The excessive use of antimicrobials has led to increasing levels of antimicrobial resistance that can spread between livestock, humans and the environment, with important One Health implications. Antimicrobial use in the livestock sector represents 73

Urban canals, as highly engineered ecosystems, exhibit distinct ecological dynamics compared to natural rivers due to altered hydrology and anthropogenic regulation. This study investigates seasonal succession in multi-trophic communities—from bacteria to fish—using environmental DNA in a navigable urban canal. We elucidate how seasonal environmental shifts and anthropogenic stressors interactively shape community structure. Our results demonstrated that seasonal hydrological regimes orchestrated community assembly through distinct mechanisms. In the dry season, nutrient accumulation (e.g., nitrogen, phosphorus) and redox conditions drove community structure. In contrast, the wet season was characterized by runoff-induced physicochemical shifts and biological pollution pulses, which exerted stronger selection pressures than organic loading alone. Notably, unlike planktonic communities that tracked these seasonal shifts, the fish assemblage exhibited temporal invariance, likely due to habitat homogenization and the dominance of tolerant generalists. These dynamics drove a functional shift from heterotrophic decomposition in the dry season to autotrophic primary production in the wet season. The phylogenetic null model analysis revealed that stochastic processes overwhelmingly dominated the community assembly. Our findings reveal predictable ecological state shifts in heavily regulated navigable urban canals, underscoring the need for season-specific management strategies targeting point-source pollution in the dry season and urban runoff control in the wet season.

Microbial inoculation is widely used to improve composting performance, yet its effectiveness hinges on inoculum composition, substrate characteristics, and composting technology, which remain poorly understood. This study compared single versus mixed inoculants across different substrates and assessed their interactions with biochar amendment and nanomembrane covering, focusing on organic matter transformation, inorganic nutrient dynamics, and biological pollution control. Mixed inoculation significantly improved heating performance in cattle manure compost compared to single strains (p < 0.05) and sustained thermophilic conditions in sludge-sawdust compost, but showed limited impact in chicken manure-sludge compost. It reduced humic acid (HA) accumulation in chicken manure-sludge compost (14.29% to −39.28%) while increasing HA content in sludge-sawdust compost (3.55–5.41 g/kg, p < 0.05). Inorganic nitrogen retention was enhanced; specifically NO3−-N concentrations rose by 175.1–222.6% in the chicken manure-sludge and by 6.7–17.9% in the sludge-sawdust compost. Microbial community analysis indicated enrichment of inoculant strains during the thermophilic phase, supporting nitrogen conservation and humification. However, inoculation increased potential pathogenic bacteria by over 51.2% across all composts and enriched predicted antibiotic resistance genes (ARGs) by 9.9–22.96% in chicken manure-sludge compost, while reducing the membrane covering’s inhibitory effect on predicted ARGs (rebound by 29.5%). Moreover, we found that the predicted ARG profiles, derived from 16S-based PICRUSt2 functional inference, covaried strongly with microbial community structure, with environmental factors such as organic carbon shaping predicted ARG dynamics mainly through indirect effects on microbial communities. These findings highlight that while mixed inoculation boosts composting efficiency, it also raises biosafety concerns. Thus, a comprehensive evaluation integrating organic, inorganic, and biological perspectives is essential before promoting thermophilic inoculants.

Phage-host interactions critically shape environmental antimicrobial resistance (AMR). Using swine manure anaerobic digestion and multi-omics (metagenomics, meta-transcriptomics, and Hi-C), we mapped the phage-bacteria arms race and its impact on AMR dynamics. We revealed that phage-mediated lysis overwhelmingly dominates transduction, while phages rarely carry antimicrobial resistance genes (ARGs), and phage-borne ARGs showed no expression, challenging the paradigm of phages as primary vectors of ARGs. Crucially, the intense on-going phage-host arms race drives the widespread presence and expression of antiviral defense systems (ADSs) in antimicrobial-resistant bacteria (ARB). These ADSs exhibit a vital ecological dual role: they protect ARBs from phage lysis promoting persistence while simultaneously suppressing horizontal gene transfer (HGT, e.g., conjugation), as validated by in vitro conjugation assays. Our findings elucidate this duality, offering a novel framework to harness phage lytic pressure and ADS-mediated HGT suppression for environmental AMR mitigation.

