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.

Zooplankton as a Transitional Host for Escherichia coli in Freshwater.

This study shows that Escherichia coli can be temporarily enriched in zooplankton under natural conditions and that these bacteria can belong to different phylogroups and sequence types (STs), including environmental, clinical, and animal isolates. We isolated 10 E. coli strains and sequenced the genomes of two of them. Phylogenetically, the two isolates were closer to strains isolated from poultry meat than to freshwater E. coli, albeit their genomes were smaller than those of the poultry isolates. After isolation and fluorescent protein tagging of strains ED1 and ED157, we show that Daphnia sp. can take up these strains and release them alive again, thus becoming a temporary host for E. coli. In a chemostat experiment, we show that this association does not prolong bacterial long-term survival, but at low abundances it also does not significantly reduce bacterial numbers. We demonstrate that E. coli does not belong to the core microbiota of Daphnia, suffers from competition by the natural Daphnia microbiota, but can profit from its carapax to survive in water. All in all, this study suggests that the association of E. coli with Daphnia is only temporary, but the cells are viable therein, and this might allow encounters with other bacteria for genetic exchange and potential genomic adaptation to the freshwater environment. IMPORTANCE The contamination of freshwater with feces-derived bacteria is a major concern regarding drinking water acquisition and recreational activities. Ecological interactions promoting their persistence are still very scarcely studied. This study, which analyses the survival of E. coli in the presence of zooplankton, is thus of ecological and water safety relevance.

Freshwater zooplankton microbiome composition is highly flexible and strongly influenced by the environment.

The association with microbes in plants and animals is known to be beneficial for host's survival and fitness, but the generality of the effect of the microbiome is still debated. For some animals, similarities in microbiome composition reflect taxonomic relatedness of the hosts, a pattern termed phylosymbiosis. The mechanisms behind the pattern could be due to co-evolution and/or to correlated ecological constraints. General conclusions are hampered by the fact that available knowledge is highly dominated by microbiomes from model species. Here, we addressed the issue of the generality of phylosymbiosis by analysing the species-specificity of microbiomes across different species of freshwater zooplankton, including rotifers, cladocerans, and copepods, coupling field surveys and experimental manipulations. We found that no signal of phylosymbiosis was present, and that the proportion of "core" microbial taxa, stable and consistent within each species, was very low. Changes in food and temperature under laboratory experimental settings revealed that the microbiome of freshwater zooplankton is highly flexible and can be influenced by the external environment. Thus, the role of co-evolution, strict association, and interaction with microbes within the holobiont concept highlighted for vertebrates, corals, sponges, and other animals does not seem to be supported for all animals, at least not for freshwater zooplankton. Zooplankton floats in the environment where both food and bacteria that can provide help in digesting such food are available. In addition, there is probably redundancy for beneficial bacterial functions in the environment, not allowing a strict host-microbiome association to originate and persist.

Antibiotic disturbance affects aquatic microbial community composition and food web interactions but not community resilience.

Notwithstanding the fundamental role that environmental microbes play for ecosystem functioning, data on how microbes react to disturbances are still scarce, and most factors that confer stability to microbial communities are unknown. In this context, antibiotic discharge into the environment is considered a worldwide threat for ecosystems with potential risks to human health. We therefore tested resilience of microbial communities challenged by the presence of an antibiotic. In a continuous culture experiment, we compared the abundance, composition and diversity of microbial communities undisturbed or disturbed by the constant addiction of tetracycline in low (10 µg/L) or intermediate (100 µg/L) concentration (press disturbance). Further, the bacterial communities in the three treatments had to face the sudden pulse disturbance of adding an allochthonous bacterium (Escherichia coli). Tetracycline, even at low concentrations, affected microbial communities by changing their phylogenetic composition and causing cell aggregation. This, however, did not coincide with a reduced microbial diversity, but was mainly caused by a shift in dominance of specific bacterial families. Moreover, the less disturbed community (10 µg/L tetracycline) was sometimes more similar to the control and sometimes more similar to heavily disturbed community (100 µg/L tetracycline). All in all, we could not see a pattern where the communities disturbed with antibiotics were less resilient to a second disturbance introducing E. coli, but they seemed to be able to buffer the input of the allochthonous strain in a similar manner as the control.

