Biology subjects

Berendonk, T. U.

Publications and source records attributed to Berendonk, T. U..

At least 19 recordsLinked to original sources

bioRxiv2026

Microbial community diversity predicts invasion resistance of freshwater biofilms against antibiotic-resistant bacteria

Rivers receive continuous inputs of antibiotic-resistant bacteria (ARB) from wastewater, agriculture, and other anthropogenic sources, yet it remains unclear whether the recipient ecological component and its microbial communities determine whether introduced ARB establish or disappear. Ecological invasion theory predicts that invasion success depends on biodiversity, community stability, and occupation of ecological niche space, but these mechanisms have rarely been evaluated together in natural microbial communities. Here, we challenged river biofilms collected from 20 sites in 12 European rivers across six countries with a model antibiotic-resistant Escherichia coli carrying a conjugative IncP-1 plasmid The invasion assays were carried out under standardized laboratory flume conditions. River biofilms differed markedly in their permissiveness to invasion despite identical invasion conditions. Higher bacterial diversity consistently accelerated invader loss rates, whereas communities containing more abundant and diverse close phylogenetic neighbours of the invader exhibited stronger exclusion during early biofilm establishment. Diversity loss during transition into the experimental system emerged as the strongest explanatory variable of invasion resistance prior to biofilm maturation, whereas Shannon diversity became the dominant predictor in mature communities. Integrating these complementary ecological dimensions substantially improved explanatory prediction of ARB persistence compared with individual predictors alone. Particularly invasion-resistant biofilms also exhibited distinct ecological community composition consistent with mature, structurally complex microbial assemblages. Together, our findings demonstrate that the establishment of ARB in the environment is not stochastic but can be predicted from measurable ecological properties of recipient microbiomes, highlighting microbial biodiversity and community organization as natural barriers to antimicrobial resistance dissemination.

microbiology↗

bioRxiv2026

From sewage to shoreline: Tracing antibiotic resistance gene trends through tropical island wastewater treatment pathways

Wastewater is a key reservoir and transmission route for antibiotic resistance genes (ARGs), enabling their spread from influent to effluent and into receiving environments. However, how combined selective pressures (antibiotics, biocides, heavy metals, pharmaceuticals) influence resistant bacteria and ARG persistence over space and time remains poorly understood. Likewise, the role of the wastewater microbiome in ARG dynamics is still unclear, as few studies integrate microbiome shifts with chemical and environmental drivers. Here, we investigated how microbiome dynamics, chemical exposures, and environmental conditions shape clinically relevant ARG dynamics from sewage to receiving environments in Guadeloupe, French Caribbean. We analysed data collected from three wastewater continuums, (hospital-based, domestic, touristic) over four campaigns (September 2021-February 2023). We characterised ARG and microbiome composition spatiotemporal patterns and used a mixed-effect model to investigate ARG associations with potential drivers, including exposome factors, microbiome dissimilarity and environmental factors. Several ARGs were negatively associated with microbiome dissimilarity (Bray-Curtis distances) (aac(6)-Ib, aph(3)-III, blaSHV, blaTEM, intI1, qnrS, sul1 and tetM). Negative associations were also observed between upstream-downstream differences in anti-inflammatory drug concentrations and the abundance of aac(6)-Ib, aph(3)-III, blaCTX-M, ermB, intI1, and tetM. In contrast, ARG relative abundance was positively associated with upstream-downstream differences in antibiotic concentrations, suggesting selection along the continuum. These findings indicate that ARG dissemination along wastewater-to-coastal pathways is shaped by opposing processes, with microbiome turnover potentially limiting ARG persistence while chemical gradients promote specific gene enrichment. The outcome is ARG-specific, with implications for antimicrobial resistance risks associated with recreational waters, seafood consumption, and coastal ecosystem interactions.

microbiology↗

bioRxiv2026

Resistance to the conjugation inhibitor AZT reveals mating partner incompatibility during plasmid transfer

