Every fifth published metagenome is not available to science.

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.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Demonstrating a Comprehensive Wastewater-Based Surveillance Approach That Differentiates Globally Sourced Resistomes.

Wastewater-based surveillance (WBS) for disease monitoring is highly promising but requires consistent methodologies that incorporate predetermined objectives, targets, and metrics. Herein, we describe a comprehensive metagenomics-based approach for global surveillance of antibiotic resistance in sewage that enables assessment of 1) which antibiotic resistance genes (ARGs) are shared across regions/communities; 2) which ARGs are discriminatory; and 3) factors associated with overall trends in ARGs, such as antibiotic concentrations. Across an internationally sourced transect of sewage samples collected using a centralized, standardized protocol, ARG relative abundances (16S rRNA gene-normalized) were highest in Hong Kong and India and lowest in Sweden and Switzerland, reflecting national policy, measured antibiotic concentrations, and metal resistance genes. Asian versus European/US resistomes were distinct, with macrolide-lincosamide-streptogramin, phenicol, quinolone, and tetracycline versus multidrug resistance ARGs being discriminatory, respectively. Regional trends in measured antibiotic concentrations differed from trends expected from public sales data. This could reflect unaccounted uses, captured only by the WBS approach. If properly benchmarked, antibiotic WBS might complement public sales and consumption statistics in the future. The WBS approach defined herein demonstrates multisite comparability and sensitivity to local/regional factors.

Pathogenic and Indigenous Denitrifying Bacteria are Transcriptionally Active and Key Multi-Antibiotic-Resistant Players in Wastewater Treatment Plants.

The global rise and spread of antibiotic resistance greatly challenge the treatment of bacterial infections. Wastewater treatment plants (WWTPs) harbor and discharge antibiotic resistance genes (ARGs) as environmental contaminants. However, the knowledge gap on the host identity, activity, and functionality of ARGs limits transmission and health risk assessment of the WWTP resistome. Hereby, a genome-centric quantitative metatranscriptomic approach was exploited to realize high-resolution qualitative and quantitative analyses of bacterial hosts of ARGs (i.e., multiresistance, pathogenicity, activity, and niches) in the 12 urban WWTPs. We found that ∼45% of 248 recovered genomes expressed ARGs against multiple classes of antibiotics, among which bacitracin and aminoglycoside resistance genes in Proteobacteria were the most prevalent scenario. Both potential pathogens and indigenous denitrifying bacteria were transcriptionally active hosts of ARGs. The almost unchanged relative expression levels of ARGs in the most resistant populations (66.9%) and the surviving ARG hosts including globally emerging pathogens (e.g., Aliarcobacter cryaerophilus) in treated WWTP effluent prioritize future examination on the health risks related to resistance propagation and human exposure in the receiving environment.

Strong impact of anthropogenic contamination on the co-occurrence patterns of a riverine microbial community.

Although the health of rivers is threatened by multiple anthropogenic stressors with increasing frequency, it remains an open question how riverine microbial communities respond to emerging micropollutants. Here, by using 16S rDNA amplicon sequencing of 60 water samples collected during different hydrological seasons, we investigated the spatio-temporal variation and the co-occurrence patterns of microbial communities in the anthropogenically impacted Jiulong River in China. The results indicated that the riverine microbial co-occurrence network had a nonrandom, modular structure, which was mainly shaped by the taxonomic relatedness of co-occurring species. Fecal indicator bacteria may survive for prolonged periods of time in river water, but they formed an independent module which had fewer interactions with typical freshwater bacteria. Multivariate analysis demonstrated that nutrients and micropollutants [i.e., pharmaceuticals and personal care products (PPCPs)] exerted combined effects in shaping α- and ß-diversity of riverine microbial communities. Remarkably, we showed that a hitherto unrecognized disruptive effect of PPCPs on the abundance variations of central species and module communities was stronger than the influence of physicochemical factors, suggesting the key role played by micropollutants for the microbial co-occurrence relationships in lotic ecosystems. Overall, our findings provide novel insights into community assembly in aquatic environments experiencing anthropogenic stresses.

Growth of Nitrosococcus-Related Ammonia Oxidizing Bacteria Coincides with Extremely Low pH Values in Wastewater with High Ammonia Content.

Ammonia oxidation decreases the pH in wastewaters where alkalinity is limited relative to total ammonia. The activity of ammonia oxidizing bacteria (AOB), however, typically decreases with pH and often ceases completely in slightly acidic wastewaters. Nevertheless, nitrification at low pH has been reported in reactors treating human urine, but it has been unclear which organisms are involved. In this study, we followed the population dynamics of ammonia oxidizing organisms and reactor performance in synthetic fully hydrolyzed urine as the pH decreased over time in response to a decrease in the loading rate. Populations of the ß-proteobacterial Nitrosomonas europaea lineage were abundant at the initial pH close to 6, but the growth of a possibly novel Nitrosococcus-related AOB genus decreased the pH to the new level of 2.2, challenging the perception that nitrification is inhibited entirely at low pH values, or governed exclusively by ß-proteobacterial AOB or archaea. With the pH shift, nitrite oxidizing bacteria were not further detected, but nitrous acid (HNO2) was still removed through chemical decomposition to nitric oxide (NO) and nitrate. The growth of acid-tolerant γ-proteobacterial AOB should be prevented, by keeping the pH above 5.4, which is a typical pH limit for the N. europaea lineage. Otherwise, the microbial community responsible for high-rate nitrification can be lost, and strong emissions of hazardous volatile nitrogen compounds such as NO are likely.

