The global spread of antimicrobial resistance (AMR) is a serious public health concern, driven by widespread antibiotic use and the global environmental circulation of antibiotic-resistant bacteria and resistance genes (ARGs). Wastewater treatment plants (WWTPs) are important sources of anthropogenic AMR entering large rivers, which serve as vital water resources but facilitate downstream dissemination. The drivers and dynamics of AMR propagation along river systems remain poorly understood. As Switzerland's longest and one of its largest rivers, the Aare, situated in the upper Rhine watershed, plays a central role in the 'water castle of Europe'. This study examines the impact of WWTP discharges, some receiving high loads of hospital effluent, on ARG distribution along the 288 km Aare river-continuum. Using quantitative PCR targeting 14 ARGs conferring resistance to eight antibiotic classes, combined with 16S rRNA gene amplicon sequencing, we conducted a high-resolution spatial survey to assess shifts in the riverine ARG content and microbiome. Concentrations of trace metals and nutrients were analyzed as tracers of anthropogenic inputs. Results revealed a progressive increase in ARG abundance downstream, driven by WWTP effluents enriched in ARGs. Effluents had 70-fold higher mean ARG concentrations than upstream waters, raising downstream levels up to 141-fold. Major tributaries such as the Reuss and Limmat sustained elevated ARG levels, while passage through lakes markedly reduced concentrations. This study provides the first detailed baseline for ARG prevalence along a large swiss river system, from pristine headwaters to pollution-affected lower reaches and insights into aquatic AMR dynamics and guidance for future monitoring.

Abstract Anaerobic carbon transformation in freshwater sediments drives substantial methane emissions globally, yet the microbial taxa linking complex carbon degradation to methane production remain poorly characterized. Here, we combined metagenomics with the first metatranscriptomic dataset from the anoxic sediments of meromictic Lake Cadagno (Swiss Alps) to identify the active microbial clades, metabolic pathways, and extrachromosomal elements (ecDNA) across a depth gradient within the upper 56 cm of sediment. We recovered 802 species-level metagenome-assembled genomes (MAGs) spanning 66 phyla and identified a Bacteroidota clade (VadinHA17) as one of the most abundant and transcriptionally active populations in the sediment. This clade encodes and transcribes a broad range of diverse glycoside hydrolases (GH), indicating a central role in complex carbohydrate degradation. Transcriptional profiles suggest that this clade ferments organic substrates to acetate and hydrogen, which are key substrates for methanogenesis. In line with this, the acetoclastic methanogen Methanothrix and hydrogenotrophic Methanoregula were among the most abundant and transcriptionally active archaea in the same depth layers. Beyond microbial genomes, we detected 86,905 viral OTUs (vOTUs) and 2,136 plasmid OTUs (pOTUs), with free viruses and plasmids accounting for 5-10% and 0.2% of all sequencing reads, respectively. Notably, plasmids and viruses associated with Bacteroidota VadinHA17 encode and transcribe GHs that could augment host carbohydrate-degrading capacity. Together, these findings reveal new details on how methane production in anoxic lake sediments emerges from a network spanning primary fermentation, methanogenesis and ecDNA-mediated metabolisms.

River biofilms are frequently exposed to invasion by antibiotic-resistant bacteria (ARB) due to episodic or chronic wastewater inputs, yet the ecological processes governing the fate of invaders and their resistance plasmids remain poorly understood. We experimentally exposed river-grown biofilms from sites differing in microbial diversity and wastewater impact to a genetically tagged ARB Escherichia coli carrying a transferable IncPα plasmid with the nptII resistance gene. Over two weeks, we tracked invader and plasmid dynamics using qPCR and plasmid-to-genome ratios as a proxy for horizontal gene transfer (HGT), complemented by 16S rRNA gene sequencing and metagenomics. Both quantification approaches yielded consistent results: the invader transiently established in all biofilms, peaking within 48 h and declining to near-background levels after 14 days. Decreasing plasmid-to-genome ratios indicated limited HGT and progressive plasmid loss. Biofilms impacted by wastewater showed slower declines, suggesting greater plasmid persistence in disturbed environments and increased abundance of specific indigenous antimicrobial resistance genes of public health concern. While the overall resistome exhibited short-lived shifts, and indigenous resistomes remained largely stable. These findings demonstrate that invader-biofilm interactions are dynamic and shaped by community context, supporting the One Health framework and highlighting how environmental conditions modulate antimicrobial resistance risks in freshwater ecosystems.

