Characteristics And Fate Of Antibiotics And Antibiotic Resistance Genes In Manure Application Soil

As emerging environmental contaminants,antibiotic resistance genes?ARGs?are different from traditional chemical pollutants.ARGs may spread via vertical transfer and horizontal transfer,making direct threat to human health through the food chain.The special transmission mechanism of ARGs does not depend on the corresponding antibiotic resistance bacteria,which further increases the environmental risk of ARGs.Antibiotic resistance has become a major global public health issue.Due to the abuse of antibiotics in animal husbandry,the potential threat of antibiobtic resistance genes could be increased because of antibiotic residue.Our group has discoverd the diversity of ARGs in livestock operations and their dissemination mechanisms to the environment.The results show that a large number of antibiotic resistant genes were input to the recipient environment.There have been many studies on the current status of resistance genes in soil,but the environmental behavior of resistance genes in soil and the influencial factors of resistance gene reduction are still lack of research.The moisture content in soil and the addition of biomass carbon may affect the reduction of antibiotic resistance genes in soil.Different types of manure and different frequency of manure applications may affect the risk of ARGs contamination caused by manure application.Farmland soil with long term application of manure and with non-manure application were collected.Control of soil moisture content and biomass carbon content were added to soil culture as control experiment.By quantitative PCR,we investigated all kinds of content level and pollution characteristics of antibiotic resistant genes and mobile genetic elements in soil samples of 0-310 d.The results showed that,in soil chronically fertilized by swine manure,two kinds of MGEs,int I1,Tn916,are detected.We also detect 9 kinds of ARGs?tetC,tetG,tetW,tetO,sul1,sul2,aadA,cmlA,floR?.Among them,aadA,floR,sul1,sul2 and cmlA genes are the dominant genes.In non-manure applicated soil,two kinds of MGEs,int I1,Tn916,are detected.We also detected 7 kinds of ARGs?tetW,tetO,sul1,sul2,aadA,cmlA,floR?.Among them,aadA is the dominant gene.Long term application of manure can significantly increase the diversity and abundance of resistant genes and mobile genetic elements in soil.Resistance genes and mobile genetic elements are decreasing in soil culture.The variation of absolute abundance is consistent with exponential fitting.The results of exponential fitting showed that with adding biomass carbon,the time of ARGs and MGEs absolute abundances are decreasing to half of the initial abundance,which is twice as the time in the two experimental groups that did not add biomass carbon.When more water is added,the half-life of ARGs and MGEs is obviously reduced.The addition of biomass carbon can slow down the reduction of ARGs and mobile genetic elements.The increase of moisture content can speed up the reduction of ARGs and mobile genetic elements.The return of straw to field may bring additional risk of resistance gene contamination.The abundance of mobile elements can indicate the degree of contamination of total resistance genes to a certain extent.There is a positive correlation between intI1 and all kinds of resistance genes.In soil culture experiments,intI1 is the main element that acts on the horizontal transfer of resistance genes in soil.The level of antibiotic contamination of soil samples in soil culture experiment in 0-310 d was investigated.10 kinds of antibiotics?ofloxacin,pefloxacin,sulfadiazine,ciprofloxacin,tetracycline,chlortetracycline,sulfadiazine,norfloxacin,enrofloxacin and salinomycin?were detected from the samples of soil culture experiments.The residual antibiotics were more than those of no manure,indicating that a certain amount of antibiotics had been imported into the soil with a long time manure application.In soil culture experiment,antibiotics in each group showed a decreasing trend in long-term culture.Enrofloxacin,ciprofloxacin,norfloxacin and chlor tetracycline have similar environmental behavior.Biomass carbon can affect the environmental behavior of antibiotics in the soil culture experiment.the content of antibiotics in the experimental group added with biomass carbon decreased more rapidly.It's because biomass carbon can enhance the microbial degradation of antibiotics in the soil,improve the microbial degradation rate of antibiotics and reduce the residue of antibiotics in the soil.In soil culture experiment,moisture had little effect on the degradation of antibiotics in soil.We collected non-manure contaminated soil,controlled soil manure and added the frequency of the controlling soil addition experiment.By quantitative PCR,we investigated content level and pollution characteristics of resistant genes and mobiled genetic elements in soil samples of 0-157 d.The results showed that,three mobile genetic elements,intI1,intI2 and Tn916,were detected in the soil with chicken manure,chicken manure fertilizer,pig manure and pig manure fertilizer.In soil added with manure,we detected 18 kinds of antibiotics(tet W,tetH,tetG,tetB/P,tetC,tetO,cmlA,cfr,floR,fexA,sul1,sul2,aadA,bla _TEM,ereA,ermA,qnrS,qnrD).In soil without addition,we detected 9 kinds of antibiotics?tet W,tetB/P,tetC,tetO,cfr,floR,fexA,sul1,sul2,aadA?.The abundance is far less than the experimental group treated with manure.Adding manure can significantly increase the content of resistance genes and mobile genetic elements in soil.The absolute abundance of resistant genes and mobile genetic elements increased rapidly in 0-7 d.After 7 d,it gradually decreases,and it can be fitted exponentially with time.The ranking risk of ARGs contamination:chicken manure>chicken manure fertilizer>pig manure>pig manure fertilizer.The sequence of mobile genetic elements contamination risk:chicken manure>pig manure>chicken manure fertilizer>pig manure fertilizer.Repeated addition of manure can significantly increase the peak of resistance genes and the absolute abundance of mobile genetic elements in chicken manure,chicken manure fertilizer and pig manure.There is a positive correlation between the absolute abundance and relative abundance of mobile genetic elements and ARGs.intI1 is the most important mobile genetic element that mediates the horizontal transfer of multiple resistant genes.Int I2 and Tn916 played a certain role in the horizontal transfer of resistance genes.In the process of horizontal transmission of resistance genes,three mobile genetic elements may have synergistic effect.This paper has investigated the reduction of resistance genes in manure soil.We also researched the effects of different frequencies and kinds of manure on the dynamic changes of resistance genes in soil.The results showed that the control of biomass carbon and water could significantly affect the reduction of resistance genes in the soil.The risk of resistance gene pollution caused by different kinds of manure and different kinds of manure on soil was different.The final conclusion is that additional biomass carbon may lead to a greater risk of resistance gene pollution,adding water can reduce the resistance genes in the soil more effectively.From the point of preventing and controlling resistant genes,pig manure should be superior to chicken manure.Composting is better than raw manure.At the same time,the frequency of manure addition should also be controlled.The mobile genetic element int I1 plays a key role in the horizontal transfer of soil resistance genes.The resistance genes in the soil with manure decreased exponentially.