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811000.9862Removal of chlortetracycline and antibiotic resistance genes in soil by earthworms (epigeic Eisenia fetida and endogeic Metaphire guillelmi). The impacts of two ecological earthworms on the removal of chlortetracycline (CTC, 0.5 and 15 mg kg(-1)) and antibiotic resistance genes (ARGs) in soil were explored through the soil column experiments. The findings showed that earthworm could significantly accelerate the degradation of CTC and its metabolites (ECTC) in soil (P < 0.05), with epigeic Eisenia fetida promoting degradation rapidly and endogeic Metaphire guillelmi exhibiting a slightly better elimination effect. Earthworms alleviated the abundances of tetR, tetD, tetPB, tetG, tetA, sul1, TnpA, ttgB and intI1 in soil, with the total relative abundances of ARGs decreasing by 35.0-44.2% in earthworm treatments at the 28th day of cultivation. High throughput sequencing results displayed that the structure of soil bacteria community was modified apparently with earthworm added, and some possible CTC degraders, Aeromonas, Flavobacterium and Luteolibacter, were promoted by two kinds of earthworms. Redundancy analysis demonstrated that the reduction of CTC residues, Actinobacteria, Acidobacteria and Gemmatimonadetes owing to earthworm stimulation was responsible for the removal of ARGs and intI1 in soil. Additionally, intI1 declined obviously in earthworm treatments, which could weaken the risk of horizontal transmission of ARGs. Therefore, earthworm could restore the CTC-contaminated soil via enhancing the removal of CTC, its metabolites and ARGs.202133798888
692110.9860Impacts of Chemical and Organic Fertilizers on the Bacterial Communities, Sulfonamides and Sulfonamide Resistance Genes in Paddy Soil Under Rice-Wheat Rotation. The responses of sulfonamides, sulfonamide-resistance genes (sul) and soil bacterial communities to different fertilization regimes were investigated by performing a field experiment using paddy soil with no fertilizer applied, chemical fertilizer applied, organic fertilizer applied, and combination of chemical and organic fertilizer applied. Applying organic fertilizer increased the bacterial community diversity and affected the bacterial community composition. Eutrophic bacteria (Bacteroidetes, Gemmatimonadetes, and Proteobacteria) were significantly enriched by applying organic fertilizer. It was also found organic fertilizer application increased sulfamethazine content and the relative abundances of sul1 and sul2 in the soil. In contrast, applying chemical fertilizer significantly increased the abundance of Nitrospirae, Parcubacteria, and Verrucomicrobia and caused no obvious changes on sul. Correlation analysis indicated that sul enrichment was associated with the increases in sulfamethazine content and potential hosts (e.g., Novosphingobium and Rhodoplanes) population. The potential ecological risks of antibiotics in paddy soil with organic fertilizer applied cannot be ignored.202236547725
692620.9858Insights into the driving factors of vertical distribution of antibiotic resistance genes in long-term fertilized soils. The prevalence of antibiotic resistance genes (ARGs) in soils has aroused wide attention. However, the influence of long-term fertilization on the distribution of ARGs in different soil layers and its dominant drivers remain largely unknown. In this study, a total of 203 ARGs were analyzed in greenhouse vegetable soils (0-100 cm from a 13-year field experiment applied with different fertilizers (control, chemical fertilizer, organic manure, and mixed fertilizer). Compared with unfertilized and chemically fertilized soils, manure application significantly increased the abundance and alpha diversity of soil ARGs, where the assembly of ARG communities was strongly driven by stochastic processes. The distribution of ARGs was significantly driven by manure application within 60 cm, while it was insignificantly changed in soil below 60 cm under different fertilization regimes. The inter-correlations of ARGs with mobile genetic elements (MGEs) and microbiota were strengthened in manured soil, indicating manure application posed a higher risk for ARGs diffusion in subsurface soil. Bacteria abundance and MGEs directly influenced ARG abundance and composition, whereas soil depth and manure application indirectly influenced ARG abundance and composition by affecting antibiotics. These results strengthen our understanding of the long-term anthropogenic influence on the vertical distribution of soil ARGs and highlight the ecological risk of ARGs in subsurface soil induced by long-term manure application.202337247491