Abstract Composting mitigates the global burden of antimicrobial resistance (AMR) in livestock manure, yet the viral biosphere’s role in resistome dynamics remains a black box. Leveraging a massive global dataset of 420 metagenomes and 42 viromes, we decipher the phage-host evolutionary arms race during ecological succession. While composting reduces total antimicrobial resistance genes (ARGs), it is bottlenecked by the selective enrichment of thermophilic antimicrobial-resistant bacteria (ARB), which dominate the residual maturation-phase resistome (∼ 60%). Among 106,387 identified viral operational taxonomic units (vOTUs), lytic interactions (39.5%) quantitatively override transduction (< 0.5%) in driving ARB decline. Correspondingly, the relative abundance of ARG-reducing vOTUs (7.24%) is orders of magnitude higher than that of vOTUs involved in transduction (0.06%) (p < 0.001). However, intense viral predation drives surviving ARB to evolve enriched antiviral defense systems (ADS) against lysis. Intriguingly, this immune shield acts as a genetic firewall, likely restricting the horizontal acquisition of ARGs via transformation or conjugation. Our findings redefine composting as an intense evolutionary arena and suggest that precision strategies targeting the thermophilic reservoir are essential for maximizing ARG mitigation and safer manure recycling.

The imbalance among nitrite supply, nitrate accumulation and aeration demand poses significant challenges in single-stage partial nitrification-anammox (PN/A) systems for stably treating high-strength anaerobically digested swine wastewater (ADSW) at low C/N ratio. Here, we propose a novel process that integrates real-time NH4+-N control with a floc-granule partitioned biomass architecture in a sequence anoxic-oxic-anoxic (SAOA) system to dynamically modulate free ammonia (FA) concentration while preventing free nitrous acid (FNA) inhibition. By maintaining an NH4+-N endpoint of 50 mg/L, FA was stabilized at 5.2 mg N/L, and FNA was effectively suppressed. Thus, the SAOA system achieved 94.14% TN removal at a loading rate of 0.24 kg N/(m3·d) and 92.71% COD at low influent COD/TN ratio of 1.71, respectively. Metagenomic and enzymatic profiling revealed a distinct ecological stratification: floccular biomass was enriched with Candidatus Kuenenia, whereas granular microenvironments favored ammonia-oxidizing bacteria (AOB), accompanied by the upregulation of key nitrification and anammox genes. Kinetic analysis of COD and NH4+-N removals revealed a stage-specific metabolic transition from carbon-driven to autotrophic nitrogen-dominated removal. This study provides mechanistically robust and scalable control paradigm for advancing simultaneous, high-efficiency nitrogen and carbon removal from nitrogen-rich and carbon-limited wastewater.

Heavy rainfall events increasingly challenge the treatment capacity and operational stability of wastewater treatment plants (WWTPs), thereby compromising their resilience in safeguarding urban aquatic environments. Nevertheless, how and to what extent heavy rainfall alerts wastewater treatment processes and DOM transformation patterns are still not well understood. This study characterized dissolved organic matter (DOM) compositional dynamics in a full-scale municipal WWTP during heavy rainfall with non-rainfall (dry-weather) baseline, integrating the analysis of excitation-emission matrix spectroscopy, UV-Vis absorbance, and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Results showed that heavy rainfall suppressed microbial degradation functions, leading to the persistence of protein-like DOM and soluble microbial products across treatment units. These effects reflect reduced hydraulic retention times and biomass washout. Despite diluted influent total phosphorus (TP, 2.5-3.0 mg/L), effluent TP concentrations (2.3-2.9 mg/L) exceeded the discharge standard of 0.5 mg/L), indicating compromised phosphorus removal efficiency. FT-ICR-MS analysis showed an increased in molecular richness and the formation of persistent phosphorus-containing formulas, which exhibited higher double bond equivalents (DBE) and lower Gibbs free energy, suggesting decreased microbial degradability. In addition, the lignin-and lipid-like molecules were enriched during heavy rainfall events. Ecological modeling confirmed stochastic DOM assembly under heavy rainfall, as evidenced by a lognormal species abundance distribution and a good fit to Sloan's neutral model. Molecular traits (e.g., N/C_wa, S/C_wa) were more effective than conventional parameters (e.g., TOC, TP) in explaining DOM compositional changes under rainfall. These results highlight a transition from biologically structured to trait-mediated stochastic DOM assembly under hydrological stress, emphasizing the necessity for adaptive wastewater treatment plant strategies, including improved phosphorus management, continuous DOM composition monitoring, and the preservation of microbial resilience, to address climate-induced disruptions.

