Integrating Multiple Bacterial Phenotypes and Bayesian Network for Analyzing Health Risks of Pathogens in Plastisphere.

Plastic pollution represents a critical threat to soil ecosystems and even humans, as plastics can serve as a habitat for breeding and refuging pathogenic microorganisms against stresses. However, evaluating the health risk of plastispheres is difficult due to the lack of risk factors and quantification model. Here, DNA sequencing, single-cell Raman-D2O labeling, and transformation assay were used to quantify key risk factors of plastisphere, including pathogen abundance, phenotypic resistance to various stresses (antibiotic and pesticide), and ability to acquire antibiotic resistance genes. A Bayesian network model was newly introduced to integrate these three factors and infer their causal relationships. Using this model, the risk of pathogen in the plastisphere is found to be nearly 3 magnitudes higher than that in free-living state. Furthermore, this model exhibits robustness for risk prediction, even in the absence of one factor. Our framework offers a novel and practical approach to assessing the health risk of plastispheres, contributing to the management of plastic-related threats to human health.

Temporal dynamics of soil bacterial network regulate soil resistomes.

Soil bacteria are diverse and form complicated ecological networks through various microbial interactions, which play important roles in soil multi-functionality. However, the seasonal effects on the bacterial network, especially the relationship between bacterial network topological features and soil resistomes remains underexplored, which impedes our ability to unveil the mechanisms of the temporal-dynamics of antibiotic resistance genes (ARGs). Here, a field investigation was conducted across four seasons at the watershed scale. We observed significant seasonal variation in bacterial networks, with lower complexity and stability in autumn, and a wider bacterial community niche in summer. Similar to bacterial communities, the co-occurrence networks among ARGs also shift with seasonal change, particularly with respect to the topological features of the node degree, which on average was higher in summer than in the other seasons. Furthermore, the nodes with higher betweenness, stress, degree, and closeness centrality in the bacterial network showed strong relationships with the 10 major classes of ARGs. These findings highlighted the changes in the topological properties of bacterial networks that could further alter antibiotic resistance in soil. Together, our results reveal the temporal dynamics of bacterial ecological networks at the watershed scale, and provide new insights into antibiotic resistance management under environmental changes.

Brand-Specific Toxicity of Tire Tread Particles Helps Identify the Determinants of Toxicity.

The widespread occurrence of tire tread particles (TPs) has aroused increasing concerns over their impacts. However, how they affect the soil fauna remains poorly understood. Here, based on systematically assessing the toxicity of TPs on soil model speciesEnchytraeus crypticusat environmentally relevant concentrations through both soil and food exposure routes, we reported that TPs affected gut microbiota, intestinal histopathology, and metabolites of the worms both through particulate- and leachate-induced effects, while TP leachates exerted stronger effects. The dominant role of TP leachates in TP toxicity was further explained by the findings that worms did not ingest TPs with a particle size of over 150 µm and actively avoided consuming TP particles. Moreover, by comparing the effects of different brands of TPs as well as new and aged TPs, we demonstrated that it was mainly TP leachates that resulted in the ubiquity of the disturbance in the worm's gut microbiota among different brands of TPs. Notably, the large variations in leachate compositions among different brands of TPs provided us a unique opportunity to identify the determinants of TP toxicity. These results provide novel insights into the toxicity of TPs to soil fauna and a reference for toxicity reduction of tires.

Aridity differentially alters the stability of soil bacterial and fungal networks in coastal and inland areas of Australia.

Despite the importance of soil bacterial and fungal communities for ecosystem services and human welfare, how their ecological networks respond to climatic aridity have yet been evaluated. Here, we collected soil samples from 47 sites across 2500 km in coastal and inland areas of eastern Australia with contrasting status of aridity. We found that the diversity of both bacteria and fungi significantly differed between inland and coastal soils. Despite the significant differences in soil nutrient availability and stoichiometry between the inland and coastal regions, aridity was the most important predictor of bacterial and fungal community compositions. Aridity has altered the potential microbial migration rates and further impacted the microbial assembly processes by increasing the importance of stochasticity in bacterial and fungal communities. More importantly, ecological network analysis indicated that aridity enhanced the complexity and stability of the bacterial network but reduced that of the fungal network, possibly due to the contrasting impacts of aridity on the community-level habitat niche breadth and overlaps. Our work paves the way towards a more comprehensive understanding of how climate changes will alter soil microbial communities, which is integral to predicting their long-term consequences for ecosystem sustainability and resilience to future disturbances.