812030.9853Insight into the fate of antibiotic resistance genes and bacterial community in co-composting green tea residues with swine manure. Green tea residues (GTRs) are byproducts of tea production and processing, and this type of agricultural waste retains nutritious components. This study investigated the co-composting of GTRs with swine manure, as well as the effects of GTRs on antibiotic resistance genes (ARGs) and the bacterial community during co-composting. The temperature and C/N ratio indicate compost was mature after processing. The addition of GTRs effectively promoted the reduction in the abundances of most targeted ARGs (tet and sul genes), mobile genetic element (MGE; intI1), and metal resistance genes (MRGs; pcoA and tcrB). Redundancy analysis (RDA) showed that GTRs can reduce the abundance of MRGs and ARGs by reducing the bioavailability of heavy metals. Network analysis shows that Firmicutes and Actinobacteria were the main hosts of ARGs and ARGs, MGEs, and MRGs shared the same potential host bacteria. Adding GTRs during composting may reduce ARGs transmission through horizontal gene transfer (HGT). GTRs affected the bacterial community, thereby influencing the variations in the ARG profiles and reducing the potential risk associated with the compost product.202032310121
700040.9853Animal manures application increases the abundances of antibiotic resistance genes in soil-lettuce system associated with shared bacterial distributions. An increasing amount of animal manures is being used in agriculture, and the effect of animal manures application on the abundance of antibiotics resistance genes (ARGs) in soil-plant system has attracted widespread attention. However, the impacts of animal manures application on the various types of bacterial distribution that occur in soil-lettuce system are unclear. To address this topic, the effects of poultry manure, swine manure or chemical fertilizer application on ARG abundance and the distribution of shared bacteria were investigated in this study. In a lettuce pot experiment, 13 ARGs and 2 MGEs were quantified by qPCR, and bacterial communities in the soil, lettuce endosphere and lettuce phyllosphere were analysed by 16S rRNA sequence analysis. The results showed that the application of poultry or swine manure significantly increased ARG abundance in the soil, a result attributed mainly to increases in the abundances of tetG and tetC. The application of poultry manure, swine manure and chemical fertilizer significantly increased ARG abundance in the lettuce endosphere, and tetG abundance was significantly increased in the poultry and swine manure groups. However, animal manures application did not significantly increase ARG abundance in the lettuce phyllosphere. Flavobacteriaceae, Sphingomonadaceae and 11 other bacterial families were the shared bacteria in the soil, lettuce endosphere, and phyllosphere. The Streptomycetaceae and Methylobacteriaceae were significantly positively correlated with intI1 in both the soil and endosphere. Chemical fertilizer application increased both the proportions of Sphingomonadaceae and tetX abundance, which were positively correlated in the endosphere. Comamonadaceae and Flavobacteriaceae were not detected in the lettuce endosphere under swine manure application. Cu was related to Flavobacteriaceae in the lettuce endosphere. Overall, poultry and swine manure application significantly increased ARG abundance in the soil-lettuce system, which might be due to the shared bacterial distribution.202134004530
811550.9853Effects of reductive soil disinfestation on potential pathogens and antibiotic resistance genes in soil. Reductive soil disinfestation (RSD) is commonly employed for soil remediation in greenhouse cultivation. However, its influence on antibiotic resistance genes (ARGs) in soil remains uncertain. This study investigated the dynamic changes in soil communities, potential bacterial pathogens, and ARG profiles under various organic material treatments during RSD, including distillers' grains, potato peel, peanut vine, and peanut vine combined with charcoal. Results revealed that applying diverse organic materials in RSD significantly altered bacterial community composition and diminished the relative abundance of potential bacterial pathogens (P < 0.05). The relative abundance of high-risk ARGs decreased by 10.7%-30.6% after RSD treatments, the main decreased ARG subtypes were AAC(3)_Via, dfrA1, ErmB, lnuB, aadA. Actinobacteria was the primary host of ARGs and was suppressed by RSD. Soil physicochemical properties, such as total nitrogen, soil pH, total carbon, were crucial factors affecting ARG profiles. Our findings demonstrated that RSD treatment inhibited pathogenic bacteria and could be an option for reducing high-risk ARG proliferation in soil.202539306413