Plasmid-mediated dissemination of antibiotic resistance genes (ARGs) is a major driver of antimicrobial resistance (AMR). The nucleoside analogue zidovudine (azidothymidine, AZT) has emerged as a promising conjugation inhibitor, yet the consequences of AZT resistance for subsequent plasmid transfer remain poorly understood. Here, we investigated how AZT resistance in donor and recipient bacteria influences conjugative plasmid transfer. Independent AZT-resistant mutants arose through nonsense mutations in two distinct thymidine metabolism genes, yjjG and tdk. Consistent with previous studies, AZT suppressed plasmid transfer 10-fold between susceptible mating partners, whereas resistance in both partners restored transfer despite continued AZT exposure. Unexpectedly, plasmid transfer was markedly reduced even in the absence of AZT when only one mating partner carried a resistance mutation, declining by up to 11.5-fold regardless of whether the resistant strain was the donor or the recipient. This mixed-resistance phenotype was rescued by supplementation with dTMP, a thymidine pathway metabolite downstream of both resistance-associated mutations, thereby increasing plasmid transfer by up to 5.88-fold and restoring transfer to levels comparable to those of susceptible-susceptible matings. In contrast, excess dTMP reduced conjugation between susceptible partners, supporting a broader role for balanced nucleotide metabolism in determining conjugation efficiency. Together, our findings identify nucleotide metabolism as a previously unrecognised determinant of conjugative plasmid transfer and suggest that the long-term efficacy of conjugation inhibitors such as AZT will depend not only on the evolution of resistance, but also on how resistance reshapes the physiological compatibility of bacterial mating partners. ImportanceAntibiotic resistance spreads rapidly because conjugative plasmids transfer resistance genes between bacteria. Drugs that inhibit plasmid transfer are therefore being explored as a new strategy to limit antimicrobial resistance, but little is known about how resistance against these compounds affects subsequent gene transfer. We show that resistance to the conjugation inhibitor zidovudine (AZT) does not simply restore plasmid transfer. Instead, transfer depends on the resistance status of both mating partners. While two susceptible or two resistant bacteria transferred plasmids efficiently, mixed susceptible-resistant pairings showed markedly reduced conjugation even in the absence of AZT. Restoring thymidine metabolism rescued this phenotype, identifying balanced nucleotide metabolism as a previously unrecognised determinant of conjugative plasmid transfer. These findings suggest that the long-term performance of conjugation inhibitors will depend not only on the evolution of resistance, but also on how resistance reshapes the physiological compatibility of bacterial mating partners, providing a new framework for understanding and designing anti-conjugation strategies.

microbiology↗

bioRxiv2026

Cycles of contamination and recovery: Combined sewer overflows drive acute but transient antimicrobial resistance exposure in an urban stream

Combined sewer overflows (CSOs) are a major pathway for untreated wastewater into urban streams, yet their role in shaping antimicrobial resistance (AMR) dynamics remains poorly understood. Here, we used high-frequency, time-resolved sampling during two storm-triggered CSO events across two monitoring locations and one stormwater-only control site in an urban stream to quantify how these disturbances affect microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) in an urban stream. CSO events caused rapid, up to two orders of magnitude, increases in bacterial, pathogen, and ARG abundance, with multiple transient peaks occurring within single overflow episodes. However, these increases were largely proportional to the total bacterial load, and most ARGs and MGEs did not change in relative abundance, indicating that CSOs primarily act as mass-transfer events rather than drivers of in situ selection. Downstream attenuation was governed by hydrological dilution despite additional CSO inputs: Both microbial and resistance signals largely returned to baseline within short time frames. This demonstrates that CSOs function as hydrologically driven pulse disturbances that generate acute but transient AMR exposure. Because CSO events lack the sustained pressure associated with continuous wastewater discharges, rapid washout prevents the long-term establishment of sewage-derived resistance. These findings highlight that AMR risk in CSO-impacted systems is driven primarily by short-term exposure rather than by persistent ecological transformation, with important implications for urban water management under increasingly extreme rainfall conditions.

microbiology↗

bioRxiv2026

Plasmid-plasmid interactions reshape extracellular vesicle cargo and gene transfer potential