The escalating crisis of antimicrobial resistance (AMR) requires innovative interventions beyond conventional antibiotics. Bacteriophages, as natural bacterial predators, represent a promising alternative. However, their influence is governed by a dynamic evolutionary continuum that determines whether they eradicate or maintain AMR populations. This work introduces a tripartite evolutionary framework to elucidate phage-host interactions: (1) an antagonistic arms race state that drives the diversification of molecular defenses; (2) a selfish protection state in which phages enhance host fitness to secure their own persistence; and (3) ecological feedback mechanisms that regulate transitions between these states. The potential for manipulating these evolutionary states to transform AMR control strategies is examined. Recent metagenomic and mechanistic evidence indicates that the arms race state provides a genetic toolkit for targeting AMR pathogens, whereas the protective state, mediated by auxiliary metabolic genes (AMGs) and superinfection exclusion, may inadvertently stabilize AMR lineages. Notably, environmental context is shown to govern the shift between kill-the-winner and piggyback-the-winner dynamics, ultimately influencing the efficiency of AMR transfer. This analysis highlights how targeted manipulation of this evolutionary triad could facilitate the development of advanced phage therapeutics and ecological engineering approaches to mitigate global AMR dissemination within the One Health framework.

Methanogenesis is a critical driver of global carbon cycling and bioenergy recovery, yet how bacteriophages modulate methane production remains poorly understood. Here, we integrated high-throughput chromosome conformation capture (Hi-C) with multi-omics to map in situ phage-host interactions through an over 440 day anaerobic digestion experiment. We captured 6100 active physical linkages, revealing that 16.3 +/- 2.1% of interactions involved auxiliary metabolic genes (AMGs). Notably, we observed a dynamic community-level compositional shift in viral life strategies driven by operational stress. Under mesophilic conditions, lysogenic piggyback-the-winner dynamics prevailed, with AMGs enhancing host competitiveness. Conversely, thermophilic conditions with high total solids stress triggered lytic kill-the-winner strategies. During this phase, AMGs supported rapid phage replication targeting overproliferating bacteria to restore the disrupted balance between acidogenesis and methanogenesis. Furthermore, we identified 529 DNA viral operational taxonomic units (vOTUs) directly infecting methanogens, alongside broad-host-range phages spanning bacterial and archaeal domains. Importantly, we detected four RNA vOTUs exclusively under thermophilic conditions, providing the first omics-based evidence of RNA phages actively infecting methanogenic archaea. These findings highlight phages as important modulators of methane production, offering a foundational framework for developing targeted phage-engineering strategies to optimize bioenergy systems and mitigate methane emissions.