Impacts of global change on the phyllosphere microbiome.

Plants form complex interaction networks with diverse microbiomes in the environment, and the intricate interplay between plants and their associated microbiomes can greatly influence ecosystem processes and functions. The phyllosphere, the aerial part of the plant, provides a unique habitat for diverse microbes, and in return the phyllosphere microbiome greatly affects plant performance. As an open system, the phyllosphere is subjected to environmental perturbations, including global change, which will impact the crosstalk between plants and their microbiomes. In this review, we aim to provide a synthesis of current knowledge of the complex interactions between plants and the phyllosphere microbiome under global changes and to identify future priority areas of research on this topic.

Microbial Multitrophic Communities Drive the Variation of Antibiotic Resistome in the Gut of Soil Woodlice (Crustacea: Isopoda).

Multitrophic communities inhabit in soil faunal gut, including bacteria, fungi, and protists, which have been considered a hidden reservoir for antibiotic resistance genes (ARGs). However, there is a dearth of research focusing on the relationships between ARGs and multitrophic communities in the gut of soil faunas. Here, we studied the contribution of multitrophic communities to variations of ARGs in the soil woodlouse gut. The results revealed diverse and abundant ARGs in the woodlouse gut. Network analysis further exhibited strong connections between key ecological module members and ARGs, suggesting that multitrophic communities in the keystone ecological cluster may play a pivotal role in the variation of ARGs in the woodlouse gut. Moreover, long-term application of sewage sludge significantly altered the woodlice gut resistome and interkingdom communities. The variation portioning analysis indicated that the fungal community has a greater contribution to variations of ARGs than bacterial and protistan communities in the woodlice gut after long-term application of sewage sludge. Together, our results showed that changes in gut microbiota associated with agricultural practices (e.g., sewage sludge application) can largely alter the gut interkingdom network in ecologically relevant soil animals, with implications for antibiotic resistance, which advances our understanding of the microecological drivers of ARGs in terrestrial ecosystem.

Semi-solid state promotes the methane production during anaerobic co-digestion of chicken manure with corn straw comparison to wet and high-solid state.

Total solid content (TS) is an important factor for biogas production during anaerobic digestion. In this study, we explored the influence of different TS (5% wet, 15% semi-solid and 25% solid state) on the relative cumulative methane production (RCMP) during anaerobic co-digestion of chicken manure with corn straw. Results showed that total ammonium nitrogen and free ammonia nitrogen concentration increased with the increase of TS. Ammonium nitrogen in treatments at 15% TS was 2.25-2.76 times as high as that at 5% TS, which was below 3 times. The highest chemical oxygen demand removal and RCMP were obtained in the treatment of 15% TS with a ratio of 2:1 chicken manure: corn straw (based on TS). The RCMP in the treatments of 15% TS were 3.63-4.59 times higher than that of 5% TS based on the volume of substrates. The abundance of Caldicoprobacter improving the degradation of corn straw was significantly positively correlated with the RCMP, and the average abundance of Caldicoprobacter at 15% TS was 8.33 and 7.02 times higher than that at 5% and 25% TS, respectively. Structural equation models analysis suggested that TS significantly impacted the RCMP by indirectly impacting free ammonia nitrogen and microbial abundance. These findings indicated semi-solid state (15% TS) decreased ammonia nitrogen releasing and improved the abundance of Caldicoprobacter, and increased RCMP during anaerobic co-digestion of chicken manure with corn straw.

Termite mound formation reduces the abundance and diversity of soil resistomes.