The continued rise of antibiotic resistance and adoption of the One Health approach necessitates reliable methods for detection and quantification of antibiotic resistance genes (ARGs) in complex environmental matrices. Here we present a consolidated set of TaqMan quantitative PCR assays for quantification of clinically relevant and emerging ARGs in complex environmental matrices. • We systematically designed five new primer sets, six TaqMan probes and verified and adapted four previously published relevant primer/probe sets from literature and evaluated their specificity in silico against current database. • For external quantification, two sets of gBlock standard libraries were designed. We experimentally validated the specificity, sensitivity, and efficiency of the assays with positive strain control DNA, negative strain control DNA, general no target controls, extraction blank controls, negative controls, and environmental test samples (i.e., metagenomic DNA from complex environmental matrices) to comprehensively assess each assays’ performance. • Optimization included iterative testing of both primer and probe concentration, annealing temperature, and annealing time. Results demonstrated robust and reliable detection and quantification of ARGs in clinical isolates and wastewater effluents with high sensitivity, specificity, and efficiency.This makes the assays suitable for surveillance in wastewater or various environmental matrices, in support of efforts to mitigate dissemination of antibiotic resistance.

In this study, we investigated the temporal and spatial quantitative changes in the concentration of antibiotic resistance gene (ARG) markers in a municipal wastewater treatment plant (WWTP). Four ARGs conferring resistance to different classes of antibiotics ( ermB, sul 1, tet [W], and bla CTXM ) and a gene used as a proxy for ARG pollution ( intl1 ) were quantified in two separate sampling campaigns covering two and half years of operation of the WWTP. First, a systematic monthly monitoring of multiple points in the inlet and the outlet revealed an absolute decrease in the concentration of all analyzed ARGs. However, the relative abundance of sul 1 and intl 1 genes relative to the total bacterial load (estimated using the universal marker 16S rDNA) increased in the outlet samples as compared to the inlet. To pinpoint the exact stage of removal and/or enrichment within the WWTP, a second sampling including the stages of the biological treatment was performed bimonthly. This revealed a distinct enrichment of sul 1 and intl 1 genes during the biological treatment phase. Moreover, the temporal and spatial variations in ARG abundance patterns within the WWTP underscored the complexity of the dynamics associated with the removal of ARGs during wastewater treatment. Understanding these dynamics is pivotal for developing efficient strategies to mitigate the dissemination of ARGs in aquatic environments. Practitioner points Regular monitoring of ARG markers in WWTPs is essential to assess temporal and spatial changes, aiding in the development of effective mitigation strategies. Understanding the dynamics of ARG abundance during biological treatment is crucial for optimizing processes and minimizing dissemination in aquatic environments. Increased relative abundance of certain ARGs highlights potential enrichment during wastewater treatment, necessitating targeted interventions. Systematic monitoring of multiple points within WWTPs can provide valuable insights into the efficacy of treatment processes in reducing ARG levels over time. The complexity of ARG abundance patterns underscores the need to develop holistic approaches to tackle antibiotic resistance in wastewater systems.

