Diversity And Abundance Of Antibiotics And ARGs In Vegetable Soil With Manure Application

Antibiotics were irreplaceable for human and animals to keep health and improve development.However,because of their misuse and abuse,many antibiotics flowed into environment,especially into soil and water environment,in the form of their original drug or metabolite with animal excrement.Their residue in environment may increase the resistant pressure on microorganism and accelerate the occurrence of antibiotics resistant bacteria.The main reason for microorganism to produce resistance is that they can carry antibiotics resistant genes(ARGs).Therefore,the use of antibiotics increased the abundance of ARGs.Vegetable soil is one most common place where using animal manure to improve soil fertility and crop production.So,it's very important and meaningful to study the occurrence and distribution of antibiotics,resistant bacteria and ARGs on vegetable soil.In this study,9 typical vegetable soils were collected from Weifang,Taian,Loude,Binzhou and Donging of Shandong Province,which were applied animal manure for many years.The purposes of this study were to study the abundance of resistant bacteria and ARGs in vegetables soil fertilized with manure for several years and to assess the correlation that may exist between the soil properties,antibiotic concentrations,resistant bacteria and ARGs.In this study,solid phase extraction(SPE)and high performance liquid chromatography(HPLC)were used to detect the abundance of antbiotics;real-time qPCR was used to detect the abundance of ARGs.Main results were as follow:(1)In all soil samples,DOX,SM2 and TYL were detected in various level.The concentration of DOX ranges from 52.37 ?g/kg to 428.16 ?g/kg in all soil samples.The concentration of SM2 ranges from 14.12 ?g/kg ~ 81.31 ?g/kg in all soil samples except S4.The concentration of TYL ranges from 70.01 ?g/kg ~ 482.36 ?g/kg in five soil samples except S1,S5,S6,S7 and S9.(2)There are many differences on the number of resistant bacteria between different antibiotics.In S0 soil without manure application,a bit resistant bacteria were detected.However,much more resistant bacteria were detected in S1~ S9 soil samples than S0.The antibiotics resistant rates of bacteria were decreased with the increase of antibiotic concentrations.The sequence of four antibiotics resistant rates were SM2,TYL,ENR and DOX from large to small in turn.(3)In the study of resistant bacteria identification,12 bacterial genus of DOX were turned out which belong 17 bacterial species.16 bacterial genus of SM2 were turned out which belong 16 bacterial species.9 bacterial genus of ENR were turned out which belong 16 bacterial species.9 bacterial genus of TYL were turned out which belong 12 bacterial species.Moreover,the dominant bacteria genus were Streptomyces,Bacillus genus,Microbacterium and Pseudomonas aeruginosa and Pseudomonas,respectively.Fuethermore,Bacillus,Flavobacterium and Pseudomonas are multidrug-resistant bacterial genus.(4)The results of PCR showed that tetW?tetM?tetO?sul1?sul2?qepA?qnrB?qnrS?ermB and ermF were detected in all soil samples.The abundance of ARGs detected by real-time qPCR were different.The richest ARGs in four kinds of ARGs were tetM,sul2,qepA and ermF,respectively.As for MEGs,the abundance of int I1 was higher than int I2.The sequence of four kinds of ARGs were sul > erm > tet > qnr from large to small in turn.(5)Pearson analysis showed that of soil propertiy of pH value was significantly correlated with resistant bacteria of ENR.The contents of heavy metals(Cu and Zn)were significantly correlated with the abundance of total qnr ARGs.Furthermore,the content of DOX was significantly correlated with the abundance of total sul,as well as the content TYL with total sul.The abundance of int I was found to be significantly correlated with the total abundance of sul and erm ARGs.