Occurrence Characteristics And Diffusion Mechanism Of Antibiotic Resistance Genes In Greenhouse Vegetable Soil With Manure Application

Bacterial resistance to antibiotics is one of the biggest threats to human and animal health in the world,antibiotic resistance genes(ARGs)as a new type of environmental pollutant have become a focus of attention.Due to the horizontal and vertical transfer of genes,ARGs can spread in a variety of environments by means of mobile genetic elements(MGEs).Antibiotics are widely used in the livestock and poultry breeding industry,which makes livestock manure become one of the main sources of antibiotic resistant bacteria(ARB)and its resistance genes in the environment.In the process of greenhouse vegetable cultivation,the amount of manure application is large and the application period is long,so the health risk caused by ARGs deserves more attention.At present,there is a lack of systematic research on the occurrence and spread risk of ARB and ARGs in greenhouse vegetable field with manure application,the factors affecting the spread of ARGs and the influence mechanism of their migration in soil-plant system are not clear.In the present study,the manure-applied greenhouse vegetable field was taken as the study object.First,soil samples were collected from the main planting areas of greenhouse vegetable fields in Shandong Province,and the ARGs and MGEs in the soil samples were fully quantitatively analyzed using high-throughput quantitative PCR technology,so as to clarify the occurrence characteristics and potential transmission and diffusion mechanisms of ARGs;plate counting and 16S r RNA gene sequencing methods were used to isolate and identify ARB in soil samples;by analyzing the correlation between the heavy metal content,physicochemical properties and ARB,ARGs in soil samples,the environmental factors affecting the distribution of ARB and ARGs in greenhouse vegetable soil were explored;the effects of two types of typical environmental factors,heavy metals(Cd and Pb)and salinity(NaCl and Na₂SO₄)on the spread and diffusion of ARGs were studied by constructing a model of the horizontal transfer of RP4 plasmid carrying ARGs between bacteria;according to the experimental results of the influence of environmental factors on the horizontal transfer of ARGs,Pb that promoted ARGs conjugative transfer was selected to study the influence mechanism of ARGs migration in soil-plant system of greenhouse field with manure application.The main results are as follows:(1)ARGs and MGEs in greenhouse vegetable soil were quantitatively detected,and the resistance mechanism was analyzed.A total of 193 ARGs in 9 main categories were detected,among them,multidrug,aminoglycoside,macrolide-lincosamide-streptogramin B(MLSB)and tetracycline resistance genes were the dominant ARGs,such as qac Edelta1-01,aad A1,aad A-01,aad A-02,aad A2-03,erm F,tet G-01,tet G-02,tet X and other ARGs were detected with high frequency and abundance in greenhouse vegetable soil.Antibiotic inactivation and efflux pump mechanism were the main action mechanisms of bacterial resistance in greenhouse vegetable soil.MGEs affected the distribution of various ARGs in greenhouse vegetable soil,the total relative abundance of MGEs was positively correlated with aminoglycoside,MLSB and tetracycline resistance genes,in addition,the transposon Tp614 had a related symbiotic relationship with a variety of ARGs.There were also symbiotic relationships among different types of ARGs,for example,the total relative abundance of aminoglycoside resistance genes was significantly correlated with chloramphenicol,MLSB,sulfonamide and tetracycline resistance genes.Soil environmental factors affect the distribution of ARGs,such as organic matter,total nitrogen,total phosphorus,p H and heavy metals(Cu,Zn,Cd,Pb)were correlated with the total relative abundance of various ARGs,redundancy analysis showed that the distribution of ARGs in the greenhouse vegetable soil was most significantly affected by MGEs.