Plasmids are key drivers of horizontal gene transfer, yet their dissemination is not limited to conjugation. Extracellular vesicles (EVs) can transport plasmid DNA, but the factors governing plasmid incorporation into EVs remain poorly understood. Here, we tested whether principles of conjugative plasmid transfer, including plasmid mobility type and plasmid-plasmid interactions, extend to EV-mediated export. Using a conjugative plasmid (pKJK5) and a mobilizable plasmid (RSF1010) in two Gram-negative hosts, we quantified plasmid incorporation into EVs under single- and dual-plasmid conditions. When present individually, the conjugative plasmid was preferentially incorporated into EVs, exceeding RSF1010 by 10-23-fold despite its lower intracellular abundance. Under co-residence, this pattern reversed: RSF1010 became enriched by 13-39-fold, while pKJK5 was reduced by 2-7-fold. Consequently, EV-associated plasmid cargo shifted to RSF1010 dominance, deviating strongly from the expected 10-fold higher pKJK5 cargo if a stochastic model based on intracellular abundance and single-plasmid conditions were applicable. We propose that mobilizable plasmids under coexistence exploit conjugative plasmid transfer machinery to access membrane-associated sites, increasing their likelihood of incorporation into EVs. Our findings demonstrate that plasmid-plasmid interactions reshape EV cargo and identify a previously unrecognized mechanism that may influence extracellular gene transfer potential in microbial communities.

microbiology↗

bioRxiv2026

Reusable immobilised quaternary ammonium particles reduce microbial and resistome burdens without promoting resistance selection during wastewater post-treatment.

Wastewater treatment plants act as convergence zones for antibiotic residues, antibiotic-resistant bacteria (ARB), and antimicrobial resistance genes (ARGs), yet conventional processes are not designed to mitigate resistance dissemination from their effluents. While chemical disinfectants are generally effective, soluble quaternary ammonium compounds (QACs) can generate subinhibitory exposure gradients that promote resistance selection and co-selection both during treatment and after release into receiving waters. Here, we evaluate a contact-restricted alternative: benzyldimethyldodecyl ammonium chloride (BDMDAC) immobilised onto hydroxyapatite microparticles as a reusable and retainable post-treatment polishing strategy. Across single-strain assays, treated wastewater exposure, and experimental community evolution, immobilised BDMDAC-functionalised particles (BDMDAC-FPs) achieved concentration-dependent antimicrobial activity without detectable biocide leaching. Optimal exposure (200 mg/L, 4 h) resulted in a ~5.5 log reduction in total bacterial abundance and removal of clinically relevant ARGs. Antimicrobial efficacy was retained after one reuse cycle, supporting operational stability. Plasmid-borne QAC ARGs did not confer protection, and no enrichment of qac-associated or non-QAC ARGs was observed. Conjugation assays demonstrated suppression of horizontal gene transfer even under suboptimal exposure, and mobility-associated markers remained stable or declined during long-term community incubation. Collectively, the data support a contact-restricted mechanism in which antimicrobial pressure is spatially confined to the particle interface, generating high local lethality while limiting diffuse subinhibitory exposure. This spatial confinement decouples antimicrobial efficacy from classical disinfectant-driven resistance selection and mobility amplification. Immobilised BDMDAC-FPs therefore provide a mechanistically distinct and evolution-conscious framework for wastewater polishing technologies.

microbiology↗

bioRxiv2025

Selective spread of mobile antibiotic resistance genes in wastewater microbiomes driven by the non-antibiotic pharmaceutical carbamazepine

