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Predation defense is an important feature of predator-prey interactions adding complexity to ecosystem dynamics. Prey organisms have developed various strategies to escape predation which differ in mode (elude vs. attack), reversibility (inducible vs. permanent), and scope (individual vs. cooperative defenses). While the mechanisms and controls of many singular defenses are well understood, important ecological and evolutionary facets impacting long-term predator-prey dynamics remain underexplored. This pertains especially to trade-offs and interactions between alternative defenses occurring in prey populations evolving under predation pressure. Here, we explored the dynamics of a microbial predator-prey system consisting of bacterivorous flagellates (Poteriospumella lacustris) feeding on Pseudomonas putida. Within five weeks of co-cultivation corresponding to about 35 predator generations, we observed a consistent succession of bacterial defenses in all replicates (n = 16). Initially, bacteria expressed a highly effective cooperative defense based on toxic metabolites, which brought predators close to extinction. This initial strategy, however, was consistently superseded by a second mechanism of predation defense emerging via de novo mutations. Combining experiments with mathematical modeling, we demonstrate how this succession of defenses is driven by the maximization of individual rather than population benefits, highlighting the role of rapid evolution in the breakdown of social cooperation.

The freshwater pearl mussel Margaritifera margaritifera is an endangered bivalve which is usually regarded as sedentary, although individual movement has been observed both vertically and horizontally. Little is known about the causes and rates of mussel movement. The objective of this study was to test the effect of microhabitat characteristics on the horizontal movement distance and rates of freshwater pearl mussels. A total of 120 mussels (length range 40–59 mm) were marked individually with passive integrated transponder tags, placed in stream microhabitats differing in their sediment composition and monitored biweekly over a period of 10 weeks. Mussels situated in sand-dominated habitats had a significantly higher mean movement rate (3.2 ± 4.2 cm/day, mean ± SD) than mussels situated in gravel-dominated (1.9 ± 2.7 cm/day) or stone-dominated habitats (1.8 ± 3.2 cm/day). The direction of the movements appeared random; however, an emigration from sandy habitats was observed, probably to avoid dislodgment from these hydraulically unstable habitats. This study demonstrates that freshwater pearl mussels can actively emigrate from unsuitable microhabitats. Once suitable streams with respect to physical, chemical, and biological quality were identified, it is therefore only necessary to identify suitable mesohabitats (area of 10–30 m2) when reintroducing or relocating mussels.

Biological invasion is a strong threat to native biodiversity, with limnic systems being especially vulnerable due to historical separation and resulting prey naivety. The prey naivety hypothesis states that native species may not be able to recognize novel predators due to a lack of common evolutionary background and, therefore, become easy targets. In a laboratory experiment, we added cues of native European bullhead (Cottus gobio Linnaeus, 1758) and invasive round goby [Neogobius melanostomus (Pallas, 1814)] to Ancylus fluviatilis Müller, 1774 originating from two different populations within the same river (one naive, one experienced towards round goby) and compared their predator avoidance behavior. Individuals from both populations recognized cues from the known predator C. gobio and reduced their locomotive activity. To round goby cues, however, naive individuals did not respond, thereby supporting the prey naivety hypothesis. Experienced individuals, in contrast, reduced their activity, suggesting a learning effect due to the co-occurrence of invasive predator and prey. At fast moving invasion fronts of highly invasive species like N. melanostomus, prey naivety can, hence, enhance their negative impact on ecosystems. Behavioral adaptation of native species resulting in predator avoidance reactions could, therefore, play an important role in ecosystem resilience and temporal invasion dynamics.

