| With the extensive use of antibiotics,aquaculture environment and livestock manure have a large number of antibiotics residues and antibiotics resistant bacteria and antibiotics resistance genes（ARGs）.Manure is recycled into agricultural production,which may bring antibiotics resistant bacteria and ARGs to soil.antibiotics resistant bacteria and ARGs are harmful to human health,becoming environment and food problems.As one of store environment and transmission mediums of antibiotics resistant bacteria and ARGs,both manure and soil play an important role.Vegetables are the basic food for humans,and the antibiotics resistant bacteria and ARGs may affect the human health and safety through food chain.Non-pathogenic bacteria are the receptor,donor and storage intermediate of resistance gene.Due to its large number and accounts for the majority of the microbial ecosystem,that is easier to occur the HGT event,non-pathogenic bacteria play an important role in the migration of ARGs.Therefore,it is necessary to study the effect of organic fertilizer on non-pathogenic resistant bacterial in soil and vegetables,and the risk of ARGs transmission,which can provide a scientific basis for the formulation of control strategies for antibiotic resistant bacteria in agricultural environment and agricultural products.This study mainly focused on soil and leafy lettuce.potting experiment,colony culture,plate count method were used to analyze the effect of organic fertilizer on levels of antibiotics resistant bacteria and multi-resistant bacteria,which were resistant to six antibiotics including cefotaxime（CTX）,erythromycin（ERM）,sulfamethoxazole（SUL）,tetracycline（TET）,doxycycline（DOX）and ciprofloxacin（CIP）respectively in soil and lettuces samples.The gene pool was constructed by gene cloning,further through the resistance gene screening and sequencing,to study the effect of organic fertilizer on integrated enzyme gene intI and 9 kinds of detection of ARGs,combination type of ARGs,type of bacteria with multi-antibiotics resistance genes.The potential level of ARGs was analyzed by plasmid extraction and polymerase chain reaction（PCR）.The results showed that:（1）Organic fertilizer could increase the number of non-pathogenic antibiotics resistant bacteria（CTr,SULr,ERMr,TETr,CIPr）in soil,DOXr was not detected both in CK and OMT soil.CTXr was the most and the number was 108 CFU·g-1.DOXr was the least and the number was 105 CFU·g-1in the absolute number of 6 kinds of antibiotics resistant bacteria after 15 d,30 d and 45 d.The divergence of CTXr,SULr,ERMr,TETr and CIPr decreased gradually between CK and OMT with the planting time.CTXr,TETr,CIPr and DOXr were significant difference at 15 d and 30 d between CK and OMT;ERMr was significant difference at 30 d between CK and OMT;and the number of antibiotics resistant bacteria was no significant difference between CK and OMT at 45 d,except for CIPr.After fertilization,the proportion of multi-antibiotics resistant bacteria increased at different time points.（2）Organic fertilizer could increase the number of non-pathogenic antibiotics resistant bacteria in lettuce.In addition to DOXr,other non-pathogenic antibiotics resistant bacteria involved in this study were prevalent in lettuce.Fertilization increased the number of non-pathogenic antibiotics resistant bacteria in lettuce except for TETr and CTXr.The difference of SULr between CK and OMT were increased;and TETr and ERMr were decreased;ERMr had no varieties;CIPr was increased,then decreased.TETr had significant difference between CK and OMT at 15 d and 30 d,and highy significant difference at 15 d.CTXr had highy significant difference at 15 d.Others were no significant difference between CK and OMT at 15 d and 30 d.At 45 d of lettuce plant,there was no significant difference.The effect of organic fertilizer on the resistance of multi-antibiotics resistant bacteria in lettuce was not much obvious,but on the other side,even if it was not fertilized,there also existed multiple drug-resistant bacteria in lettuce.（3）IntI was common in soil and lettuce and was not related to fertilizer.Applying organic manure increased the detection rate of intⅠ,tet and sul genes in soil and lettuce.Different kinds of tet genes were different and there was no obvious law.The detection rate of sul genes showed sull>sul2.（4）After applying organic manure in soil,the detection rate of tetC in tet genes was significantly increased,followed by tetG,tetB,tetE,tetA and tetM,tetL remained little changed.The difference was significant at 30 d,and the detection rate was almost same at 45 d.（5）After applying organic manure in lettuce,the detection rate of tetG in tet genes was significantly increased,followed by tetM and tetB.The tetL and tetA in OMT were less than those in CK and tetC was not checked out.With the increase of planting time,the effect of fertilization on tet genes in lettuce decreased,even reduced the detection rate of tet genes,and had significant effect on sul.（6）Application of organic fertilizer could incease the detection rate of multiple antibiotic resistance genes,the detection rate of intⅠ in soil and lettuce.（7）Application of organic fertilizer could incease the composite type of antibiotics resistant bacteria containing multiple antibiotic resistance genes in soil and lettuce,changed the combination type and abundance of multi-antibiotic resistance genes in lettuce.The multi-antibiotics resistant strains in soil were mainly Pseudomonas sp.,brevibacterium sp.and Arthrobacter sp.both in CK and OMT.The multi-antibiotic resistant strains in lettuce were mainly Pseudomonas sp.and brevibacterium sp.both in CK and OMT,and the introduced strains were mainly Arthrobacter sp..（8）The intⅠ gene and sul,tet resistance gene were detected in plasmids in the selected non-pathogenic multi-antibiotics resistant bacteria.The detection rate of intⅠin soil and lettuce was 100%.Therefore,the resistance genes in soil and lettuce might have a risk of horizontal transmission,which also required further study of the characteristics of these plasmids. |
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