Carbamazepine (CBZ), a widely used anticonvulsant, is a persistent aquatic micropollutant that withstands biodegradation and accumulates in wastewater-impacted environments. While non-antibiotic pharmaceuticals, including CBZ, have been shown to stimulate horizontal gene transfer (HGT) under laboratory conditions, their effects in complex microbial communities remain poorly understood. Here, we applied an experimental evolution framework to wastewater microbiomes exposed to a gradient of environmentally relevant CBZ concentrations (0-50 {micro}g/mL) for three days. CBZ reduced growth of the community in a concentration-dependent manner. Still, community composition was largely unaffected by CBZ exposure. However, antibiotic resistance gene (ARG)- and mobile genetic element (MGE)-specific responses emerged. Among the tested 26 ARGs, four clinically relevant ones (blaCMY, blaOXA-48, blaCTX-M, dfrA1) increased in relative abundance in a dose-dependent manner, correlating with proliferation of IncP and IncW plasmids and the transposon IS26, pointing to enhanced HGT as the underlying mechanism. In contrast, only ermF abundance rose independently of MGEs, suggesting direct selection. Other ARGs and MGEs, including integron integrase intI1 and IncQ plasmids, showed no consistent CBZ response. Effects were strongest at subinhibitory concentrations (0.05-5 {micro}g/mL), matching upper levels detected in surface waters. These findings demonstrate that CBZ can selectively promote AMR dissemination through both HGT and direct selection, but in a targeted rather than community-wide manner. Our results highlight the need to consider non-antibiotic pharmaceuticals in AMR surveillance and environmental risk assessment frameworks, as they may shape the environmental resistome beyond antibiotic exposure.

microbiology↗

bioRxiv2025

Environmental reservoir of resistance genes for the last resort antibiotic Cefiderocol.

Antibiotic resistance poses a global public health threat. Cefiderocol, a recently introduced siderophore cephalosporin, employs a "Trojan Horse" mechanism by exploiting bacterial iron uptake systems for cell entry. Yet, resistant clinical isolates are already observed in clinics and resistance mechanisms are difficult to characterize. Here, we applied functional metagenomics to identify cefiderocol resistance genes. Functional metagenomic DNA libraries from diverse environmental samples collected across several countries were expressed in a cefiderocol-sensitive Escherichia coli host. This yielded four resistant clones with DNA originating from wastewater or freshwater DNA libraries. The identified antibiotic resistance genes (ARGs) causing an increase in cefiderocol minimum inhibitory concentrations encoded for beta-lactamases (VEB-3, OXA-372 homolog and YbxI homolog) and a partial penicillin binding protein homolog. Three of four shared closest homologs in pathogenic bacteria. One ARG was associated with a mobile genetic element and was broadly distributed across all wastewater samples from every country surveyed. This study underscores the critical importance of environmental surveillance for ARGs, particularly for novel agents like cefiderocol with limited understanding of resistance mechanisms.

microbiology↗

bioRxiv2025

Suppressing selection for antibiotic resistance in the environment: A transparent, ecology-based approach to predicted no-effect concentrations

Selection for antibiotic resistance has been demonstrated at low, environmentally-relevant antibiotic concentrations. Over the past decade, the concept of minimum selective concentrations (MSC) has been adopted in environmental regulation to define maximum permissible antibiotic concentrations. Such empirically determined MSC values often fail to reflect the complexity of natural communities, where susceptibility and resistance-associated fitness costs vary widely across species. To address this limitation, computational approaches have been developed to predict no-effect concentrations for selection of antibiotic resistance (PNECres) from routinely collected minimum inhibitory concentration (MIC) data. However, these approaches often lack a strong ecological basis, undermining confidence in their predictions. Here, we propose a simple but biologically consistent framework to derive PNECres values by integrating MIC data with probabilistic estimates of resistance-related fitness costs. Our results suggest that current regulatory environmental threshold concentrations should be lowered by at least one order of magnitude to guard against selection for antibiotic resistance.