Methanogenesis is a critical driver of global carbon cycling and bioenergy recovery, yet how bacteriophages modulate methane production remains poorly understood. Here, we integrated high-throughput chromosome conformation capture (Hi-C) with multi-omics to map in situ phage-host interactions through an over 440 day anaerobic digestion experiment. We captured 6100 active physical linkages, revealing that 16.3 ± 2.1% of interactions involved auxiliary metabolic genes (AMGs). Notably, we observed a dynamic community-level compositional shift in viral life strategies driven by operational stress. Under mesophilic conditions, lysogenic piggyback-the-winner dynamics prevailed, with AMGs enhancing host competitiveness. Conversely, thermophilic conditions with high total solids stress triggered lytic kill-the-winner strategies. During this phase, AMGs supported rapid phage replication targeting overproliferating bacteria to restore the disrupted balance between acidogenesis and methanogenesis. Furthermore, we identified 529 DNA viral operational taxonomic units (vOTUs) directly infecting methanogens, alongside broad-host-range phages spanning bacterial and archaeal domains. Importantly, we detected four RNA vOTUs exclusively under thermophilic conditions, providing the first omics-based evidence of RNA phages actively infecting methanogenic archaea. These findings highlight phages as important modulators of methane production, offering a foundational framework for developing targeted phage-engineering strategies to optimize bioenergy systems and mitigate methane emissions.

Antimicrobial resistance (AMR) poses a critical global health challenge, with livestock manure acting as a significant environmental reservoir for antimicrobial resistance genes (ARGs). Anaerobic digestion (AD) is a pivotal process for mitigating ARG dissemination at the livestock-environment-human interface. This study aims to elucidate the global dynamics of ARGs in AD systems, focusing on virus-host interactions and arms race, to identify actionable strategies for AMR control. We analyzed 205 metagenomic (4.5 Tb) and 36 meta-transcriptomic (640 Gb) datasets, including 15 newly generated datasets, revealing that pig manure AD harbors the highest ARG abundance (0.668 ARGs/16S rRNA), while AD systems generally exhibit limited transcriptional activation of ARGs. We constructed a viral dataset for livestock manure AD (GVD_LMAD), comprising 59,316 DNA and 727 RNA viral operational taxonomic units (vOTUs). Virus-host interactions established by CRISPR-Cas spacer, tRNA and homology matches revealed 889 lytic infections of antimicrobial-resistant bacteria (ARB) compared to only 18 ARG transduction events. Further analysis showed that the relative abundance of vOTUs assigned to the reduction role (4.11% ± 3.19%) was substantially higher than that of reproduction (0.72% ± 0.64%) and transduction (0.19% ± 0.30%), demonstrating that, among viral processes, lysis outweighs transduction in contributing to ARG abundance reduction in AD. Furthermore, an antiviral defense system (ADS) catalogue (GADSC_LMAD), derived from 2760 high-quality metagenome-assembled genomes (MAGs) containing 39,307 ADS, with ADS prevalence in ARB (7.8 ± 6.0 per MAG), indicating an intensified virus-host arms race in AD that may shield ARB from phage lysis. The resulting CRISPR-Cas immune network with expressed spacers targets foreign ARG-carrying sequences (primarily plasmids and ICEs), suggesting a mechanism that restricts horizontal gene transfer (HGT) via conjugation and transformation, despite shielding ARB from phage lysis. Collectively, these findings highlight that viral communities significantly contribute to ARG reduction through phage lysis relative to transduction, while the ADS-mediated arms race, despite protecting ARB, constructs a biological firewall that potentially limits HGT of ARGs. This study provides novel insights into virus-host dynamics as a key mechanism for controlling ARG dissemination in AD systems.

Beyond abiotic factors, microbial interactions are critical yet understudied regulators of environmental antibiotic resistance gene (ARG) spread, whose neglect hinders control efforts. We synthesize their dual roles in ARG dissemination and advocate for integrated advanced methodologies combining tracking, modeling, and validation to decode interaction networks. Moreover, we propose novel interventions strategies that range from molecular disruption to network re-engineering, leveraging ecological insights to mitigate resistance spread across environmental compartments.

