Spread And Transfer Of Antibiotics And Antibiotic Resistance In The Livestock Manure-soil-vegetable Endophytic System

Veterinary antibiotics(VAs)have been widely used to treat animal diseases and promote animal growth in livestock production because of their positive effects.Many studies have demonstrated that antibiotics,antibiotic-resistant bacteria(ARB)and antibiotic resistance genes(ARGs)were usually excreted via animal manures and spread into soil with the land application of manure.Some environmental and public health risks are highly concerned such as the pollution of antibiotic residues,development of ARB,spread and diffusion of ARGs.Compared to antibiotics,ARGs are more persistent and recalcitrant in environemnts,easier to be transferred through horizontal gene transfer processes,which result in large production of multiple antibiotic resistance(MAR)and/or even development into “super drug-resistant bacteria” seriously threatening human health.Therefore,ARGs have been considered as a kind of pollutants.As an important reservoir of ARB and ARGs,animal manure could lead to a significant increase of antibiotic resistance in soil environment through manure fertilization process.However,little information is available about the spread and transfer of antibiotic resistance from manure to plant endophytic systems during crops planting.In the present study,the concentrations of antibiotics and ARGs in animal manure and fertilized soil,the dynamic processes of antibiotic resistance during simple composting,and the changes of antibiotic resistance in vegetable endophytic systems under antibiotic or multiple antibiotic-resistant bacteria(MRB)exposure were investigated in detail by using the techniques of real-time fluorescence quantitative PCR(qPCR),functional gene array,plate enumeration and high-throughput sequencing,etc.The innovative results are shown as follows:1.Distribution of antibiotics and ARGs in animal manure and fertilized soilTo evaluate the potential risks of antibiotics pollution in agricultural systems,the distribution of antibiotics and ARGs in animal manures and manure-fertilized agricultural soils were studied by using ASE-UPLC/MS detection techniques,qPCR and a high-throughput functional gene array(GeoChip 5.0).Several antibiotics were simultaneously present in manure or fertilized soil samples except the non manure-amended soil.While there were more ARGs identified in soil than manure samples by GeoChip.Especially abundant ARGs were detected in the soil sample without manure-amended.qPCR detection indicated a higher ARGs abundance in manure than soil samples.2.Dynamic changes in antibiotic resistance during simple composting of animal manureThe behaviors of fourteen antibiotics,fifteen types of ARGs,and MRB were monitored in chicken and swine manure under simple composting conditions.As shown in the results,most drugs were degraded at the end of the 60-day compost,while some individual antibiotics were still detected due to their relatively high initial concentrations and stabilities.As determined by selective plate counting,the counts of cultivated aerobic heterotrophic MRB decreased two orders of magnitude,but the proportions of MRB to total cultivable heterotrophic bacteria increased from 0.35% to 1.8% in chicken manure and from 0.08% to 0.45% in swine manure after composting.Among the fifteen ARGs quantified by qPCR,the relative abundances of most ARGs generally decreased one or two orders of magnitude after 60 days of the composting treatment.However,the genes tetA,tetM and sul2 in chicken manure and sul1,qnrD,aacC2 and mefA in swine manure increased to 2–100 times of their initial levels by the end of the compost.3.Influence of antibiotic exposure on antibiotic resistance in vegetable endophytic systemsA series of hydroponic pakchoi experiments were conducted to explore plant growth,antibiotic uptake,and the development of antibiotic resistance in endophytic systems under the exposure of tetracycline,cephalexin,and sulfamethoxazole at 50% minimum inhibitory concentration(MIC)levels and MIC levels,respectively.The three types of antibiotics promoted pakchoi growth at 50% MIC values and the target antibiotics were absorbed by the treated vegetables.Additionally,the rates of antibiotic-resistant endophytic bacteria(AREB)to total cultivable endophytic bacteria(TCEB)significantly increased with the antibiotics accumulation in the plants.The detection and quantification of ARGs indicated that four types,tetX,blaCTX,and sul1 and sul2,which correspond to tetracycline,cephalexin,and sulfamethoxazole resistance,respectively,were present in the pakchoi endophytic systems and their concentrations increased with the exposed antibiotic concentrations.4.Transfer and colonization of manure-sourced MRB and ARGs in vegetable endophytic systems through soil and plant rhizosphereTo investigate whether the manure–sourced MRB and ARGs could be transferred and even colonized in the vegetable endophytic systems,pot experiments were performed with pakchoi or lettuce cultivation with the following treatments:(1)non-treated,(2)chicken manure-treated,(3)MRB-treated and(4)organic fertilizer-treated.Manure or MRB significantly increased the rates of multiple antibiotic-resistant endophytic bacteria(MREB)to TCEB in vegetable.Further,16 S rRNA gene sequencing and the community analysis indicated that Pseudomonas sp.and Alcaligenes sp.from manure could enter and colonize in the vegetable tissues through MBR amendment.Manure and MRB application increased the abundance of ARGs in endophytic bacteria.The fact that some bacteria with multiple antibiotic resistance were detected in harvested vegetables after growing in MRB-amended soil,which implied a potential threat to human health through eating the edible parts of vegetables.