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bioRxiv2024

Fish are poor sentinels for surveillance of riverine AMR

Effective surveillance of antimicrobial resistance (AMR) in the environment is crucial for assessing the human and animal health risk of AMR pollution. Wastewater treatment plants (WWTPs) are one of the main sources of AMR pollutants discharged into water bodies. One important factor for assessing the risks associated with such pollution is the colonization potential of the resistant bacteria (ARB) and resistance genes (ARGs) from the environment into human or animal microbiomes upon exposure. This study explores whether fish can act as sentinels for surveillance of AMR pollution in general and specifically the human colonization potential of ARB in rivers impacted by WWTP effluents. Two riverine fish species, Brown trout, and European bullhead, were sampled up- and downstream a German WWTP. The two fish species were chosen due to their different lifestyles: Trout are mainly actively swimming in the water phase, while bullheads are sedentary and river sediment-associated. The bacterial microbiomes and resistomes of fish gills, skin, and feces were compared with those of the respective river water and sediment up- and downstream of the WWTP. Microbiomes of both fish mirrored the changes in river water and sediment downstream of the WWTP, with significant shifts in bacterial community composition, particularly an increase in Proteobacteria and Verrucomicrobia. However, increases in ARG abundances observed in water and sediment downstream of the WWTP were not reflected in any of the fish-associated resistomes. This indicates that while the fish microbiome is sensitive to environmental changes, resistomes of poikilothermic animals such as fish are less responsive to colonization by ARB originating from WWTPs and may not serve as effective sentinels for assessing AMR pollution and colonization risks in freshwater environments. This study highlights the complexity of using wildlife as indicators for environmental AMR pollution and suggests that other species are better suited for surveillance efforts. HighlightsO_LIWWTP effluent affected downstream river microbiome composition of water and sediment C_LIO_LIWWTP effluent significantly increased the level of six tested ARGs downstream C_LIO_LIChanges in environmental microbiome composition were mirrored in trout and bullhead C_LIO_LINo effect of the WWTP effluent on resistomes and individual ARG abundance in fishes C_LI

microbiology↗

bioRxiv2024

Jellyfish blooms - an overlooked hotspot and potential vector for the transmission of antimicrobial resistance in marine environments

Jellyfish, and gelatinous zooplankton (GZ) in general, represent an important component of marine food webs. Certain GZ species are capable of generating massive blooms of severe environmental impact. These blooms are often followed by a sudden collapse of the entire population, introducing considerable amounts of organic matter (GZ-OM) in the oceans interior. GZ-OM represents an abundant substrate to promote bacterial growth and copious colonizable surface for microbial interactions. Hence we hypothesized that this GZ-OM serves as a yet overlooked hotspot for transmitting antimicrobial resistance genes (ARGs) in marine environments. For this we experimentally evolved and analyzed marine microbial communities in microcosms in presence and absence of OM from scyphozoan Aurelia aurita s.l. and ctenophore Mnemiopsis leidyi. Communities evolved under GZ-OM exposure displayed an up to 4-fold increase in relative ARG and an up to 10-fold increase in abundance of horizontally transferable mobile genetic elements (MGEs) per 16S rRNA gene copy compared to the controls. This trait was consistent across ARG and MGE classes and independent of the GZ species, suggesting that the underlying mechanism is indeed based on the general influx of nutrients and colonizable surfaces. Potential ARG carriers included known key GZ-OM degraders, but also genera containing potential pathogens hinting towards an increased risk of ARG transfer to pathogenic strains. Here, Vibrio were pinpointed as potential key species directly associated with several significantly elevated ARGs and MGEs. Subsequent whole-genome sequencing of a Vibrio isolate from the microcosm experiment revealed the genetic potential for the mobilization and transfer of ARGs in GZ-OM degrading microbial consortia. With this study, we established the first link between two emerging issues of marine coastal zones, jellyfish blooms and AMR spread, both likely increasing in projected future ocean scenarios.

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bioRxiv2023

Tradeoffs of increasing temperatures for the spread of antimicrobial resistance in river biofilms

