Spatial And Temporal Distribution Characteristics And Source Analysis Of Antibiotic-Resistant Genes In The Water Environment In The Lanzhou Section Of The Yellow River

The overuse of antibiotics has led to the generation of antibiotic resistance genes（ARGs）induced by residual antibiotics in the environment.The water environment is rich in microbial species and vulnerable to contamination by residual antibiotics,which has become one of the important reservoirs of ARGs.Lanzhou is an important node city in the upper reaches of the Yellow River,and the ecological safety in the Lanzhou section of the Yellow River basin is not only related to the drinking water safety of residents on both sides of the river,but also affects the sustainable development of the province and the downstream society.However,the distribution pattern of ARGs and potential pollution sources in the Lanzhou section of the Yellow River basin are still unclear.Therefore,in this study,sampling sites were set up in Lanzhou section of the Yellow River basin,and a variety of environmental media（surface water,sediment,point source effluent,and non-point source soil）were selected as the study objects.Surface water and sediment samples were collected from eight dry and tributary cross-sections,two riparian effluent outfalls as point source pollution samples,and seven farmland and mountain soils as non-point source pollution samples.In this study,fluorescence quantitative PCR was used to quantify the abundance of bacterial 16S r DNA（V3～V4 region）,eukaryotic microbial 18S r DNA（V4 region）,tetracycline resistance genes（tet M,tet X）,macrolide resistance genes（erm B,erm F,ere A）,sulfonamide resistance genes（sul1,sul2）,and mobile genetic element integrase genes（int I1）and transposon genes（tnp A-04）.The aims were to clarify the abundance,diversity and distribution characteristics of antibiotic resistance genes in the Lanzhou section of the Yellow River basin comprehensively,and to reveal the correlation between antibiotic resistance genes and various environmental factors coupled with multivariate statistical analysis,providing important reference data for the traceability analysis of antibiotic-resistance genes.The results of the study showed that:1.Through the analysis of the occurrence of antibiotic resistance genes in various environmental media（surface water,non-point source soil,point source effluent and sediment）in the Lanzhou section of the Yellow River basin,it was found that the detection rates of seven ARGs and two mobile gene elements（MGEs）selected for detection in this study were100%in the above four environmental medias.The average total absolute abundance of ARGs and MGEs in surface and bottom sediments of Lanzhou section of the Yellow River was 4.99×10¹⁰ and 4.49×10¹⁰ copies/g,respectively.The average total absolute abundance of MGES was 5.56×10⁸ and 2.05×10⁸ copies/g.The average abundance of ARGs and MGEs in non-point source soils was 1.55×10¹¹ and 3.29×10⁹ copies/g,respectively.The average total absolute abundance of ARGs in point source water in wet and dry seasons was 1.04×10¹¹ and3.03×10¹³ copies/L,respectively.The mean total absolute abundance of MGEs was 3.23×10¹⁰and 4.68×10¹⁰ copies/L,respectively.The results showed that the total absolute abundance of ARGs and MGEs in the four environmental media was in the order of surface water>point source effluent,and non-point source soil>sediment.Among the four different media,the absolute abundance of sulfa ARGs is the highest,and the highest abundance of sul2 is sul1 in all other media except sediments.2.Based on the analysis of the occurrence of antibiotic-resistance genes in surface water in Lanzhou section of the Yellow River,the abundance of ARGs and MGEs in all sampling points in wet season was higher than that in dry season.The average total absolute abundance of ARGs in wet season and dry season was 2.69×10¹⁴ and 1.53×10¹⁴ copies/L,respectively.The mean total absolute abundance of MGEs was 5.48×10¹² and 1.99×10¹¹ copies/L,respectively.The absolute abundance of sulfonamides ARGs in different river sections is the highest,and the absolute abundance of macrolides and tetracycline ARGs is 2 to 5 orders of magnitude lower than that of sulfonamides ARGs.These results indicate that ARGs have a strong transmissibility in river water environment,which is difficult to be reduced naturally once acquired,and human disturbance will accelerate the spread and diffusion of ARGs.The abundance of ARGs in wet season is higher than that in dry season,which is probably because a large number of ARGs carrying particles enter the water body due to the erosion of surface rainwater runoff in wet season.3.Through the analysis of the correlation between antibiotic resistance genes and environmental factors in various environmental media（surface water,non-point source soil,point source effluent and sediment）in the Lanzhou section of the Yellow River basin,There were significant positive correlations between macrolide ARGs（erm B,erm F,ere A）,tetracycline ARGs（tet M,tet X）,sul2,tnp A-04,int I1,DOC and TN in non-point source soil（P<0.05）.DOC,NH₄⁺,NO₃^-and TN in point source water were significantly positively correlated with erm B,int I1 and tnp A-04（P<0.05）.The above results showed that most ARGs were highly correlated with the number of bacteria,the number of eukaryotic microorganisms,DOC and nutrient content levels of TN,TP,NO₃^-and NH₄⁺,indicating that nutrient content and microbial abundance may be one of the important environmental factors regulating the occurrence of ARGs.At the same time,there was a significant positive correlation between most ARGs and int I1,suggesting that int I1 may be the main driving factor for ARGs propagation and diffusion.4.Through structural equation model analysis of environmental factors,microbial abundance,ARGs and MGEs in various environmental media（surface water,non-point source soil,point source effluent and sediment）in the Lanzhou section of the Yellow River basin.The standardized path coefficients were-0.087,-0.004 and-0.095,respectively.The MGEs in surface water,non-point source soil,point source effluent and sediment had a positive and direct influence on ARGs level in surface water,and the standardized path coefficients were 1.009,0.084,0.030 and 0.106,respectively,among which the MGEs in surface water had the largest direct influence on ARGs level in surface water.These results indicate that exogenous and endogenous ARGs contribute little to the overall level of ARGs in water,but large input of MGEs may enhance ARGs enrichment in water,and the increase of MGEs abundance caused by exogenous non-point source pollution and endogenous sediment disturbance may be the main driving factors of ARGs enrichment in water.In summary,this study explored the spatial and temporal distribution characteristics of ARGs and their leading environmental influencing factors in the Lanzhou section of the Yellow River Basin,and analyzed the differences in the composition and distribution of ARGs in sediments,non-point source soil and point source effluent of the Lanzhou section of the Yellow River Basin,indicating that ARGs have a strong ability to spread in the water environment of the Yellow River,and the interference of human activities will accelerate the diffusion of ARGs.It is concluded that ARGs enrichment in surface water in Lanzhou section of the Yellow River is probably caused by the interaction of exogenous non-point source pollution and endogenous sediments.This study provides data reference for environmental and ecological risk assessment of ARGs in Lanzhou section of the Yellow River Basin,and also provides important decision-making basis for strengthening ecological protection and management in the upper reaches of the Yellow River and promoting high-quality development of the whole basin.