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The information on the effects of ZnONPs on soil biological processes and the resulting changes of ecosystem function is limited in wheat straw return agricultural soil. This study investigated the effects of ZnONPs on soil heterotrophic respiration and biochemical properties in wheat straw return farmland soil. An 84-day microcosm incubation experiment was implemented after the exposure of different doses of ZnONPs (0, 100, and 500 mg kg−1 dry soil) in a greenhouse under the temperature of 25 ± 3 °C. Soil heterotrophic respiration and soil enzyme activities were detected. Microbial community composition and diversity were analyzed by Illumina MiSeq platform. Heterotrophic respiration was significantly inhibited by ZnONPs at 500 mg kg−1. The activities of β-glucosidase, cellulose, and cellobiohydrolase decreased under high-level exposure of ZnONPs. The labile Zn2+ assimilated by soil microorganism was increased by 331% and 707%, respectively. Bacterial communities in the soil are more sensitive to ZnONPs than fungal ones. The changes of heterotrophic respiration were closely correlated with soil pH values and microbial compositions, especially fungal communities. Although ascomycetes were inhibited by ZnONPs, their abundance was the highest within soil fungal community. The inhibition of heterotrophic respiration was associated with the variation of soil fungal communities, although the bacterial communities are sensitive to ZnONPs. The microbiome modulation is necessary for the management of the decomposition of returned wheat straw in the field contaminated by ZnONPs. The effects of ZnONPs on soil ecological process should be considered when the application of fertilizers and pesticides which contain ZnONPs.

The increasing and simultaneous pollution of plastic debris and antibiotic resistance in aquatic environments makes plastisphere a great health concern. However, the development process of antibiotic resistome in the plastisphere is largely unknown, impeding risk assessment associated with plastics. Here, we profiled the temporal dynamics of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and microbial composition in the plastisphere from initial microbial colonization to biofilm formation in urban water. A total of 82 ARGs, 12 MGEs, and 63 bacterial pathogens were detected in the plastisphere and categorized as the pioneering, intermediate, and persistent ones. The high number of five MGEs and six ARGs persistently detected in the whole microbial colonization process was regarded as a major concern because of their potential role in disseminating antibiotic resistance. In addition to genomic analysis, D2O-labeled single-cell Raman spectroscopy was employed to interrogate the ecophysiology of plastisphere in a culture-independent way and demonstrated that the plastisphere was inherently more tolerant to antibiotics than bacterioplankton. Finally, by combining persistent MGEs, intensified colonization of pathogenic bacteria, increased tolerance to antibiotic, and potential trophic transfer into a holistic risk analysis, the plastisphere was indicated to constitute a hot spot to acquire and spread antibiotic resistance and impose a long-term risk to ecosystems and human health. These findings provide important insights into the antibiotic resistome and ecological risk of the plastisphere and highlight the necessity for comprehensive surveillance of plastisphere.

Protists, an integral component of soil microbiome, are one of the main predators of bacteria. Bacteria can produce toxic secondary metabolites, e.g., antibiotics to fight stress under the predation pressure of protists; however, impacts of antibiotics on the profile of protists in soils remain unclear. Here, we constructed a microcosm incubation to investigate the effects of two common antibiotics, oxytetracycline and ciprofloxacin, on the protistan and bacterial communities in an arable soil. Rhizaria were the most abundant protist supergroup, followed by Amoebozoa, Stramenopiles, and Aveolata. Among trophic functional groups, consumers were predominant within the protistan community. The protistan alpha-diversity was not significantly changed, while the bacterial alpha-diversity was decreased under the pressure of antibiotics. Nevertheless, the antibiotic exposure considerably reduced the relative abundance of protistan lineages in Rhizaria and Amoebozoa, which were the dominant supergroups of protistan consumers, while increased the relative abundance of other consumer and phototrophic protists. Altogether, we provide novel experimental evidence that the bacterivorous consumers, an important functional group of protists, were more sensitive to antibiotics than other functional groups. Our findings have potential implications for the induced alterations of protistan community and their ecological functions under the scenarios of projected increasing global antibiotic usage.

Tread particles (TPs) from vehicle tires are widely distributed in soil ecosystems; therefore, there is an urgent need to evaluate their effects on soil biota. In the present study, the soil worm Enchytraeus crypticus was incubated for 21 days in soil microcosms containing increasing concentrations of TPs (0, 0.0048%, 0.024%, 0.12%, 0.6%, and 3% of dry soil weight). High concentrations of zinc (Zn, 9407.4 mg kg-1) and polycyclic aromatic hydrocarbons (PAHs, 46.8 mg kg-1) were detected in the TPs, which resulted in their increased concentrations in soils amended with TPs. We demonstrated that TPs had an adverse effect on the survival (decreased by more than 25%) and reproduction (decreased by more than 50%) of the soil worms. Moreover, TP exposure disturbed the microbiota of the worm guts and surrounding soil. In addition, a covariation between bacterial and fungal communities was observed in the worm guts after exposure to TPs. Further analysis showed that TP exposure caused an enrichment of microbial genera associated with opportunistic pathogenesis in the worm guts. The combined results from this study indicate that TPs might threaten the terrestrial ecosystem by affecting soil fauna and their gut microbiota.

The discovery of comammox Nitrospira being capable of complete oxidising ammonia to nitrate radically challenged the conventional concept of two-step nitrification. However, the response of comammox Nitrospira to nitrification inhibitors (NIs) and their role in soil nitrification remain largely unknown, which has hindered our ability to predict the efficiency of NIs in agroecosystems. We evaluated the effect of four NIs, 2-chloro-6-(trichloromethyl) pyridine (nitrapyrin), 3,4-dimethylpyrazole phosphate (DMPP), allylthiourea (ATU) and dicyandiamide (DCD) on the growth of comammox Nitrospira, ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in two pasture and arable soils. The amendment of nitrogen fertiliser significantly increased soil nitrate concentrations over time, indicating a sustaining nitrification activity in both soils. The addition of all the four NIs effectively reduced the production of nitrate in both soils, but to varying degrees during incubation. The abundances of comammox Nitrospira clade A were significantly increased by addition of nitrogen fertilisers and significantly impeded by the four NIs in the pasture soil, but their abundances were only remarkably hindered by nitrapyrin in the arable soil. All the four NIs obviously inhibited the AOB abundances in both soils. Except for DMPP, the other three NIs effectively suppressed the AOA abundances in both soils. We provided new evidence that growth of comammox Nitrospira clade A can be stimulated by nitrogen fertilisers and inhibited by various nitrification inhibitors, suggesting their potential role in nitrification of agricultural soils.

The importance of diet in regulating the gut microbiome of globally distributed and functionally important soil generalist invertebrates such as collembolans remain poorly understood. Here, we studied a model collembolan (Folsomia candida) and found that diet (bacteria, plant litters, yeast, mixed food) is a critical factor in regulating the microbial diversity and community composition of this important soil organism. Collembolans fed with litter exhibited the lowest bacterial diversity and were dominated by Ochrobactrum. Conversely, collembolans fed with mixed diets resulted in the highest bacterial diversity. Our findings further suggest that microbial communities associated with different diets are linked to different levels of collembolan fitness. For example, the relative abundance of the genera of unclassified Thermogemmatisporaceae, Brevibacillus, and Novosphingobium were positively correlated with growth of the collembolans. Together, our work provides evidence that diet is a major force controlling the gut microbiome of collembolans, and is a good environmental predictor for collembolan growth, with implications for ecosystem functioning in terrestrial environments.