This study investigated the effects of chlortetracycline (CTC) and copper (Cu), individually and in combination, on the transfer and expression of antibiotic resistance genes (ARGs) in anaerobic digestion (AD). Utilizing metagenomics and high-throughput quantitative PCR (HT-qPCR), we found that high concentrations of Cu (400 mgL (-1)) and CTC (80 mgL (-1)) significantly enriched the relative abundance of ARGs attributed to the changes of bacterial community (p < 0.05), whereas lower concentrations (Cu: 40 mgL (-1); CTC: 8 mgL (-1)) had minimal effects. The species abundance distribution and neutral community models indicated that antibiotic resistome is determined by stochastic processes, and the abundance of ARGs is determined by a limited number of core ARGs, showing the resilience to the selection of antibiotics and heavy metals. Reverse transcription HT-qPCR indicated that most ARGs were silent or expressed at low levels; however, regardless of the concentration, CTC enhanced the expression of ARGs, particularly those linked to tetracycline ribosome protection. In contrast, high Cu levels inhibited ARGs expression due to its non-selective toxicity, which was evidenced by a failure to produce methane. CTC and Cu both showed limited impacts on the potential mobility of ARGs shown by metagenomics, although TET significantly increased the conjugation frequency reflected by conjugation assays (p < 0.05). ARGs carried by virus only accounted for 0.26 % +/- 0.10 % of the total, with no evidence of phage-mediated transduction, and phage lysis significantly contributed to ARGs reduction. While CTC and Cu showed limited effects on phage-carrying ARGs, they notably inhibited phage lytic activity, as indicated by virus-host ratios and phage lysis tests, further leading to the enrichment of ARGs in AD system. Our findings provide novel insights into the ARGs transfer under selective pressure of antibiotics and heavy metals in AD.

Bacteriophages are pivotal in shaping microbial communities, but their structural and functional responses to antibiotic stress in aerobic biofilms remain underexplored. This study aims to fill this void by providing a comprehensive understanding of how viral communities in aerobic biofilms adapt to increasing antibiotic pressures through interactions with their bacterial hosts. Three lab-scale aerobic biofilm systems were established and operated for 577 days, two of those were exposed to increasing influent concentrations of oxytetracycline (OTC) and streptomycin (STM), respectively. The dynamics of the biofilm virome under antibiotic stress was revealed by metagenomic sequencing. Results showed that the virome in aerobic biofilms displayed a high percentage (98.7 %) of unknown bacteriophages, indicating considerable viral diversity. As for the hosts of phages, a total of 1741 bacteriophage contigs were associated with 660 distinct bacterial hosts. In antibiotic-treated systems, broad-host-range generalist bacteriophages accounted for over 17.95 % (STM) and 17.90 % (OTC), compared to 14.32 % in the control. Furthermore, viral community did not carry diverse antibiotic resistance genes, which only accounted for 0.34 % of the resistome. Additionally, it did not regulate the number of resistant bacteria by activating the lytic and lysogenic cycles in this study. This indicated that the contribution of transduction to the horizontal spread of resistant determinants is very limited in the aerobic biofilm. Under antibiotic stress, viral auxiliary metabolic genes compensated for incomplete metabolic pathways in host cells, particularly those related to carbohydrate, amino acid, and cofactor metabolism. These genes likely offer dual benefits to bacterial hosts by repairing antibiotic-induced cellular damage and supporting energy generation, thereby providing adaptive advantages for bacterial survival and proliferation under antibiotic selection pressure. This study uncovers the complex interactions between bacteriophages, their hosts, and environmental pressures. It suggests that viral communities in these environments compensate for functional metabolism rather than promote resistance development under antibiotic stress, providing new insights into the potential roles of bacteriophages in the regulation of microbial-driven processes.

Treated wastewater from wastewater treatment plants (WWTPs) is a major contributor to the transfer of antibiotic resistance genes (ARGs) into urban rivers. However, the role of viral communities in this process remains poorly understood. This study focused on North Canal in Beijing, China, which receives over 80 % of its water from treated wastewater, to investigate the impact of viral communities on ARGs transfer. Results showed significant seasonal variation in the abundance and composition of ARGs, with 30 high-risk ARGs detected, accounting for 1.50 % ± 1.28 % of total ARGs. The assembly of ARGs in North Canal followed a stochastic process of homogenizing dispersal, with conjugative mobility playing a key role in horizontal gene transfer with Pseudomonas as primary host for HGT. The potential conjugative mobility of ARGs is significantly higher in wet season (69.4 % ± 17.3 %) compared to dry season (42.9 % ± 17.1 %), with conjugation frequencies ranging from 1.18 × 10-6 to 2.26 × 10-4. Viral species accumulation curves approaching saturation indicated the well captured viral diversity, and no phages carrying ARGs were found among 27,523 non-redundant viral operational taxonomic units. Most of the phages (89.2 % ± 3.8 %) were lytic in North Canal, which were observed to contribute to ARGs reduction by lysing their host bacteria, reflected by higher virus-host ratio and demonstrated by the phage lysis assays in treated wastewater and receiving river. We provided compelling evidence that phage-host interactions can reduce ARGs through host lysis, highlighting their potential role in mitigating ARG transmission in urban rivers receiving treated wastewater.

