The Mechanism Of Promoting Antibiotic Resistance Gene Horizontal Transfer By Typical Pollutants In Water Environment

In the past several decades,the serious threat of antibiotic resistance to environmental pollution and global public health has attracted widespread attention from scholars at home and abroad.With the increase of population and the acceleration of industrialization,the increase of pollutants in the water environment has promoted the spread of antibiotic resistance genes to varying degrees.However,a large number of studies have found that in addition to antibiotics,many other typical pollutants such as heavy metals,disinfectants,ionic liquids,etc.,can work independently or in combination with other pollutants to promote the spread of antibiotic resistance genes in the water environment.At present,most studies on the effects of pollutants in the water environment on the horizontal transfer of resistance genes are mostly described in the phenomenon,and how the selective pressure generated by pollutants affects the binding,transfer and conversion of resistance genes between bacterial species is currently unclear.In this work,we first study the effect of heavy metals at environmental level on promoting horizontal transfer of antibiotic resistance genes,and further analyze the mechanism of action by tracking the dynamic process of plasmid horizontal transfer and the changes of bacterial cell membrane.Secondly,the imidazole ionic liquid[C_4,6,8MIM][Br]are selected as selective pressure to study the effects of different alkyl side chain lengths on its transfer frequency.Analyze the mechanism of action of ionic liquids on extracellular polymers in the system during horizontal transfer.The main results shows:1.In addition to the inhibition of horizontal transfer of antagonistic genes with the increase of concentration of Cd,the heavy metals Cu,Pb and Zn induce the increase of the horizontal transfer frequency of the resistance plasmid and promote the spread of antibiotic resistance genes in water environment.Among them,Cu（5.0μg/L）has the strongest ability to increase the horizontal transfer of plasmid RP4.At the same time,Cu increases the minimum inhibitory concentration（MIC）of the transconjugants to 8antibiotics and 4 heavy metals,indicating that the horizontal transfer of the multi-resistant plasmid RP4 induce by Cu cause multiple resistance to the indigenous acceptor bacteria in the environment.2.Quantitative analyze the absolute abundance of the tnpR gene（copies/mL water）and the absolute abundance of the traF gene（copies/mL water）by tracking the dynamic changes of donor bacteria,recipient bacteria and transconjugants in the junction transfer system.Correlation with Cu concentration demonstrate that Cu promotes horizontal transfer of the plasmid RP4-mediated resistance gene.Transmission electron microscopy（TEM）and flow cytometry（FCM）confirme that Cu increases the permeability of bacterial cell membranes in horizontal transfer systems,leading to damage of cell membranes,which in turn promote antibiotic resistance genes between bacteria.Horizontal transfer.In addition,horizontal transfer experiments with Cu-induced antibiotic and heavy metal combined resistance shows that Cu exposure significantly promoted the transfer of joint resistance between antibiotics and heavy metals in trans-bacteria.3.Imidazole ionic liquid[C_4,6,8MIM][Br]promote the horizontal transfer frequency of antibiotic resistance genes in the pure bacterial junction transfer system,and also promote the resistance genes other than antibiotics in the experiment.The longer the ionic liquid of the side chain alkyl chain,the higher the frequency of promoting horizontal transfer of the resistance gene.The protein content of the extracellular polymer in the horizontal transfer system increase with the increase of the concentration of the ionic liquid,and the polysaccharide content don`t change significantly,indicating that the ionic liquids have had a certain effect with the extracellular protein.