Growing evidences have demonstrated that heavy metal contamination can promote the proliferation of antibiotic resistance genes (ARGs) via co-selection. However, effects of nano-metal-materials on the occurrence and level of ARGs in the soil and plant, have not been fully explored. To gain insights into this impact, we conducted a pot experiment by adding nano‑silver particles (AgNPs) as a stimuli and Ag ion (AgNO3) and tetracycline as a comparison. By using high throughput quantitative PCR, our results indicated that application of AgNPs (~20 nm and ~50 nm) at a concentration of 100 ppm resulted in no significant changes in the abundance of ARGs in either soil or phyllosphere (P > 0.05). Nevertheless, the overall pattern of resistome, especially in soil, was shifted following AgNPs application, with a significance increase in the relative abundance of efflux pumps genes, which is an important mechanism for co-selection of ARGs by heavy metals. By comparison, Ag ion at an equivalent Ag mass of AgNPs markedly increased ARGs abundance and shifted ARGs profile in soil, indicating that free Ag ion had a stronger impact on ARGs than AgNPs. These findings provide new insights in assessing the risks of manufactured nanomaterials accumulated in the environment. Keywords: Nano-materials, Silver nanoparticles, Antibiotic resistance, Co-selection, Plant microbiome

Loss of biodiversity is a major threat to the ecosystem processes upon which society depends. Natural ecosystems differ in their resistance to invasion by alien species, and this resistance can depend on the diversity in the system. Little is known, however, about the barriers that microbial diversity provides against microbial invasion. The increasing prevalence of antibiotic-resistant bacteria is a serious threat to public health in the 21st century. We explored the consequences of the reduction in soil microbial diversity for the dissemination of antibiotic resistance. The relationship between this diversity and the invasion of antibiotic resistance was investigated using a dilution-to-extinction approach coupled with high-capacity quantitative PCR. Microbial diversity was negatively correlated with the abundance of antibiotic-resistance genes, and this correlation was maintained after accounting for other potential drivers such as incubation time and microbial abundance. Our results demonstrate that high microbial diversity can act as a biological barrier resist the spread of antibiotic resistance. These results fill a critical gap in our understanding of the role of soil microbial diversity in the health of ecosystems.

Growing evidence points to the pivotal role of the environmental factors in influencing the transmission of antibiotic resistance genes (ARGs) and the propagation of resistant human pathogens. However, our understanding of the ecological and evolutionary environmental factors that contribute to development and dissemination of antibiotic resistance is lacking. Here, we profiled a wide variety of ARGs using the high-throughput quantitative PCR analysis in 61 soil samples collected from ocean and river beaches, which are hotspots for human activities and platforms for potential transmission of environmental ARGs to human pathogens. We identified the dominant abiotic and biotic factors influencing the diversity, abundance and composition of ARGs in these ecosystems. A total of 110 ARGs conferring resistance to eight major categories of antibiotics were detected. The core resistome was mainly affiliated into β-lactam and multidrug resistance, accounting for 66.9% of the total abundance of ARGs. The oprJ gene conferring resistance to multidrug was the most widespread ARG subtype detected in all the samples. The relative abundances of total ARGs and core resistome were significantly correlated with salinity-related properties including electrical conductivity and concentrations of sodium and chloride. Random forest analysis and structural equation modelling revealed that salinity was the most important factor modulating the distribution patterns of beach soil ARGs after accounting for multiple drivers. These findings suggest that beach soil is a rich reservoir of ARGs and that salinity is a predominant factor shaping the distribution patterns of soil resistome.

Antibiotic resistance genes (ARGs) have emerged as a global health concern. A large volume of work has already been devoted to ARGs in aquatic ecosystems. However, ARG dispersal patterns in air remain to be largely unknown despite of its greater role in transmission. This work aims to investigate time-resolved airborne spread of ARGs and their corresponding subtype bacterial carriers in highly polluted air. Time-resolved air samples (20 m3 each with three samples) were collected using a high volume sampler (1 m3/min) every 4 h continuously (both day and night) during low (14–93 μg/m3) and high PM2.5 (36–205 μg/m3) pollution times (over 6 days with a total of 69 air samples) in Beijing. All air samples were subjected to 16S rRNA sequence analysis for 39 ARG subtypes. Pure culturable bacterial isolates from Beijing and Shijiazhuang were Sanger sequenced for species identification and also subjected to high throughput ARG subtype detection. ARG and its subtype relative abundances in the air were observed to differ greatly (up to 3 folds for abundance) both day and night, and the blaTEM gene was found to lead the ARG abundance. For an early morning time, the multi-drug resistant NDM-1 gene was detected up to 30% of total ARG abundance in highly polluted air. Identified as a major NDM-1 and vanB gene carrier, Bacillus halotolerans were also shown to disseminate more ARG subtypes. On another front, tnpA and intI1 were shown to vary greatly in abundance, while the sul3 gene was found widespread among the culturable Bacillus isolates in the air. Principal component analysis (PCA) showed different gene co-occurrence networks for different PM2.5 pollution episodes, e.g., tnpA and intI1 for gene transfer and integration, respectively, were found more abundant for the high PM2.5 pollution episode. This study highlights a serious yet previously unidentified public health threat from time-resolved airborne spread of ARGs. Further work is urgently warranted to track the sources of ARGs for their optimized control during high PM2.5 pollution episodes. Keywords: Antibiotic resistance genes, Particulate matter, Air pollution, Time-resolved ARGs

Liming is a common agronomic practice used for alleviating soil acidification to improve plant growth. However, it is still unclear how liming can affect the gut microbiota composition of soil fauna, and subsequently the nutrient cycling and litter decomposition mediated by soil fauna. In the present study the effect of liming on the gut microbiota of two types of soil fauna, Folsomia candida, and Enchytraeus crypticus was investigated by using 16S rRNA gene high-throughput sequencing. The results revealed that there are differences between the gut microbial communities of the two types of soil fauna as well as between the gut microbiome of the soil fauna and the surrounding soil. Enterobacteriaceae and Bacillaceae were the predominant families in the gut microbiota of E. crypticus, while Rickettsiaceae and Moraxellaceae were the predominant families in the gut microbiota of F. candida. Liming affected the gut microbiota of E. crypticus at both the taxonomical and core microbiota level. The gut microbiota of F. candida was not affected by liming. Structural equation models suggest that 97% of the variation in the E. crypticus gut microbiota could be explained by liming-induced changes in soil properties and the soil microbial community. The indirect effects of liming, caused by a shift in the soil microbial community, contributed more in reshaping the gut microbiota of E. crypticus than the direct effects of the changed soil properties did. These findings suggest that the effects of liming on the gut microbiota composition in soil fauna are species-specific and are likely dependent on the response of the host to changes in soil properties and the soil microbial community.

Microbial symbiosis is essential for the normal development and growth of hosts. Past attention has mostly been paid to its effects on plants and vertebrates. The effects of environmental pressures such as antibiotics on the microbiome of soil fauna remain largely elusive. We used bacterial 16S rRNA gene high-throughput sequencing to examine the response of microbiome of soil invertebrate Enchytraeus crypticus to oral tetracycline exposure. After two-week exposure, tetracycline-free oat was used as food to monitor the restoration of E. crypticus microbiome. The results showed that Proteobacteria, Actinobacteria and Planctomycetes were the three dominant phyla in all samples, Rhizobiaceae and Kaistia were the most abundant family and genus in all samples, respectively. After 14 days tetracycline exposure, Planctomycetes declined dramatically from 33.05% to 3.28% (P = 0.016), but Actinobacteria elevated substantially from 2.47% to 23.65% (P = 0.004). The alpha-diversity of microbial community increased significantly after tetracycline exposure compared to the control (P = 0.014). Terminating tetracycline exposure led to the recovery of E. crypticus microbiome back to the background level within 14 days. Our results suggest that while tetracycline can disturb the microbiome in E. crypticus significantly, the effects of the antibiotic on E. crypticus microbiome may not be permanent but reversibly diminish after stopping exposure for a period of time. The results may contribute to extending our understanding of the effect of antibiotics on microbiome of soil invertebrates. Capsule The microbiome of E. crypticus exposed to tetracycline is perturbed and reversibly restored after terminating the exposure.

