Preliminary Study On The Effects And Mechanisms Of Nanomaterials On Antibiotic Resistance Genes In Landfill Leachate Microbes

Residual antibiotics have accelerated the induction of substantial antibiotic resistance bacterial（ARB）and antibiotics resistance genes（ARGs）in the environment,due to widespread usage of antibiotics recent years.ARGs with persistent stability can be disseminated and proliferated through horizontal genes transfer（HGT）,which poses great potential threat to human health.In the same way,application of nanomaterials can be seen everywhere in daily supplies with the speedy development of nanotechnology,which could lead to great potential release of nanoparticles into environment,becoming one of emerging contaminants to ecology.However,there are few researches dealing with the impacts of nanomaterials on ARGs so far.Besides,the scientific problem concerning the impacts of nanoparticles on the dissemination of ARGs is vague.Thereby,in order to bridge the gap knowledge involving in the impacts and mechanism of nanomaterials on the ARGs in environment compartment with multiply contaminant.This research investigated the impacts of different nanomaterials（including metal NPs,metal oxide NPs and SWNT）on the microbes with ARGs from young landfill leachate.Besides,two typical drug-resistant strains were used to verify the results.The main conclusions and results are as follows:1.Characterization of nanomaterials and their effects on microbes（1）By using inductively coupled plasma spectrometer（ICP）,the dissolved ions of nanomaterials(Zn²⁺and Cu²⁺)were determined.It was found that the higher concentration of nanomaterials would release more ions,while the ratio of the dissolved ions decreased with the increase of the exposure concentration of nanomaterials in the system.According to the exposure experiment of nanomaterials on Vibrio fischeri,it could preliminarily judge that the toxicity sequence for nanomaterials was Cu NPs>ZnO NPs>SWNT>Zn NPs>CuO NPs>TiO₂ NPs.（2）The growth curve of the microorganism was fitted by Logistic Model,It was found that SWNT and TiO₂ NPs were helpful to stimulate the growth of microorganism,while Cu NPs,Zn NPs,ZnO NPs and CuO NPs inhibited the growth of microorganism,of which the ZnO NPs inhibition effect was the largest.Further,SEM images show that the nanomaterials can be adsorbed tightly on the surface of microbes,but it does not damage the surface structure of microbes.2.Study on the effect of different nanomaterials on the transmission of antibiotic resistance genes（ARGs）in microbes.（1）The exposure experiments of microbes from landfill leachate showed that the presence of nanomaterials was helpful to reduce the resistance genes（compared with the blank group）,and the decrease extent increased with the increase of the concentration of nanomaterials.Among them,the action intensity of ZnO NPs is the greatest,and that of SWNTs and TiO₂ NPs are the least.（2）The results of redundancy analysis（RDA）showed that there was a negative correlation between the target gene and the exposure concentration and time,and the exposure time had the greatest influence on the target resistance gene sul1.However,exposure concentration had the greatest effect on the target gene intl1.3.Preliminary investigation on the mechanism of nanomaterials affecting resistance genes from the community structure and Cell level.（1）High throughput sequencing and q-PCR gene quantification were used to analyze the community structure and ARGs in the reaction system,respectively.It was found that the main microbes in the reaction system were Bacillus,and the resistant genes in the system were sul1,aadA1 and intl1,which did not change with the species of nanomaterials.It is assumed that the existence and distribution of resistance genes are dominated by the population structure.（2）It was found that both Zn²⁺and Cu²⁺could inhibit the target resistance genes according to the exposure experiment of microorganism to ions extracted from the nanomaterials,which was consistent with the law of the effect of the nanomaterials on the resistance genes.These results suggest that the release of ions is one of the main factors that influence the resistance genes of nanomaterials.In addition,quantitative analysis of microbial extracellular polymers affected by nanomaterials showed that the increase of extracellular polymers was helpful to slow down the decrease of antibiotic resistance genes in nanomaterial system.（3）Variation partitioning analysis（VPA）for the effects of reactive oxygen species,extracellular polymers,dissolved ions and microbial growth inhibition rate on target gene changes.The results show that microbial growth inhibition rate has the highest explanatory weight（22.44%）in all variables.The second is ion dissolution and reactive oxygen species,and the explanatory weights for genetic changes were15.63%and 14.73%,respectively.The results showed that the inhibition of microorganism growth by nanoparticles was the main mechanism for explaining the decrease of antibiotic resistance genes.（4）Two typical drug-resistant strains（i.e.Alcaligenes faecalis and Escherichia coli）were used to verify the above-mentioned results,showing that CuO NPs and ZnO NPs could inhibit the target resistance gene obviously.And the effect of nanomaterials on the resistance genes of microbes was verified by pure bacteria experiment.Through analyzing the interaction and mechanism between emerging pollutants,this study is helpful to understand the law of resistance gene migration and transformation under the condition of complex pollution.It provides a theoretical basis for reducing the adverse effects of nanomaterials and controlling the propagation and diffusion of antibiotic resistant genes.