The microbiome associated with two Synechococcus ribotypes at different levels of ecological interaction.

Planktonic cyanobacteria belonging to the genus Synechococcus are ubiquitously distributed in marine and fresh waters, substantially contributing to total carbon fixation on a global scale. While their ecological relevance is acknowledged, increasing resolution in molecular techniques allows disentangling cyanobacteria's role at the micro-scale, where complex microbial interactions may drive the overall community assembly. The interplay between phylogenetically different Synechococcus clades and their associated bacterial communities can affect their ecological fate and susceptibility to protistan predation. In this study, we experimentally promoted different levels of ecological interaction by mixing two Synechococcus ribotypes (MW101C3 and LL) and their associated bacteria, with and without a nanoflagellate grazer (Poterioochromonas sp.) in laboratory cultures. The beta-diversity of the Synechococcus-associated microbiome in laboratory cultures indicated that the presence of the LL ribotype was the main factor determining community composition changes (41% of total variance), and prevailed over the effect of protistan predation (18% of total variance). Our outcomes also showed that species coexistence and predation may promote microbial diversity, thus highlighting the underrated ecological relevance of such micro-scale factors.

Constitutive presence of antibiotic resistance genes within the bacterial community of a large subalpine lake.

The fate of antibiotic resistance genes (ARGs) in environmental microbial communities is of primary concern as prodromal of a potential transfer to pathogenic bacteria. Although of diverse origin, the persistence of ARGs in aquatic environments is highly influenced by anthropic activities, allowing potential control actions in well-studied environments. However, knowledge of abundance and space-time distribution of ARGs in ecosystems is still scarce. Using quantitative real-time PCR, we investigated the presence and the abundance of twelve ARGs (against tetracyclines, ß-lactams, aminoglycosides, quinolones and sulphonamides) at different sampling sites, depths and seasons, in Lake Maggiore, a large subalpine lake, and in the area of its watershed. We then evaluated the correlation between each ARG and a number of ecological parameters in the water column in the deepest part of the lake. Our results suggest the constitutive presence of at least four ARGs within the bacterial community with a high proportion of bacteria potentially resistant to tetracyclines and sulphonamides. The presence of these ARGs was independent of the total bacterial density and temperature. The dynamics of tet(A) and sulII genes were, however, positively correlated with dissolved oxygen and negatively to chlorophyll a, suggesting that the resistant microbes inhabit specific niches. These observations indicate that the lake is a reservoir of antibiotic resistances, highlighting the need of a deeper understanding of the sources of ARGs and the factors allowing their persistence in waters.

Where do the antibiotic resistance genes come from? A modulated analysis of sources and loads of resistances in Lake Maggiore.

Antibiotic resistance genes (ARGs) are abundant in aquatic ecosystems affected by human activities. Understanding the fate of ARGs across different ecosystems is essential because of the significant role aquatic environments play in the cycle of antibiotic resistance. We quantified selected ARGs in Lake Maggiore, its main tributaries, and the effluent of the main wastewater treatment plant (WWTP) discharging directly into the lake. We linked their dynamics to the different anthropogenic impacts in each tributary's watershed. The dynamics of tetA in the lake were influenced by those of the rivers and the WWTP effluent, and by the concentration of N-NH4, related to anthropogenic pollution, while sul2 abundance in the lake was not influenced by any water inflow. The dynamics of the different ARGs varied across the different rivers. Rivers with watersheds characterized by high population density, touristic activities, and secondary industries released more ARGs, while ermB correlated with higher numbers of primary industries. This study suggests a limited contribution of treated wastewater in the spread of ARGs, indicating as prevalent origin other sources of pollution, calling for a reconsideration on what are considered the major sources of ARGs into the environment.

Anthropogenic pollution may enhance natural transformation in water, favouring the spread of antibiotic resistance genes.

