| Environmental pollution caused by bacterial resistance and spread is the focus of global attention.Two-dimensional black phosphorus(2D-BP)nanomaterials have been widely used in many fields due to their unique physical and chemical properties.The use of 2D-BP nanomaterials and environmental residues may cause bacteria to develop resistance to them,and may also increase the risk of transmission of resistance genes.This article studied the changes in drug resistance and related mechanisms of bacterial resistance after repeated exposure to sub-inhibitory concentrations of 2D-BP nanomaterials;the effects of different concentrations of 2DBP on the horizontal conjugative transfer of resistance genes mediated by RP4 plasmids and related mechanisms.The main results and conclusions of the study are as follows:(1)Escherichia coli K-12 MG1655 developed resistance to it after repeated exposure to sub-inhibitory concentrations of 2D-BP nanomaterials.The emergence of bacterial drug resistance is related to the decrease of the permeability of the bacterial cell membrane and the efflux of 2D-BP into the cell.The level of reactive oxygen species(ROS)produced by drug-resistant bacteria increased significantly,and the relative expression abundance of oxidative stress genes and SOS response genes increased significantly.It indicates that the resistance of 2D-BP bacteria may be related to the proper production of ROS and the protective effect of SOS response.(2)The analysis of the whole genome resequencing results of 2D-BP-resistant bacteria(BP-res)showed that the production of bacterial resistance is related to the production of prophages that enhance the adaptation of bacterial cells to the external environment.The existence of gene islands that help bacteria adapt to the external environment and mediate the acquisition of drug resistance also promotes the emergence of bacterial drug resistance.Bacterial resistance is related to 1939 related gene pathways such as carbohydrate metabolism and membrane transport annotated by KEGG function.Bacterial drug resistance is mainly related to the 3326 biological process genes annotated by GO function,which are mainly related to metabolism and cell process genes.Bacterial resistance is related to the 53 resistance-related genes annotated by ARDB.Bacterial resistance is related to the three genetic mutation genes of BP-res bacterial strains(dmd A,mnt P and gyr A).The analysis of the prokaryotic transcription results of BP-sen and BP-res bacteria showed that the bacterial resistance is related to the regulation of specific expression genes of BP-res strains.And bacterial resistance is related to the regulation of up-regulation of differentially expressed genes in 204 BP-res strains enriched by GO function annotation,and the regulation of up-regulation of differentially expressed genes in 182 BP-res strains enriched in KEGG function annotation.(3)The 2D-BP nanomaterials of 1.9?125 mg/L promoted the RP4 plasmidmediated transfer of resistance genes,while the 2D-BP of 250 mg/L significantly inhibited the transfer of resistance genes.The effect of 2D-BP nanomaterials on resistant gene binding and transfer is positively correlated with the production of ROS in the donor and recipient bacterial cells.High concentrations of 2D-BP may destroy cell components to produce high levels of ROS and inhibit binding transfer.A moderate concentration of 2D-BP may affect the permeability of the cell membrane to produce an appropriate amount of ROS and increase the binding and transfer of plasmids.Fourier transform infrared spectroscopy analysis of the donor and recipient bacterial cells showed that the 2D-BP nanomaterials mainly act on proteins in the donor bacterial cells and lipids in the recipient bacterial cells.This affects the horizontal transfer of resistance genes mediated by the RP4 plasmid in bacterial cells.(4)The relative m RNA expression analysis and correlation analysis of the target gene showed that 2D-BP at 1.9 mg/L promoted conjugative transfer by increasing the m RNA expression of genes oxy R,omp F,mar A,omp C and lex A.15.6 mg/L 2D-BP nanomaterials promoted conjugative transfer by increasing the m RNA expression of rec A,trf Ap,Ruv A,rpo S and sox R.31.3 mg/L and 125 mg/L of 2D-BP can promote the effect of kor A,sox S,osm C,tra J,pol B and rec A,sox S,kor A,osm C,and tra J m RNA expression enhancement respectively.The 250 mg/L 2D-BP may reduce the expression of related target genes and inhibit the horizontal conjugative transfer of resistance genes.Pearson correlation analysis showed that the concentration of 2D-BP nanomaterials and the relative abundance of target gene m RNA have an obvious positive correlation,and there is also a certain positive correlation between target genes.Different concentrations of 2D-BP may affect the expression of different target genes in the conjugative transfer system to different degrees,thereby affecting the plasmid-mediated horizontal conjugative transfer of resistance genes. |
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