Study On Antibiotic Resistance Genes Pollution And Microbial Diversity In Environmental Media

Objective: As the world continues to enter the "post-antibiotic era",the issue of antibiotic resistance has become an increasingly serious public health problem.Latest research results indicate that the environment is a key factor affecting the widespread spread of antibiotic resistant bacteria and the frequent occurrence of antibiotic-resistant pathogens.However,there is still a lack of systematic research on antibiotic resistance pollution in environmental media.In the present study,three typical environmental types of antibiotic resistance pollution in urban rivers,lakes and dairy farms were inluded as the research object,the occurrence and distribution characteristics of antibiotic resistance genes were analyzed,and the relationship between antibiotic resistance and microbial communities was estimeted.Our study results will provide a strong scientific basis for the prevention and control of antibiotic resistance pollution in environmental media.Methods:1.Water and soil samples were collected from Hutuo River,Longquan Lake and dairy farm of Shijiazhuang,respectively.After extracting the genomic DNA of the sample,11 ARGs and class I integrators and transposon gene(Int1,Tnp A-IS4)were quantitatively detected by real-time PCR.The differences in the detection rate,absolute concentration and relative concentration of ARGs between the samples and the correlation with Int1 and Tnp A-IS4 were analyzed2.Microbial community diversity and species composition were detected by Pac Bio 16 S full-length amplicon sequencing,and the relationship between microbial community and ARGs was analyzed.3.Species classification and CARD functional gene annotation of samples were performed using next-generation metagenomic sequencing,and the differences among different groups were examined.Results:1.Real-time PCR results showed that the detection rate,absolute and relative concentrations of ARGs in water were significantly higher than those in soil.The ARGs pollution level in the Hutuo River and Longquan Lake were roughly flat,but significantly higher than those in the dairy farm,while there was no significant difference in ARGs pollution levels in the soil around dairy farms.The pollution levels of 11 ARGs in environmental media were positively correlated with Int1 and Tnp A-IS4.2.The results of Pac Bio 16 S full-length amplicon sequencing showed that the Alpha diversity of soil microbial communities was higher than that of water samples,while the Alpha diversity of Hutuo River and Longquan Lake was higher than that of pond water next to dairy farms.The species composition of microbial communities differed significantly between the Hutuo River,Longquan Lake and dairy farms,and these differential species were closely related to ARGs concentrations.3.The results of metagenomic sequencing showed that Proteobacteria,Actinobacteria and Bacteroides were the dominant phylums in water and soil samples.There were significant differences in ARO and species composition between water and soil samples.There were also statistical differences in ARO,Resistance＿formated and species composition between the Hutuo River,Longquan Lake and the pond next to the dairy farm.The number of ARO in the pond water was the highest,followed by the Hutuo River,the lowest in Longquan Lake,and the number of Resistance＿formated in the pond water and the Hutuo River was higher than in Longquan Lake.Conclusions:1.The Hutuo River,Longquan Lake and dairy farms are highly polluted by 11 kinds of ARGs,and the trend of water >soil,and Hutuo River >Longquan Lake >dairy farms was found.Acquired antibiotic resistance mediated by mobile gene elements is the main way of causing high-level of ARGs pollution in environmental media..2.Microbial community diversity was closely related to the distribution of ARGs.Significant differences were observed in the species composition of microbial communities in different sample types.Different types of environments may be of importance reason for the pollution level of ARGs in environmental media.3.The results of metagenomic sequencing showed that,the pollution patterns and microbial communities of resistance genes between water and soil,as well as Hutuo River,Longquan Lake and dairy farms were statistically different,which further verified the above results.