Effects Of Heavy Metal Stress On The Horizontal Transfer Of ARGs In Artificial Wetland

It is found that antibiotic resistance genes,as a new type of pollutants,will bring potential health risks and safety hazards to human health and ecological environment,and the existence of heavy metal pollution in the environment may be one of the factors affecting the spread of antibiotic resistance genes.Exploring the stress effect of heavy metal resistance gene transmission and trying to analyze the causes from the molecular level can provide theoretical support for eliminating the safety risks of antibiotic resistance genes.Based on the "Natural Science Foundation Project of Shandong Province(ZR2021MD025)and Jinan Water Science and Technology Project(JNSWKJ202102)",this thesis focuses on the analysis of the effect of heavy metal stress on the horizontal transfer of antibiotic resistance genes,and attempts to explore the causes of the effect at the molecular level.Combined with ecological treatment technology of constructed wetland,the effect of horizontal transfer of heavy metal stress antibiotic resistance gene on ecological treatment of constructed wetland was explored.The main contents of this thesis are as follows:(1)Screening strains.By screening for strains that transmit antibiotic resistance genes via conjugation transfer,Three donor strains,Morganella morganii MP63,Enterobacter roggenkampii POL1 and Enterobacter cloacae POL9,and one recipient strain,Escherichia coli 25 DN.(2)To analyze the effect of horizontal transfer of common heavy metal resistance genes in water environment.Heavy metal contaminants include copper(Cu),zinc(Zn),arsenic(As),mercury(Hg),cadmium(Cd),chromium(Cr),tellurium(Te),and silver(Ag).The results showed that different types and concentrations of heavy metals had different effects on conjugation transfer between different strains.No copper(Cu)or zinc(Zn)was found to affect conjugation transfer between tested strains,but the addition of specific concentrations of arsenic(As),mercury(Hg)or tellurium(Te)could reduce conjugation transfer conjugation number and inhibit the growth of strains.The addition of specific concentration of chromium(Cr)significantly increased the number of zygotes but inhibited the growth of strains,while the addition of specific concentration of cadmium(Cd)or silver(Ag)significantly increased the number of zygotes but did not inhibit the growth of strains.(3)The combinations of thalli with significant influence of heavy metal stress on conjugation and transfer were selected for transcriptome sequencing.log2(Control/Trial)>1 and Q-value(padj)<0.05 was the screening condition.Transcriptome analysis results showed that the expressions of some antibiotic resistance genes and heavy metal tolerance genes of 25 DN strain were increased under heavy metal chromium(Cr)and cadmium(Cd)stress,and chromium(Cr)promoted the expression of transfer-related genes.There was no significant difference in the expression of resistance genes between POL1 and POL9 strains under antibiotic and heavy metal stress conditions.Antibiotic stress,or antibiotic and heavy metal chromium(Cr)stress caused significant changes in the transcription levels of some antibiotics and heavy metals in MP63 strain.(4)Construct three different combinations of constructed wetlands for joint transfer verification.The results showed that there was conjugative transfer of polymyxin E,tetracycline,ciprofloxacin,ampicillin and erythromycin resistance genes with Escherichia coli as receptor bacteria in natural water.The conjugative transfer test of donor bacterium Morganella morganii MP63 and recipient bacterium Escherichia coli 25 DN in constructed wetlands showed that: 1)Different wetland model combinations had the ability to inhibit conjugative transfer of antibiotic resistance genes;2)Chromium(Cr)can promote conjugation and transfer of polymyxin E and ciprofloxacin magnitude resistance genes in constructed wetlands,and increase the number of zygotes;3)The addition of chromium(Cr)in constructed wetlands had inhibitory effect on conjugation transfer of tetracycline,ampicillin and erythromycin resistance genes,and the inhibitory effect of combination 1 and combination 3 was more obvious.In summary,this study found that heavy metal stress can significantly affect the horizontal transfer of antibiotic resistance genes and the expression of related genes,and the research results can provide theoretical support for the indirect co-transfer of different bacteria under heavy metal stress,and also provide ideas,theoretical basis and practical guidance for exploring the influence of heavy metal stress on the spread of antibiotic resistance genes in constructed wetlands.