Antibiotic resistance genes (ARGs), prevalent across multiple environmental media, threaten human health worldwide and are considered emerging environmental contaminants. Earthworm gut, a niche for bacteria to survive, represents a potential reservoir for ARGs in soil. However, the compositions of ARGs in the earthworm gut microbiota remain elusive, especially under field conditions. In this study, we applied high-throughput quantitative PCR to profile the ARGs in the gut microbiota of earthworms after chronic exposure to fertilizers. To elucidate the factors that impact the ARGs composition, the bacterial community of gut microbiota, mobile genetic elements (MGEs), soil (nutrients, heavy metals, and antibiotics) and the properties of gut content (pH and nutrients) were analyzed. A total of 98 subtypes among 9 major types of ARGs, and 3 different MGEs were detected in the gut microbiota of earthworms. Organic fertilizer (sewage sludge and chicken manure) application significantly increased the diversity and abundance of ARGs. Of the 1123 identified operational taxonomic units (OTUs) at 97% similarity cutoff, most of them were assigned to Firmicutes (55.5%) and Proteobacteria (33.6%) in earthworm gut microbiota. Long-term organic fertilization slightly changed the microbiota composition, but did not impact the diversity. Partial redundancy analysis (pRDA) revealed that bacterial community, combined with environmental factors (soil and gut content properties) and MGEs, explained 72% of the variations of ARGs in the earthworm gut. Furthermore, the co-occurrence pattern between ARGs and MGEs indicated that horizontal gene transfer via MGEs may occur in the earthworm gut. These findings improve the current understanding of the dynamics of soil fauna-associated ARGs and the gut microbiota of earthworms may be an underappreciated hotspot for ARGs in the environment. Keywords: Antibiotic resistome, Gut microbiota, Soil fauna, Sewage sludge, Chicken manure

Phyllosphere contains various microorganisms that may harbor diverse antibiotic resistance genes (ARGs). However, we know little about the composition of antibiotic resistome and the factors influencing the diversity and abundance of ARGs in the phyllosphere. In this study, 16S rRNA gene amplicon sequencing and high-throughput quantitative PCR approaches were employed to investigate the effects of long-term (over 10 years) organic fertilization on the phyllosphere bacterial communities and antibiotic resistome. Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes dominated in the phyllosphere bacterial communities. Long-term application of sewage sludge and chicken manure altered the phyllosphere bacterial community composition, with a remarkable decrease in bacterial alpha-diversity. A total of 124 unique ARGs were detected in the phyllosphere. The application of sewage sludge and chicken manure significantly increased the abundance of ARGs, with a maximum 2638-fold enrichment. Variation partitioning analysis (VPA) together with network analysis indicated that the profile of ARGs is strongly correlated with bacterial community compositions. These results improve the knowledge about the diversity of plant-associated antibiotic resistome and factors influencing the profile of ARGs in the phyllosphere.

The widespread occurrence of tire particles (TPs) in soils has attracted considerable attention due to their potential threats. The assemblage of bacteria and associated antibiotic resistant genes (ARGs) on TPs is yet largely unknown, especially under the stress of soil pollutants. In the present study, TPs were incubated in soils with or without the stress of heavy metal (Cu2+) or/and antibiotic (tetracycline), and bacterial community and ARG profile on TPs and in soils were explored using high-throughput sequencing and high-throughput quantitative PCR. Results indicated that bacterial community structure on TPs was significantly different from the surrounding soils, with a lower diversity, and significantly shifted by heavy metal and antibiotic exposure. Additionally, a diverse set of ARGs were detected on TPs, and their abundances were significantly increased under the stress of heavy metal and antibiotic, revealing a strong synergistic effect. Moreover, a good fit was observed for the correlation between bacterial community and ARG profile on TPs. Taken together, this study, for the first time, demonstrates that TPs can provide a novel niche for soil bacteria and soil resistome, and heavy metal and antibiotic exposure may potentially increase the abundance of ARGs on TPs, threatening soil ecosystems and human health.

Drinking water treatment techniques are used globally in the context of water security and public health, yet they are not applicable to antibiotic resistance gene (ARG) contamination. Using high-throughput quantitative PCR, we analyzed the prevalence and diversity of ARGs and mobile genetic elements (MGEs) in water supplies. A total of 224 ARGs and MGEs were detected in all sampling sites. Absolute abundance and detected number of ARGs decreased significantly (P < 0.05) in sand filter water after drinking water treatment and increased thereafter at point-of-use (household tap water). Changes in the composition and diversity of the bacterial community were observed in water samples at different steps. A significant correlation (P < 0.001) between microbial communities and ARG profiles was observed, and variance in ARG profiles could be primarily attributed to community composition (11.9%), and interaction between community composition, environmental factors and MGEs (30.7%). A network analysis was performed, and the results showed eight bacterial phyla were significantly correlated with nine different classes of ARGs, suggesting the potential bacterial host for ARGs. This study suggested that although the absolute abundance of ARGs decreased after treatment of drinking water treatment plants (DWTPs), the rebounded of ARGs in the water distribution system should not be neglected.

Microorganisms associated with plants may alter the traits of the human microbiome important for human health, but this alteration has largely been overlooked. The plant microbiome is an interface between plants and the environment, and provides many ecosystem functions such as improving nutrient uptake and protecting against biotic and abiotic stress. The plant microbiome also represents a major pathway by which humans are exposed to microbes and genes consumed with food, such as pathogenic bacteria, antibiotic-resistant bacteria, and antibiotic-resistance genes. In this review we highlight the main findings on the composition and function of the plant microbiome, and underline the potential of plant microbiomes in the dissemination of antibiotic resistance via food consumption or direct contact.

The human pathogenic bacteria (HPB) in animal feces may disseminate to agricultural soils with their land application as organic fertilizer. However, the knowledge about the impacts of different sources and rates of animal manures on the temporal changes of soil HPB remains limited, which hamper our ability to estimate the potential risks of their land application. Here, we constructed an HPB database including 565 bacterial strains. By blasting the 16 S rRNA gene sequences against the database we explored the occurrence and fate of HPB in soil microcosms treated with two rates of swine, poultry or cattle manures. A total of 30 HPB were detected in all of manure and soil samples. Poultry manure at the high level obviously improved the abundance of soil HPB. The application of swine manure could introduce concomitant HPB into the soils. Of which, Pseudomonas syringae pv. syringae B728a and Escherichia coli APEC O78 may deserve more attention because of their survival for a few days in manured soils and being possible hosts of diverse antibiotic resistance genes (ARGs) as revealed by co-occurrence pattern. Bayesian source tracking analysis showed that the HPB derived from swine manure had a higher contribution to soil pathogenic communities than those from poultry or cattle manures in early days of incubation. Mantel test together with variation partitioning analysis suggested that bacterial community and soil physicochemical properties were the dominant factors determining the profile of HPB and contributed 64.7% of the total variations. Overall, our results provided experimental evidence that application of animal manures could facilitate the potential dissemination of HPB in soil environment, which should arouse sufficient attention in agriculture practice and management to avoid the threat to human health.