Aquatic ecosystems are crucial in the antimicrobial resistance cycle. While intracellular DNA has been extensively studied to understand human activity's impact on antimicrobial resistance gene (ARG) dissemination, extracellular DNA is frequently overlooked. This study examines the effect of anthropogenic water pollution on microbial community diversity, the resistome, and ARG dissemination. We analyzed intracellular and extracellular DNA from wastewater treatment plant effluents and lake surface water by shotgun sequencing. We also conducted experiments to evaluate anthropogenic pollution's effect on transforming extracellular DNA (using Gfp-plasmids carrying ARGs) within a natural microbial community. Chemical analysis showed treated wastewater had higher anthropogenic pollution-related parameters than lake water. The richness of microbial community, antimicrobial resistome, and high-risk ARGs was greater in treated wastewaters than in lake waters both for intracellular and extracellular DNA. Except for the high-risk ARGs, richness was significantly higher in intracellular than in extracellular DNA. Several ARGs were associated with mobile genetic elements and located on plasmids. Furthermore, Gfp-plasmid transformation within a natural microbial community was enhanced by anthropogenic pollution levels. Our findings underscore anthropogenic pollution's pivotal role in shaping microbial communities and their antimicrobial resistome. Additionally, it may facilitate ARG dissemination through extracellular DNA plasmid uptake.

Grazing resistant freshwater bacteria profit from chitin and cell-wall-derived organic carbon.

The rise of grazing resistant planktonic bacteria in freshwater lakes during vernal phytoplankton blooms is favoured by predation of heterotrophic nanoflagellates (HNF). The spring period is also characterized by increased availability of organic carbon species that are in parts derived from cellular debris generated during bacterivory or viral lysis, such as peptidoglycan, chitin and their subunit N-acetylglucosamine (NAG). We tested the hypothesis that two dominant grazing resistant bacterial taxa, the ac1 tribe of Actinobacteria (ac1) and filamentous bacteria from the LD2 lineage (Saprospiraceae), profit from such carbon sources during periods of intense HNF predation. The abundances of ac1 and LD2 rose in parallel with HNF, and disproportionally high fractions of cells from both lineages were involved in NAG uptake. Members of ac1 and LD2 were significantly more enriched after NAG addition to lake water. However, highest growth rates of both bacterial lineages were found on chitin and peptidoglycan. Moreover, the direct or indirect transfer of organic carbon from peptidoglycan to LD2 filaments could be demonstrated. We thus provide evidence that these taxa may benefit twofold from protistan predation: by removal of their competitors, and by specific physiological adaptations to utilize carbon sources that are released during grazing or viral lysis.

Co-treatment of landfill leachate with urban wastewater by chemical, physical and biological processes: Fenton oxidation preserves autochthonous bacterial community in the activated sludge process.

The impact of Fenton oxidation (FO) and Air stripping (AS) pre-treatments on the bacterial community of a biological activated sludge (B-AS) process for the co-treatment of mature landfill leachate (MLL) and urban wastewater (UWW) was assessed. In this work high-throughput sequencing was used to identify changes in the composition of the bacterial communities when exposed to different landfill leachate's pre-treatments. The combination of FO and AS to increase biodegradability (BOD5/COD) and reduce ammonia concentration (NH3) respectively, allowed to successfully operate the B-AS and effectively treat MLL. In particular, BOD5/COD resulted to be the key factor for bacterial community shifting. The microbiological community of the B-AS, mainly composed by the phylum Bacteroidota (Saprospiraceae, PHOS-HE51, Chitinophagaceae) after FO pre-treatment, shifted to Pseudomonadota (Caulobacteraceae and Hyphomicrobiaceae) when FO was not used. At the same time a drastic reduction in BOD5 removal was observed (90%-58%). On the other hand, high NH3 concentration affected the abundance of the family Saprospiraceae, known to play a key role in the degradation of complex organic compounds in B-AS. The results obtained suggest that a suitable combination of pre-treatments can reduce the negative effect of MLL on the B-AS process, reducing the pressure on autochthonous bacteria and therefore the acclimatization time of the biological process.

Increased similarity of aquatic bacterial communities of different origin after antibiotic disturbance.

