A pan-genomic approach reveals novel Sulfurimonas clade in the ferruginous meromictic Lake Pavin.

The permanently anoxic waters in meromictic lakes create suitable niches for the growth of bacteria using sulphur metabolisms like sulphur oxidation. In Lake Pavin, the anoxic water mass hosts an active cryptic sulphur cycle that interacts narrowly with iron cycling, however the metabolisms of the microorganisms involved are poorly known. Here we combined metagenomics, single-cell genomics, and pan-genomics to further expand our understanding of the bacteria and the corresponding metabolisms involved in sulphur oxidation in this ferruginous sulphide- and sulphate-poor meromictic lake. We highlighted two new species within the genus Sulfurimonas that belong to a novel clade of chemotrophic sulphur oxidisers exclusive to freshwaters. We moreover conclude that this genus holds a key-role not only in limiting sulphide accumulation in the upper part of the anoxic layer but also constraining carbon, phosphate and iron cycling.

Functional diversity of microbial eukaryotes in a meromictic lake: Coupling between metatranscriptomic and a trait-based approach.

The advent of high-throughput sequencing has led to the discovery of a considerable diversity of microbial eukaryotes in aquatic ecosystems, nevertheless, their function and contribution to the trophic food web functioning remain poorly characterized especially in freshwater ecosystems. Based on metabarcoding data obtained from a meromictic lake ecosystem (Pavin, France), we performed a morpho-physio-phenological traits-based approach to infer functional groups of microbial eukaryotes. Metatranscriptomic data were also analysed to assess the metabolic potential of these groups across the diel cycle, size fraction, sampling depth, and periods. Our analysis highlights a huge microbial eukaryotic diversity in the monimolimnion characterized by numerous saprotrophs expressing transcripts related to sulfur and nitrate metabolism as well as dissolved and particulate organic matter degradation. We also describe strong seasonal variations of microbial eukaryotes in the mixolimnion, especially for parasites and mixoplankton. It appears that the water mixing (occurring during spring and autumn) which benefits photosynthetic host communities also promotes parasitic fungi dissemination and over-expression of genes involved in the zoospore phototaxis and stage transition in the parasitic cycle. Mixoplanktonic haptophytes over-expressing photosynthesis-, endocytosis- and phagosome-linked genes under nutrient limitation also suggest that phagotrophy may provide them an advantage over non-phagotrophic phytoplankton.

FreshOmics: A manually curated and standardized -omics database for investigating freshwater microbiomes.

Freshwater is a critical resource for human survival but severely threatened by anthropogenic activities and climate change. These changes strongly impact the abundance and diversity of the microbial communities which are key players in the functioning of these aquatic ecosystems. Although widely documented since the emergence of high-throughput sequencing approaches, the information on these natural microbial communities is scattered among thousands of publications and it is therefore difficult to investigate the temporal dynamics and the spatial distribution of microbial taxa within or across ecosystems. To fill this gap and in the FAIR principles context we built a manually curated and standardized microbial freshwater -omics database (FreshOmics). Based on recognized ontologies (ENVO, MIMICS, GO, ISO), FreshOmics describes 29 different types of freshwater ecosystems and uses standardized attributes to depict biological samples, sequencing protocols and article attributes for more than 2487 geographical locations across 71 countries around the world. The database contains 24,808 sequence identifiers (i.e., Run_Id / Exp_ID, mainly from SRA/DDBJ SRA/ENA, GSA and MG-RAST repositories) covering all sequence-based -omics approaches used to investigate bacteria, archaea, microbial eukaryotes, and viruses. Therefore, FreshOmics allows accurate and comprehensive analyses of microbial communities to answer questions related to their roles in freshwater ecosystems functioning and resilience, especially through meta-analysis studies. This collection also highlights different sort of errors in published works (e.g., wrong coordinates, sample type, material, spelling).

Plasmidome analysis of a hospital effluent biofilm: Status of antibiotic resistance.

Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered >350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem.

Microsporidia are a large group of obligate intracellular eukaryotic parasites related to Fungi. Recent studies suggest that their diversity has been greatly underestimated and little is known about their hosts other than metazoans, and thus about their impact on the communities at the base of the food web. In this work, we therefore studied the diversity of Microsporidia over one year and identified potential new hosts in small-sized fractions (<150 µm) in a lake ecosystem using a metabarcoding approach coupled with co-occurrence networks and tyramide signal amplification-fluorescent in situ hybridization. Our analysis shows a great Microsporidia diversity (1 472 OTUs), with an important part of this diversity being unknown. Temporal variations of this diversity have been observed, which might follow temporal variations of their potential hosts such as protists and microzooplankton. New hosts among them were identified as well as associations with phytoplankton. Indeed, repeated infections were observed in Kellicottia (rotifers) with a prevalence of 38% (infected individuals). Microsporidia inside a Stentor (ciliate) were also observed. Finally, potential infections of the diatom Asterionella were identified (prevalence <0.1%). The microsporidian host spectrum could be therefore even more important than previously described, and their role in the functioning of lake ecosystems is undoubtedly largely unknown.