Toward a Comprehensive Strategy to Mitigate Dissemination of Environmental Sources of Antibiotic Resistance.

Antibiotic resistance is a pervasive global health threat. To combat the spread of resistance, it is necessary to consider all possible sources and understand the pathways and mechanisms by which resistance disseminates. Best management practices are urgently needed to provide barriers to the spread of resistance and maximize the lifespan of antibiotics as a precious resource. Herein we advise upon the need for coordinated national and international strategies, highlighting three essential components: (1) Monitoring, (2) Risk Assessment, and (3) Mitigation of antibiotic resistance. Central to all three components is What exactly to monitor, assess, and mitigate? We address this question within an environmental framework, drawing from fundamental microbial ecological processes driving the spread of resistance.

Inactivation of Antibiotic Resistant Bacteria and Resistance Genes by Ozone: From Laboratory Experiments to Full-Scale Wastewater Treatment.

Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

Physical extraction of microorganisms from water-saturated, packed sediment.

Microbial characterization of aquifers should include samples of both suspended and attached microorganisms (biofilms). We investigated the effect of shear, sonication, and heat on the extraction of microorganisms from water-saturated, packed sediment columns containing established biofilms. Shear was studied by increasing flow velocity of the column eluent, sonication by treating the columns with ultrasound at different power levels, and heat by warming up the column eluent to different temperatures. Effluent cell concentrations were used as a measure of extraction efficiency. Dissolved organic carbon and adenosine tri-phosphate (ATP) concentrations were used to corroborate cell-extraction results. Additionally, ATP was used as an indicator of cell-membrane integrity. Extraction quality was determined by comparing terminal-restriction fragment length polymorphism (T-RFLP) profiles of extracted bacterial communities with destructively sampled sediment-community profiles. Sonication and heat increased the extraction efficiency up to 200-fold and yielded communities comparable to the sediment community. These treatments showed high potential for in-situ application in aquifers.

Environmental microbiome diversity and stability is a barrier to antimicrobial resistance gene accumulation.

When antimicrobial resistant bacteria (ARB) and genes (ARGs) reach novel habitats, they can become part of the habitat's microbiome in the long term if they are able to overcome the habitat's biotic resilience towards immigration. This process should become more difficult with increasing biodiversity, as exploitable niches in a given habitat are reduced for immigrants when more diverse competitors are present. Consequently, microbial diversity could provide a natural barrier towards antimicrobial resistance by reducing the persistence time of immigrating ARB and ARG. To test this hypothesis, a pan-European sampling campaign was performed for structured forest soil and dynamic riverbed environments of low anthropogenic impact. In soils, higher diversity, evenness and richness were significantly negatively correlated with relative abundance of >85% of ARGs. Furthermore, the number of detected ARGs per sample were inversely correlated with diversity. However, no such effects were present in the more dynamic riverbeds. Hence, microbiome diversity can serve as a barrier towards antimicrobial resistance dissemination in stationary, structured environments, where long-term, diversity-based resilience against immigration can evolve.

Chemical extraction of microorganisms from water-saturated, packed sediment.

Microbial characterization of aquifers should combine collection of suspended and attached microorganisms (biofilms). This study investigated chemical extraction of microorganisms from water-saturated, packed sediment containing established biofilms. It compares the use of different detachment-promoting agent (DPA) solutions with tap water as eluent in column experiments. Extraction efficiency was determined from cell concentrations in the column effluent. Adenosine triphosphate concentrations were measured to confirm cell extraction and as an indicator of cell membrane integrity. Quality of extracted bacterial communities was assessed by comparing their terminal restriction fragment length polymorphism profiles with destructively sampled sediment-community profiles. Extraction efficiency increased more than 8-fold when deionized water, D-amino acids, or enzymes were used as a DPA. Community profiles recovered by individual DPA solutions showed more pronounced differences at the level of rare microbial groups, whereas abundant groups appeared ubiquitous across treatments. These results suggest that comparison of communities extracted by different DPAs can provide improved information on the occurrence of rare microbial groups in biofilms.

Differential effects of wastewater treatment plant effluents on the antibiotic resistomes of diverse river habitats.

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.

Synthetic oligonucleotides as quantitative PCR standards for quantifying microbial genes.

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.

Hydrodynamic regimes modulate nitrogen fixation and the mode of diazotrophy in Lake Tanganyika.