Stochastic or deterministic processes control the bacterial community assembly in waters and their understanding is a fundamental question to correctly manage aquatic environments exposed to the release of antibiotics from anthropogenic sources. It has been suggested that microdiversity (i.e. the rare biosphere) convers freshwater communities with stability, meaning that previously rare taxa bloom when the community is disturbed. Since there might be a seed bank of similar, but not abundant, bacterial taxa in different waters, we tested whether a disturbance by an antibiotic cocktail would increase similarity in bacterial communities from different freshwater systems (a wastewater effluent and two lakes). In a continuous culture set-up in chemostats, we show that disturbance with antibiotics causes communities from different environments to become more similar. Once the antibiotic pressure is released the communities tend to become more dissimilar again. This shows that there is a similar shift in community composition even in waters from very different origins when they are disturbed by antibiotics, even at low concentrations. Antibiotics impact the bacterial communities at the cell and the community level, independently by the original degree of anthropogenic stress they are adapted to, altering the original phenotypes, genotypes, and the relations between bacteria.

Class 1 integron and related antimicrobial resistance gene dynamics along a complex freshwater system affected by different anthropogenic pressures.

The risk for human health posed by polluted aquatic environments, and especially those carrying antibiotic resistance genes (ARGs) of clinical interest, is still debated. This is because of our limited knowledge of the dynamics of antimicrobial resistance in the environment, the selection mechanisms underlying the spread of ARGs, and the ecological factors potentially favoring their return to humans. The Class 1 integron is one of the most effective platforms for the dissemination of ARGs. In this study we investigated a freshwater system consisting of a lake-river-lake continuum, determining the abundance of class 1 integrons and their associated ARGs by a modulated metagenomic approach. Bacterial abundance and community composition were used to identify the potential carriers of class 1 integrons and their associated ARGs over a period of six months. Class 1 integrons and their ARG cargoes were significantly more abundant in riverine sampling sites receiving treated wastewater. Further, class 1 integrons carried ARGs ranked at the highest risk for human health (e.g., catB genes), in particular, genes encoding resistance to aminoglycosides. Genera of potential pathogens, such as Pseudomonas and Escherichia-Shigella, were correlated with class 1 integrons. The lake-river-lake system demonstrated a clear relationship between the integrase gene of class 1 integrons (intI1) and anthropogenic impact, but also a strong environmental filtering that favored the elimination of intI1 once the human derived stressors were reduced. Overall, the results of this study underline the role class 1 integrons as proxy of anthropogenic pollution and suggest this genetic platform as an important driver of aminoglycoside resistance genes, including high risk ARGs, of potential concern for human health.

Rapid successions affect microbial N-acetyl-glucosamine uptake patterns during a lacustrine spring phytoplankton bloom.

The vernal successions of phytoplankton, heterotrophic nanoflagellates (HNF) and viruses in temperate lakes result in alternating dominance of top-down and bottom-up factors on the bacterial community. This may lead to asynchronous blooms of bacteria with different life strategies and affect the channelling of particular components of the dissolved organic matter (DOM) through microbial food webs. We followed the dynamics of several bacterial populations and of other components of the microbial food web throughout the spring phytoplankton bloom period in a pre-alpine lake, and we assessed bacterial uptake patterns of two constituents of the labile DOM pool (N-acetyl-glucosamine [NAG] and leucine). There was a clear genotypic shift within the bacterial assemblage, from fast growing Cytophaga-Flavobacteria (CF) affiliated with Fluviicola and from Betaproteobacteria (BET) of the Limnohabitans cluster to more grazing resistant AcI Actinobacteria (ACT) and to filamentous morphotypes. This was paralleled by successive blooms of viruses and HNF. We also noted the transient rise of other CF (related to Cyclobacteriaceae and Sphingobacteriaceae) that are not detected by fluorescence in situ hybridization with the general CF probe. Both, the average uptake rates of leucine and the fractions of leucine incorporating bacteria were approximately five to sixfold higher than of NAG. However, the composition of the NAG-active community was much more prone to genotypic successions, in particular of bacteria with different life strategies: While 'opportunistically' growing BET and CF dominated NAG uptake in the initial period ruled by bottom-up factors, ACT constituted the major fraction of NAG active cells during the subsequent phase of high predation pressure. This indicates that some ACT could profit from a substrate that might in parts have originated from the grazing of protists on their bacterial competitors.