Global river systems are grappling with severe pollution from antibiotic resistance genes (ARGs), with river-reservoir (R-R) systems being a common feature in urban waterways. The intensified extreme rainfall events triggered by global climate change exacerbate the spread of ARGs posed by non-point source pollution and combined sewage overflows. This study employs a metagenomics approach to decipher the profile of ARGs and virus-host interactions driving their transfer under heavy rainfall in North Canal, Beijing, with extensive R-R systems. Results indicated that R-R systems contributed to ARGs reduction despite continuous discharge of treated wastewater into the North Canal. The ARGs assembly is predominantly governed by stochastic process, and heavy rainfall enhances the dispersal capability. Nonetheless, the deterministic process determined the assembly of both microbial and viral community. Heavy rainfall not only significantly increased the abundance and diversity of ARGs within the rivers with minimal change in the reservoir, but also promotes the horizontal gene transfer of ARGs with higher conjugative mobility. Although the species accumulation curves approached saturation, no viruses carrying ARGs were detected among the 23,835 non-redundant viral operational taxonomic units (vOTUs), and lytic phage-ARB interactions drove the ARGs reduction with higher VHRs, highlighting its contribution to the reduction of ARGs in R-R system after heavy rainfall.

The relationship between chemodiversity and microbial succession in wastewater treatment plants (WWTPs) is highly intricate and bidirectional. The specific contribution of the microbial community to changes in the composition of dissolved organic matter (DOM) within different biological treatment units remains unclear, as does the reciprocal influence of DOM composition on microbial succession. In this study, spectroscopy ((Excitationemission matrix) EEM-PARAFAC, Ultraviolet (UV) -spectrum, Fourier transform infrared spectrometer (FT-IR)), Liquid chromatograph mass spectrometer (LC-MS) and Fourier transform ion cyclotron resonance (FT-ICR) MS along with high -throughput sequencing technology were used to explore the relationship between chemodiversity and microbial succession in WWTPs concerning seasonal changes. The results showed that WWTPs with anaerobic/anoxic/oxic (A 2 O) processes can metabolize and transform most of the wastewater DOM, and the anaerobic unit has the highest removal rate for fluorescence DOM (FDOM, 14.07%64.43%); the anaerobic unit increased aliphatic/proteins and lignin -like molecules but decreased relative intensity, while the anoxic unit removed unsaturated hydrocarbons, aromatic structures, and lignin -like substances. The impact of seasonal changes on the composition and removal of FDOM and DOM in wastewater treatment is significant, and the variations that occur during different seasons affect microbial activity, as well as the production, degradation, and transformation of organic compounds throughout the wastewater treatment process. Network analysis shows that Parcubacteria_genera_incertae_sedis plays a crucial role in DOM chemodiversity, highlighting the crucial contribution of microbial communities to both the structure and operation of the entire DOM network. The results in this study could provide some theoretical and practical basis for guiding the process optimization of WWTPs. (c) 2024 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Occurrence and transfer of antibiotic resistance genes (ARGs) was investigated concerning sludge particle size in a typical wastewater treatment plant, and the roles of vertical (VGT) and horizontal gene transfer (HGT) in the spread of ARGs were explored. Results showed that although membrane bioreactor (MBR) effectively reduced the relative abundance of ARGs in the water phase, it concurrently enriched ARGs in MBR sludge, particularly for the largest-size particles (>150 mu m). A decreasing trend in the relative abundance of ARGs was observed along with the decrease of sludge particle size, and larger-size particle sludge (>106 mu m) formed a relatively stable composition of ARGs, while ARGs on smaller-size particle sludge (6.5-106 mu m) fluctuate rapidly. Particle size does not affect the abundance distribution patterns or assembly mechanisms of ARGs as deterministic processes. The smallest-size particles were the primary attachment site for bacterial pathogens with highest diversity. Larger-size particle sludge (>106 mu m) showed higher frequency of HGT, with Proteobacteria as the dominant hosts for this process.