The factors that govern the geographical distribution of nitrogen fixation are fundamental to providing accurate nitrogen budgets in aquatic environments. Model-based insights have demonstrated that regional hydrodynamics strongly impact nitrogen fixation. However, the mechanisms establishing this physical-biological coupling have yet to be constrained in field surveys. Here, we examine the distribution of nitrogen fixation in Lake Tanganyika - a model system with well-defined hydrodynamic regimes. We report that nitrogen fixation is five times higher under stratified than under upwelling conditions. Under stratified conditions, the limited resupply of inorganic nitrogen to surface waters, combined with greater light penetration, promotes the activity of bloom-forming photoautotrophic diazotrophs. In contrast, upwelling conditions support predominantly heterotrophic diazotrophs, which are uniquely suited to chemotactic foraging in a more dynamic nutrient landscape. We suggest that these hydrodynamic regimes (stratification versus mixing) play an important role in governing both the rates and the mode of nitrogen fixation.

Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples.

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

Bacterial chitin hydrolysis in two lakes with contrasting trophic statuses.

Chitin, which is a biopolymer of the amino sugar glucosamine (GlcN), is highly abundant in aquatic ecosystems, and its degradation is assigned a key role in the recycling of carbon and nitrogen. In order to study the significance of chitin decomposition in two temperate freshwater lakes with contrasting trophic and redox conditions, we measured the turnover rate of the chitin analog methylumbelliferyl-N,N'-diacetylchitobioside (MUF-DC) and the presence of chitinase (chiA) genes in zooplankton, water, and sediment samples. In contrast to the eutrophic and partially anoxic lake, chiA gene fragments were detectable throughout the oligotrophic water column and chiA copy numbers per ml of water were up to 15 times higher than in the eutrophic waters. For both lakes, the highest chiA abundance was found in the euphotic zone--the main habitat of zooplankton, but also the site of production of easily degradable algal chitin. The bulk of chitinase activity was measured in zooplankton samples and the sediments, where recalcitrant chitin is deposited. Both, chiA abundance and chitinase activity correlated well with organic carbon, nitrogen, and concentrations of particulate GlcN. Our findings show that chitin, although its overall contribution to the total organic carbon is small (~0.01 to 0.1%), constitutes an important microbial growth substrate in these temperate freshwater lakes, particularly where other easily degradable carbon sources are scarce.

Local conditions structure unique archaeal communities in the anoxic sediments of meromictic Lake Kivu.

Meromictic Lake Kivu is renowned for its enormous quantity of methane dissolved in the hypolimnion. The methane is primarily of biological origin, and its concentration has been increasing in the past half-century. Insight into the origin of methane production in Lake Kivu has become relevant with the recent commercial extraction of methane from the hypolimnion. This study provides the first culture-independent approach to identifying the archaeal communities present in Lake Kivu sediments at the sediment-water interface. Terminal restriction fragment length polymorphism analysis suggests considerable heterogeneity in the archaeal community composition at varying sample locations. This diversity reflects changes in the geochemical conditions in the sediment and the overlying water, which are an effect of local groundwater inflows. A more in-depth look at the archaeal community composition by clone library analysis revealed diverse phylogenies of Euryarchaeota and Crenarachaeota. Many of the sequences in the clone libraries belonged to globally distributed archaeal clades such as the rice cluster V and Lake Dagow sediment environmental clusters. Several of the determined clades were previously thought to be rare among freshwater sediment Archaea (e.g., sequences related to the SAGMEG-1 clade). Surprisingly, there was no observed relation of clones to known hydrogentrophic methanogens and less than 2 % of clones were related to acetoclastic methanogens. The local variability, diversity, and novelty of the archaeal community structure in Lake Kivu should be considered when making assumptions on the biogeochemical functioning of its sediments.

Wastewater bypass is a major temporary point-source of antibiotic resistance genes and multi-resistance risk factors in a Swiss river.

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.

Microeukaryotic gut parasites in wastewater treatment plants: diversity, activity, and removal.

BACKGROUND: During wastewater treatment, the wastewater microbiome facilitates the degradation of organic matter, reduction of nutrients, and removal of gut parasites. While the latter function is essential to minimize public health risks, the range of parasites involved and how they are removed is still poorly understood. RESULTS: Using shotgun metagenomic (DNA) and metatranscriptomic (RNA) sequencing data from ten wastewater treatment plants in Switzerland, we were able to assess the entire wastewater microbiome, including the often neglected microeukaryotes (protists). In the latter group, we found a surprising richness and relative abundance of active parasites, particularly in the inflow. Using network analysis, we tracked these taxa across the various treatment compartments and linked their removal to trophic interactions. CONCLUSIONS: Our results indicate that the combination of DNA and RNA data is essential for assessing the full spectrum of taxa present in wastewater. In particular, we shed light on an important but poorly understood function of wastewater treatment - parasite removal. Video Abstract.

Long-read metagenomic sequencing reveals shifts in associations of antibiotic resistance genes with mobile genetic elements from sewage to activated sludge.

BACKGROUND: There is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents. RESULTS: The abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75-90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40-73 to 31-68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35-13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4-2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating vertical gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination. CONCLUSIONS: Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention. Video abstract.