Wastewater treatment plants (WWTPs) are key sources of antimicrobial resistance genes (ARGs) that could influence the resistomes of microbial communities in various habitats of the receiving river ecosystem. However, it is currently unknown which habitats are most impacted and whether ARGs, like certain chemical contaminants, could be accumulated or enriched in the river ecosystem. We conducted a systematic metagenomic survey on the antibiotic resistomes of WWTP effluent, four riverine habitats (water, suspended particles, sediment, epilithic biofilm), and freshwater amphipod gut microbiomes. The impact of WWTP effluent on the downstream habitats was assessed in nine Swiss rivers. While there were significant differences in resistomes across habitats, the wastewater resistome was more similar to the resistome of receiving river water than to the resistomes of other habitats, and river water was the habitat most strongly impacted by the WWTPs effluent. The sulfonamide, beta-lactam, and aminoglycoside resistance genes were among the most abundant ARGs in the WWTP effluents, and especially aadA, sul1, and class A beta-lactamase genes showed significantly increased abundance in the river water of downstream compared to upstream locations (p < 0.05). However, this was not the case for the sediment, biofilm, and amphipod gut habitats. Accordingly, evidence for accumulation or enrichment of ARGs through the riverine food web was not identified. Our study suggests that monitoring riverine antimicrobial resistance determinants could be conducted using “co-occurrence” of aadA, sul1, and class A beta-lactamase genes as an indicator of wastewater-related pollution and should focus on the water as the most affected habitat.

Real-time quantitative PCR (qPCR) has been widely used to quantify gene copy numbers in microbial ecology. Despite its simplicity and straightforwardness, establishing qPCR assays is often impeded by the tedious process of producing qPCR standards by cloning the target DNA into plasmids. Here, we designed double-stranded synthetic DNA fragments from consensus sequences as qPCR standards by aligning microbial gene sequences (10–20 sequences per gene). Efficiency of standards from synthetic DNA was compared with plasmid standards by qPCR assays for different phylogenetic marker and functional genes involved in carbon (C) and nitrogen (N) cycling, tested with DNA extracted from a broad range of soils. Results showed that qPCR standard curves using synthetic DNA performed equally well to those from plasmids for all the genes tested. Furthermore, gene copy numbers from DNA extracted from soils obtained by using synthetic standards or plasmid standards were comparable. Our approach therefore demonstrates that a synthetic DNA fragment as qPCR standard provides comparable sensitivity and reliability to a traditional plasmid standard, while being more time- and cost-efficient.

As motivation to address environmental dissemination of antimicrobial resistance (AMR) is mounting, there is a need to characterize mechanisms by which AMR can propagate under environmental conditions. Here we investigated the effect of temperature and stagnation on the persistence of wastewater-associated antibiotic resistance markers in riverine biofilms and the invasion success of genetically-tagged Escherichia coli. Biofilms grown on glass slides incubated in-situ downstream of a wastewater treatment plant effluent discharge point were transferred to laboratory-scale flumes fed with filtered river water under potentially stressful temperature and flow conditions: recirculation flow at 20 °C, stagnation at 20 °C, and stagnation at 30 °C. After 14 days, quantitative PCR and amplicon sequencing were used to quantify bacteria, biofilms diversity, resistance markers (sul1, sul2, ermB, tetW, tetM, tetB, blaCTX-M-1, intI1) and E. coli. Resistance markers significantly decreased over time regardless of the treatment applied. Although invading E. coli were initially able to colonize the biofilms, its abundance subsequently declined. Stagnation was associated with a shift in biofilm taxonomic composition, but there was no apparent effect of flow conditions or the simulated river-pool warming (30 °C) on AMR persistence or invasion success of E. coli. Results however indicated that antibiotic resistance markers in the riverine biofilms decreased under the experimental conditions in the absence of exposure to external inputs of antibiotics and AMR.

Untreated combined sewage (bypass) is often discharged by wastewater treatment plants to receiving rivers during stormwater events, where it may contribute to increased levels of antibiotic resistance genes (ARGs) and multi-resistance risk factors (multi-resistant bacteria and multi-resistance genomic determinants (MGDs)) in the receiving water. Other contamination sources, such as soil runoff and resuspended river sediment could also play a role during stormwater events. Here we report on stormwater event-based sampling campaigns to determine temporal dynamics of ARGs and multi-resistance risk factors in bypass, treated effluent, and the receiving river, as well as complimentary data on catchment soils and surface sediments. Both indicator ARGs (qPCR) and resistome (ARG profiles revealed by metagenomics) indicated bypass as the main contributor to the increased levels of ARGs in the river during stormwater events. Furthermore, we showed for the first time that the risk of exposure to bypass-borne multi-resistance risk factors increase under stormwater events and that many of these MGDs were plasmid associated and thus potentially mobile. In addition, elevated resistance risk factors persisted for some time (up to 22 h) in the receiving water after stormwater events, likely due to inputs from distributed overflows in the catchment. This indicates temporal dynamics should be considered when interpreting the risks of exposure to resistance from event-based contamination. We propose that reducing bypass from wastewater treatment plants may be an important intervention option for reducing dissemination of antibiotic resistance.

