Effect Of Copper And Nanoplastic Contamination With Different Moieties On The Enrichment And Horizontal Transfer Of Antibiotic Resistance Genes

China is a major producing and using country of antibiotics,with serious misuse of antibiotics for medical and livestock use,which has led to increasing bacterial resistance due to the misuse of antibiotics.Bacteria andpathogens can acquire antibiotic resistance genes(ARGs)from the environment through horizontal gene transfer(HGT),leading to an increase in the abundance of ARGs in the environment and making it easier for pathogens to acquire drug resistance or even multidrug resistance,thereby posing a serious health risk to humans.Several studies have shown that heavy metal Cu contamination may promote horizontal transfer of ARGs,but the mechanism remains to be further investigated Microplastics may act as carriers of horizontal transfer of ARGs and promote horizontal transfer of drug-resistant genes,but no reports on how nanoplastics with different moieties affect horizontal transfer of ARGs.In this study,the effects of traditional and novel pollutants on antibiotic resistance gene production were explored from the environment and laboratory.Based on this,the mechanisms by which heavy metal Cu pollution and microplastic pollution affect the horizontal transfer of genes were analyzed.(1)Bacterial communities in agricultural fields were analyzed using 16S gene sequencing and their changes were presented in stacked histograms.The results showed that the abundance of Proteobacteria,Acidobacteria,Bacteroidetes,Gemmatimonadetes,and Firmicutes showed an increasing trend and reached the maximum at Cu concentration of 400 mg/kg.The abundance of species such as Chloroflexi,Verrucomicrobia,Nitrospirae,Chlorobi,Actinobacteria and WS3 decreased with the increase of Cu concentration.Yet,abundance of species such as Planctomycetes is not influenced by Cu concentration(2)The sequencing data were compared with the KEGG database,and then gene prediction was performed to analyze the effect of heavy metal contamination on antibiotic resistance genes at the genetic level.The results showed that the abundance of both ARGs and HGT-related genes increased with the increase of Cu concentration.This is because the heavy metal Cu promotes the production of excessive ROS in bacteria,which induces the SOS and DNA repair response,disrupts the cell membrane and secretion system of bacteria,and thus promotes the horizontal transfer of drug resistance genes.(3)The data of related genes and bacterial genera with higher abundance were pooled to analyze the correlation between related genes and dominant bacteria.Bacterial communities whose abundance increased with Cu concentration were positively correlated with ARGs and HGT-related genes;bacterial communities whose abundance decreased with Cu concentration were negatively correlated with antibiotic resistance genes and gene-level transfer-related genes.(4)This experiment used pUC119 plasmid carrying ampicillin resistance and Escherichia coli DH5α to construct ARGs transformation system to study the effects of PS-base,PS-COOH,PS-NH2 and the coupling of these nanoscale plastics with Cu on HGT.The results showed that:PS-base and carboxy PS-COOH had a promoting effect on HGT,2.1 times and 2.5 times at 500 mg/L,respectively.While PS-NH2 had an inhibiting effect on E.coli,8 times at 500 mg/L.and the nanoplastic coupled with Cu further promoted the horizontal transfer of ARGs.(5)The potential mechanism of aging nanoplastic particles affecting ARGs transformation was further revealed by macro-transcriptome sequencing,free radical detection and bactericidal assay.The results showed that PS-base and PS-COOH promoted ARGs transformation mainly because the nanoplastics coerced the bacteria to produce ROS,which disrupted the bacterial cell membrane,thus inducing the bacterial SOS and DNA repair response and altering the bacterial secretion system,which in turn promoted the horizontal transfer of drug-resistant genes.While the inhibitory effect of PS-NH2 on transformation was mainly due to the PS-NH2 is highly toxic to DH5α,and 97.9%of bacteria in the transformation system were killed by PS-NH2.