River microbial communities regularly act as the first defense barrier against the spread of antimicrobial resistance genes (ARG) that enter environmental microbiomes through wastewaters. However, how the invasion dynamics of wastewater-born ARGs into river biofilm communities will shift due to increasing average and peak temperatures worldwide through climate change remains unknown. Here we aimed at elucidating the effects of increasing temperatures on both, the natural river biofilm resistome, as well as the river biofilms invadability by foreign, wastewater-born ARGs. To achieve this, natural biofilms were grown in a pristine German river and transferred to artificial laboratory recirculation flume systems at three different temperatures (20{degrees}C, 25{degrees}C, 30{degrees}C). Already after one week of acclimatization to the temperatures, significant increases in the abundance of most naturally occurring ARGs were detected in the biofilms exposed to the highest temperature. Thereafter, biofilms were exposed to a single pulse of wastewater and the invasion dynamics of wastewater-born ARGs were analyzed over a period of two weeks. While initially after one day ARGs were able to invade all biofilms successfully and in equal proportions, the foreign invading ARGs were lost at a far increased rate at 30{degrees}C over time. ARG levels dropped to the initial natural levels at 30{degrees}C after 14 days. Contrary at the lower temperatures ARGs remained far elevated and certain ARGs were able to establish themselves in the biofilms. Overall, we here demonstrate tradeoffs of increasing temperature between increases in naturally occurring and faster loss dynamics of invading ARGs.

microbiology↗

bioRxiv2023

Cigarette smoking promotes the spread of antimicrobial resistance in the human lung and the environment

While immediate health risks of cigarette smoking are well-established, indirect health impacts of cigarette-derived pollutants through proliferation of antimicrobial resistance (AMR) among bacteria remain understudied. Here, exposure to cigarette smoke condensate at relevant concentrations resulted in >2-fold elevated transfer rates of a multi-drug-resistance encoding plasmid between Pseudomonas strains in artificial lung sputum medium. This effect was connected to elevated reactive oxygen species production as part of the bacterial stress response when exposed to cigarette-derived toxicants. Similar results were obtained under exposure to cigarette ash leachate in environmental medium. Further, used cigarette filters enriched in toxic residues were submerged in a wastewater stream, and colonized by altered microbial communities compared to unused filters. These communities were significantly enriched in pathogens and AMR. Hence, filters could facilitate hitchhiking of high-risk bacteria to novel environments. We demonstrate that cigarette-derived compounds can promote the spread of AMR within the human lung and natural environments.

microbiology↗

bioRxiv2023

Microbiome diversity: A barrier to the environmental spread of antimicrobial resistance?

BackgroundIn the environment, microbial communities are constantly exposed to invasion by antimicrobial resistant bacteria (ARB) and their associated antimicrobial resistance genes (ARGs) that were enriched in the anthroposphere. A successful invader has to overcome the biotic resilience of the habitat, which is more difficult with increasing biodiversity. The capacity to exploit resources in a given habitat is enhanced when communities exhibit greater diversity, reducing opportunities for invaders, leading to a lower persistence. In the context of antimicrobial resistance (AMR) dissemination, exogenous ARB reaching a natural community may persist longer if the biodiversity of the autochthonous community is low, increasing the chance of ARGs to transfer to community members. Reciprocally, high microbial diversity could serve as a natural long-term barrier towards invasion by ARB and ARGs. ResultsTo test this hypothesis, a sampling campaign across seven European countries was carried out to obtain 172 environmental samples from sites with low anthropogenic impact. Samples were collected from contrasting environments: stationary structured forest soils, or dynamic river biofilms and sediments. Microbial diversity and relative abundance of 27 ARGs and 5 mobile genetic element marker genes were determined. In soils, higher diversity, evenness and richness were all significantly negatively correlated with the relative abundance of the majority (>85%) of ARGs. Furthermore, the number of detected ARGs per sample was inversely correlated with diversity. However, no such effects were found for the more dynamic, regularly mixed rivers. Conclusions: In conclusion, we demonstrate that diversity can serve as barrier towards AMR dissemination in the environment. This effect is mainly observed in stationary, structured environments, where long-term, diversity-based resilience against invasion can evolve. Such barrier effects can in the future be exploited to limit the environmental proliferation of AMR.

microbiology↗

bioRxiv2022

Exposure to environmental stress decreases the resistance of river microbial communities towards invasion by antimicrobial resistant bacteria