Soil-plant microbiome plays a critical role in the regulation of terrestrial ecosystem function and service, including biogeochemical cycling and primary production. The lack of knowledge regarding the differences in microbial functional traits, i.e. the functional genes related to carbon (C), nitrogen (N), phosphorus (P) and sulfur (S) cycles, between soil and plant microbiomes hampers our prediction of the terrestrial nutrient cycling processes under the pressure of anthropogenic disturbance. Herein, a quantitative microbial element cycling (QMEC) method and amplicon sequencing was employed to characterize CNPS cycling genes and microbial communities in soil and plant samples collected from peri-urban farmland with high anthropogenic disturbance and forest ecosystem with minimal disturbance. The soil-plant system harbored a diverse array of CNPS cycling genes, which were significantly more abundant in soil than in phyllosphere. The overall CNPS gene profiles in farmland samples was distinct from that of forest samples in both soil and plant phyllosphere. Farmland samples had a lower abundance of CNPS cycling genes than forest samples, indicating that intensive agricultural management practices may consequently compromise the biogeochemical cycling potential of nutrients. Significant positive correlations between the abundance of CNPS cycling genes and microbial diversity were observed in phyllosphere microbiome but not in soil, suggesting that the functional redundancy in soil microbiome may be higher than that of phyllosphere microbiome. Taken together, we provide experimental evidence for the substantial impacts of anthropogenic disturbance on CNPS cycling genes in the soil-plant system and necessitate future efforts to unravel the plant microbiome diversity and functionality under the pressure of global changes.

Growing evidence suggests that livestock manure used as organic fertilizer in agriculture may lead to the potential propagation of antibiotic resistance genes (ARGs) from “farm to fork.” However, little is known about the impacts of manure fertilization on the incidence of ARGs in the plant-associated microbiomes (including rhizosphere, endosphere, and phyllosphere), which hampers our ability to assess the dissemination of antibiotic resistance in the soil-plant system. Here, we constructed a pot experiment to explore the effects of poultry and cattle manure applications on the shifts in the resistome in the plant microbiome of harvested cherry radish. A total of 144 ARGs conferring resistance to eight major classes of antibiotics were detected among all the samples. Rhizosphere and phyllosphere microbiomes harbored significantly higher diversity and abundance of ARGs than did root endophytic microbiomes of cherry radish. Manure application significantly increased the abundance of ARGs in the rhizosphere and phyllosphere but not in the endophytes of the root, which is the edible part of cherry radish. Soil and plant microbiomes changed dramatically after manure applications and clustered separately according to different sample types and treatments. Structural equation modeling revealed that bacterial abundance was the most important factor modulating the distribution patterns of soil and plant resistomes after accounting for multiple drivers. Taken together, we provide evidence that enrichment of the resistome in the rhizosphere and phyllosphere of cherry radish is more obvious than with the endosphere after manure application, suggesting that manure amendment might not enhance the dissemination of ARGs into the root of vegetables in the pot experiment. IMPORTANCE Our study provides important evidence that manure application increased the occurrence of ARGs in the rhizosphere and phyllosphere of cherry radish, compared with that in the endophytic bacterial microbiota of root, which is the edible part of cherry radish. Our findings suggest that although manure amendment is a significant route of ARGs entering agricultural soils, these manure-derived ARGs may be at low risk of migrating into the endophytes of root vegetables.

Land application of animal manures is a major dissemination route for antibiotic resistance genes (ARGs) in agro-ecosystems, which are a potential risk to public health if they migrate into the food chain. Despite the fact that animal manure is a rich reservoir of ARGs our knowledge of the impacts of different types and amounts of animal manures on the temporal succession of a wide array of ARGs and potential ARG-hosts remains limited. Here, we constructed soil microcosms amended with two levels of swine, poultry or cattle manures to explore the time-course patterns of ARGs, mobile genetic elements (MGEs) including integrons and transposons, and bacterial communities. The high-throughput quantitative PCR detected 260 unique ARGs, and the application of all manure types, especially at the higher concentration (80 mg manure g−1 soil), significantly increased the diversity and abundances of ARGs and MGEs. The abundance of ARGs in manured soils declined over time, but was still higher than that in untreated soils after 120 days, indicating the persistence of ARGs in manured soils. The next-generation sequencing revealed a clear shift in the bacterial community compositions of manured soils during the incubation. Mantel test and network analysis revealed that the ARG profile was strongly correlated with the bacterial community compositions. Variation partitioning analysis, and structure equation models, further indicated that bacterial phylogeny played a primary role in shaping the ARG profiles in manured soils. However, the significant correlation between the abundances of MGEs and ARGs suggested that the potential effects of horizontal gene transfer on the persistence of ARGs should not be overlooked. In addition, soil properties, which were strongly affected by the added manures, could also affect the ARG patterns. These findings demonstrated the temporal patterns and dissemination risk of ARGs in manured soils, which might contribute to the development of effective strategies to minimise the spread of ARGs in agro-ecosystems.

Gut microbiota contribute to host health. Numerous recent studies have focused on the survival and reproduction of nontarget soil animals exposed to the toxicity of silver nanoparticles (AgNPs) but changes in the gut microbiota due to nanoparticle toxicity are largely unknown. Here, we examine some effects of AgNPs and silver nitrate (ionic Ag) on the gut microbiota of the common soil collembolan Folsomia candida using Illumina sequencing and concomitant changes in antibiotic resistance genes (ARGs) of the gut microbiota using high-throughput quantitative PCR. A large number of Ag accumulated in Ag-exposed individuals after 28 days and ionic Ag significantly inhibited the reproduction of the collembolan (by 19.3%). Exposure to AgNPs disturbed the composition of the collembolan gut bacterial community, resulting in dysbiosis of the gut microbiota. However, the dominant microbiota was shared among different treatments. In addition, AgNPs exposure did indeed reduce the incidence of ARGs in the collembolan gut microbiota. A weak relationship was identified between gut bacterial communities and ARG profiles. These results extend our knowledge regarding the role of the gut microbiota in assessing the soil ecotoxicology of AgNPs.

Microbiota in urban green spaces underpin ecosystem services that are essential to environmental health and human wellbeing. However, the factors shaping the microbial communities in urban green spaces, especially those associated with turf grass phyllosphere, remain poorly understood. The lack of this knowledge greatly limits our ability to assess ecological, social and recreational benefits of urban green spaces in the context of global urbanization. In this study, we used amplicon sequencing to characterize soil and grass phyllosphere bacterial communities in 40 urban green spaces and three minimally disturbed national parks in Victoria, Australia. The results indicated that urbanization might have shown different impacts on soil and grass phyllosphere microbial communities. The bacterial diversity in soil but not in grass phyllosphere was significantly higher in urban green spaces than in national parks. Principal coordinate analysis revealed significant differences in the overall patterns of bacterial community composition between urban green spaces and national parks for both soil and grass phyllosphere. Industrial development, as represented by the number of industries in the region, was identified as a key driver shaping the bacterial community profiles in urban green spaces. Variation partitioning analysis suggested that industrial factors together with their interaction with other factors explained 20% and 28% of the variances in soil and grass phyllosphere bacterial communities, respectively. The findings highlight the importance of industrial development in driving the spatial patterns of urban microbiomes, and have important implication for the management of microbiomes in urban green spaces.

