soils;but significantly inhibited the activities in the earthworm-inoculated soils,and the inhibitory effect was positively correlated with the exposure concentration.The exposure responses of earthworm enzyme activity to PVC-MPs varied with enzyme types: catalase showed a trend of "inhibition-stimulation";glutathione transferase showed a trend of "stimulation-inhibition-recovery";acetylcholinesterase showed a trend of "inhibitionstimulation-recovery";and malondialdehyde content continued to increase.(2)PVC-MPs exposure had no significant effect on the microbial activity,Mc Intosh index and Shannon index in the earthworm-uninoculated soils,but showed a significant inhibition in the earthworm-inoculated soils.PVC-MPs exposure had a significant inhibitory effect on the microbial activity of earthworm gut,and the inhibitory intensity was significantly positively correlated with the exposure concentration.However,there was no significant effect of PVC-MPs exposure on the Mc Intosh index and Shannon index in the earthworm guts.(3)PVC-MPs exposure significantly decreased the number of bacterial 16 S r RNA genes and the relative abundance of Acidobacteria in the earthworm-uninoculated soils,but increased the richness of bacterial communities and the relative abundance of Actinobacteria.PVC-MPs exposure had no significant effect on the number and abundance of bacteria in earthworm-inoculated soils,but significantly increased the relative abundance of Bacteroidetes.Exposure to PVC-MPs for 21 d significantly increased the number of bacteria but decreased the richness of bacterial community in the in the earthworm guts.The relative abundances of Proteobacteria and Bacteroidetes increased by 1.16 and 1.11 times,respectively;while the relative abundances of Firmicutes and Acidobacteria decreased by11.21% and 18.09%,respectively.(4)The results of metagenomic analysis showed that PVC-MPs exposure increased the diversity and abundance of ARGs in the earthworm-uninoculated soils.Among which the abundance of tetracenomycin and chloramphenicol resistance genes increased by 4.18 and9.04 times,respectively,and tcm A,tet V,opr M and acr A increased by 2.13～9.04 times.Inoculation with earthworms significantly increased the diversity and abundance of ARGs in the soil,among which the abundance of β-lactam,polymyxin,quinolone and tetracycline resistance genes increased by 1.65～5.66 times.PVC-MPs exposure increased the diversity and abundance of ARGs in the earthworm-inoculated soils,in which the abundance of aminoglycoside and β-lactam resistance genes increased by 3.86 and 2.58 times,respectively.The abundance of some ARGs subtypes(i.e.,van X,aac(2’)-I,mex T,CGB-1,and ksg A)increased by 1.70～3.00 times.q PCR results showed that PVC-MPs exposure increased the absolute abundance and relative abundance of tet A and aad A in both the earthwormuninoculated and earthworm-inoculated soils.In addition,exposure to PVC-MPs slightly increased the abundance of mobile genetic elements(MGEs)in both the earthwormuninoculated and earthworm-inoculated soils.Inoculation with earthworm increased the diversity and abundance of MGEs by 2.47 and 2.21 times,respectively.(5)The diversity and abundance of ARGs in earthworm guts increased by 1.60 and 1.38 times after exposure to PVC-MPs for 21 d.Of which the abundance of aminoglycoside,bacitracin,β-lactam,chloramphenicol,fosmidomycin,macrolide-lincomycin-streptomycin,and puromycin resistance genes increased by 1.54～18.25 times,and the abundance of mdt B,ros A,van X,mex I and ceo B increased by 2.91～40.37 times.q PCR results showed that PVCMPs exposure increased the absolute abundance of sul1 and aad A in the earthworm guts.Exposure to PVC-MPs for 21 d reduced the diversity and abundance of MGEs in the earthworm guts,and the abundance of transposase,integrase and insertion element significantly decreased.The correlation analysis showed that the abundance changes of ARGs in the soil may be related to MGEs,while the abundance changes of ARGs in the earthworm guts may not be related to MGEs.In summary,this study revealed the response of microbial community structure,functional diversity,enzyme activity,and diversity and abundance of ARGs in the soils and earthworm guts after exposure to the PVC-MPs.The research results can not only elucidate the spread of ARGs in the MPs-polluted soil-earthworm ecosystem,but also provide a scientific basis for the ecological risk assessment of MPs-polluted soil.