801760.9853Dose-Dependent Effect of Tilmicosin Residues on ermA Rebound Mediated by IntI1 in Pig Manure Compost. The impact of varying antibiotic residue levels on antibiotic resistance gene (ARG) removal during composting is still unclear. This study investigated the impact of different residue levels of tilmicosin (TIM), a common veterinary macrolide antibiotic, on ARG removal during pig manure composting. Three groups were used: the CK group (no TIM), the L group (246.49 ± 22.83 mg/kg TIM), and the H group (529.99 ± 16.15 mg/kg TIM). Composting removed most targeted macrolide resistance genes (MRGs) like ereA, ermC, and ermF (>90% removal), and reduced ermB, ermX, ermQ, acrA, acrB, and mefA (30-70% removal). However, ermA increased in abundance. TIM altered compost community structure, driving succession through a deterministic process. At low doses, TIM reduced MRG-bacteria co-occurrence, with horizontal gene transfer via intI1 being the main cause of ermA rebound. In conclusion, composting reduces many MRG levels in pig manure, but the persistence and rebound of genes like ermA reveal the complex interactions between composting conditions and microbial gene transfer.202541011454
812370.9851The effect of bulk-biochar and nano-biochar amendment on the removal of antibiotic resistance genes in microplastic contaminated soil. Biochar amendment has significant benefits in removing antibiotic resistance genes (ARGs) in the soil. Nevertheless, there is little information on ARGs removal in microplastic contaminated soil. Herein, a 42-day soil microcosm experiment were carried out to study how two coconut shell biochars (bulk- and nano-size) eliminate soil ARGs with/without microplastic presence. The results showed that microplastic increased significantly the numbers and abundances of ARGs in soil at 14d of cultivation. And, two biochars amendment effectively inhibited soil ARGs spread whether or not microplastic was present, especially for nano-biochar which had more effective removal compared to bulk-biochar. However, microplastic weakened soil ARGs removal after applying same biochar. Two biochars removed ARGs through decreasing horizontal gene transfer (HGT) of ARGs, potential host-bacteria abundances, some bacteria crowding the eco-niche of hosts and promoting soil properties. The adverse effect of microplastic on ARGs removal was mainly caused by weakening mobile genetic elements (MGEs) removal, and by changing soil properties. Structural equation modeling (SEM) analysis indicated that biochar's effect on ARGs profile was changed by its size and microplastic presence through altering MGEs abundances. These results highlight that biochar amendment is still an effective method for ARGs removal in microplastic contaminated soil.202437907163
692380.9850Soil types influence the fate of antibiotic-resistant bacteria and antibiotic resistance genes following the land application of sludge composts. Sewage sludge was generally considered a significant reservoir of antibiotic resistance genes (ARGs) and could enter agricultural systems as fertilizer after composting. Soil types and the discrepancy of sludge composts could have influenced the fate of antibiotic-resistant bacteria (ARB) following the land application of sludge composts, which deserved to be clarified. Thus, the fate of ARB and ARGs following the land application of three types of sludge composts (A, B, and C) to three different soils (red soil, loess, and black soil) was investigated. The results showed that tetX, which was enriched the most during composting, did not affect the soil resistome, whereas tetG did. Soil types influenced the dynamics of ARB and ARGs significantly, whereas no significant difference was observed among compost types. The advantage of reducing ARGs during the composting process in compost B did not extend to land application. Land application of composts influenced the microbial community significantly at the early stage, but the microbial community returned to the control pattern gradually. Changes in the microbial community contributed more to the dynamics of ARGs in red and black soil compared with other factors, including co-selection from heavy metals, horizontal gene transfer, biomass and environmental factors, whereas horizontal gene transfer, reflected by intI1 levels, contributed the most in loess.201829793114
691790.9850Response characteristics of antibiotic resistance genes and bacterial communities during agricultural waste composting: Focusing on biogas residue combined with biochar amendments. This research investigated biogas residue and biochar addition on antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and changes in bacterial community during agricultural waste composting. Sequencing technique investigated bacterial community structure and ARGs, MGEs changes. Correlations among physicochemical factors, ARGs, MGEs, and bacterial community structure were determined using redundancy analysis. Results confirmed that biochar and biogas residue amendments effectively lowered the contents of ARGs and MGEs. The main ARGs detected was sul1. Proteobacteria and Firmicutes were the main host bacteria strongly associated with the dissemination of ARGs. The dynamic characteristics of the bacterial community were strongly correlated with pile temperature and pH (P < 0.05). Redundancy and network analysis revealed that nitrate, intI1, and Firmicutes mainly affected the in ARGs changes. Therefore, regulating these key variables would effectively suppress the ARGs spread and risk of compost use.202336657587