Termites are pivotal ecosystem engineers in tropical and subtropical habitats, where they construct massive nests ('mounds') that substantially modify soil properties and promote nutrient cycling. Yet, little is known about the roles of termite nesting activity in regulating the spread of antimicrobial resistance (AMR), one of the major Global Health challenges. Here, we conducted a large-scale (> 1500 km) investigation in northern Australia and found distinct resistome profiles in termite mounds and bulk soils. By profiling a wide spectrum of ARGs, we found that the abundance and diversity of antibiotic resistance genes (ARGs) were significantly lower in termite mounds than in bulk soils (P < 0.001). The proportion of efflux pump ARGs was significantly lower in termite mound resistome than in bulk soil resistome (P < 0.001). The differences in resistome profiles between termite mounds and bulk soils may result from the changes in microbial interactions owing to the substantial increase in pH and nutrient availability induced by termite nesting activities. These findings advance our understanding of the profile of ARGs in termite mounds, which is a crucial step to evaluate the roles of soil faunal activity in regulating soil resistome under global environmental change.

Precipitation increases the abundance of fungal plant pathogens in Eucalyptus phyllosphere.

Understanding the current and future distributions of plant pathogens is critical to predict the plant performance and related economic benefits in the changing environment. Yet, little is known about the roles of environmental drivers in shaping the profiles of fungal plant pathogens in phyllosphere, an important habitat of microbiomes on Earth. Here, using a large-scale investigation of Eucalyptus phyllospheric microbiomes in Australia and the multiple linear regression model, we show that precipitation is the most important predictor of fungal taxonomic diversity and abundance. The abundance of fungal plant pathogens in phyllosphere exhibited a positive linear relationship with precipitation. With this empirical dataset, we constructed current and future atlases of phyllosphere plant pathogens to estimate their spatial distributions under different climate change scenarios. Our atlases indicate that the abundance of fungal plant pathogens would increase especially in the coastal regions with up to 100-fold increase compared with the current abundance. These findings advance our understanding of the distributions of fungal plant pathogens in phyllospheric microbiomes under the climate change, which can improve our ability to predict and mitigate their impacts on plant productivity and economic losses.

Termite mounds reduce soil microbial diversity by filtering rare microbial taxa.

Termites are ubiquitous insects in tropical and subtropical habitats, and some of them construct massive nests ('mounds'), which substantially promote substrate heterogeneity by altering soil properties. Yet, the role of termite nesting process in regulating the distribution and diversity of soil microbial communities remains poorly understood, which introduces uncertainty in predictions of ecosystem functions of termite mounds in a changing environment. Here, by using amplicon sequencing, we conducted a survey of 134 termite mounds across >1500 km in northern Australia and found that termite mounds significantly differed from bulk soils in the microbial diversity and community compositions. Compared with bulk soils, termite nesting process decreased the microbial diversity and the relative abundance of rare taxa. Rare taxa had a narrower habitat niche breadth than dominant taxa and might be easier to be filtered by the potential intensive microbial competition during the nesting processes. We further demonstrated that the shift in pH induced by termite nesting process was a major driver shaping the microbial community profiles in termite mounds. Together, our work provides novel evidence that termite nesting is an important process in regulating soil microbial diversity, which advances our understanding of the functioning of termite mounds.

Fates of Antibiotic Resistance Genes in the Gut Microbiome from Different Soil Fauna under Long-Term Fertilization.

Applying organic fertilizers has been well documented to facilitate the dissemination of antibiotic resistance genes (ARGs) in soil ecosystems. However, the role of soil fauna in this process has been seldom addressed, which hampers our ability to predict the fate of and to manage the spread of ARGs. Here, using high-throughput quantitative polymerase chain reaction (HT-qPCR), we examined the effect of long-term (5-, 8-, and 10-year) fertilization treatments (control, inorganic fertilizers, and mixed fertilizers) on the transfer of ARGs between soil, nematodes, and earthworms. We found distinct fates for ARGs in the nematodes and earthworms, with the former having higher enriched levels of ARGs than the latter. Fertilization impacted the number and abundance of ARGs in soil, and fertilization duration altered the composition of ARGs. Shared ARGs among soil, nematodes, and earthworm guts supported by a fast expectation-maximization microbial source tracking analysis demonstrated the trophic transfer potential of ARGs through this short soil food chain. The transfer of ARGs was reduced by fertilization duration, which was mainly ascribed to the reduction of ARGs in the earthworm gut microbiota. This study identified the transfer of ARGs in the soil-nematode-earthworm food chain as a potential mechanism for a wider dissemination of ARGs in the soil ecosystem.