Antibiotic-resistant bacteria and antibiotic resistance gene (ARGs) loads dissipate through sewage treatment plants to receiving aquatic environments, but the mechanisms that mitigate the spread of these ARGs are not well understood due to the complexity of full-scale systems and the difficulty of source tracking in downstream environments. To overcome this problem, we targeted a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR), whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs and receiving aquatic ecosystems. We analyzed a large set of physicochemical measurements, concomitant with the cultivation of total and cefotaxime-resistant Escherichia coli, microbial community analyses, and quantitative PCR (qPCR)/digital droplet PCR (ddPCR) quantification of selected ARGs and mobile genetic elements (MGEs). The MABR removed most of the sewage-derived organic carbon and nitrogen, and simultaneously, E. coli, ARG, and MGE levels dropped by approximately 1.5- and 1.0-log unit mL(-1), respectively. Similar levels of E. coli, ARGs, and MGEs were removed in the reservoir, but interestingly, unlike in the MABR, the relative abundance (normalized to 16S rRNA gene-inferred total bacterial abundance) of these genes also decreased. Microbial community analyses revealed the substantial shifts in bacterial and eukaryotic community composition in the reservoir relative to the MABR. Collectively, our observations lead us to conclude that the removal of ARGs in the MABR is mainly a consequence of treatment-facilitated biomass removal, whereas in the stabilization reservoir, mitigation is linked to natural attenuation associated with ecosystem functioning, which includes abiotic parameters, and the development of native microbiomes that prevent the establishment of wastewater-derived bacteria and associated ARGs.IMPORTANCE Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs. We evaluated ARB and ARG dynamics across the raw-sewage-MABR-effluent trajectory, concomitant with evaluation of microbial community composition and physicochemical parameters, in an attempt to identify mechanisms associated with ARB and ARG dissipation. We found that removal of ARB and ARGs in the MABR was primarily associated with bacterial death or sludge removal, whereas in the reservoir it was attributed to the inability of ARBs and associated ARGs to colonize the reservoir due to a dynamic and persistent microbial community. The study demonstrates the importance of ecosystem functioning in removing microbial contaminants from wastewater. Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs.

Selection for antibiotic resistance at very low antibiotic concentrations has been demonstrated for individual antibiotics in single species experiments. Furthermore, selection in these focal strains is reduced when taking place in complex microbial community context. However, in the environment, bacteria are rarely exposed to single, but rather complex mixtures of selective agents. Here, we explored how the presence of a second selective agent affects selection dynamics between isogenic pairs of focal E. coli strains, differing exclusively in a single resistance determinant, in the absence and presence of a model wastewater community across a gradient of antibiotics. An additional antibiotic that exclusively affects the model wastewater community, but to which the focal strains are resistant to, was chosen as the second selective agent. This allowed exploring how inhibition alters the community’s ability to reduce selection. In the presence of the community, the selection coefficient at specific antibiotic concentrations was consistently decreased compared to the absence of the community. While pressure through the second antibiotic significantly decreased the activity and diversity of the community, its ability to reduce selection was consistently maintained at levels comparable to those recorded in absence of the second antibiotic. This indicates that the observed effects of community context on selection dynamics are rather based on competitive or protective effects between the focal strains and a small proportion of bacteria within the community, than on general competition for nutrients. These findings have implications for our understanding of the evolution and selection for multi-drug resistant strains.

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.

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 method’s 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.

To understand the dynamics of antimicrobial resistance (AMR), in a One-Health perspective, surveillance play an important role. Monitoring systems already exist in the human health and livestock sectors, but there are no environmental monitoring programs. Therefore there is an urgent need to initiate environmental AMR monitoring programs nationally and globally, which will complement existing systems in different sectors. However, environmental programs should not only identify anthropogenic influences and levels of AMR, but they should also allow for identification of transmissions to and from human and animal populations. In the current review we therefore propose using antimicrobial resistant Escherichia coli as indicators for monitoring occurrence and levels of AMR in the environment, including wildlife.

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 ofE. coliinto the biofilms were then monitored for 14 days. Despite an initially successful introduction ofE. coliinto 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.

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.

Treated wastewater is a major pathway by which antibiotic resistance genes (ARG) enter aquatic ecosystems. However, knowledge gaps remain concerning the dissemination of specific ARG and their association with bacterial hosts. Here, we employed shotgun metagenomics to track ARG and taxonomic markers in river biofilms along a gradient of fecal pollution depicted by crAssphage signatures. We found strong evidence for an impact of wastewater effluents on both community composition and resistomes. In the light of such simultaneity, we employed a model comparison technique to identify ARG-host relationships from nonassembled metagenomic DNA. Hereby, a major cause of spurious associations otherwise encountered in correlation-based ARG-host analyses was suppressed. For several families of ARG, namely those conferring resistance to beta-lactams, particular bacterial orders were identified as candidate hosts. The found associations of

