Disseminations Of Antibiotic Resistant Bacteria And Antibiotic Resistance Genes In Soil-plant System

Over the past decades,the world has increased antimicrobial resistance(AMR)in diverse environments due to the overuse and misuse of antibiotics for human therapy and livestock production.Understanding the distribution and dissemination of antibiotic resistant bacteria(ARB)and antibiotic resistance gene(ARG)is necessary to control and solve the problem of AMR.With the introduction of the One Health concept(i.e.ARG transmission across environment-animal-human),recent studies not only focus on the spread of ARB and ARG in sole media,but also pay more attention to the ARB and ARG transmission across the barrier of ecological niches.The soilplant system is very important,because it is a hub which connects the natural ecological environment,animal and human health.Understanding the mechanism of ARB and ARGs spread in the soil-plant system is the basis for effective control of the AMR in environment,animal and human.Although there have been many studies on soil resistome,the impact on soil indigenous bacterial community caused by the spread of foreign ARB is still unclear.Contrast to extensive studies on antibiotic resistome in soil,water,and air,only limited examples are available for plant resistome,and most of them only focus on the ARGs distribution.The direct evidence that ARB and ARG transmission in the soil-plant system is still missing.Moreover,the main source of plant resistome is also poor understood.Herein,this study conducts systematic research on the mechanism of ARB and ARG spread in soil-plant system based on bioinformatics approaches and laboratory techniques.First,we explored the abundance changes of foreign ARB spreading to the soil,and also assessed the impacts of soil indigenous bacterial community caused by foreign ARB.Then,we provided the direct evidence that ARB and their ARG could spread in the soil-plant system.Finally,we revealed the relative contribution of the inoculum source to the plant microbiota and resistome.This study would help us understand the transmission of ARB and their ARGs in soi-plant system and provide an important theoretical basis for controlling the spread of AMR in the environment.According to the possible spread direction of foreign ARB and ARG after entering the soil-plant system,this study first studied the abundance changes of foreign ARB introduced into the soil,and also comprehensively evaluated its impact on the structure and function of soil indigenous bacterial community,as well as the effect of antibiotic pollution on these impacts.Herein,using a microcosm experiment we studied the abundance changes of a model ARB(multidrug-resistant Escherichia coli)and soil bacterial community composition and function(presented with niche structure and niche breadth)in the response to the model ARB amendment in the absence and presence of tetracycline contamination.Results demonstrated that the abundance of foreign ARB decreased significantly regardless of tetracycline.The ARB amendment increased the diversity and niche breadth and altered the composition and niche structure of the soil bacterial community.Tetracycline contamination further enhanced these impacts probably via increasing the survival of foreign ARB in soil.Interestingly,the ARB-induced changes in the bacterial community composition and function were synchronized,which might be driven by the substantial changes in some core taxa(Proteobacteria,Bacteroidetes,Chloroflexi,and Patescibacteria).Furthermore,the impacts of the foreign ARB on soil bacterial community lasted longer than the survival of foreign ARB in tetracycline-uncontaminated and low contaminated soils,demonstrating that the amendment of foreign ARB into soil likely challenges the stability of the soil bacterial community in a relatively long period.After foreign ARB are introduced into the soil,they can survive for some time despite a significant decline in abundance.So,foreign ARB and their ARG may pose the risk to spread to plants growing in the soil.In order to provide the direct evidence that the ARB and their ARG could disseminate into plants interior,a simple hydroponic system was further constructed,in which E.coli with dual fluorescence labeling and RP4 plasmids was used as the representative of drug-resistant bacteria.Aimed to explore the influence of soil bacteria on this process,this study also sat up an experimental group with soil bacteria amendment and a control group without soil bacteria.Quantitative PCR combined with confocal laser scanning microscopy(CLSM)were used to visually characterize the distribution of ARB and their ARG in the hydroponic solution and the inside and outside of the plant.At the same time,by fluorescence-activated cytometry sorting(FACS)combined with 16 s high-throughput sequencing,the composition of the bacterial community obtained ARG through horizontal gene transfer(HGT)in the hydroponic system was analyzed.The results showed that exogenous multi-drug resistant E.coli could migrate from the hydroponic solution to the inside of the plant,and transfer the RP4 plasmid to the plant endophytic bacteria via HGT.The presence of soil bacteria can inhibit the colonization of exogenous multidrug-resistant E.coli in plants,however,it surprisingly promoted the carrying RP4 plasmid to spread to plant endophytic bacteria.In the presence of soil bacteria,the structure of plant bacterial community carrying the RP4 plasmid was changed significantly,and the number of core bacteria carrying the RP4 plasmid shared between the interior and exterior of the plant was also increased.These results indicated that soil bacteria,especially the soil Proteobacteria,might first obtain the RP4 plasmid from the multidrug-resistant E.coli,and then translocate into plant tissues,promoting the spread of RP4 plasmid in plant endophytic bacteria.It is worth noting that the exogenous multidrug-resistant E.coli might spread the their ARG to human and plant pathogenic bacteria in plants via HGT.Then,this study further analyzed the main environmental sources of plant resistome and their transmission routes.To this end,we collected lettuce and organic fertilizer,air,soil and irrigation water,and conducted metagenomic sequencing and data analysis.Comparing with the SARG database for ARG annotation,the distribution characteristics of ARGs in different parts of lettuce,as well as organic fertilizer,air,soil and irrigation water were revealed.Combining metaphlan2 to analyze the bacterial community structure of different parts of lettuce,organic fertilizer,air,soil and irrigation water.FEAST analysis was used to analyze the main sources of drug-resistant genes and microorganisms in different parts of plants.Procrustes test based on BrayCurtis distance explored the relationship between ARGs and bacterial community.Finally,MEGAHIT(v 1.2.9)was used to assemble the metagenomic sequence,Prodigal(v 2.6.3)was used to predict the ORF in the metagenomic sequence.Diamond,SARG and ICEberg databases were used to annotate the host of ARGs combined with Mobile genetic element(MGE)at the species level.Results showed that the detected number and abundance of overall ARGs from lettuce phyllosphere was the largest,and multidrug resistance genes were the predominant ARG types in the lettuce(regardless of roots and leaves).Irrigation water and soil are the main sources of lettuce resistome.The ARGs of irrigation water mainly spread from water to leaf endosphere through phyllosphere,then transferred from leaf endosphere to root endosphere via plant tissuse.The ARGs of soil might mainly spread from soil to root endosphere via rhizosphere,then transferred from root endosphere to leaf endosphere by plant tissuse.The source distribution of ARGs lettuce was not consistent with the bacterial community,which might be due to higher tendency of ARG-HGT in the plant microbiome.Pseudomonas was the main host of ARGs that co-occurred with MGEs(i.e.,ARGs flanked with at least one MGE on the contig).These results revealed the main external environmental sources and propagation paths of plant resistome,which could provide a theoretical basis for controlling the spread of plant resistome and protect human health and environment.