Environmental microbiomes are constantly exposed to invasion events through foreign, antibiotic resistant bacteria that were enriched in the anthropic sphere. However, the biotic and abiotic factors, as well as the natural barriers that determine the invasion success of these invader bacteria into the environmental microbiomes are poorly understood. A great example of such invasion events are river microbial communities constantly exposed to resistant bacteria originating from wastewater effluents. Here, we aim at gaining comprehensive insights into the key factors that determine their invasion success with a particular focus on the effects of environmental stressors, regularly co-released in wastewater effluents. Understanding invasion dynamics of resistant bacteria is crucial for limiting the environmental spread of antibiotic resistance. To achieve this, we grew natural microbial biofilms on glass slides in rivers for one month. The biofilms were then transferred to laboratory, recirculating flume systems and exposed to a single pulse of a model resistant invader bacterium (E. coli) either in presence or absence of stress induced by Cu2+. The invasion dynamics of E. coli into the biofilms were then monitored for 14 days. Despite an initially successful introduction of E. coli into the biofilms, independent of the imposed stress, over time the invader perished in absence of stress. However, under stress c the invading strain successfully established and proliferated in the biofilms. Noteworthy, the increased establishment success of the invader coincided with a loss in microbial community diversity under stress conditions, likely due to additional niche space becoming available for the invader.

microbiology↗

bioRxiv2022

The global groundwater resistome: core ARGs and their dynamics - an in silico re-analysis of publicly available groundwater metagenomes

Despite the importance of groundwater as a drinking water resource, currently, no comprehensive picture regarding the global levels of antibiotic resistance genes (ARGs) in groundwater environments exists. Moreover, the biotic and abiotic factors that shape the groundwater resistome on the global scale remain to be explored. Herein, we attempted to fill this knowledge gap through in silico re-analysis of publicly available global groundwater metagenomes. First, nine ARGs encoding resistance to aminoglycosides (aadA, aph(3), and ant(3)), sulfonamides (sul1 and sul2), {beta}-lactams (blaOXA and blaTEM), tetracyclines (tet(C)) and macrolides (msr(E)) were identified to constitute the core groundwater resistome with high detection and abundance levels. Second, the global drivers of groundwater resistome composition were identified by applying a structural equation model with mixed effects to disentangle the individual contributions of each abiotic and biotic factor. Most notably, global effects of the origin of groundwater samples on the resistome were detected with samples from high-income countries (HICs) constantly displaying lower ARG and mobile genetic element (MGE) abundances than those from low-and-middle-income countries (LMICs). While these effects were consistent across antibiotic classes, biotic factors such as interactions of the groundwater microbiome with fungal or bacterial natural producers of antibiotics, or the co-occurrence of ARGs on mobile genetic elements (MGEs) played significant roles in shaping abundance patterns of resistance towards individual antibiotic classes. Only few ARGs correlated to individual bacterial genera, with microbial community composition in general weakly associated with resistome composition. In conclusion, we provide a first global picture of the resistome of low-anthropogenic impacted groundwater environments and the underlying anthropogenic and biotic drivers shaping it, which can be used as a baseline in future surveillance of antibiotic resistance.

microbiology↗

bioRxiv2022

Quantification of the mobility potential of antibiotic resistance genes through multiplexed ddPCR linkage analysis