Seasonal microbial dynamics in the ocean inferred from assembled and unassembled data: a view on the unknown biosphere.

In environmental metagenomic experiments, a very high proportion of the microbial sequencing data (> 70%) remains largely unexploited because rare and closely related genomes are missed in short-read assemblies. The identity and the potential metabolisms of a large fraction of natural microbial communities thus remain inaccessible to researchers. The purpose of this study was to explore the genomic content of unassembled metagenomic data and test their level of novelty. We used data from a three-year microbial metagenomic time series of the NW Mediterranean Sea, and conducted reference-free and database-guided analysis. The results revealed a significant genomic difference between the assembled and unassembled reads. The unassembled reads had a lower mean identity against public databases, and fewer metabolic pathways could be reconstructed. In addition, the unassembled fraction presented a clear temporal pattern, unlike the assembled ones, and a specific community composition that was similar to the rare communities defined by metabarcoding using the 16S rRNA gene. The rare gene pool was characterised by keystone bacterial taxa, and the presence of viruses, suggesting that viral lysis could maintain some taxa in a state of rarity. Our study demonstrates that unassembled metagenomic data can provide important information on the structure and functioning of microbial communities.

A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems.

Microsporidia are obligate intracellular eukaryotic parasites known to parasitize many species of the animal kingdom as well as some protists. However, their diversity is underestimated, in part as a consequence of the failure of 'universal' primers to detect them in metabarcoding studies. Besides, due to the inconsistency between taxonomy and phylogenetic data, available databases may assign incorrectly sequences obtained with high-throughput sequencing. In this work, we developed a comprehensive reference database which positions microsporidian SSU rRNA gene sequences within a coherent ranked phylogenetic framework. We used this phylogenetic framework to study the microsporidian diversity in lacustrine ecosystems, focusing on < 150 µm planktonic size fractions. Our analysis shows a high diversity of Microsporidia, with the identification of 1531 OTUs distributed within seven clades, of which 76% were affiliated to clade IV2 and 20% to clade I (nomenclature presented hereby). About a quarter of the obtained sequences shared less than 85% identity to the closest known species, which might represent undescribed genera or families infecting small hosts. Variations in the abundance of Microsporidia were recorded between the two lakes sampled and across the sampling period, which might be explained by spatio-temporal variations of their potential hosts such as microeukaryotes and metazooplankton.

Reconstruction of plasmids by shotgun sequencing from environmental DNA: which bioinformatic workflow?

Plasmids play important roles in microbial evolution and also in the spread of antibiotic resistance. Plasmid sequences are extensively studied from clinical isolates but rarely from the environment with a metagenomic approach focused on the plasmid fraction referred to as the plasmidome. A clear challenge in this context is to define a workflow for discriminating plasmids from chromosomal contaminants existing in the plasmidome. For this purpose, we benchmarked existing tools from assembly to detection of the plasmids by reference-free methods (cBar and PlasFlow) and database-guided approaches. Our simulations took into account short-reads alone or combined with moderate long-reads like those actually generated in environmental genomics experiments. This benchmark allowed us to select the best tools for limiting false-positives associated to plasmid prediction tools and a combination of reference-guided methods based on plasmid and bacterial databases.

Protist taxonomic and functional diversity in soil, freshwater and marine ecosystems.

Protists dominate eukaryotic diversity and play key functional roles in all ecosystems, particularly by catalyzing carbon and nutrient cycling. To date, however, a comparative analysis of their taxonomic and functional diversity that compares the major ecosystems on Earth (soil, freshwater and marine systems) is missing. Here, we present a comparison of protist diversity based on standardized high throughput 18S rRNA gene sequencing of soil, freshwater and marine environmental DNA. Soil and freshwater protist communities were more similar to each other than to marine protist communities, with virtually no overlap of Operational Taxonomic Units (OTUs) between terrestrial and marine habitats. Soil protists showed higher γ diversity than aquatic samples. Differences in taxonomic composition of the communities led to changes in a functional diversity among ecosystems, as expressed in relative abundance of consumers, phototrophs and parasites. Phototrophs (eukaryotic algae) dominated freshwater systems (49% of the sequences) and consumers soil and marine ecosystems (59% and 48%, respectively). The individual functional groups were composed of ecosystem- specific taxonomic groups. Parasites were equally common in all ecosystems, yet, terrestrial systems hosted more OTUs assigned to parasites of macro-organisms while aquatic systems contained mostly microbial parasitoids. Together, we show biogeographic patterns of protist diversity across major ecosystems on Earth, preparing the way for more focused studies that will help understanding the multiple roles of protists in the biosphere.