The spread of antibiotic resistance genes (ARG) occurs widely through plasmid transfer majorly facilitated via bacterial conjugation. To assess the spread of these mobile ARG, it is necessary to develop appropriate tools to estimate plasmid transfer rates under different environmental conditions. Process-based models are widely used for the estimation of plasmid transfer rate constants. Empirical studies have repeatedly highlighted the importance of subtle processes like delayed growth, the maturation of transconjugants, the physiological cost of plasmid carriage, and the dependence of conjugation on the culture′s growth stage. However, models used for estimating the transfer rates typically neglect them. We conducted virtual mating experiments to quantify the impact of these four typical structural model deficits on the estimated plasmid transfer rate constants. We found that under all conditions, the plasmid cost and the lag phase in growth must be taken into account to obtain unbiased estimates of plasmid transfer rate constants. We observed a tendency towards the underestimation of plasmid transfer rate constants when structurally deficient models were fitted to virtual mating data. This holds for all the structural deficits and mating conditions tested in our study. Our findings might explain an important component of the negative bias in model predictions known as the plasmid paradox. We also discuss other structural deficits that could lead to an overestimation of plasmid transfer rate constants and we demonstrate the impact of ill-fitted parameters on model predictions.

Single mutations can confer resistance to antibiotics. Identifying such mutations can help to develop and improve drugs. Here, we systematically screen for candidate quinolone resistance-conferring mutations. We sequenced highly diverse wastewater E. coli and performed a genome-wide association study (GWAS) to determine associations between over 200,000 mutations and quinolone resistance phenotypes. We uncovered 13 statistically significant mutations including 1 located at the active site of the biofilm dispersal gene bdcA and 6 silent mutations in the aminoacyl-tRNA synthetase valS. The study also recovered the known mutations in the topoisomerases gyrase (gyrA) and topoisomerase IV (parC). In summary, we demonstrate that GWAS effectively and comprehensively identifies resistance mutations without a priori knowledge of targets and mode of action. The results suggest that mutations in the bdcA and valS genes, which are involved in biofilm dispersal and translation, may lead to novel resistance mechanisms.

Environmental science and pollution research : ESPR (2021). doi:10.1007/s11356-021-17691-0, Published by Springer, Berlin ; Heidelberg

The reuse of treated wastewater (e.g. for irrigation) is a common practice to combat water scarcity problems world-wide. However, the potential spread of opportunistic pathogens and fecal contaminants like Enterococci within the subsoil could pose serious health hazards. Additional sources (e.g., leaky sewer systems, livestock farming) aggravate this situation. This study contributes to an understanding of pathogen spread in the environment, using a combined modelling and experimental approach. The impact of quartz sediment and certain wastewater characteristics on the dissemination of Enterococcus faecalis JH2–2 is investigated. The transport processes of advection-dispersion and straining were studied by injecting conservative saline tracer and fluorescent microspheres through sediment packed columns, and evaluating resulting breakthrough curves using models. Similarly, simultaneously occurring reactive processes of microbial attachment, decay, respiration and growth were studied by injecting Enterococcus faecalis JH2–2 suspended in water with or without dissolved oxygen (DO) and nutrients through sediment, and evaluating resulting inlet and outlet concentration curves. The processes of straining, microbial decay and growth, were important when DO was absent. Irreversible attachment was important when DO was present. Sensitivity analysis of each parameter was conducted, and field scale behavior of the processes was predicted, to facilitate future work.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression., A comparative genomics study of nine Paramecium species reveals successful invasion of genes by transposable elements in their germline genomes, showing that the internal eliminated sequences (IESs) followed an evolutionary trajectory remarkably similar to that of spliceosomal introns.

Strict vertical (parent-to-offspring) transmission of symbionts may be associated with benevolence and mutualism, because host reproduction and symbiont transmission are linked. Nonetheless, many such symbionts do reduce host fitness costs and the factors driving their evolution are unclear. Using microcosm populations ofCaedibacter taeniospiralis, a vertically transmitted bacterial symbiont of the protozoanParamecium tetraurelia, we investigated evolutionary change under permissive conditions (26°C) and under heat stress (32°C), over 150 host generations. We found evidence for symbiont adaptation to heat stress, involving increased infection persistence and, consequently, higher rates of vertical transmission. For certain genetic backgrounds, heat-stress adaptation was associated with higher proliferation of infected lines at 26°C, suggesting cost-free evolution of super-generalists. Despite a general decrease in symbiont virulence relative to ancestral strains, infected evolved lines still showed substantial reductions (>20%) in population growth. There was no sign of a temperature-specific reduction in virulence or protection of the host against heat stress. Our results show that obligately vertically transmitted symbionts can still qualify as ‘parasites’ and evolve adaptations that do not help their hosts. Long-term infection persistence might require additional mechanisms, such as the symbiont-mediated ’killer trait’ in this system, allowing the selective elimination of uninfected individuals in the population.