A Metabarcoding Protocol to Analyze Coastal Planktic Communities Collected by Desalination Plant Filters: From Sampling to Bioinformatic Exploratory Analyses.

The temporal dynamics of coastal planktic communities can be disclosed through DNA metabarcoding on the filters of reverse-osmosis desalination plants. Here, we describe the steps that are necessary to process the filters in order to create the subsamples used for DNA extraction and the bioinformatic pipeline to perform the first exploratory analyses on this kind of dataset.

PET particles raise microbiological concerns for human health while tyre wear microplastic particles potentially affect ecosystem services in waters.

Although abundant and chemically peculiar, tyre wear microplastic particles (TWP) and their impact on the microbial communities in water are largely understudied. We tested in laboratory based semi-continuous cultures the impact of TWP and of polyethylene terephthalate (PET) derived particles (following a gradient of relative abundance) on the pathobiome (the group of potential human pathogenic bacteria) of a freshwater microbial community exposed to contamination by the effluent of a urban wastewater treatment plant, for a period of 28 days. We could define the modulated impact of the two types of microplastic particles: while PET does not favour bacterial growth, it offers a refuge to several potential pathogens of allochthonous origin (from the treated sewage effluent), TWP act as an additional carbon source, promoting the development and the massive growth of a biofilm composed by fast-growing bacterial genera including species potentially harmful and competitive in abating biodiversity in surface waters. Our results demonstrate the different ecological role and impact on freshwater environments of TWP and PET particles, and the need to approach the study of this pollutant not as a whole, but considering the origin and the chemical composition of the different particles.

Contribution of plasmidome, metal resistome and integrases to the persistence of the antibiotic resistome in aquatic environments.

Wastewater treatment plants (WWTPs) are among the main hotspots of antibiotic resistance genes (ARGs) in the environment. Previously, we demonstrated that, by increasing anthropogenic pollution, the antibiotic resistome persisted in the microbial community of rivers and lakes, independently by changes in community composition. In this study, we reanalysed the data to test for the relation of metal resistance genes (MRGs), plasmids, and integrons to the persistence of the antibiotic resistome. The experiment consisted in replicated co-cultures of riverine or lacustrine microbial communities and WWTP effluents in different proportions. Samples before (T0) and after a short period of incubation (TF) were collected and community metagenomic data were obtained by shotgun sequencing. The data were processed to annotate MRGs, plasmids, and integrases. The integrases stabilized in the aquatic environment following the degree of contamination with effluent water (in particular in one site), whereas MRGs and plasmids showed stochastic trajectories. These results confirm the potential correlation between integrons and anthropogenic pollution, and the reliability of intI1 as a pollution marker. Only in one site MRGs, plasmids, and ARGs were correlated, highlighting their partial contribution to the persistence of ARGs in surface waters.

Antibiotic resistance genes correlate with metal resistances and accumulate in the deep water layers of the Black Sea.

Seas and oceans are a global reservoir of antibiotic resistance genes (ARGs). Only a few studies investigated the dynamics of ARGs along the water column of the Black Sea, a unique environment, with a peculiar geology, biology and history of anthropogenic pollution. In this study, we analyzed metagenomic data from two sampling campaigns (2013 and 2019) collected across three different sites in the Western Black Sea at depths ranging from 5 to 2000 m. The data were processed to annotate ARGs, metal resistance genes (MRGs) and integron integrase genes. The ARG abundance was significantly higher in the deep water layers and depth was the main driver of beta-diversity both for ARGs and MRGs. Moreover, ARG and MRG abundances strongly correlated (r = 0.95). The integron integrase gene abundances and composition were not influenced by the water depth and did not correlate with ARGs. The analysis of the obtained MAGs showed that some of them harbored intI gene together with several ARGs and MRGs, suggesting the presence of multidrug resistant bacteria and that MRGs and integrons could be involved in the selection of ARGs. These results demonstrate that the Black Sea is not only an important reservoir of ARGs, but also that they accumulate in the deep water layers where co-selection with MRGs could be assumed as a relevant mechanism of their persistence.