The introduction of antibiotic-resistant bacteria into riverine systems through the discharge of wastewater treatment plant (WWTP) effluent and agricultural waste poses significant health risks. Even when not pathogenic, these bacteria can act as reservoirs for antibiotic resistance genes (ARGs), transferring them to pathogens that infect humans and animals. In this study, we used fluorescence in situ hybridization, qPCR, and metagenomics to investigate how anthropogenic activities affect microbial abundance and the resistome along the Holtemme River, a small river in Germany, from near-pristine to human-impacted sites. Our results showed higher bacterial abundance, a greater absolute and relative abundance of ARGs, and a more diverse ARG profile at the impacted sites. Overall, the ARG profiles at these sites reflected antibiotic usage in Germany, with genes conferring resistance to drug classes such as beta-lactams, aminoglycosides, folate biosynthesis inhibitors, and tetracyclines. There were also variations in the ARG profiles of the impacted sites. Notably, there was a high abundance of the oxacillin resistance gene OXA-4 at the downstream site in the river. In the metagenome assembly, this gene was associated with a contig homologous to small plasmids previously identified in members of the Thiotrichaceae. The likely in-situ host of the putative plasmid was a close relative of Thiolinea (also known as Thiothrix) eikelboomii, a prominent member of WWTP microbiomes worldwide. Our results show that the effluent from WWTPs can introduce bacteria into the environment that act as shuttle systems for clinically relevant ARG.

Extensive dam construction has resulted in the formation of distinctive river-reservoir interconnected system, which also produced a river-reservoir gradient worldwide. However, dynamic characteristics of planktonic microorganism and its response mechanisms to combined pollutants in river-reservoir system remain extremely scarce. Here, a representative river-reservoir system was chosen to comprehensively investigate the dynamic characteristics of planktonic bacterial and micro-eukaryotic communities, as well as their response mechanisms to water quality parameters (WPs) and combined pollutants including (nutrients (Nuts), metal ions (MIs), polycyclic aromatic hydrocarbons (PAHs), organophosphorus pesticides (OPPs) and antibiotics (Ants)). Our results indicated that the river-reservoir gradient exerted more influences on planktonic bacterial and micro-eukaryotic communities compared with seasonal factor. Along the gradient, reservoir system weakened the interaction between bacteria and micro-eukaryotes, while it strengthened their mutualistic relationship between bacteria and micro-eukaryotes. Assembly mechanism of bacterial community shifted from the deterministic process to stochastic process along the gradient, while the assembly mechanism of micro-eukaryotic community was stochastic processes in the river-reservoir system. The river-reservoir gradient could affect the driving role of some WPs, Nuts, MIs, PAHs, OPPs and Ants on the bacteria and micro-eukaryotes to some extent. From the perspective of direct effect, the Nuts had a negative impact on bacterial community in the river-reservoir system, while Nuts was the predominant factor facilitating micro-eukaryotic community in the river-reservoir system. From the perspective of comprehensive effect, OPPs and Nuts could respectively dominate bacterial community in the river system and reservoir system, while the corresponding dominant factors for micro-eukaryotic community were the Nuts and Ants respectively. In general, the OPPs and Ants could constantly pose negative impacts on the bacterial community and micro-eukaryotic community along the river-reservoir gradient. Therefore, while carrying out the control of combined pollutants in basins, it is necessary to give priority to strengthening the ecological risk management of emerging pollutants such as organophosphorus pesticides and antibiotics.