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 (

In the present study, we investigated the impact of treated wastewater (TWW) irrigation on the prevalence of antibiotic resistance genes (ARGs) in subsoil pore-water, a so-far under-appreciated matrix. We hypothesized that TWW irrigation increases ARG prevalence in subsoil pore-water. This hypothesis was tested using a multiphase approach, which consisted of sampling percolated subsoil pore-water from lysimeter-wells of a real-scale TWW-irrigated field, operated for commercial farming practices, and controlled, laboratory mesocosms irrigated with freshwater or TWW. We monitored the abundance of six selected ARGs (sul1, blaOXA-58, tetM, qnrS, blaCTX-M-32 and blaTEM), the intI1 gene associated with mobile genetic elements and an indicator for anthropogenic pollution and bacterial abundance (16S rRNA gene) by qPCR. The bacterial load of subsoil pore water was independent of both, irrigation intensity in the field study and irrigation water type in the mesocosms. Among the tested genes in the field study, sul1 and intI1 exhibited constantly higher relative abundances. Their abundance was further positively correlated with increasing irrigation intensity. Controlled mesocosm experiments verified the observed field study results: the relative abundance of several genes, including sul1 and intI1, increased significantly when irrigating with TWW compared to freshwater irrigation. Overall, TWW irrigation promoted the spread of ARGs and intI1 in the subsoil pore-water, while the bacterial load was maintained. The combined results from the real-scale agricultural field and the controlled lab mesocosms indicate that the dissemination of ARGs in various subsurface environments needs to be taken into account during TWW irrigation scenarios.Graphical abstractHighlightsTWW irrigation intensity and sul1 & intI1 abundance correlate in a real-scale fieldARGs & intI1 increase in subsoil pore-water during TWW irrigation in mesocosmsNo increase of ARGs & intI1 in freshwater irrigated mesocosmsTWW irrigation does not affect the bacterial load of subsoil pore-water

Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? Here, we reveal that metagenomes from no fewer than 20% of the papers found in our literature search, published between 2016 and 2019, were not deposited in a repository or were simply inaccessible. The proportion of inaccessible data within the literature has been increasing year-on-year. Noncompliance with Open Data is best predicted by the scientific discipline of the journal. The number of citations, journal type (e.g., Open Access or subscription journals), and publisher are not good predictors of data accessibility. However, many publications in high–impact factor journals do display a higher likelihood of accessible metagenomic data sets. Twenty-first century science demands compliance with the ethical standard of data sharing of metagenomes and DNA sequence data more broadly. Data accessibility must become one of the routine and mandatory components of manuscript submissions—a requirement that should be applicable across the increasing number of disciplines using metagenomics. Compliance must be ensured and reinforced by funders, publishers, editors, reviewers, and, ultimately, the authors., Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? This Essay reveals that every fifth published metagenome is not available to science, and asks whether data accessibility should be considered a mandatory component of manuscript submission.

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely distributed in the environment where they represent potential public health threats. Quantitative PCR (qPCR) is a suitable approach to detect and quantify ARGs in environmental samples. However, the comparison of gene quantification data between different laboratories is challenging since the data are predominantly obtained under non-harmonized conditions, using different qPCR protocols. This study aimed at carrying out an inter-laboratory calibration in order to assess the variability inherent to the qPCR procedures for quantification of ARGs. With this aim, samples of treated wastewater collected in three different countries were analysed based on common DNA extract pools and identical protocols as well as distinct equipment, reagents batches, and operators. The genes analysed were the 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 and the artificial pNORM1 plasmid containing fragments from the seven targeted genes was used as a reference. The 16S rRNA gene was the most abundant, in all the analysed samples, followed by intI1, sul1, qnrS, and blaTEM, while blaCTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, blaTEM and qnrS the inter-laboratory variation was below 28% (3-8%, 6-18%, 8-21%, 10-24%, 15-28%, respectively). While it may be difficult to fully harmonize qPCR protocols due to equipment, reagents and operator variations, the inter-laboratory calibration is an adequate and necessary step to increase the reliability of comparative data on ARGs abundance in different environmental compartments and/or geographic regions.

Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

High throughput sequencing-based metagenomic analysis and network analysis were applied to investigate the broad-spectrum profiles of ARGs in landfill leachate from 12 cities in China. In total, 526 ARG subtypes belonging to 21 ARG types were detected with abundances ranging from 1.1 × 10-6 to 2.09 × 10-1 copy of ARG/copy of 16S rRNA gene. 68 ARG subtypes that accounted for 73.4%-93.4% of the total ARG abundances were shared by all leachate samples. The four most abundant ARGs, sul1, sul2, aadA and bacA can be served as ARG indicators to quantitatively predict the total abundances by linear functions (r2 = 0.577-0.819, P < 0.001). No distinct regional distribution pattern of the ARGs was observed among different cities in China, while the ARG compositions of the leachate were clearly distinct from those of other environmental sample types. Nearly 90% ARG subtypes in the anaerobic digestion sludge from sewage treatment plants (STPADS) were shared by the leachate and the abundances of leachate and STPADS ARGs generalists accounted for 84.5% and 87.7% of total abundances in these two types of anaerobic samples, respectively. Furthermore, Procrustes analysis suggested that microbial community composition might be the determining factor of ARG compositions in landfill leachate. ARGs within the same type or among the different types showed higher incidences of non-random co-occurrence and 17 genera might be potential hosts of multiple ARGs. This study highlighted that landfill leachate is an important reservoir of various ARGs and provided a useful reference for the surveillance and risk management of ARGs in landfill environments.

The accelerated spread of antibiotic resistance genes (ARG) in the environment occurs mainly through plasmid transfer facilitated via bacterial conjugation. To predict and efficiently counteract the problems associated with ARG transmission, it is important to estimate conjugation rates under different experimental conditions. The classical models typically used to estimate parameters for mating experiments, while pragmatic in calculating growth and plasmid transfer, often ignore processes such as the reduction in growth due to plasmid bearing costs and are non-inclusive of environmental influences like temperature effects. Here, we present a process-based numerical model taking into account the fitness cost associated with plasmid carriage and temperature dependencies in vertical and horizontal gene transfer processes. Observations from liquid culture conjugation experiments using Escherichia coli and the plasmid pB10 were used to validate our proposed model. We present a comparison between the parameters estimated using the existing and the proposed model. Uncertainties in the estimated parameters were quantified using classical and advanced Bayesian methods. For our mating experiments, we found that at temperatures between 20 and 37 °C, the plasmid bearing costs reduced the growth rates by > 35%. The temperature dependency model of conjugation showed a good fit (mean absolute percentage error < 10%) independent of the bacteria and the plasmid under study. The proposed model simultaneously estimates growth and plasmid transfer rate constants for all three strains (donor, recipient, and transconjugant). Simultaneous estimation of growth and conjugation parameters is particularly useful to estimate the spread of ARG when one of the mating partners inhibits the growth of the other, which is common in multi-species mating or when the incurred plasmid costs are situation dependent (e.g., increased plasmid cost in a mating environment) as observed in this study.

Ordinary differential equations (ODE) are at the center of many environmental models. The R software is increasingly used to implement ODE-based models boosted by add-on packages providing numerical solvers. This paper introduces an R package called rodeo to facilitate the implementation of ODE-based models. It addresses a standardized model notation and documentation as well as the automatic generation of efficient, solver-compliant Fortran or R source code. The package has the potential to make a multitude of environmental models, more comprehensible, reusable, and exchangeable.

Es wurde die Dynamik der Antibiotikaresistenz-Gene (ARG) und die zugehorige mikrobielle Diversitat in der Kanalisation, dem Zulauf und Ablauf einer Klaranlage untersucht. Hierzu wurden Untersuchungen in einem Zeitraum von zwei Jahren durchgefuhrt und Proben saisonal genommen. Basierend auf Analysen der quantitativen PCR (qPCR) zeigen unsere Ergebnisse ein saisonales Muster fur die relativen Haufigkeiten der ARG, mit hoheren ARG Konzentrationen im Herbst und Winter. Es wurden keine signifikanten Unterschiede in den relativen Konzentrationen der ARG zwischen Kanalisation, Zulauf und Ablauf der Klaranlage detektiert, aber die mikrobielle Gemeinschaft war deutlich verschieden. Dies deutet darauf hin, dass die ARG nicht im dominanten Teil der mikrobiellen Gemeinschaft der Klaranlage vertreten sind. Eine Ausnahme bildeten die Konzentrationen fur das Resistenzgen vanA, fur welches erhohte relative Konzentrationen im Ablauf detektiert wurden.