Contribution of microplastic particles to the spread of resistances and pathogenic bacteria in treated wastewaters.

Microplastic Particles (MPs) are ubiquitous pollutants widely found in aquatic ecosystems. Although MPs are mostly retained in wastewater treatment plants (WWTPs), a high number of MPs reaches the open waters potentially contributing to the spread of pathogenic bacteria and antibiotic resistance genes in the environment. Nowadays, a limited number of studies have focused on the role of MPs as carriers of potentially pathogenic and antibiotic resistant bacteria in WWTPs. Thus, an investigation on the community composition (by 16S rRNA gene amplicon sequencing) and the abundance of antibiotic and metal resistance genes (by qPCR) of the biofilm on MPs (the plastisphere) and of planktonic bacteria in treated (pre- and post-disinfection) wastewaters was performed. MPs resulted to be very similar in terms of type, color, size, and chemical composition, before and after the disinfection. The bacterial community on MPs differed from the planktonic community in terms of richness, composition, and structure of the community network. Potentially pathogenic bacteria generally showed higher abundances in treated wastewater than in the biofilm on MPs. Furthermore, among the tested resistance genes, only sul2 (a common resistance gene against sulfonamides) resulted to be more abundant in the plastisphere than in the planktonic bacterial community. Our results suggest that the wastewater plastisphere could promote the spread of pathogenic bacteria and resistance genes in aquatic environment although with a relatively lower contribution than the wastewater planktonic bacterial community.

Antarctic coastal nanoplankton dynamics revealed by metabarcoding of desalination plant filters: Detection of short-term events and implications for routine monitoring.

One of the main requirements of any sound biological monitoring is the availability of long term and, possibly, temporal data with a high resolution. This is often difficult to be achieved, especially in Antarctica, due to a variety of logistic constraints, which make continuous sampling and monitoring activities generally unfeasible. Here we focus on the 5 µm filters used in the desalination plant of the Italian research base "Mario Zucchelli" in the Terra Nova Bay area (Ross Sea, Antarctica) to evaluate intra-annual coastal nanoplankton dynamics. These filters, together with others of larger mesh sizes, are used to decrease the amount of organisms and debris in the input seawater before the desalination processes take place, hence automatically collect the plankton present in the water column around the desalination system intake. We have used a DNA metabarcoding approach to characterize the communities retained by filters' sets collected in January 2012 and 2013. Intra-annual dynamics were disclosed with an unprecedented detail, that would not have been possible by using standard sampling approaches, and highlighted the importance of extreme, stochastic events such as katabatic wind pulses, which triggered dramatic, short-term shifts in coastal nanoplankton composition. This method, by combining a cost-effective sampling and molecular techniques, may represent a viable solution for long-term monitoring programs focusing on Antarctic coastal communities.

Spatial distribution of antibiotic and heavy metal resistance genes in the Black Sea.

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are worldwide considered as emerging contaminants of large interest, and a primary threat to human health. It is becoming clear that the environment plays a central role in the transmission, spread, and evolution of antibiotic resistance. Although marine systems have been largely investigated, only a few studies have considered the presence of ARGs in meso- and bathypelagic waters. To date, no molecular based studies have yet been made to investigate the occurrence of ARGs in the Black Sea, the largest meromictic basin in the world, receiving water from a number of important European rivers and their residues of anthropogenic activities in permanently stratified mesopelagic water masses. In this study, we determined the presence and the abundance of five ARGs (blaCTXM, ermB, qnrS, sul2, tetA) and of the heavy metal resistance gene (HMRG) czcA, in different sampling sites in the eastern and western Black Sea, at several depths (up to 1000 m) and various distances from the shoreline. Three ARGs (blaCTXM, sul2, and tetA) and czcA were present in at least 43% of the analysed samples, whereas ermB and qnrS were never detected. In particular, sul2 abundances increased significantly in coastal location, whereas tetA increased with sampling depth. These findings point out the Black Sea as a source of ARGs and HMRGs distributed along the whole water column.