8100100.9850Composting temperature directly affects the removal of antibiotic resistance genes and mobile genetic elements in livestock manure. The high antibiotic resistance gene (ARGs) contents in livestock manure pose a potential risk to environment and human health. The heap composting with an ambient temperature and thermophilic composting are two methods for converting livestock manure into fertilizer. This study investigated the variations in ARGs and mobile genetic elements (MGEs) and revealed potential mechanisms for ARGs removal using the two composting methods. The ARGs abundance were enriched by 44-fold in heap composting, among them, the macrolide-resistance genes increased significantly. On the contrary, the ARGs were removed by 92% in thermophilic composting, among them, tetracycline-resistance genes decreased by 97%. The bacterial hosts of ARGs were associated with the variations of ARGs and MGEs. The tetO was correlated with the most diverse bacteria in heap composting, and Bacteroidetes was the major host bacteria. While tetT was correlated with the most diverse bacteria in thermophilic composting, and Proteobacteria was the major host bacteria. Structural equation models showed that the enrichment of ARGs in heap composting was mainly correlated with bacterial communities, whereas, the removal of ARGs in thermophilic composting was directly affect by MGEs. Composting temperature directly affected the variations in ARGs. Higher and lower temperatures significantly decreased and increased, respectively, ARGs and MGEs abundance levels.202235306090
8117110.9850Composting of oxytetracycline fermentation residue in combination with hydrothermal pretreatment for reducing antibiotic resistance genes enrichment. Hydrothermal pretreatment can efficiently remove the residual antibiotics in oxytetracycline fermentation residue (OFR), but its effect on antibiotic resistance genes (ARGs) during composting remains unclear. This study compared the shifts in bacterial community and evolutions in ARGs and integrons during different composting processes of OFRs with and without hydrothermal pretreatment. The results demonstrated that hydrothermal pretreatment increased the bacterial alpha diversity at the initial phase, and increased the relative abundances of Proteobacteria and Actinobacteria but decreased that of Bacteroidetes at the final phase by inactivating mycelia and removing residual oxytetracycline. Composting process inevitably elevated the abundance and relative abundance of ARGs. However, the increase in ARGs was significantly reduced by hydrothermal pretreatment, because the removal of oxytetracycline decreased their potential host bacteria and inhibited their horizontal gene transfer. The results demonstrated that hydrothermal pretreatment is an efficient strategy to reduce the enrichment of ARGs during the OFR composting.202033099099
7992120.9850Impact of bioaccessible pyrene on the abundance of antibiotic resistance genes during Sphingobium sp.- and sophorolipid-enhanced bioremediation in soil. Soils are exposed to various types of chemical contaminants due to anthropogenic activities; however, research on persistent organic pollutants and the existence of antibiotic resistance genes (ARGs) is limited. To our knowledge, the present work for the first time focused on the bioremediation of soil co-contaminated with pyrene and tetracycline/sulfonamide-resistance genes. After 90 days of incubation, the pyrene concentration and the abundance of the four ARGs (tetW, tetM, sulI, and sulII) significantly decreased in different treatment conditions (p<0.05). The greatest pyrene removal (47.8%) and greatest decrease in ARG abundance (from 10(-7) to 10(-8) ARG copies per 16S rRNA copy) were observed in microcosms with a combination of bacterial and sophorolipid treatment. Throughout the incubation, pyrene bioaccessibility constantly declined in the microcosm inoculated with bacteria. However, an increased pyrene bioaccessibility and ARG abundance at day 40 were observed in soil treated with sophorolipid alone. Tenax extraction methods and linear correlation analysis indicated a strong positive relationship between the rapidly desorbing fraction (Fr) of pyrene and ARG abundance. Therefore, we conclude that bioaccessible pyrene rather than total pyrene plays a major role in the maintenance and fluctuation of ARG abundance in the soil.201526164069