Abstract Background The abundance of clinically important antibiotic resistance determinants in wastewater has raised concerns regarding their dissemination into the environment and transmission to animals and humans. However, the high bacterial diversity and complexity of environmental microbiomes makes it challenging to comprehensively detect and quantify clinically relevant resistance determinants. To tackle this outstanding technical bottleneck, classic phenotype-based approaches vastly underestimate the diversity of clinically relevant resistance in environmental reservoirs, while increasingly applied genotyping-based approaches alone usually fail to capture information on the resistance phenotype, viability, and activities of resistant bacteria. Results Here, we developed phenotypic metagenomics as a novel high-throughput approach to couple selective cultivation (phenotyping) and metagenomics (genotyping) to overcome these difficulties of classic phenotype or genotype-based approaches. Using this new method, we were able to track the invasion of wastewater-born multi-resistant opportunistic pathogens into river sediment and epilithic biofilm in 9 Swiss rivers downstream of wastewater treatment plants (WWTPs). The results show that WWTPs release clinically important carbapenem-resistant opportunistic pathogens and promotes the spread of extended-spectrum beta-lactamase resistance genes in the receiving river water. Especially, epilithic biofilms were identified as an unrecognized environmental reservoir of wastewater-associated carbapenem resistant bacteria, which appear to also invade the gut of amphipods, a group of generalist invertebrate in local rivers. We further used metagenome-assembled genomes to characterize phenotypically resistant bacteria and their extended-spectrum beta-lactamase and carbapenemase resistance genes to confirm our finding that wastewater effluent increases resistance levels of extended-spectrum beta-lactamases in the receiving river water. Conclusions This study demonstrates that where culture plating and culture-independent metagenomics fail individually, their complementary combination allows tracking multi-resistant bacteria as they invade river habitats. The proof-of-concept approach that couple resistance phenotyping and genotyping provides a roadmap for sensitive, standardized, and cost-effective surveillance of the clinically relevant aspects of environmental resistomes. The findings call for a thorough life cycle assessment of the selection and transmission of last-resort antibiotic resistance determinants from clinical to environmental reservoirs.

Wastewater treatment plants are ‘hotspots’ for antibiotic resistance genes and bacteria but sewer overflows exacerbate the problem by disseminating them into rivers. Swiss researchers suggest ways to minimise these effects.

River networks are one of the main routes by which the public could be exposed to environmental sources of antibiotic resistance, that may be introduced e.g. via treated wastewater. In this study, we applied a comprehensive integrated analysis encompassing mass-flow concepts, chemistry, bacterial plate counts, resistance gene quantification and shotgun metagenomics to track the fate of the resistome (collective antibiotic resistance genes (ARGs) in a microbial community) of treated wastewater in two Swiss rivers at the kilometer scale. The levels of certain ARGs and the class 1 integron integrase gene (intI1) commonly associated with anthropogenic sources of ARGs decreased quickly over short distances (2-2.5 km) downstream of wastewater discharge points. Mass-flow analysis based on conservative tracers suggested this decrease was attributable mainly to dilution but ARG loadings frequently also decreased (e.g., 55.0-98.5 % for ermB and tetW) over the longest studied distances (6.8 and 13.7 km downstream). Metagenomic analysis confirmed that ARG of wastewater-origin did not persist in rivers after 5 ~ 6.8 km downstream distance. sul1 and intI1 levels and loadings were more variable and even increased sharply at 5 ~ 6.8 km downstream distance on one occasion. While input from agriculture and in-situ positive selection pressure for organisms carrying ARGs cannot be excluded, in-system growth of biomass is a more probable explanation. The potential for direct human exposure to the resistome of wastewater-origin thus appeared to typically abate rapidly in the studied rivers. However, the riverine aquatic resistome was also dynamic, as evidenced by the increase of certain gene markers downstream, without obvious sources of anthropogenic contamination. This study provides new insight into drivers of riverine resistomes and pinpoints key monitoring targets indicative of where human sources and exposures are likely to be most acute.