Manure Application Did Not Enrich Antibiotic Resistance Genes in Root Endophytic Bacterial Microbiota of Cherry Radish Plants.

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.

Oxytetracycline and Ciprofloxacin Exposure Altered the Composition of Protistan Consumers in an Agricultural Soil.

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.

Temporal Dynamics of Antibiotic Resistome in the Plastisphere during Microbial Colonization.

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.

Dysbiosis in the Gut Microbiota of Soil Fauna Explains the Toxicity of Tire Tread Particles.

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.

Adsorbed Sulfamethoxazole Exacerbates the Effects of Polystyrene (∼2 µm) on Gut Microbiota and the Antibiotic Resistome of a Soil Collembolan.

Microplastics pollution in the environment is now receiving worldwide attention; however, the effects of copollution of antibiotics and microplastics on the gut microbiome of globally distributed and functionally important nontarget soil animals remain poorly understood. We studied a model collembolan (Folsomia candida) and found that the ingestion of microplastics (polystyrene, 2-2.9 µm) substantially altered the gut microbiome, antibiotic resistance gene (ARG) profile, and the isotopic fractionation in the soil collembolan tissue. Importantly, collembolans exposed to polystyrene microplastics loaded with sulfamethoxazole (MA) presented a distinctive gut microbiome, ARG profile, and isotopic fractionation compared to those exposed to polystyrene alone (MH). We observed that the abundance of ARGs and mobile genetic elements (MGEs) in the MA-treated collembolan guts was significantly higher than in the MH and the control treatments. There were also strong interactions between the gut microbiome and ARGs in the collembolan guts. We further found that bacterial ß-diversity correlated significantly with the δ13C and δ15N values in collembolan body tissues. Together, our results indicate that changes in isotopic fractionation and ARG profiles in the collembolan were induced by the changes in gut microbiota and suggest that microplastics from diverse sources may have profound influences on soil fauna and soil food webs.

Organic Carbon Amendments Affect the Chemodiversity of Soil Dissolved Organic Matter and Its Associations with Soil Microbial Communities.

The "4 per mil" initiative recognizes the pivotal role of soil in carbon resequestration. The need for evidence to substantiate the influence of agricultural practices on chemical nature of soil carbon and microbial biodiversity has become a priority. However, owing to the molecular complexity of soil dissolved organic matter (DOM), specific linkages to microbial biodiversity have eluded researchers. Here, we characterized the chemodiversity of soil DOM, assessed the variation of soil bacterial community composition (BCC), and identified specific linkages between DOM traits and BCC. Sustained organic carbon amendment significantly ( P < 0.05) increased total organic matter reservoirs, resulted in higher chemodiversity of DOM and emergence of recalcitrant moieties (H/C < 1.5). In the meantime, sustained organic carbon amendment shaped the BCC to a more eutrophic state while long-term chemical fertilization directed the BCC toward an oligotrophic state. Meanwhile, higher connectivity and complexity were observed in organic carbon amendment by DOM-BCC network analysis, indicating that soil microbes tended to have more interaction with DOM molecules after organic matter inputs. These results highlight the potential for organic carbon amendments to not only build soil carbon stocks and increase their resilience but also mediate the functional state of soil bacterial communities.

Impact of Wastewater Treatment on the Prevalence of Integrons and the Genetic Diversity of Integron Gene Cassettes.

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.

Exposure of a Soil Collembolan to Ag Nanoparticles and AgNO3 Disturbs Its Associated Microbiota and Lowers the Incidence of Antibiotic Resistance Genes in the Gut.

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.

Land Use Influences Antibiotic Resistance in the Microbiome of Soil Collembolans Orchesellides sinensis.

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.