Lignite could be used to promote methane production during high-solid anaerobic co-digestion (HS-AcoD) of pig manure, however, the effects of lignite amendment on the fate of ARGs during HS-AcoD are unknown. Here, we explored the influence of lignite (0%, 8%, 16%, 32%, and 64%) on the fate of ARGs during HS-AcoD of pig manure. The results showed that 16% lignite reduced the absolute abundance of ARGs by 28.71% compared with the 0% lignite treatment. Variation partitioning analysis suggested the combined effect of microbial community, mobile genetic elements (MGEs) and environmental factors was the major driver shaping the pattern of ARGs. The potential hosts of ARGs were Bifidobacterium, Lactobacillus, Tissierella and Streptococcus. Structural equation models analysis suggested lignite indirectly impacted the pattern of ARGs by significantly reducing the abundance of microbial community and MGEs. These findings give an insight into the mechanistic understanding of the lignite influence on the reduction of ARGs during HS-AcoD.

The increasing prevalence of antibiotic resistance is a global threat to public health. Antibiotic resistance genes (ARGs) in soil have been demonstrated to be able to enter food chain. Strategies to mitigate the spread of antibiotic resistance from soil to crops are of great importance for food safety and human health. Soil amendment with biochar is a widely used approach to improve soil fertility. However, the impact of biochar on mitigating antibiotic resistance of soil and organically produced vegetables that are eaten raw is largely unknown. To gain insights into this impact, pot experiments planting Brassica chinensis L. in soil amended with biochar were conducted and antibiotic resistance genes (ARGs) were characterized by HT-qPCR targeting almost all major classes of ARGs and 10 mobile genetic elements (MGEs) marker genes. A total of 131 ARGs and 9 MGEs in soil and phyllosphere samples were identified. After biochar amendment, the abundance of ARGs was significantly decreased in non-planted soil. By comparison, no significant decrease of ARGs was found in rhizosphere and phyllosphere, indicating that vegetable planting affected antibiotic resistome in biochar-amended soil. To understand this effect, bacterial phylogeny structures within soil and phyllosphere were analyzed and found to correlate with their respective resistome, indicating that planting can influence the effect of biochar on soil antibiotic resistome by altering microbial community compositions. Structure equation models further revealed that a higher bacterial diversity corresponded to a decreased ARGs content. These results suggested that biochar amendment alone was not sufficient enough to alleviate ARGs level in planted soil and crops, instead, maintaining or increasing soil microbial diversity is potentially more useful in mitigating ARG spread and accumulation.

Although the roles of earthworms and soil collembolans in the transport of microplastics have been studied previously, the effects of the soil biota at different trophic levels and interspecific relationships remain poorly understood. Here, we examine three soil microarthropod species to explore their effects on the transport of microplastics. The selected Folsomia candida and Hypoaspis aculeifer are extensively used model organisms, and Damaeus exspinosus is a common and abundant indigenous species in China. A model food chain (prey-collembolan and predator-mite) was structured to test the role of the predator-prey relationship in the transport of microplastics. Commercial Polyvinyl chloride (PVC) particles (Diameter: 80-250 μm) were selected as the test microplastics, because large amounts of PVC have persisted and accumulated in the environment. Synchronized soil microarthropods were held in plates for seven days to determine the movement of microplastics. The 5000 microplastic particles were carefully placed in the center of each plate prior to the introduction of the animals. Our results clearly show that all three microarthropod species moved and dispersed the microplastics in the plates. The 0.54%, 1.8% and 4.6% of the added microplastic particles were moved by collembolan, predatory mite and oribatid mite, respectively. Soil microarthropods (0.2 cm) transported microplastic particles up to 9 cm. The avoidance behavior was observed in the collembolans in respect of the microplastics. The predatory -prey relationship did promote the transport of microplastics in the plates, increasing transport by 40% compared with the effects of adding single species (P .05). Soil microarthropods commonly occur in surface soils (0-5 cm) and, due to their small body size, they can enter soil pores. Our results therefore suggest that the movement of microplastics by soil microarthropods may influence the exposure of other soil biota to microplastics and change the physical properties of soils.

Effects of microplastics on aquatic organisms have been widely studied in recent years but effects on soil biota, and especially on the gut microbiota of soil animals, remain poorly understood. An experiment was therefore conducted using the common soil collembolan Folsomia candida exposed to microplastics for 56 days to investigate the effects of plastics on gut microbiota, growth, reproduction and isotopic turnover of collembolans in the soil ecosystem. A diverse microbial community was observed in the collembolan gut, consisting of (at phylum level) Actinobacteria (∼44%), Bacteroidetes (∼30%), Proteobacteria (∼12%) and Firmicutes (∼11%). Distinctly different bacterial communities and lower microbial diversity were found in the collembolan gut compared with the surrounding soil. We also found that exposure to microplastics significantly enhanced bacterial diversity and altered the microbiota in the collembolan gut. Moreover, collembolan growth and reproduction were significantly inhibited (by 16.8 and 28.8%, respectively) and higher δ15N and δ13C values were observed in the tissues after exposure to microplastics. These results indicate that exposure to microplastics may impact non-target species via changes in their microbiota leading to alteration of isotopic and elemental incorporation, growth and reproduction. The collembolan gut microbial data acquired fill a gap in our knowledge of the ecotoxicity of microplastics.

Manure application is a common practice that not only adds nutrients and organic matter to arable soils for crop growth, but also introduces antibiotic resistance genes (ARGs), posing a potential risk to human health. To investigate the mechanisms underlying the spread of ARGs in manured soil, especially the impact of manure-borne and indigenous soil microorganisms, a microcosm experiment with four specially designed treatments over a period of two months was conducted, including soil, soil with irradiated pig manure, irradiated soil with pig manure, and soil with pig manure. A total of 240 unique ARGs were detected via a high-throughput quantitative PCR (HT-qPCR) targeting almost all major classes of ARGs. Manure application significantly increased the diversity and abundance of ARGs in soil (P < 0.01), and also markedly shifted the bacterial composition that was significantly correlated with ARGs profiles. Manure-borne microorganisms contributed largely to the elevation of ARGs due to both the addition of manure-borne antibiotic resistant bacteria (ARB) in soil and potential horizontal gene transfer (HGT) via mobile genetic elements (MGEs) from manure-borne ARB to indigenous soil microorganisms. In contrast, indigenous soil microorganisms were demonstrated to prevent the dissemination of ARGs from manure to soil. The reason could be due to that indigenous soil microorganisms prevented the invasion and establishment of manure-borne ARB in soil. The abundance of ARG in manured soil decreased over time, but was still higher than that in control soil, indicating the persistence of ARGs in manured soil. These findings may shed light on the mechanisms underlying the spread and fate of ARGs in manured soil and also clues for ARGs mitigation.

Struvite recovered from wastewater is a renewable source of phosphorus and nitrogen and can be used as fertilizer for plant growth. However, antibiotics and resistome can be enriched in the struvite derived from wastewater. Robust understanding of the potential risks after struvite application to soils has remained elusive. Here, we profiled antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in struvite, soil, rhizosphere and phyllosphere of Brassica using high-throughput quantitative PCR. A total of 165 ARGs and 10 MGEs were detected. Application of struvite was found to increase both the abundance and diversity of ARGs in soil, rhizosphere and phyllosphere. In addition, ARGs shared exclusively between Brassica phyllosphere and struvite were identified, indicating that struvite was an important source of ARGs found in phyllosphere. Furthermore, OTUs shared between rhizosphere and phyllosphere were found to significantly correlate with ARGs, suggesting that microbiota in leaf and root could interconnect and ARGs might transfer from struvite to the surface of plants via rhizosphere using bacteria as spreading medium. These findings demonstrated that struvite as an organic fertilizer can facilitate the spread of antibiotic resistance into human food chain and this environment-acquired antibiotic resistance should be put into human health risk assessment system.