A comprehensive monitoring of a broad set of antibiotics in the final effluent of wastewater treatment plants (WWTPs) of 7 European countries (Portugal, Spain, Ireland, Cyprus, Germany, Finland, and Norway) was carried out in two consecutive years (2015 and 2016). This is the first study of this kind performed at an international level. Within the 53 antibiotics monitored 17 were detected at least once in the final effluent of the WWTPs, i.e.: ciprofloxacin, ofloxacin, enrofloxacin, orbifloxacin, azithromycin, clarithromycin, sulfapyridine, sulfamethoxazole, trimethoprim, nalidixic acid, pipemidic acid, oxolinic acid, cefalexin, clindamycin, metronidazole, ampicillin, and tetracycline. The countries exhibiting the highest effluent average concentrations of antibiotics were Ireland and the southern countries Portugal and Spain, whereas the northern countries (Norway, Finland and Germany) and Cyprus exhibited lower total concentration. The antibiotic occurrence data in the final effluents were used for the assessment of their impact on the aquatic environment. Both, environmental predicted no effect concentration (PNEC-ENVs) and the PNECs based on minimal inhibitory concentrations (PNEC-MICs) were considered for the evaluation of the impact on microbial communities in aquatic systems and on the evolution of antibiotic resistance, respectively. Based on this analysis, three compounds, ciprofloxacin, azithromycin and cefalexin are proposed as markers of antibiotic pollution, as they could occasionally pose a risk to the environment. Integrated studies like this are crucial to map the impact of antibiotic pollution and to provide the basis for designing water quality and environmental risk in regular water monitoring programs, We thank all the wastewater treatment plant staff and other collaborators who made this work possible. We would also thank Virginie Keller and Andrew Johnson for their help in the data processing. Funding: This work was financed by the Water JPI through the national funding agencies supporting the consortium WaterJPI/0001/2013 STARE—“Stopping Antibiotic Resistance Evolution” (Cyprus, RPF; Germany, BMBF; Spain, MINECO; Finland, AKA; Ireland, EPA; Norway, RCN; Portugal, FCT). Authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124 and 2017-SGR-1404-Water and Soil Quality Unit). S.R.-M. acknowledges the Ramon y Cajal program (RYC-2014-16707). I.V.-M. was supported by the FCT grant SFRH/BPD/87360/2012. Laura Rodriguez de la Ballina is acknowledged for her help in the design of graphical abstract.

Airplane sanitary facilities are shared by an international audience. We hypothesized the corresponding sewage to be an extraordinary source of antibiotic-resistant bacteria (ARB) and resistance genes (ARG) in terms of diversity and quantity. Accordingly, we analyzed ARG and ARB in airplane-borne sewage using complementary approaches: metagenomics, quantitative polymerase chain reaction (qPCR), and cultivation. For the purpose of comparison, we also quantified ARG and ARB in the inlets of municipal treatment plants with and without connection to airports. As expected, airplane sewage contained an extraordinarily rich set of mobile ARG, and the relative abundances of genes were mostly increased compared to typical raw sewage of municipal origin. Moreover, combined resistance against third-generation cephalosporins, fluorochinolones, and aminoglycosides was unusually common (28.9%) among Escherichia coli isolated from airplane sewage. This percentage exceeds the one reported for German clinical isolates by a factor of 8. Our findings suggest that airplane-borne sewage can effectively contribute to the fast and global spread of antibiotic resistance.

Bacterial resistance against the last-resort antibiotic colistin is of increasing concern on a global scale. Wastewater is suspected to be one of the pathways by which resistant bacteria and the respective genes are disseminated. We employed a metagenomics approach to detect and quantify colistin resistance genes in raw municipal wastewater sampled at 9 locations all over Germany (14 samples in total, collected in 2016/2017). Our data support the findings of earlier studies according to which the prevalence of the colistin resistance gene mcr-1 is still low. However, we were able to demonstrate that the total prevalence of colistin resistance genes is dramatically underestimated if the focus is put on that specific gene alone. In comparison to mcr-1, other gene variants like mcr-3 and mcr-7 proved to be 10 to 100 times more abundant in samples of untreated wastewater. The average relative abundances expressed as copies per 16S rRNA gene copies were 2.3×10-3 for mcr-3, 2.2×10-4 for mcr-4, 3.0×10-4 for mcr-5, and 4.4×10-4 for mcr-7. While these four gene variants were ubiquitous in all 14 samples, mcr-1 was detected only once at a relative abundance of 1.4×10-5. Our results suggest a high risk of increasing incidence of colistin resistance as large amounts of mcr genes are continuously disseminated to diverse microbial communities via the wastewater path.