Seasonality of archaeal proteorhodopsin and associated Marine Group IIb ecotypes (Ca. Poseidoniales) in the North Western Mediterranean Sea.

The Archaea Marine Group II (MGII) is widespread in the world's ocean where it plays an important role in the carbon cycle. Despite recent discoveries on the group's metabolisms, the ecology of this newly proposed order (Candidatus Poseidoniales) remains poorly understood. Here we used a combination of time-series metagenome-assembled genomes (MAGs) and high-frequency 16S rRNA data from the NW Mediterranean Sea to test if the taxonomic diversity within the MGIIb family (Candidatus Thalassarchaeaceae) reflects the presence of different ecotypes. The MAGs' seasonality revealed a MGIIb family composed of different subclades that have distinct lifestyles and physiologies. The vitamin metabolisms were notably different between ecotypes with, in some, a possible link to sunlight's energy. Diverse archaeal proteorhodopsin variants, with unusual signature in key amino acid residues, had distinct seasonal patterns corresponding to changing day length. In addition, we show that in summer, archaea, as opposed to bacteria, disappeared completely from surface waters. Our results shed light on the diversity and the distribution of the euryarchaeotal proteorhodopsin, and highlight that MGIIb is a diverse ecological group. The work shows that time-series based studies of the taxonomy, seasonality, and metabolisms of marine prokaryotes is critical to uncover their diverse role in the ocean.

Transient Dynamics of Archaea and Bacteria in Sediments and Brine Across a Salinity Gradient in a Solar Saltern of Goa, India.

The microbial fluctuations along an increasing salinity gradient during two different salt production phases - initial salt harvesting (ISH) phase and peak salt harvesting (PSH) phase of Siridao solar salterns in Goa, India were examined through high-throughput sequencing of 16S rRNA genes on Illumina MiSeq platform. Elemental analysis of the brine samples showed high concentration of sodium (Na+) and chloride (Cl-) ions thereby indicating its thalassohaline nature. Comparison of relative abundance of sequences revealed that Archaea transited from sediment to brine while Bacteria transited from brine to sediment with increasing salinity. Frequency of Archaea was found to be significantly enriched even in low and moderate salinity sediments with their relative sequence abundance reaching as high as 85%. Euryarchaeota was found to be the dominant archaeal phylum containing 19 and 17 genera in sediments and brine, respectively. Phylotypes belonging to Halorubrum, Haloarcula, Halorhabdus, and Haloplanus were common in both sediments and brine. Occurence of Halobacterium and Natronomonas were exclusive to sediments while Halonotius was exclusive to brine. Among sediments, relative sequence frequency of Halorubrum, and Halorhabdus decreased while Haloarcula, Haloplanus, and Natronomonas increased with increasing salinity. Similarly, the relative abundance of Haloarcula and Halorubrum increased with increasing salinity in brine. Sediments and brine samples harbored about 20 and 17 bacterial phyla, respectively. Bacteroidetes, Proteobacteria, and Chloroflexi were the common bacterial phyla in both sediments and brine while Firmicutes were dominant albeit in sediments alone. Further, Gammaproteobacteria, Alphaproteobacteria, and Deltaproteobacteria were observed to be the abundant class within the Proteobacteria. Among the bacterial genera, phylotypes belonging to Rubricoccus and Halomonas were widely detected in both brine and sediment while Thioalkalispira, Desulfovermiculus, and Marinobacter were selectively present in sediments. This study suggests that Bacteria are more susceptible to salinity fluctuations than Archaea, with many bacterial genera being compartment and phase-specific. Our study further indicated that Archaea rather than Bacteria could withstand the wide salinity fluctuation and attain a stable community structure within a short time-frame.

Assessing the response of micro-eukaryotic diversity to the Great Acceleration using lake sedimentary DNA.