The demand for organic food products, especially for organic vegetables has been growing rapidly in the last few decades. However, the risk of introducing more antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) to the vegetables by organic production procedures has long been overlooked. In our study, by using high-throughput quantitative PCR and 16sRNA Illumina sequencing technology, we investigated the abundance and diversity of ARGs and the microbial communities in conventionally (CPL) and organically produced lettuce (OPL). A total of 134 ARGs were detected in the phyllosphere and leaf endophyte of the samples. Absolute copy numbers of ARGs in phyllosphere were 8-fold higher in the OPL than in CPL. We also observed a significant difference in the microbial communities between OPL and CPL, and a lower diversity of both phyllosphere and leaf endophytic bacteria in OPL than in CPL. The Mantel test and variation partitioning analysis (VPA) suggested that the profile of ARGs is strongly affected by bacterial community compositions. Network analysis between ARGs and bacterial taxa indicated that eight bacterial families were implicated to be the potential hosts of ARGs. These results provide insights into the impacts of organic farming on the profiles of bacterial and ARG compositions in vegetables. Keyword: Antibiotic resistance genes, Phyllosphere, Vegetables, Network analysis

It has been proved that nanoplastics can effectively adsorb pollutants and thus influence their behavior and availability. The combined toxic effects of nanoplastic and its adsorbed pollutant on the soil fauna are still not well known. We used high-throughput quantitative PCR to explore the effects of oral nanoscale polystyrene and tetracycline exposure on antibiotic resistance genes in the soil invertebrate Enchytraeus crypticus, and used bacterial 16S rRNA gene amplification sequencing to examine the response of the microbiome of E. crypticus. After 14 days of tetracycline and nanoscale polystyrene exposure, we terminated exposure and monitored the restoration of ARGs and microbiome in the E. crypticus. Results showed that the number of ARGs, especially macrolide-lincosamide-streptogramin B (MLSB), tetracycline ARGs, as well as multidrug ARGs, increased with exposure to nanoscale polystyrene and tetracycline. The abundance of Aminoglycoside and Beta_Lactamase ARGs in E. crypticus also significantly increased. The exposure significantly perturbed the abundance of families Microbacteriaceae, Streptococcaceae, Enterobacteriaceae, Rhodocyclaceae and Sphinomonadaceae. After terminating exposure for 14 days, the diversity and abundance of ARGs were not completely restored, while the microbiome was not permanently changed but reversibly impacted.

The emergence, evolution and spread of antibiotic resistance genes (ARGs) in the environment represent a global threat to human health. Our knowledge of antibiotic resistance in human-impacted ecosystems is rapidly growing with antibiotic use, organic fertilization and wastewater irrigation identified as key selection pressures. However, the importance of biological interactions, especially predation and competition, as a potential driver of antibiotic resistance in the natural environment with limited anthropogenic disturbance remains largely overlooked. Stress-affected bacteria develop resistance to maximize competition and survival, and similarly bacteria may develop resistance to fight stress under the predation pressure of protists, an essential component of the soil microbiome. In this article, we summarized the major findings for the prevalence of natural ARGs on our planet and discussed the potential selection pressures driving the evolution and development of antibiotic resistance in natural settings. This is the first article that reviewed the potential links between protists and the antibiotic resistance of bacteria, and highlighted the importance of predation by protists as a crucial selection pressure of antibiotic resistance in the absence of anthropogenic disturbance. We conclude that an improved ecological understanding of the protists-bacteria interactions and other biological relationships would greatly expand our ability to predict and mitigate the environmental antibiotic resistance under the context of global change.

The increasing antimicrobial resistance in manure-amended soil can potentially enter food chain, representing an important vehicle for antibiotic resistance genes (ARGs) transmission into human microbiome. However, the pathways for transmission of ARGs from soil to plant remain unclear. Here, we explored the impacts of poultry and cattle manure application on the patterns of resistome in soil and lettuce microbiome including rhizosphere, root endosphere, leaf endosphere and phyllosphere, to identify the potential transmission routes of ARGs in the soil-plant system. After 90 days of cultivation, a total of 144 ARGs were detected in all samples using high-throughput quantitative PCR. Rhizosphere soil samples harbored the most diverse ARGs compared with other components of lettuce. Cattle manure application increased the abundance of ARGs in root endophyte, while poultry manure application increased ARGs in rhizosphere, root endophyte and phyllosphere, suggesting that poultry manure may have a stronger impact on lettuce resistomes. The ARG profiles were significantly correlated with the bacterial community, and the enrichment of soil and plant resistomes was strongly affected by the bacterial taxa including Solibacteres, Chloroflexi, Acidobacteria, Gemm-1 and Gemmatimonadetes, as revealed by the network analyses. Moreover, the overlaps of ARGs between lettuce tissues and soil were identified, which indicated that plant and environmental resistomes are interconnected. Our findings provide insights into the transmission routes of ARGs from manured soil to vegetables, and highlight the potential risks of plant resistome migration to the human food chain. Keywords: Antibiotic resistance genes, Manure-amended soil, Plant resistome, Endophyte, Phyllosphere

The plant microbiome represents a crucial pathway for human exposure to environmental antibiotic resistance. However, little information is available regarding the plant associated resistome in human-related environments at a larger scale. Here, by high-throughput quantitative-PCR chip-based array and amplicon sequencing, we characterized antibiotic resistance genes (ARGs) and bacterial communities in plant and soil microbiomes from human highly disturbed peri-urban farmland and less disturbed forest at a watershed scale. A total of 71 ARGs were detected in the phyllosphere, which covered almost all the major recognized classes of antibiotics that are administered commonly to humans and animals. The overall pattern of the plant associated resistome in intensive anthropogenic influenced farmland was significantly different from that of forest environments (PERMANOVA, P

Sewage sludge and manure are common soil amendments in crop production; however, their impact on the abundance and diversity of the antibiotic resistome in soil remains elusive. In this study, by using high-throughput sequencing and high-throughput quantitative PCR, the patterns of bacterial community and antibiotic resistance genes (ARGs) in a long-term field experiment were investigated to gain insights into these impacts. A total of 130 unique ARGs and 5 mobile genetic elements (MGEs) were detected and the long-term application of sewage sludge and chicken manure significantly increased the abundance and diversity of ARGs in the soil. Genes conferring resistance to beta-lactams, tetracyclines, and multiple drugs were dominant in the samples. Sewage sludge or chicken manure applications caused significant enrichment of 108 unique ARGs and MGEs with a maximum enrichment of up to 3845 folds for mexF. The enrichment of MGEs suggested that the application of sewage sludge or manure may accelerate the dissemination of ARGs in soil through horizontal gene transfer (HGT). Based on the co-occurrence pattern of ARGs subtypes revealed by network analysis, aacC, oprD and mphA-02, were proposed to be potential indicators for quantitative estimation of the co-occurring ARGs subtypes abundance by power functions. The application of sewage sludge and manure resulted in significant increase of bacterial diversity in soil, Proteobacteria, Acidobacteria, Actinobacteria and Chloroflexi were the dominant phyla (>10% in each sample). Five bacterial phyla (Chloroflexi, Planctomycetes, Firmicutes, Gemmatimonadetes and Bacteroidetes) were found to be significantly correlated with the ARGs in soil. Mantel test and variation partitioning analysis (VPA) suggested that bacterial community shifts, rather than MGEs, is the major driver shaping the antibiotic resistome. Additionally, the co-occurrence pattern between ARGs and microbial taxa revealed by network analysis indicated that four bacterial families might be potential hosts of ARGs. These results may shed light on the mechanism underlining the effects of amendments of sewage sludge or manure on the occurrence and dissemination of ARGs in soil. Keywords: Antibiotic resistance genes, Bacterial pathogens, Horizontal gene transfer, Network analysis, Sewage sludge

