| Antibiotic resistance genes(ARGs)can disseminate in the environment via gene horizontal transfer(HGT),which seriously threatens ecological security and human health.Horizontal gene transfer can occur between different bacterial species,which exacerbates the environmental risk of ARGs.In recent years,the influence of various environmental factors on the proliferation and transfer of ARGs has become a hot research topic.Nanoplastic is an emerging environmental pollutant,and it is still unclear how the nanoplastics impact the dissemination of ARGs in the environment.It is of great practical significance to study the effect of nanoplastics on the horizontal transfer of ARGs,due to the fact that nanoplastics and ARGs co-occur in the environment.Therefore,this study employed two horizontal transfer models,transformation and conjugation,to explore the influence of polystyrene nanoplastics on the dissmination of ARGs,and to reveal the associated mechanisms for the promoted horizontal transfer of ARGs by nanoplastics.The main results are as follows:(1)Nanoplastics can significantly promote the transformation of antibiotic resistance genes mediated by plasmid.Polystyrene plastic microspheres(with sizes of 75 nm and 30μm,respectively)were selected to investigate the effect of micro(nano)plastics on the transformation of antibiotic resistance genes under a transformation system of p UC19 and Escherichia coli DH5α.The results showed that microplastic particles with the size of 30 μm had no significant effect on the transformation of plasmid,while the nanoplastic particles with size of 75 nm exerted a significant stimulating effect on the plasmid transformation.After 4 h exposed to nanoplastics,the efficiency of the uptake and transformation of Escherichia coli for plasmid p UC19 was significantly enhanced.When the concentration of nanoplastics was 500 mg / L,the transformation efficiency was increased by 5.4 times compared to the control group.Moreover,the agarose gel electrophoresis also confirmed the existence of intact p UC19 plasmid in the transformants.(2)The mechanisms for the promoted ARGs transformation by nanoplastics were revealed.Nanoplastics stimulated cells to produce a large number of intracellular reactive oxygen species,and induced the antioxidant reaction of cells,which lead to the increase of Superoxide dismutase(SOD),Catalase(CAT)and Glutathione(GSH)in cells.Meanwhile,nanoplastics also improved the permeability of cell membrane and formed holes on the surface of cell membrane,which facilitated the entry of exogenous DNA into the cell.Transcriptome analysis showed that under the influence of nanoplastics,the m RNA expression of oxidative stress,cell membrane and DNA repair related genes were upregulated,suggesting that the stress response of cells may also promote the transformation of ARGs.(3)Nanoplastics can significantly promote the conjugation transfer of antibiotic resistance genes mediated by plasmid.Polystyrene nanoplastic(particle size 75 nm)was selected to study the effect of nanoplastics on the conjugation of antibiotic resistance genes,using a constructed conjugation system of Escherichia coli K12 as a donor strain(contain RP4 plasmid)and Escherichia coli CV-601 as a recipient strain.The results showed that the conjugation frequency of RP4 plasmid between the same species of Escherichia coli increased with the increased concentration of nanoplastics.Under the conditions of 28 ℃and 8 h conjugation time,50 mg/L nanoplastics increased the conjugation frequency by 5.5times compared to the control group.(4)The mechanisms for the promoted conjugation transfer of ARGs by nanoplastics were revealed.The introduction of nanoplastics into the conjugation system induced both donor and recipient bacteria to generate a large number of intracellular reactive oxygen species,which caused certain damage to the cell membrane and improved the permeability of the cell membrane.Under the environmental pressure of nano plastics,the expression of antioxidant systems in the donor and recipient bacteria were totally different.The donor bacteria increased the contents of SOD and GSH,while the receptor bacteria only increased GSH content.In addition,nanoplastics affected the m RNA expression of RP4 plasmid conjugative transfer related genes,and facilitated the formation of "conjugation bridge" between the donor and recipient bacteria,thus promoting the conjugative transfer of ARGs. |
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