Long-term time series have provided evidence that anthropogenic pressures can threaten lakes. Yet it remains unclear how and the extent to which lake biodiversity has changed during the Anthropocene, in particular for microbes. Here, we used DNA preserved in sediments to compare modern micro-eukaryotic communities with those from the end of the 19th century, i.e., before acceleration of the human imprint on ecosystems. Our results obtained for 48 lakes indicate drastic changes in the composition of microbial communities, coupled with a homogenization of their diversity between lakes. Remote high elevation lakes were globally less impacted than lowland lakes affected by local human activity. All functional groups (micro-algae, parasites, saprotrophs and consumers) underwent significant changes in diversity. However, we show that the effects of anthropogenic changes have benefited in particular phototrophic and mixotrophic species, which is consistent with the hypothesis of a global increase of primary productivity in lakes.

Honeybee gut microbiota dysbiosis in pesticide/parasite co-exposures is mainly induced by Nosema ceranae.

Honeybees ensure a key ecosystem service by pollinating many agricultural crops and wild plants. However, in the past few decades, managed bee colonies have been declining in Europe and North America. Researchers have emphasized both parasites and pesticides as the most important factors. Infection by the parasite Nosema ceranae and exposure to pesticides can contribute to gut dysbiosis, impacting the honeybee physiology. Here, we examined and quantified the effects of N. ceranae, the neonicotinoid thiamethoxam, the phenylpyrazole fipronil and the carboxamide boscalid, alone and in combination, on the honeybee gut microbiota. Chronic exposures to fipronil and thiamethoxam alone or combined with N. ceranae infection significantly decreased honeybee survival whereas the fungicide boscalid had no effect on uninfected bees. Interestingly, increased mortality was observed in N. ceranae-infected bees after exposure to boscalid, with synergistic negative effects. Regarding gut microbiota composition, co-exposure to the parasite and each pesticide led to decreased abundance of Alphaproteobacteria, and increased abundance of Gammaproteobacteria. The parasite also induced an increase of bacterial alpha-diversity (species richness). Our findings demonstrated that exposure of honeybees to N. ceranae and/or pesticides play a significant role in colony health and is associated with the establishment of a dysbiotic gut microbiota.

A Pediococcus strain to rescue honeybees by decreasing Nosema ceranae- and pesticide-induced adverse effects.

Honeybees ensure a key ecosystemic service by pollinating many agricultural crops and wild plants. However, since few decades, managed bee colonies have declined worldwide. This phenomenon is considered to be multifactorial, with a strong emphasis on both parasites and pesticides. Infection by the parasite Nosema ceranae and exposure to pesticides can contribute to adverse effects, resulting in a perturbation of the honeybee physiology. We thus hypothesized that probiotic treatment could be promising to treat or prevent these disturbances. The aim of this study was to evaluate the effects of probiotics on N. ceranae-infected and intoxicated honeybees (by the insecticide thiamethoxam and the fungicide boscalid). For this purpose, experiments were conducted with five probiotics. Among them, Pediococcus acidilactici (PA) showed the best protective effect against the parasite and pesticides. PA significantly improved the infected honeybee lifespan as prophylactic and curative treatments (respectively 2.3 fold and 1.7 fold). Furthermore, the exposure to pesticides induced an increase of honeybee mortality compared with the control group (p < .001) that was restored by the PA treatment. Despite its beneficial effect on honeybee lifespan, the PA administration did not induce changes in the gut bacterial communities (neither in abundance or diversity). N. ceranae and the pesticides were shown to deregulate genes involved in honeybee development (vitellogenin), immunity (serine protease 40, defensin) and detoxification system (glutathione peroxidase-like 2, catalase), and these effects were corrected by the PA treatment. This study highlights the promising use of PA to protect honeybees from both pathogens and pesticides.

Monitoring of enterovirus diversity in wastewater by ultra-deep sequencing: An effective complementary tool for clinical enterovirus surveillance.

In a one-year (October 2014-October 2015) pilot study, we assessed wastewater monitoring with sustained sampling for analysis of global enterovirus (EV) infections in an urban community. Wastewater was analysed by ultra-deep sequencing (UDS) after PCR amplification of the partial VP1 capsid protein gene. The nucleotide sequence analysis showed an unprecedented diversity of 48 EV types within the community, which were assigned to the taxonomic species A (n = 13), B (n = 23), and C (n = 12). During the same period, 26 EV types, of which 22 were detected in wastewater, were identified in patients referred to the teaching hospital serving the same urban population. Wastewater surveillance detected a silent circulation of 26 EV types including viruses reported in clinically rare respiratory diseases. Wastewater monitoring as a supplementary procedure can complement clinical surveillance of severe diseases related to non-polio EVs and contribute to the final stages of poliomyelitis eradication.

Genomic ecology of Marine Group II, the most common marine planktonic Archaea across the surface ocean.