We lack empirical evidence for the relative importance of microbial functional attributes vs taxonomic attributes in regulating specified soil processes related to carbon (C) and nitrogen (N) cycling, which has hindered our ability to predict the responses of ecosystem multifunctionality to environmental changes. Here, we collected soil samples from a long-term experimental field with eight inorganic and organic fertilization treatments and evaluated the linkage between microbial functional attributes (abundance of functional genes), taxonomic attributes (microbial taxonomic composition), and soil processes including soil respiration, denitrification and nitrification. Long-term fertilization had no significant effect on the bacterial or fungal alpha-diversity. The treatments of chicken manure and sewage sludge addition significantly altered the rates of soil respiration, denitrification and nitrification, which were significantly correlated with the abundances of relevant functional genes. Random forest model indicated that the abundance of functional genes was the main diver for the rate of soil processes. The predominant effect of microbial functional attributes in driving soil processes was maintained when simultaneously accounting for multiple abiotic (total C, total N and soil pH) and biotic drivers (bacterial and fungal community structure), indicating that microbial functional attributes were the predominant driver predicting the rate of soil respiration, denitrification and nitrification. Our results suggested the importance of developing a functional gene-centric framework to incorporate microbial communities into biogeochemical models, which may provide new insights into the biodiversity-functions relationship and have implications for future management of the consequences of biodiversity loss for ecosystem multifunctionality.

Soil microbial communities play a central role in driving multiple ecosystem functions and ecological processes that are key to maintaining the plant productivity. However, we lack sound evidence for the linkage between soil microbial diversity and plant productivity, which hinders our ability to predict the consequences of microbial diversity loss for food security under the context of global environmental change. Here, we used the dilution-to-extinction approach to test the consequences of soil microbial diversity loss for the aboveground plant biomass in a glasshouse experiment. Compared with original soils, the bacterial alpha-diversity (Observed operational taxonomic units and Shannon index) significantly decreased in treatments with serially diluted inoculum. Principal coordinates analysis showed that the overall bacterial community compositions (beta-diversity) in original soils were clearly separated from the treatments with serially diluted inoculum. The aboveground biomass of lettuce harvested from the original soils was significantly higher than that from the sterilized soils regardless of the inoculation. The ordinary least squares regression model showed a significant linear relationship between the plant biomass and bacterial alpha-diversity, indicating that reduction in soil microbial diversity could result in a significant decline in the biomass of lettuce. No significant correlation was observed between plant biomass and soil processes including soil basal respiration and denitrification rates. Structural equation models suggested that the effects of soil microbial diversity on the plant biomass were maintained even when simultaneously accounting for other drivers (soil properties and biological processes). Our study provides experimental evidence that soil microbial diversity is important to the maintenance of the plant productivity and suggests that the functional redundancy in soil microbial communities may be overestimated especially in the agroecological system.

Plant microbiome, as the second genome of plant, and the interface between human and environmental microbiome, represents a potential pathway of human exposure to environmental pathogens and resistomes. However, the impact of host identity on the profile of resistomes in plant phyllosphere is unclear and this knowledge is vital for establishing a framework to evaluate the dissemination of antibiotic resistance via the plant microbiome. Here, we explored the phyllosphere microbiome and resistomes in 12 selected plant species. By using High-throughput quantitative PCR, we identified a total of 172 unique resistance genes in plant phyllosphere microbiome, which was significantly divergent from the profile of resistomes in associated soils (Adonis, P 0.01). Host identity had a significant effect on the plant resistome, which was mainly attributed to the dissimilarity of phyllosphere bacterial phylogeny across different plants. We identified a core set of plant resistomes shared in more than 80% of samples, which accounted for more than 64% of total resistance genes. These plant core resistomes conferred resistance to antibiotics that are commonly administered to humans and animals. Our findings extend our knowledge regarding the resistomes in plant phyllosphere microbiome and highlight the role of host identity in shaping the plant associated antibiotic resistance genes.

Nitrification inhibitors have been reported to reduce nitrous oxide emission and nitrate leaching in agricultural systems. The effects of repeated applications of urea alone or in combination with nitrification inhibitors on nitrogen (N) cycling microbes involved in nitrification and denitrification together with non-targeted microbes are not well understood. Therefore, the objective of this study was to investigate the effects of repeated application of urea and DMPP on soil physio-chemistry, ammonia oxidizers and total bacteria in the soil. We collected soil samples from a 4.5-year field experiment under crop rotation with repeated application of seven treatments, namely control (CK), Urea (U), Urea + DMPP (UE) applied at 40, 80 and 120 kg N ha−1, each treatment with three replicates. Ammonia-oxidizing bacteria (AOB) gene copy numbers increased as the N application rate increased (from 0 to 120 kg N ha−1). The use of DMPP significantly reduced AOB and nirK gene copy numbers compared to urea alone at an application rate of 120 kg N ha−1. There was no treatment effect on the abundance of ammonia-oxidizing archaea (AOA), Comammox clade A and B, nosZ and bacterial 16S rRNA genes. The community composition of AOB and AOA changed with N addition and use of DMPP but increasing N addition rate changed the composition of AOB only. Addition of N increased potential nitrification rates at 80 and 120 kg N ha−1. There was no significant treatment effect on the relative abundance of bacteria at the phylum level. This experiment demonstrated that the application of N (with or without DMPP) at rates lower than 120 kg N ha−1 would not result in significant impacts on soil archaeal and bacterial ecology.

Numerous studies have investigated the composition and diversity of antibiotic resistance genes (ARGs) in multiple environments but the pattern of ARGs in field-collected soil fauna remains poorly understood. In the present study soil collembolans were collected from six sites with three different land use types (parkway land, park land, and arable land) and 285 ARGs and 10 mobile genetic elements (MGEs) in the microbiome of these "wild" collembolans were quantified by high-throughput quantitative PCR. A total of 76 unique ARGs and 5 MGEs were detected. There were significant differences between collection sites in the antibiotic resistome in the collembolans. Land use significantly altered the distribution patterns of collembolan ARGs. Thirty shared ARGs and three shared MGEs were identified. The co-occurrences of shared resistomes were largely random, and more positive relationships were found in the coassociation network. Partial redundancy analysis confirms that the changes in bacterial communities explained 27.77% of the variation in ARGs. These findings suggest that resistance genes are pervasive in the microbiome associated with the field collembolan and the activity of the collembolans may contribute to the spread and dissemination of resistance genes in the environment, an aspect of ARGs that has until now been largely overlooked.

The integron platform allows the acquisition, expression, and dissemination of antibiotic resistance genes within gene cassettes. Wastewater treatment plants (WWTPs) contain abundant resistance genes; however, knowledge about the impacts of wastewater treatment on integrons and their gene cassettes is limited. In this study, by using clone library analysis and high-throughput sequencing, we investigated the abundance of class 1, 2, and 3 integrons and their corresponding gene cassettes in three urban WWTPs. Our results showed that class 1 integrons were most abundant in WWTPs and that wastewater treatment significantly reduced the abundance of all integrons. The WWTP influents harbored the highest diversity of class 1 integron gene cassettes, whereas class 3 integron gene cassettes exhibited highest diversity in activated sludge. Most of the gene cassette arrays detected in class 1 integrons were novel. Aminoglycoside, beta-lactam, and trimethoprim resistance genes were highly prevalent in class 1 integron gene cassettes, while class 3 integrons mainly carried beta-lactam resistance gene cassettes. A core class 1 integron resistance gene cassette pool persisted during wastewater treatment, implying that these resistance genes could have high potential to spread into environments through WWTPs. These data provide new insights into the impact of wastewater treatment on integron pools and highlight the need for surveillance of resistance genes within both class 1 and 3 integrons. IMPORTANCE Wastewater treatment plants represent a significant sink and transport medium for antibiotic resistance bacteria and genes spreading into environments. Integrons are important genetic elements involved in the evolution of antibiotic resistance. To better understand the impact of wastewater treatment on integrons and their gene cassette contexts, we conducted clone library construction and high-throughput sequencing to analyze gene cassette contexts for class 1 and class 3 integrons during the wastewater treatment process. This study comprehensively profiled the distribution of integrons and their gene cassettes (especially class 3 integrons) in influents, activated sludge, and effluents of conventional municipal wastewater treatment plants. We further demonstrated that while wastewater treatment significantly reduced the abundance of integrons and the diversity of associated gene cassettes, a large fraction of integrons persisted in wastewater effluents and were consequentially discharged into downstream natural environments.

