Effects Of Quorum Sensing On Horizontal Transfer Of ARGs In Microplastic Biofilms

Microplastics and antibiotic resistance gene(ARGs)contamination are hot topics in the field of ecological environment this year.Microplastic biofilms are considered to be a factor in the spread of ARGs due to their specific ecological loci.In the process of biofilm formation,quorum sensing signal molecules are generated at higher density,and when the signal molecules reach the threshold,they regulate the formation of biofilm and the horizontal transfer of ARGs.In this study,LLE-UPLC-MS/MS was used to screen the quorum sensing signal molecules secreted by microplastic biofilms in the reaction system of Gram-negative and positive bacteria respectively,and the regulation effect of quorum sensing signal molecules and inhibitors on ARGs transformation was explored by adding exogenous quorum sensing signal molecules and inhibitors.Through transcriptomic sequencing,to explore the regulation of quorum sensing signal molecules and signal molecule inhibitors quorum sensing genes in microplastic biofilms.Studying the mechanism of quorum sensing on the horizontal transfer of ARGs in microplastic biofilms can provide a new way to control the spread of resistance genes.The research results of this paper are as follows:(1)An internal standard method for the simultaneous determination of 11 AHLs signal molecules secreted by biofilm quorum sensing was established by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Optimized for fast and accurate quantitative analysis of signaling molecules.The established analytical method was used to analyze the quorum sensing of microplastic biofilms,and the effect of quorum sensing signal molecules on biofilm formation was analyzed.There are three signal molecules of C10-HSL.The method detected the most signal molecules on the microplastic PE,and the concentration of C12-HSL was the highest.These three signaling molecules can promote the formation of biofilms.This provides a theoretical basis for studying the effect of quorum sensing on the formation of microplastic biofilms.(2)C8-HSL,C12-HSL and 3-OXo-C10-HSL quorum-sensing signal molecules could promote the conversion frequency of ARGs in the biofilm and natural substrate of bacillus subtilis microplastics.C4-HSL,3OC12-HSL and N-3-OH-C12-HSL,as exogenous signal molecules of Gram-negative acinetobacter patacinetobacter,promoted the horizontal metastasis of ARGs within a certain range,and the effect of signal molecules on the frequency of horizontal metastasis of ARGs was weakened beyond a certain concentration.The conversion frequency of the natural matrix group was similar to that of the microplastic group,and the overall value increased slightly compared with the control group.Furanone,2(5H)-furanone,indole and coumarin all inhibited ARGs metastasis to varying degrees.On quartz sand,the four inhibitors all had inhibition effect on ARGs transformation frequency,which was less than that on microplastic biofilm.(3)Quantitative fluorescence PCR was used to quantify the effects of quorum sensing related genes,quorum sensing signal molecules and signal molecule inhibitors on the expression level of quorum sensing related genes in the process of horizontal transfer.Eight target genes(Las I,las R,rhll,rhl R,lux I,lux R,lux S,sdi A)were increased in the treatment of different concentration of quorum sensing signal molecules,and C8-HSL significantly affected las R and lux R.C12-HSL significantly affected lux I and lux R,while 3OC10-HSL significantly affected las R,rhll and lux I.Quorum-sensing signaling molecules regulate bacterial biofilm formation by controlling gene expression,which ultimately promotes the horizontal transfer of ARGs on microplastic biofilms.