Effects Of Manure Application On The Levels Of Antibiotic Resistance Genes In A Greenhouse Soil And Vegetables

Antibiotic resistance genes(ARGs)have been considered as new environmental pollutants.In recent years,more and more studies shown that the increase and diffusion of drug resistance in environmental pathogenic bacteria will pose potential harm to the health of humans and animals,therefore,it has attracted much attention in the field of global environmental research.The application of antibiotic-treated animal manure is a major resource of ARGs into the soil.Previous studies have demonstrated that manure-derived ARGs and pathogenic organisms rapidly decline over time following manure application,however,the impacts of long-term and repeated application of animal manure on the diversity and abundance of soil ARGs remains limited.There were few studies on ARGs pollution in Shanxi,so we sampled the greenhouse soils and vegetables with long-term dairy cattle and chicken manure application.Here,we investigated the diversity and abundance of ARGs in greenhouse soils with 13 years dairy cattle and chicken manure(DM and CM)application,and the half-life of antibiotic resistant bacteria(ARB)isolated from manured soils in Taiyuan,China.In addition,the control soil samples were collected from an abandoned field without known history of manure application adjacent to the agricultural field,which were all three repetitions.Furthermore,vegetable samples were collected from DM to detect the diversity and abundance of ARGs.The main results of the research were described as follows:1.In this study,six antibiotics including oxytetracycline(OTC),chlortetracycline(CTC),cefpiramide(CPM),cefuroxime(CUM),norfloxacin(NOR),and erythromycin(ETM)were quantified in manure soils collected from greenhouse by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The long-term manure application significantly improved the level of residual antibiotics in the soil,in addition,the effect of CM was more pronounced than DM.These findings also indicated that both DM and CM significantly improve the levels of residual antibiotics,and the abundances of ARGs,intI1 and soil organic carbon and total nitrogen strongly correlated each other.2.Our study combined the experiment of microbial culture and the quantitative PCR to discovered that the half-life of antibiotic resistant bacteria(ARB)was different,and the correlations of ARGs,intI1 and tnpA genetic elements extracted from ARB with culture times were different.The shortest half-life was 0.74 d in anti-CPM strain and the longest half-life of anti-NOR strain was 9.75 d.Besides,the strong link was found between the population of isolated ARB and the half-life(r = 0.908,P = 0.012).There was significantly positive link between total ARGs and intI1,but there were different correlations between MGEs(intI1 and tnpA)and each specific ARGs.3.We studied the ARGs of three vegetables(pakchoi,baby cabbage and summer radish)in DM through the quantitative PCR.The results showed that 4 ARGs were found in these vegetables,including tetA,tetE,tetX,tet G,and the relative abundance of these genes in vegetables was higher than in soil.Although a few ARGs were detected in vegetables,which indicated that the potential damage to human health would be caused by vegetables in soils due to manure application.In general,our finding suggested that the long-term and repeated application of animal manure treated with antibiotic caused ARGs pollution to the soil environment.Both the levels of antibiotics and the stability of ARGs might determine the diversity and abundance of ARGs in manured soils.