Gut microbiota make an important contribution to host health but the effects of environmental pressures on the gut microbiota of soil fauna are largely uncharacterized. Here, we examine the effects of norfloxacin and oxytetracycline on the gut microbiome of the common soil collembolan Folsomia candida and concomitant changes in the incidence of antibiotic resistance genes (ARGs) in the gut and in growth of the collembolan. Exposure to 10 mg antibiotics kg

Wastewater treatment plants (WWTPs) contain diverse antibiotic resistance genes (ARGs), and thus are considered as a major pathway for the dissemination of these genes into the environments. However, comprehensive evaluations of ARGs dynamic during wastewater treatment process lack extensive investigations on a broad spectrum of ARGs. Here, we investigated the dynamics of ARGs and bacterial community structures in 114 samples from eleven Chinese WWTPs using high-throughput quantitative PCR and 16S rRNA-based Illumina sequencing analysis. Significant shift of ARGs profiles was observed and wastewater treatment process could significantly reduce the abundance and diversity of ARGs, with the removal of ARGs concentration by 1–2 orders of magnitude. Whereas, a considerable number of ARGs were detected and enriched in effluents compared with influents. In particular, seven ARGs mainly conferring resistance to beta-lactams and aminoglycosides and three mobile genetic elements persisted in all WWTPs samples after wastewater treatment. ARGs profiles varied with wastewater treatment processes, seasons and regions. This study tracked the footprint of ARGs during wastewater treatment process, which would support the assessment on the spread of ARGs from WWTPs and provide data for identifying management options to improve ARG mitigation in WWTPs. Keywords: Antibiotic resistance genes, Human pathogens, Horizontal gene transfer, Human health, Urban environment

Struvite recovered from wastewater is promising for recycling phosphorus into soil as fertilizers. However, struvite application may prompt the proliferation of antibiotic resistance in soil and plant. This study examined the impacts of struvite application and biochar amendment on integrons abundance and gene cassette contexts in rhizosphere soil and phyllosphere using quantitative PCR and clone library analysis. Microcosm experiments revealed that class 1 integron was the most prevalent in all samples, with higher concentration and higher relative abundance in rhizosphere than those in phyllosphere. The majority of resistance gene cassettes were associated with genes encoding resistance to aminoglycosides, beta-lactams and chloramphenicols. Struvite application significantly increased the genetic diversity of antibiotic resistance gene cassettes in both rhizosphere and phyllosphere. However, biochar amendment attenuated the increasing effect of struvite application exerting on the class 1 integron antibiotic resistance gene cassette pool in phyllosphere. These findings highlighted human activities to be the source of integron gene cassette pool and raised the possibility of using biochar amendment as an alternative mean for mitigating antibiotic resistance in environments.

With the rapid development of urbanization and industrialization, the peri-urban areas are often the sites for waste dumps, which may exacerbate the occurrence and spread of antibiotic resistance from waste to soil bacteria. However, the profiles of antibiotic resistomes and the associated factors influencing their dissemination in peri-urban areas have not been fully explored. Here, we characterized the antibiotic resistance genes (ARGs) in peri-urban arable and pristine soils in four seasons at the watershed scale, by using high-throughput qPCR. ARGs in peri-urban soils were diverse and abundant, with a total of 222 genes were detected in the peri-urban soil samples. The arable soil harbored more diverse ARGs compared to the pristine soils, and nearly all the ARGs detected in the pristine soils were also detected in the farmlands. A random forest prediction showed that the overall patterns of ARGs clustered closely with the landuse type. Mantel test and partial redundancy analysis indicated that bacterial community variation is a major contributor to antibiotic resistome alteration. Significant positive correlation was found between the abundance of ARGs and mobile genetic elements (MGEs), suggesting potential mobility of ARGs in peri-urban areas. Our results extend knowledge of the resistomes compositions in peri-urban areas, and suggest that anthropogenic activities driving its spatial and temporal distribution.

Landfills are so far the most common practice for the disposals of municipal solid waste (MSW) worldwide. Since MSW landfill receives miscellaneous wastes, including unused/expired antibiotics and bioactive wastes, it gradually becomes a huge potential bioreactor for breeding antibiotic resistance. Antibiotic resistance genes (ARGs) in landfill can flow to the environment through leakage of landfill leachate and pose a risk to public health. Using high throughput quantitative Polymerase Chain Reaction (HT-qPCR), we investigated the prevalence, diversity of ARGs and its association with various mobile genetic elements (MGEs) in MSW landfill groundwater. Totally 171 unique ARGs (belonging to 9 ARG types, encompassing 3 major resistance mechanisms) and 8 MGEs (6 transposase genes, and 2 integron-integrase genes) were identified. The normalized abundance of ARG was ranging from 0.24 to 5.66 copies/cell with multidrug, beta-lactams and tetracycline resistance genes being the most abundant ARG types. The co-occurrence pattern and significant correlation between MGEs and ARGs, indicated that MGEs may play an important role in the persistence and proliferation of ARGs. A Mantel test and Procrustes analysis suggested that ARG profiles were significantly correlated with bacterial community. Variation partitioning analysis (VPA) further demonstrated that bacterial community shifts contribute 65.8% of the total ARG variations. Additionally network analysis revealed that 15 bacterial taxa at family level might be the potential hosts of ARGs. These findings provide evidence that groundwater near MSW landfill is an underappreciated hotspot of antibiotic resistance and contribute to the spread of ARGs via the flowing contaminated groundwater.

Urban green spaces are closely related to the activities and health of urban residents. Turf grass and soil are two major interfaces between the environmental and human microbiome, which represent potential pathways for the spread of antibiotic resistance genes (ARGs) from environmental to human microbiome through skin-surface contact. However, the information regarding the prevalence of ARGs in urban green spaces and drivers in shaping their distribution patterns remain unclear. Here, we profiled a wide spectrum of ARGs in grass phyllosphere and soils from 40 urban parks across Greater Melbourne, Australia, using high throughput quantitative PCR. A total of 217 and 218 unique ARGs and MGEs were detected in grass phyllosphere and soils, respectively, conferring resistance to almost all major classes of antibiotics commonly used in human and animals. The plant microbiome contained a core resistome, which occupied >84% of the total abundance of ARGs. In contrast, no core resistome was identified in the soil microbiome. The difference between plant and soil resistome composition was attributed to the difference in bacterial community structure and intensity of environmental and anthropogenic influence. Most importantly, the abundance of ARGs in urban green spaces was significantly positively related to industrial factors including total number of business, number of manufacturing, and number of electricity, gas, water and waste services in the region. Structural equation models further revealed that industrial distribution was a major factor shaping the ARG profiles in urban green spaces after accounting for multiple drivers. These findings have important implications for mitigation of the potential risks posed by ARGs to urban residents. Keywords: Resistome, Urban green spaces, Phyllosphere, Anthropogenic activities, Public health