In stratified lakes, methane-oxidizing bacteria (MOB) are strongly mitigating methane fluxes to the atmosphere by consuming methane entering the water column from the sediments. MOB communities in lakes are diverse and vertically structured, but their spatio-temporal dynamics along the water column as well as physico-chemical parameters and interactions with other bacterial species that drive the community assembly have so far not been explored in depth. Here, we present a detailed investigation of the MOB and bacterial community composition and a large set of physico-chemical parameters in a shallow, seasonally stratified, and sub-alpine lake. Four highly resolved vertical profiles were sampled in three different years and during various stages of development of the stratified water column. Non-randomly assembled MOB communities were detected in all compartments. We could identify methane and oxygen gradients and physico-chemical parameters like pH, light, available copper and iron, and total dissolved nitrogen as important drivers of the MOB community structure. In addition, MOB were well-integrated into a bacterial-environmental network. Partial redundancy analysis of the relevance network of physico-chemical variables and bacteria explained up to 84% of the MOB abundances. Spatio-temporal MOB community changes were 51% congruent with shifts in the total bacterial community and 22% of variance in MOB abundances could be explained exclusively by the bacterial community composition. Our results show that microbial interactions may play an important role in structuring the MOB community along the depth gradient of stratified lakes.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum β-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Wastewater treatment plants (WWTPs) are implicated as hotspots for the dissemination of antibacterial resistance into the environment. However, the in situ processes governing removal, persistence, and evolution of resistance genes during wastewater treatment remain poorly understood. Here, we used quantitative metagenomic and metatranscriptomic approaches to achieve a broad-spectrum view of the flow and expression of genes related to antibacterial resistance to over 20 classes of antibiotics, 65 biocides, and 22 metals. All compartments of 12 WWTPs share persistent resistance genes with detectable transcriptional activities that were comparatively higher in the secondary effluent, where mobility genes also show higher relative abundance and expression ratios. The richness and abundance of resistance genes vary greatly across metagenomes from different treatment compartments, and their relative and absolute abundances correlate with bacterial community composition and biomass concentration. No strong drivers of resistome composition could be identified among the chemical stressors analyzed, although the sub-inhibitory concentration (hundreds of ng/L) of macrolide antibiotics in wastewater correlates with macrolide and vancomycin resistance genes. Contig-based analysis shows considerable co-localization between resistance and mobility genes and implies a history of substantial horizontal resistance transfer involving human bacterial pathogens. Based on these findings, we propose future inclusion of mobility incidence (M%) and host pathogenicity of antibiotic resistance genes in their quantitative health risk ranking models with an ultimate goal to assess the biological significance of wastewater resistomes with regard to disease control in humans or domestic livestock.

Aquatic ecosystems serve as a dissemination pathway and a reservoir of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG). In this study, we investigate the role of the bacterial sporobiota to act as a vector for ARG dispersal in aquatic ecosystems. The sporobiota was operationally defined as the resilient fraction of the bacterial community withstanding a harsh extraction treatment eliminating the easily lysed fraction of the total bacterial community. The sporobiota has been identified as a critical component of the human microbiome, and therefore potentially a key element in the dissemination of ARG in human-impacted environments. A region of Lake Geneva in which the accumulation of ARG in the sediments has been previously linked to the deposition of treated wastewater was selected to investigate the dissemination of tet(W) and sul1, two genes conferring resistance to tetracycline and sulfonamide, respectively. Analysis of the abundance of these ARG within the sporobiome (collection of genes of the sporobiota) and correlation with community composition and environmental parameters demonstrated that ARG can spread across the environment with the sporobiota being the dispersal vector. A highly abundant OTU affiliated with the genus Clostridium was identified as a potential specific vector for the dissemination of tet(W), due to a strong correlation with tet(W) frequency (ARG copy numbers/ng DNA). The high dispersal rate, long-term survival, and potential reactivation of the sporobiota constitute a serious concern in terms of dissemination and persistence of ARG in the environment.