Antibiotic resistance genes (ARGs) are widely disseminated within microbiomes of the human, animal, and environmental spheres. There is a clear need for global monitoring and risk assessment initiatives to evaluate the risks of ARGs towards human health. Therefore, not only ARG abundances within a given environment, but also their mobility, hence their ability to spread to human pathogenic bacteria needs to be quantified. Consequently, methods to accurately quantify the linkage of ARGs with mobile genetic elements are urgently needed. We developed a novel, sequencing-independent method for assessing ARG mobility by combining multiplexed droplet digital PCR (ddPCR) on DNA sheared into short fragments with statistical analysis. This allows quantifying the physical linkage between ARGs and mobile genetic elements, here demonstrated for the sulfonamide ARG sul1 and the Class1 integron integrase gene intI1. The methods efficiency is demonstrated using mixtures of model DNA fragments with either linked and unlinked target genes: Linkage of the two target genes can be accurately quantified based on high correlation coefficients between observed and expected values (R2) as well as low mean absolute errors (MAE) for both target genes, sul1 (R2=0.9997, MAE=0.71%, n=24) and intI1 (R2=0.9991, MAE=1.14%, n=24). Furthermore, we demonstrate that the chosen fragmentation length of DNA during shearing allows adjusting the rate of false positives and false negative detection of linkage. The applicability of the developed method for environmental samples is further demonstrated by assessing the mobility of sul1 across a wastewater treatment plant. The presented method allows rapidly obtaining reliable results within hours. It is labor- and costefficient and does not rely on sequencing technologies. Furthermore, it has a high potential to be scaled up to multiple targets. Consequently, it merits consideration to be included within global AMR surveillance initiatives for assessing ARG mobility. Author AbstractAntibiotic resistance represents a major problem in treating bacterial infections and endangers public health. Antibiotic resistant genes (ARGs) can spread among microbes in humans, animals, and the environment, thanks to mobile genetic elements. These are genetic structures involved in the mobility of genetic information and hence microbial traits. Methods that allow simultaneously quantifying the abundance of ARGs together with their association/linkage with mobile genetic elements are fundamental for assessing the risk they pose to human health, as mobility increases their likelihood to spread to human pathogens. Here we developed a novel method that allows to quantify the abundance and the linkage between ARGs and mobile genetic elements. The method relies on droplet digital PCR (ddPCR) technology performed on fragmented environmental DNA (of a chosen size), combined with statistical analysis. We found that the method accurately quantifies the linkage between the two targets in model and environmental DNA. The method is delivering rapid, labor- and cost-efficient results as it does not rely on technology that require prior bioinformatics knowledge and can be included in future monitoring frameworks.

microbiology↗

bioRxiv2022

Microbiome and resistome dynamics along a sewage-effluent-reservoir continuum underline the role of natural attenuation in effluent receiving reservoirs

This study assessed temporal dynamics of total and antibiotic resistant fecal bacterial indicators and antibiotic resistance genes (ARG) along a sewage-effluent-reservoir continuum, in an experimental system consisting of a sewage-fed membrane-aerated bioreactor (MABR) whose effluent fed a 4500 L polypropylene basin that mimicked an effluent storage reservoir. We applied a multidisciplinary approach that coupled physicochemical analyses, cultivation of total and cefotaxime-resistant E. coli, microbiome (bacterial and eukaryotic) analysis and qPCR/ddPCR quantification of selected ARGs. Total and cefotaxime-resistant E. coli loads dropped by approximately 1.5 log units in both the MABR and the reservoir, but the relative reduction (normalized to 16S rRNA genes) in both E. coli and ARGs was higher in the reservoir. Reservoir microbiomes were significantly different from those in the MABR, and were characterized by temporal shifts and periodic algal (Chlorophyta) blooms that were coupled to oxygen and pH fluctuations. Collectively, the data indicates that the decrease in E. coli and ARGs in the MABR was primarily facilitated by sludge removal, whereas in the reservoir, it was predominantly associated with microbial community shifts. The study highlights the capacity of ecological interactions in mitigating antibiotic resistance in both engineered and natural ecosystems. ImportanceAntibiotic resistance is considered one of the most significant public health predicaments of the 21st century, and there is growing evidence that anthropogenically impacted environments such as those receiving raw and treated sewage can contribute to this phenomenon. In this study, we evaluated the dynamics of total and antibiotic resistant fecal pathogen indicators and antibiotic resistance genes along a sewage-treated wastewater-effluent reservoir continuum, concurrent to evaluation of microbial community composition and physicochemical parameters. Results indicate that both the treatment bioreactor and the effluent storage reservoir removed resistant bacteria and antibiotic resistance encoding genes. However, in the reactor removal was primarily linked to physical processes, whereas in the storage reservoir it appeared to be strongly facilitated by ecological interactions. The study highlights the capacity of aquatic ecosystems to alleviate antibiotic resistance, and suggests that ecological processes in aquatic ecosystems can be harnessed to mitigate antibiotic resistance.

microbiology↗