Effects And Mechanisms Of Typical Pollutants In Soil On Conjugative Transfer Of Antibiotic Resistance Genes

The horizontal transfer of antibiotic resistance genes（ARGs）in soil has been threatening the health and safety of soil ecosystem,and even causing great health risks to human health due to the contact migration between soil,water and plants.Heavy metals,antibiotics,disinfectants,nanoparticles and other pollutants in soil can impact on the horizontal transfer of ARGs,which are related to the multi-medium interface behavior and process of ARGs among soil water phase,bacterium and pollutants.However,at present,much attention has been paid to the abundance and migration of ARGs in soil,the mechanisms of typical pollutants（such as heavy metals,PAHs and surfactants）and their compound pollutants affecting ARGs conjugative transfer in soil are not clear.Therefore,this paper centers on the interface process,interface properties and gene expression of ARGs in the indirect conjugative transfer between donor and recipient strains,establishes an intergeneric conjugative transfer system with E.coli（RP4）as donor strain and Klebsiella Trevisan as recipient strain,and chooses Cd²⁺and anion surfactants sodium dodecyl benzene sulfonate（SDBS）and phenanthrene as typical soil pollutants.This paper focuses on the effects and mechanisms of the above pollutants and their combined pollutants on the conjugation of ARGs in the donor-recipient strains and soil water phase.The research results are expected to provide theoretical support for controlling and running down the ARGs in soil,so as to ensure the health and safety of soil ecosystem and reduce the risk of human health.The following valuable research results are obtained in this paper:（1）It was found that SDBS（10 mg/L～80 mg/L）and phenanthrene（1 mg/L）promoted the conjugation of RP4 and increased the conjugative frequency by 2 to 4 bacteria,and the conjugative frequency was as low as 0.32×10^-6.（2）It was clear that the cell interface properties of donor-recipient bacterium were positively correlated with RP4 conjugative transfer.for example,the eicosapentaenoic acid in bacterial fatty acid methyl ester was positively correlated with CSH（R²=0.86）,and both of them were positively correlated with conjugative transfer frequency.（3）The mechanisms of SDBS and phenanthrene promoting the frequency of RP4conjugation were explored.SDBS（10 mg/L～80 mg/L）and phenanthrene（1 mg/L）increased the hydrophobicity of bacterial cell interface（1.4-1.8 times）and the ability of biofilm formation（1.7～1.8 times）,thus increasing the conjugation transfer frequency of RP4.In addition,the above concentrations of SDBS and phenanthrene promoted the production and accumulation of reactive oxygen species（1.25-1.3 times）and the expression of outer membrane porin（1.4-1.5 times）,which led to the increase of cell membrane permeability by 1.2-1.3 times and promoted the conjugation and transfer of RP4.However,the effect of Cd²⁺（0.1 mg/L～2 mg/L）is inverse.（4）SDBS and phenanthrene activated the m RNA expression of trb B by inhibiting the m RNA expression of kor A and kor B（0.023-fold,0.020-fold）,thus promoting the contact between donor and recipient bacteria;promoting the transfer and replication of RP4 by inhibiting the expression of kor B and trb A（0.026-fold,0.021-fold）;and promoting the occurrence of conjugate metastasis by increasing the expression of tra F（159,2.68-fold）.And the effect of Cd²⁺on conjugation is just the opposite.（5）It was confirmed that the transfer frequency of RP4 in soil with moderate heavy metal pollution was higher than that mild heavy metal pollution.On the one hand,heavy metals（Zn,Mn,Cu）in the soil increase the hydrophobicity of the cell is conducive to the entry of RP4,thus increasing the conjugation frequency.