8101130.9850Enhanced removal of antibiotic resistance genes and mobile genetic elements during swine manure composting inoculated with mature compost. Livestock manure is a major source of antibiotic resistance genes (ARGs) that enter the environment. This study assessed the effects of inoculation with mature compost (MC) on the fates of ARGs and the bacterial community during swine manure composting. The results showed that MC prolonged the thermophilic period and promoted the decomposition of organic matter, which was due to the rapid growth and reproduction of thermophilic bacteria (Bacillus, Thermobifida, and Thermobacillus). MC significantly reduced the relative abundances of ARGs (1.02 logs) and mobile genetic elements (MGEs) (1.70 logs) after composting, especially sulfanilamide resistance genes. The total ARGs removal rate was 1.11 times higher in MC than the control. Redundancy analysis and structural equation modeling showed that horizontal gene transfer mediated by MGEs (ISCR1 and intI1) was the main direct factor related to the changes in ARGs during composting, whereas the C/N ratio and pH were the two most important indirect factors. Network analysis showed that members of Firmicutes comprising Romboutsia, Clostridisensu_stricto_1, and Terrisporobacter were the main bacterial hosts of ARGs and MGEs. MC reduced the risk of ARGs transmission by decreasing the abundances of bacterial hosts. Thus, MC is a promising strategy for reducing the proliferation risk of ARGs.202133858100
8021140.9849The profile of antibiotic resistance genes in pig manure composting shaped by composting stage: Mesophilic-thermophilic and cooling-maturation stages. The variation of antibiotic resistance genes (ARGs) and influential factors in pig manure composting were investigated by conducting simulated composting tests using four different supplement materials (wheat straw, corn straw, poplar sawdust and spent mushroom). The results show that the relative abundance of total ARGs increased by 0.19-1.61 logs after composting, and tetX, sulI, sulII, dfrA1 and aadA were the major contributors. The variations of ARG profiles and bacterial communities throughout the composting were clearly divided into mesophilic-thermophilic and cooling-maturation stages in all tests, while different supplement materials did not exert a noticeable influence. Network analysis demonstrated the diversity of bacterial hosts for ARGs, the existence of multiple antibiotic resistant bacteria, and the weak correlations between ARGs and physicochemical factors in the composting piles. Of note, integron intI1 and Mycobacterium (a potential pathogen) were positively correlated with eight and four ARGs, respectively, that displayed increased abundance after composting.202032109697
7538150.9849Short-term thermophilic treatment cannot remove tetracycline resistance genes in pig manures but exhibits controlling effects on their accumulation and spread in soil. In this work, a microcosm experiment was conducted to merely mimic thermophilic phase in aerobic composting with pig manures in order to explore: (i) the effect of thermophilic phase in composting on the abundances of tetracycline resistance genes (TRGs); and (ii) the impacts of the treated manures on the abundances of TRGs in soil. It was found that 4days of thermophilic process reduced the abundance of TRGs in pig manures by ∼1 lg unit compared to the samples without treatments, suggesting that other phases in composting may play significant roles in removal of TRGs. Once pig manures with thermophilic treatment were applied to soil, TRGs abundances decreased to the levels in unfertilized soil. With correlation analyses, it was concluded that pig manure derived tetracycline-resistant bacteria (TRB) and nutrients exerted different effects on TRGs abundances in soil. In conclusion, short-term thermophilic treatment cannot remove tetracycline resistance genes in pig manures but exhibits controlling effects on their accumulation and spread in soil. Nutrients enrichment in soil following manuring of treated pig manures, together with a large proportion of gram-positive TRB left in treated pig manures with less risk to TRGs spread, contributed to the controlling effects.201728715744
8098160.9848Elimination of antibiotic resistance genes and human pathogenic bacteria by earthworms during vermicomposting of dewatered sludge by metagenomic analysis. This study used a metagenomic approach to investigate the effects of earthworms on ARGs and HPB during the vermicomposting of dewatered sludge. Results showed that 139 types of ARGs were found in sludge vermicompost, affiliated to 30 classes. Compared with the control, the total abundance of ARGs in sludge vermicompost decreased by 41.5%. Moreover, the types and sequences of plasmids and integrons were also decreased by vermicomposting. Proteobacteria and Actinobacteria were the most dominant hosts of ARGs in sludge vermicompost. In addition, earthworms reduced the total HPB abundance and modified their diversity, thus leading to higher abundance of Enterobacteriaceae in sludge vermicompost. However, the sludge vermicompost was still ARG and HPB enriched, indicating a remaining environmental risk for agricultural purpose. The observed change of microbial community and the reduction of mobile genetic elements caused by earthworm activity are the main reasons for the alleviation of ARG pollution during vermicomposting.202031787516