(2)The abundance and species of cultivable ARB in greenhouse vegetable soil were analyzed,and the relationship between ARB and environmental factors was discussed.The resistance rates of bacteria to different antibiotics in the soil were different,in general,the order of the average resistance rate of bacteria to antibiotics was sulfamethazine>erythromycin>vancomycin>ampicillin>kanamycin>ciprofloxacin>tetracycline>chloramphenicol.A total of 100 strains of ARB belonging to 22 genera were screened out from soil samples,among them,ampicillin,chloramphenicol,tetracycline,vancomycin,sulfamethazine,erythromycin,ciprofloxacin and kanamycin resistant bacteria were screened out 10,11,11,19,13,10,11 and15 strains,respectively.In all ARB,Pseudomonas and Bacillus were the dominant genera,which were resistant to 5 and 8 antibiotics,respectively.According to the results of species similarity identification,some bacteria were resistant to different kinds of antibiotics,which may be multiple ARB.Soil environmental factors can affect the abundance of ARB,such as organic matter,total nitrogen,total phosphorus,p H and heavy metals(Cu,Zn,Cd,Pb)were correlated with the abundance of ARB,among them,p H was mainly significantly negatively correlated with ARB abundance.(3)The effects of environmental factors,heavy metals(Pb and Cd)and salinity(NaCl and Na₂SO₄)on the horizontal transfer of ARGs between Escherichia coli and its possible mechanisms were studied.The results showed that Cd,NaCl and Na₂SO₄ at the set concentration range generally reduced the conjugative transfer frequency of RP4 plasmid carrying ARGs between bacteria within genera,under the action of NaCl and Na₂SO₄,the membrane of the tested bacteria did not change significantly,and the m RNA expression levels of oxidative stress and SOS reaction of the donor bacteria were down regulated,Cd reduced the conjugative transfer of RP4 plasmid may be due to the serious damage to the bacterial membrane.Different concentrations of Pb significantly promoted the conjugative transfer frequency of RP4 plasmid carrying ARGs between bacteria within genera,the mechanism of Pb promoting the horizontal transfer of ARGs may be caused by the formation of pores on the cell membrane surface of the tested strain,which increases the permeability of the bacterial membrane,and the up-regulation of m RNA expression levels of oxidative stress and SOS response genes of the tested strains under the action of Pb.(4)The effects of Pb on the migration and transformation of ARGs in greenhouse vegetable soil-plant system with manure application were further studied.A variety of ARGs were detected in the soil,lettuce root endophytes and leaf endophytes.The relative abundance of ARGs in soil was higher than that in root endophytes,and the relative abundance of ARGs in leaf endophytes was the lowest.In a certain concentration range,Pb promoted the enrichment of various ARGs in root endophytes,and promoted the enrichment of beta-Lactamase and other types of ARGs in soil,but reduced the total relative abundance of ARGs in leaf endophytes.Pb affected the microbial diversity in soil,roots and leaves,after Pb treatment,the Chao1 index,Shannon index and Simpson index of the root increased significantly.Pb significantly changed the diversity of microbial community in roots,which was consistent with the total relative abundance of ARGs,this may be an important reason for the change of ARGs.In all samples,there was a significant correlation between the total relative abundance of MGEs and the total relative abundance of various types of ARGs,and through network co-occurrence analysis,it was found that some MGEs,ARGs and bacterial genera in soil,root and leaf had significant symbiotic relationships under the action of Pb,indicating that ARGs had a variety of potential host bacteria,and MGEs may affect the migration and transformation of ARGs in soil-plant system.This study systematically explored the occurrence characteristics of ARGs and ARB in greenhouse vegetable soil with manure application,and clarified that soil physicochemical properties and heavy metal pollution of greenhouse vegetable soil have different degrees of influence on the distribution of ARGs and ARB,and the key role of MGEs in the distribution of ARGs in greenhouse vegetable soil.Environmental factors of NaCl,Na₂SO₄ and Cd generally reduced the risk of horizontal transfer of ARGs among bacteria within genera,while Pb in the environmental pollution level promoted the horizontal transfer of ARGs among bacteria within genera and promoted the accumulation and migration of some ARGs in soil and endophytic bacteria of vegetable roots.The results of this study can provide a basis for the risk assessment of ARGs spread in greenhouse vegetable fields.