| Disinfection is an essential link in the water purification process,Disinfection by-products(DBPs)generated in the disinfection process has always been a research hotspot in the field of water safety.As more and more DBPs are detected in various water environments around the world,Especially New Coronavirus pneumonia.(COVID-19)since the outbreak of the epidemic,the load of disinfectants in the environment has increased,and the ecological risk of DBPs has to attract attention.The spread of bacterial drug resistance poses a great threat to the ecological environment and human health.Plasmid mediated bacterial drug resistance gene conjugative transfer is the most important way of bacterial drug resistance transmission,and many environmental factors can promote the process of plasmid conjugative transfer.Based on the long-term coexistence of DBPs and antibiotic resistance genes(ARGs)in the water environment.This paper intends to study the propagation law of typical carbon and nitrogen-containing DBPs on the conjugative transfer of ARGs,analyze its mechanism,and clarify the ecological risk within the actual low concentration range of typical DBPs in the water environment.In this paper,taking typical DBPs containing carbon trichloromethane and nitrogen dichloroacetonitrile as the research objects,the RP4 plasmid mediated conjugative transfer model was constructed to explore the effects and laws of low concentrations of trichloromethane and dichloroacetonitrile in water environment on conjugation transfer,by detecting the level of reactive oxygen species(ROS),the permeability of cell membrane,the observation of bacterial surface morphology,combined with genome-wide RNA sequencing and proteomic analysis,the mechanism of ARGs conjugative transfer promoted by trichloromethane and dichloroacetonitrile was revealed.The results show that:(1)Low concentrations of trichloromethane and dichloroacetonitrile in water environment can significantly promote the conjugative transfer of ARGs,in which the concentration of trichloromethane is 25 μg/L,dichloroacetonitrile concentration is 10 μg/L When was bonded for 16 h,the promoting effect was the most significant,and the maximum bonding transfer frequency could be increased by 5～6 folds.(2)Trichloromethane and dichloroacetonitrile can significantly induce the production of ROS in donor and recipient bacteria cells.Under the experimental conditions,the level of ROS in donor and recipient bacteria cells increased by1.3～1.8 folds.After adding ROS scavenger,the production of ROS in bacterial cells was significantly reduced,and the plasmid conjugative transfer frequency induced by trichloromethane or dichloroacetonitrile was significantly reduced,which proved that trichloromethane and dichloroacetonitrile Acetonitrile induced excessive production of ROS in bacterial cells is the main but not the only potential mechanism for promoting conjugation and metastasis.In addition,trichloromethane and dichloroacetonitrile also induced the increase of a series of core gene expression and protein abundance related to ROS production and SOS response,in which the expression of key genes ahp F,kat G,sod A,sox S and trx C related to ROS production increased by 1.30～3.78 folds and 1.47～2.07 folds,respectively;The expression of core genes rec A,rec F and uvr A related to SOS response increased by 1.24～1.71 folds and 1.19～1.49 folds respectively;The abundance of core protein sod B related to SOS reaction increased by 1.23 and 1.18 folds,respectively.(3)Both trichloromethane and dichloroacetonitrile can increase the permeability of donor and recipient bacterial cell membrane,which can be significantly increased by 1.3～1.6 folds under experimental conditions.Under transmission electron microscope,trichloromethane and dichloroacetonitrile can cause certain damage to cell membrane and increase the fusion of cell membrane between bacteria.The expression and protein abundance of core genes related to bacterial cell membrane are in trichloromethane and dichloroacetonitrile The expression of ompA,ompC,ompF were up-regulated by 1.43,1.06,1.70 folds and1.63,1.89 and 1.33 folds,respectively;The lipopolysaccharide synthesis gene waa A increased by 1.66 and 1.44 folds,respectively;The abundance of osmotic stress response protein oms E increased by 1.14 and 1.21 folds,respectively.(4)Trichloromethane and dichloroacetonitrile can significantly up regulate the expression of key genes involved in pili formation and ATP synthesis in donor and recipient bacteria.Under trichloromethane experimental conditions,the expression of fim C,fim D,fim G and fim H genes involved in pili formation can be increased by about 2.2～2.8 folds,and the transcription level of six ATP coding genes(atp A-atp H)can be increased by about 1.3～1.7 folds.In addition,the expression of key genes on RP4 plasmid also changed significantly under the action of trichloromethane and dichloroacetonitrile.The expression of conjugation transfer transcription genes tra G,tra I and tra M increased by 1.35～1.48 folds under exposure to dichloroacetonitrile.In conclusion,typical DBPs trichloromethane and dichloroacetonitrile can significantly promote the conjugative transfer of RP4 plasmid under long-term low concentration exposure.The mechanism is mainly manifested in the production of intracellular ROS,SOS response,increase of cell membrane permeability,up regulation of gene expression related to pili production,ATP synthesis and RP4 plasmid,and increase of protein abundance.This study provides a theoretical basis for the scientific evaluation of the potential biological effects and ecological risks of DBPs in the water environment by studying the laws and mechanisms of typical DBPs promoting ARGs conjugative transfer. |
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