Here we used flow cytometry (FCM) and filtration paired with amplicon sequencing to determine the abundance and composition of small low nucleic acid (LNA)-content bacteria in a variety of freshwater ecosystems. We found that FCM clusters associated with LNA-content bacteria were ubiquitous across several ecosystems, varying from 50 to 90% of aquatic bacteria. Using filter-size separation, we separated small LNA-content bacteria (passing 0.4 µm filter) from large bacteria (captured on 0.4 µm filter) and characterized communities with 16S amplicon sequencing. Small and large bacteria each represented different sub-communities within the ecosystems’ community. Moreover, we were able to identify individual operational taxonomical units (OTUs) that appeared exclusively with small bacteria (434 OTUs) or exclusively with large bacteria (441 OTUs). Surprisingly, these exclusive OTUs clustered at the phylum level, with many OTUs appearing exclusively with small bacteria identified as candidate phyla (i.e. lacking cultured representatives) and symbionts. We propose that LNA-content bacteria observed with FCM encompass several previously characterized categories of bacteria (ultramicrobacteria, ultra-small bacteria, candidate phyla radiation) that share many traits including small size and metabolic dependencies on other microorganisms.

The introduction of antibiotics for both medical and non-medical purposes has had a positive effect on human welfare and agricultural output in the past century.However, there is also an important ecological legacy regarding the use of antibiotics and the consequences of increased levels of these compounds in the environment as a consequence of their use and disposal.This legacy was investigated by quantifying two antibiotic resistance genes (ARG) conferring resistance to tetracycline (tet(W)) and sulfonamide (sul1) in bacterial seed bank DNA in sediments.The industrial introduction of antibiotics caused an abrupt increase in the total abundance of tet(W) and a steady increase in sul1.The abrupt change in tet(W) corresponded to an increase in relative abundance from ca. 1960 that peaked around 1976.This pattern of accumulation was highly correlated with the abundance of specific members of the seed bank community belonging to the phylum Firmicutes.In contrast, the relative abundance of sul1 increased after 1976.This correlated with a taxonomically broad spectrum of bacteria, reflecting sul1 dissemination through horizontal gene transfer.The accumulation patterns of both ARGs correspond broadly to the temporal scale of medical antibiotic use.Our results show that the bacterial seed bank can be used to look back at the historical usage of antibiotics and resistance prevalence.

The introduction of antibiotics for both medical and non-medical purposes has had a positive effect on human welfare and agricultural output in the past century. However, there is also an important ecological legacy regarding the use of antibiotics and the consequences of increased levels of these compounds in the environment as a consequence of their use and disposal. This legacy was investigated by quantifying two antibiotic resistance genes (ARG) conferring resistance to tetracycline (tet(W)) and sulfonamide (sul1) in bacterial seed bank DNA in sediments. The industrial introduction of antibiotics caused an abrupt increase in the total abundance of tet(W) and a steady increase in sul1. The abrupt change in tet(W) corresponded to an increase in relative abundance from ca. 1960 that peaked around 1976. This pattern of accumulation was highly correlated with the abundance of specific members of the seed bank community belonging to the phylum Firmicutes. In contrast, the relative abundance of sul1 increased after 1976. This correlated with a taxonomically broad spectrum of bacteria, reflecting sul1 dissemination through horizontal gene transfer. The accumulation patterns of both ARGs correspond broadly to the temporal scale of medical antibiotic use. Our results show that the bacterial seed bank can be used to look back at the historical usage of antibiotics and resistance prevalence.