The Evolution Of TRGs,SRGs And β-RGs In Black Soil Under Freeze-Thaw Condition

Antibiotic resistance genes(ARGs)is a new emerged pollutant,while its spread poses an emerging public health problem.Horizontal gene transfer mediated by mobile genetic elements(MGEs)is the major contributor to the spread of ARGs in the environment.In this study,a livestock farm in Dulbert Mongolian Autonomous County,Daqing City,Heilongjiang Province was selected as the research object.The geographic information system(GIS)was used to visualize the spatial distribution characteristics of soil in the livestock farm under the two states of freezing and thawing.To reveal the difference in relative aboundance and environmental behavior of ARGs under seasonal freeze-thaw condition,it provides a basic theoretical basis for further protection of black soil areas and soil remediation.Methods:(1)Combined with GIS,frozen and thawed soil at the depth of 10-cm and 50cm were collected from the livestock farm.(2)The fluorescence quantitative PCR technology was used to detect the relative aboundance of 22 ARGs and 13 MGEs in frozen soil and 28 ARGs and 19 MGEs in thawed soil,and the comprehensive score was ranked based on Principal Component Analysis(PCA).ArcMap was used to output the pollution cloud map of ARGs in the different soil layers under freeze-thaw conditions and the spatial distribution characteristics were analyzed.(3)The correlation between ARGs and MGEs under freeze-thaw conditions were discussed by Pearson index method and network analysis.Results:(1)The relative abundance of ARGs in different soil layers was significantly different.The relative abundance of ARGs in frozen soil samples was higher than that in thawed soil samples,and the relative abundance of blaTEM in soil samples was the highest(0.47).In frozen soil and thawed soil samples,the relative abundance of ARGs in 50-cm depth soil layer was higher than that in 10-cm depth soil layer.In frozen soil samples,the top three principal component contributions in the 10-cm depth soil layer were tetX>sul2>sul1,and the percentage of blaTEM,sul1,sul2,tetW and tetX clustered in the first quadrant was 82.3%.The top three principal component contributions in the 50-cm depth soil layer were sul2>tetX>blaTEM,and the first quadrant was sull,sul2 and tetX,accounting for 77.5%.In thawed soil samples,the top three principal component contributions in the 10-cm depth soil layer were sull>sul2>tetAP,and the percentage of sull,sul2,tetAP,tetM and tetX in the first quadrant was 89.7%.The top three principal component contributions in the 50-cm depth soil layer were blaTEM>sul2>tetX,and the percentage of blaTEM in the first quadrant was the highest(61.1%).The overall sample analysis indicated that blaTEM,sul1,sul2 and tetX ranked top.(2)The relative abundance of total MGEs in frozen soil samples was higher than that in thawed soil samples,which was consistent with the trend of ARGs detection results.In frozen soil samples,the top three contributions of principal components in 10-cm depth soil layer were TRB-C>intI1>tnpA-04,TRB-C accounted for 75.7%in the first quadrant.TRB-C>tnpA-06>intI1 ranked the top three in the principal component contribution of 50-cm depth soil layer,TRB-C and intI1 accounted for 87.9%in the first quadrant,which were the main MGEs.In thawed soil samples,the top three contributions of MGEs in the 10-cm depth soil layer were TRB-C>intI1>tnpA-04,and the proportion of intI1 and intI3 in the first quadrant was 84%.The top three contributions of MGEs in the 50-cm depth soil layer were intI1>intI3>tnpA-04,and the proportion of intI1,intI3,tnpA-04 and TRB-C in the first quadrant was 98.7%.(3)Correlation analysis showed that the relative abundance of most ARGs in frozen soil samples was moderately or highly correlated with MGEs.In the 10-cm depth soil layer of frozen soil,intI1,TRB-C,TRA-A,tnpA-03,tnpA-06,intI2 and TP614 were clustered into one group,and the remaining MGEs were clustered into one group.In the 50-cm depth soil layer,TRB-C,tnpA-04,tnpA-06,intI1,tnpA-07 and intI2 were clustered into one group,and the remaining MGEs were clustered into one group.In the 10-cm depth soil layer,IS26,IncQ-oriV,ISCR1,intI3,ISCR2,TN916 and tnpA-04 were similar,and the remaining MGEs were similar.In 50-cm depth soil layer,intI3,intIl,tnpA-02,IS613,IncP-oriT,IncQ-oriV,intI2,tnpA-04 and tnpA-07 were similar,and other MGEs were similar.Conclusion:This study comprehensively revealed the temporal and spatial evolution of ARGs in black soil under freeze-thaw conditions.blaTEM the dominant resistance gene in pasture.The total relative abundance of ARGs was significantly different under freeze-thaw conditions.The total abundance of ARGs in frozen soil was higher than that in thawed soil,and the relative abundance of ARGs in 50-cm depth soil layer was higher than that in 10-cm depth soil layer.The spatial and temporal distribution of MGEs was consistent with ARGs,and showed a high correlation.Therefore,it is speculated that TRB-C mediates the horizontal transfer of blaTEM in the surface soil of the pasture,and intIl and TRB-C mediate the spread of blaTEM in the biosphere at the depth of 50 cm.The lower ambient temperature in winter may be more conducive to the proliferation and transmission of ARGs in soil,and the horizontal transfer induced by MGEs aggravates the population exposure risk of ARGs.ARGs in soil may be transmitted to humans through direct contact or indirect animal-environmenthuman pathways.Therefore,this study provides theoretical guidance and scientific basis for the potential risk assessment of soil ecosystems in black soil areas,and is crucial for ensuring land productivity and agricultural product safety.