The Effect And Mechanisms Of Pheromone-responsive Plasmid-mediated Conjugative Transfer Of Antibiotic Resistance Genes In Enterococcus Faecalis Promoted By Bisphenols

Since the introduction of antibiotics in the 1920 s,it has been a great success in treating bacterial infections and has saved countless lives.However,as the use of antibiotics has become more widespread,their negative effects have also emerged,for example the abuse of antibiotic caused antibiotic resistance in bacteria posing a serious threat to human health.Bacteria acquire antibiotic resistance by two main ways.The first one is that bacteria that are inherently antibiotic resistant are screened out or generate gene mutation to be resistant to antibiotics by selective pressure from the massive use of antibiotics.The second one is bacteria acquire mobile genetic elements carrying resistance genes through horizontal gene transfer.The latter make more bacteria acquire resistance.Mobile genetic elements include plasmids,transposons,insertion sequences and phages.The horizontal transfer of resistance genes mediated by plasmid play an important role in the transmission of resistance genes.Enterococcus faecalis(E.faecalis)is a Gram-positive bacterium that is widespread in the natural water environment.The conjugative transfer of ARGs in E.faecalis mediated by pheromone-responsive plasmid(PRP)has a relatively high frequency and efficiency compared to that of R and F plasmids.The pCF10 plasmid is one of the most representative pheromone-responsive plasmids and its conjugative transfer is an efficient way to cause rapid spread of antibiotic resistance genes in E.faecalis.Research in the area of drug-resistant bacteria has been focused on the spread of resistance genes due to antibiotic misuse,and in recent years studies have begun to pay close attention to the effect of non-antibiotic factors such as nanomaterials and metal ions on the widespread spread of resistance genes,but there are no studies about the effect of bisphenols,which are present at high concentrations in natural water,on the horizontal transfer of resistance genes.Bisphenol A(BPA)was once one of the most produced chemicals in the world,and it was widely used in the manufacture of a wide range of industrial and household products.Because of its chemical similarity to the natural estrogen 17β-estradiol,it can interfere with the human endocrine system and cause many endocrine-related diseases.As a result,several countries have issued bans on the production and use of BPA.As BPA has been phased out of industrial production,a number of BPA analogues,which are structurally similar to BPA,have gradually taken their place in industry.Recent studies have shown that bisphenol analogues also have a disruptive effect on the human endocrine system because of the similarity of chemical structure between bisphenol A analogues and BPA.They both contain two phenolic rings in their structural formula,differing only in the substituent groups of the different bisphenol analogues.Since bisphenols,as environmental estrogenic pollutants,have a similar chemical conformation to pheromones,we speculated bisphenols could also influence PRP-mediated conjugative transfer.Based on the above analysis,we propose the hypothesis that bisphenols can facilitate PRP pCF10-mediated antibiotic resistance gene conjugative transfer and have a pheromone effect.In this study,we developed a model of PRP-mediated antibiotic resistance gene conjugative transfer,selected several bisphenols with high content in the natural water environment and relatively high application in industry as research objects,investigated the effect of various bisphenols on antibiotic resistance gene conjugative transfer in E.faecalis,analyzed the factors affecting PRP conjugative transfer under the effect of BPA,and analyzed the mechanism of PRP conjugative transfer by bisphenol A from the genetic,protein and morphological aspects.The main findings are as follows.A pheromone-responsive plasmid-mediated conjugative transfer model of antibiotic resistance genes in E.faecalis was constructed using the pCF10 plasmid.The model is relatively stable and can be used to evaluate the effects of environmental estrogens on the conjugative transfer of PRP resistance genes.Several environmental estrogenic contaminants(e.g.Bisphenol A,Bisphenol S,Bisphenol F,Tetrabromobisphenol A,Tetrachlorobisphenol A and Bisphenol AF),which are abundantly distributed in the natural water environment or widely used in industry,significantly promoted pCF10 plasmid-mediated conjugative transfer.Bisphenol A concentration,bacterial density,mating time,mating temperature and the ratio of donor and recipient bacteria could directly affect conjugative transfer of antibiotic resistance genes.The mechanism of BPA promoting the conjugative transfer of ARGs mediated by pCF10 plasmid was not the widely demonstrated pathway of activating bacterial oxidative antioxidant systems and thereby affecting bacterial cell membrane permeability BPA could promote the high expression of the pheromone-encoding gene ccfA,thereby increase the content of pheromone c CF10,which puts the conjugative system in an induced state.BPA also promotes the increasing expression of the prgB gene,which encodes an adhesion protein.BPA subsequently enhancing interbacterial adhesion is also an important mechanism of which BPA affects conjugative transfer of ARGs.BPA is also chemically similar to natural hormones and has a pheromone effect.A method was constructed to evaluate the biological effects of bisphenols based on the conjugative transfer of antibiotic resistance genes,through which the biological effects of the bisphenols involved in this study were quantified in the following order:bisphenol A >tetrabromobisphenol A >bisphenol AF> bisphenol F > pheromone cCF10 >bisphenol S.In this study,we have demonstrated that BPA at environmental concentrations can promote pheromone-mediated conjugative transfer of antibiotic resistance genes mediated by plasmid pCF10,and have conducted a preliminary investigation into the mechanisms by which BPA affects pCF10 plasmid conjugative transfer.In addition to direct impact on human health,BPA increases the abundance of ARGs in the environment,making the spread of antibiotic resistance genes even more critical.This study will enhance society’s knowledge and understanding of the biological effects of environmental estrogen pollutants and fill the research gap in the field of ecological safety of BPA,a traditional environmental pollutant.It will provide a theoretical basis for harmonious coexistence between humans and the ecological environment,and may provide technical support to achieve the ultimate goal of monitoring and controlling the transmission elements of BPA and ARGs.