Combined Toxicity And Resistance Of Microplastics And Norfloxacin On Escherichia Coli

Microplastics have been increasing wide concern about their harmful influence on the environment,and their co-existence with environmental pollutants could exacerbate the damage to organisms and even ecosystems.Antibiotic resistance has been another global environmental concern,the abuse of antibiotics has led to widespread pollution with resistant genes and resistant bacteria,which has become a serious threat to both clinical treatment and human health.The environmental co-existence of microplastics and antibiotics will contribute to combined stress on bacterial survival,and may exacerbate the antibiotic resistance contamination in the environment.Hence,there is significant environmental significance for investigating the combined toxicity and combined resistance effects on bacteria by mixtures of microplastics and antibiotics.In this paper,polystyrene microplastics（PS）of different particle sizes and functional groups and norfloxacin（NOR）were selected as test compounds to investigate the dose-responses of single pollutants and binary mixtures on E.coli growth,endogenous and exogenous resistance,discuss the mechanism of action based on adsorption effect,intracellular reactive oxygen species and cell membrane permeability,and then the combined toxic and resistance actions were judged by interactive effects.The main findings were as follows:1)The tested mixtures exhibited hormesis effects with low concentrations promoting and high concentrations inhibiting the growth of E.coli.The combined toxic effects of the mixtures were observed to be similar to the dose-response of NOR,the combined toxic effects of the mixtures were found to be similar to the dose-response of NOR,and the growth inhibition rates and the type of combined toxic actions by the mixtures exposure on E.coli were mainly related to the free NOR concentration and the intracellular ROS level.According to single toxicity tests,all six PSs inhibited E.coli growth and the toxic effects of PS were particle size and functional group dependent.2)The promotion of resistance mutations in E.coli by the tested mixture was PS particle size and functional group dependent.The increased concentration of PS in the mixture contributed to higher mutation frequency,while increased concentration of NOR caused increased and then decreased on mutation frequency,where ROS levels were major factor which inducing mutations in bacteria.The combined mutation actions by binary mixtures of PS and NOR(EC₁₀,EC₇₀)on E.coli tended to synergistic effects,which indicated that the mixtures of microplastics and antibiotics could have greater potential to accelerate the emergence of antibiotic resistance in bacteria.Furthermore,the combined mutation actions between PS and NOR on E.coli were associated with corresponding combined toxic effects.3)All the tested mixtures promoted the RP4 plasmid conjugation transfer,and exhibited PS particle size and functional group dependence.The combined conjugative transfer actions between PS and NOR were judged to be synergistic based on the interactive effects,which PS and NOR could synergistically increase the efficiency of resistance gene horizontal transfer between bacteria,indicating that NOR exposure may further aggravate the dissemination of antibiotic resistance induced by PS.In addition,increased intracellular ROS levels as well as cell membrane permeability facilitated the plasmid conjugative transfer.The single determination demonstrated the particle size and functional group dependence of PS single exposure on plasmid-mediated dissemination of resistance genes between E.coli.Thus,microplastics exposure could promote the emergence and dissemination of resistance genes,as well as the co-existence of microplastics and antibiotics could aggravate the risk of bacterial resistance in the environment.