6999170.9848Different impacts of manure and chemical fertilizers on bacterial community structure and antibiotic resistance genes in arable soils. Both manure and chemical fertilizers are widely used in modern agriculture. However, the impacts of different fertilizers on bacterial community structure and antibiotic resistance genes (ARGs) in arable soils still remain unclear. In this study, high-throughput sequencing and quantitative PCR were employed to investigate the bacterial community structure, ARGs and mobile genetic elements (MGEs) influenced by the application of different fertilizers, including chemical fertilizers, piggery manure and straw ash. The results showed that the application of fertilizers could significantly change the soil bacterial community and the abundance of Gaiella under phylum Actinobacteria was significantly reduced from 12.9% in unfertilized soil to 4.1%-7.4% in fertilized soil (P < 0.05). It was also found that the application of manure could cause a transient effect on soil resistome composition and the relative abundance of ARGs increased from 7.37 ppm to 32.10 ppm. The abundance of aminoglycoside, sulfonamide and tetracycline resistance genes greatly increased after manure fertilization and then gradually returned to normal levels with the decay of some intestinal bacteria carrying ARGs. In contrast, the application of chemical fertilizers and straw ash significantly changed the bacterial community structure but exerted little effect on soil resistome. Overall, the results of this study illustrated the different effects of different fertilizers on the soil resistome and revealed that the changes of soil resistome induced by manure application mainly resulted from alteration of bacteria community rather than the horizontal gene transfer.201728898777
6927180.9847Manure application: A trigger for vertical accumulation of antibiotic resistance genes in cropland soils. The application of livestock manure increases the dissemination risk of antibiotic resistance genes (ARGs) in farmland soil environment. However, the vertical migration behavior and driving factor of ARGs in manured soil under swine manure application remains undefined. Here, the dynamics of ARGs, mobile genetic elements (MGEs) and bacterial communities in different soil depths (0 - 80 cm) with long-term swine manure application were tracked and conducted using real-time qPCR. Results showed that long-term application of swine manure remarkably facilitated the vertical accumulation of ARGs and MGEs, in particular that the relative abundance of bla(ampC) showed significant enrichment with increasing depth. ARGs abundance was similar in the three fields with long-term application of swine manure. (p＞0.05). Procrustes analysis indicated that microbial communities were the dominant drivers of ARGs variation in topsoil, and the changes of environmental factors played a vital role in vertical migration ARGs in cropland soils. Additionally, the variation patterns of high-risk ARGs (i.e., bla(ampC), bla(TEM-1)) were influenced by the dominant bacteria (Actinomycetes) and pH. This study illustrated that the swine manure application promoted the vertical migration of ARGs, including multidrug resistance determinants, highlighting the ecological risk caused by long-term manure application.202235483148
7535190.9847The effects of pig manure application on the spread of tetracycline resistance in bulk and cucumber rhizosphere soils: a greenhouse experiment. It is important to understand the dynamics of tetracycline-resistant bacteria (TRB) and tetracycline resistance genes (TRGs) in bulk and rhizosphere soils for evaluating the spread of TRGs from pig manure to human. In this work, a greenhouse experiment was conducted to investigate the difference in abundance of TRB, tetracycline-resistant Escherichia coli (TRE), tetracycline-resistant Pseudomonas spp. (TRP), and TRGs between bulk and cucumber rhizosphere soils. The application of pig manure resulted in the long-term persistence of TRB, TRE, TRP, and TRGs in bulk soil and rhizosphere of cucumber for at least 65 days. Pig manure application dose was the major driving force in altering the abundances of TRB and TRE, whereas TRP was disturbed mainly by compartment (bulk soil or rhizosphere). Both TRE and the percentage of TRE in bulk and rhizosphere soils increased linearly with an increase in dose of pig manure. The exponential relationships between pig manure dose and TRP along with TRP percentage were also noted. There were significant differences in the relative abundances of TRGs between bulk and cucumber rhizosphere soils, suggesting the use of pig manure exerted a more lasting impact on the spread of TRGs in the rhizosphere than in the bulk soil.201728222270