Planktonic Archaea have been detected in all the world's oceans and are found from surface waters to the deep sea. The two most common Archaea phyla are Thaumarchaeota and Euryarchaeota. Euryarchaeota are generally more common in surface waters, but very little is known about their ecology and their potential metabolisms. In this study, we explore the genomic ecology of the Marine Group II (MGII), the main marine planktonic Euryarchaeota, and test if it is composed of different ecologically relevant units. We re-analyzed Tara Oceans metagenomes from the photic layer and the deep ocean by annotating sequences against a custom MGII database and by mapping gene co-occurrences. Our data provide a global view of the distribution of Euryarchaeota, and more specifically of MGII subgroups, and reveal their association to a number of gene-coding sequences. In particular, we show that MGII proteorhodopsins were detected in both the surface and the deep chlorophyll maximum layer and that different clusters of these light harvesting proteins were present. Our approach helped describing the set of genes found together with specific MGII subgroups. We could thus define genomic environments that could theoretically describe ecologically meaningful units and the ecological niche that they occupy.

Viruses as key reservoirs of antibiotic resistance genes in the environment.

Antibiotic resistance is a rapidly growing health care problem globally and causes many illnesses and deaths. Bacteria can acquire antibiotic resistance genes (ARGs) by horizontal transfer mediated by mobile genetic elements, where the role of phages in their dissemination in natural environments has not yet been clearly resolved. From metagenomic studies, we showed that the mean proportion of predicted ARGs found in prophages (0-0.0028%) was lower than those present in the free viruses (0.001-0.1%). Beta-lactamase, from viruses in the swine gut, represented 0.10 % of the predicted genes. Overall, in the environment, the ARG distribution associated with viruses was strongly linked to human activity, and the low dN/dS ratio observed advocated for a negative selection of the ARGs harbored by the viruses. Our network approach showed that viruses were linked to putative pathogens (Enterobacterales and vibrionaceae) and were considered key vehicles in ARG transfer, similar to plasmids. Therefore, these ARGs could then be disseminated at larger temporal and spatial scales than those included in the bacterial genomes, allowing for time-delayed genetic exchanges.

Temporal covariation of epibacterial community and surface metabolome in the Mediterranean seaweed holobiont Taonia atomaria.

An integrative multi-omics approach allowed monthly variations for a year of the surface metabolome and the epibacterial community of the Mediterranean Phaeophyceae Taonia atomaria to be investigated. The LC-MS-based metabolomics and 16S rDNA metabarcoding data sets were integrated in a multivariate meta-omics analysis (multi-block PLS-DA from the MixOmic DIABLO analysis) showing a strong seasonal covariation (Mantel test: p < 0.01). A network based on positive and negative correlations between the two data sets revealed two clusters of variables, one relative to the 'spring period' and a second to the 'summer period'. The 'spring period' cluster was mainly characterized by dipeptides positively correlated with a single bacterial taxon of the Alteromonadaceae family (BD1-7 clade). Moreover, 'summer' dominant epibacterial taxa from the second cluster (including Erythrobacteraceae, Rhodospirillaceae, Oceanospirillaceae and Flammeovirgaceae) showed positive correlations with few metabolites known as macroalgal antifouling defences [e.g. dimethylsulphoniopropionate (DMSP) and proline] which exhibited a key role within the correlation network. Despite a core community that represents a significant part of the total epibacteria, changes in the microbiota structure associated with surface metabolome variations suggested that both environment and algal host shape the bacterial surface microbiota.

Diversity, spatial distribution and activity of fungi in freshwater ecosystems.

High-throughput sequencing has given new insights into aquatic fungal community ecology over the last 10 years. Based on 18S ribosomal RNA gene sequences publicly available, we investigated fungal richness and taxonomic composition among 25 lakes and four rivers. We used a single pipeline to process the reads from raw data to the taxonomic affiliation. In addition, we studied, for a subset of lakes, the active fraction of fungi through the 18S rRNA transcripts level. These results revealed a high diversity of fungi that can be captured by 18S rRNA primers. The most OTU-rich groups were Dikarya (47%), represented by putative filamentous fungi more diverse and abundant in freshwater habitats than previous studies have suggested, followed by Cryptomycota (17.6%) and Chytridiomycota (15.4%). The active fraction of the community showed the same dominant groups as those observed at the 18S rRNA genes level. On average 13.25% of the fungal OTUs were active. The small number of OTUs shared among aquatic ecosystems may result from the low abundances of those microorganisms and/or they constitute allochthonous fungi coming from other habitats (e.g., sediment or catchment areas). The richness estimates suggest that fungi have been overlooked and undersampled in freshwater ecosystems, especially rivers, though they play key roles in ecosystem functioning as saprophytes and parasites.