Fecal and skin microbiota of two rescued Mediterranean monk seal pups during rehabilitation.

IMPORTANCE: This study showed that during the rehabilitation of two rescued Mediterranean monk seal pups (Monachus monachus), the skin and fecal bacterial communities showed similar succession patterns between the two individuals. This finding means that co-housed pups share their microbiomes, and this needs to be considered in cases of infection outbreaks and their treatment. The housing conditions, along with the feeding scheme and care protocols, including the admission of antibiotics as prophylaxis, probiotics, and essential food supplements, resulted in bacterial communities with no apparent pathogenic bacteria. This is the first contribution to the microbiome of the protected seal species of M. monachus and contributes to the animal's conservation practices through its microbiome.

Increased contribution of parasites in microbial eukaryotic communities of different Aegean Sea coastal systems.

Background-Aim: Protistan communities have a major contribution to biochemical processes and food webs in coastal ecosystems. However, related studies are scarce and usually limited in specific groups and/or sites. The present study examined the spatial structure of the entire protistan community in seven different gulfs and three different depths in a regional Mediterranean Sea, aiming to define taxa that are important for differences detected in the marine microbial network across the different gulfs studied as well as their trophic interactions. Methods: Protistan community structure analysis was based on the diversity of the V2-V3 hypervariable region of the 18S rRNA gene. Operational taxonomic units (OTUs) were identified using a 97% sequence identity threshold and were characterized based on their taxonomy, trophic role, abundance and niche specialization level. The differentially abundant, between gulfs, OTUs were considered for all depths and interactions amongst them were calculated, with statistic and network analysis. Results: It was shown that Dinophyceae, Bacillariophyta and Syndiniales were the most abundant groups, prevalent in all sites and depths. Gulfs separation was more striking at surface corroborating with changes in environmental factors, while it was less pronounced in higher depths. The study of differentially abundant, between gulfs, OTUs revealed that the strongest biotic interactions in all depths occurred between parasite species (mainly Syndiniales) and other trophic groups. Most of these species were generalists but not abundant highlighting the importance of rare species in protistan community assemblage. Conclusion: Overall this study revealed the emergence of parasites as important contributors in protistan network regulation regardless of depth.

The Reliability of Metagenome-Assembled Genomes (MAGs) in Representing Natural Populations: Insights from Comparing MAGs against Isolate Genomes Derived from the Same Fecal Sample.

The recovery of metagenome-assembled genomes (MAGs) from metagenomic data has recently become a common task for microbial studies. The strengths and limitations of the underlying bioinformatics algorithms are well appreciated by now based on performance tests with mock data sets of known composition. However, these mock data sets do not capture the complexity and diversity often observed within natural populations, since their construction typically relies on only a single genome of a given organism. Further, it remains unclear if MAGs can recover population-variable genes (those shared by >10% but <90% of the members of the population) as efficiently as core genes (those shared by >90% of the members). To address these issues, we compared the gene variabilities of pathogenic Escherichia coli isolates from eight diarrheal samples, for which the isolate was the causative agent, against their corresponding MAGs recovered from the companion metagenomic data set. Our analysis revealed that MAGs with completeness estimates near 95% captured only 77% of the population core genes and 50% of the variable genes, on average. Further, about 5% of the genes of these MAGs were conservatively identified as missing in the isolate and were of different (non-Enterobacteriaceae) taxonomic origin, suggesting errors at the genome-binning step, even though contamination estimates based on commonly used pipelines were only 1.5%. Therefore, the quality of MAGs may often be worse than estimated, and we offer examples of how to recognize and improve such MAGs to sufficient quality by (for instance) employing only contigs longer than 1,000 bp for binning.IMPORTANCE Metagenome assembly and the recovery of metagenome-assembled genomes (MAGs) have recently become common tasks for microbiome studies across environmental and clinical settings. However, the extent to which MAGs can capture the genes of the population they represent remains speculative. Current approaches to evaluating MAG quality are limited to the recovery and copy number of universal housekeeping genes, which represent a small fraction of the total genome, leaving the majority of the genome essentially inaccessible. If MAG quality in reality is lower than these approaches would estimate, this could have dramatic consequences for all downstream analyses and interpretations. In this study, we evaluated this issue using an approach that employed comparisons of the gene contents of MAGs to the gene contents of isolate genomes derived from the same sample. Further, our samples originated from a diarrhea case-control study, and thus, our results are relevant for recovering the virulence factors of pathogens from metagenomic data sets.

Gut Microbiota of Five Sympatrically Farmed Marine Fish Species in the Aegean Sea.

In this study, we hypothesized that sympatrically grown farmed fish, i.e. fish which experience similar environmental conditions and nutritionally similar diets, would have more convergent gut microbiota. Using a "common garden" approach, we identified the core microbiota and bacterial community structure differences between five fish species farmed in the same aquaculture site on the west coast of the Aegean Sea, Greece. The investigated individuals were at similar developmental stages and reared in adjacent (< 50 m) aquaculture cages; each cage had 15 kg fish m-3. The diets were nutritionally similar to support optimal growth for each fish species. DNA from the midgut of 3-6 individuals per fish species was extracted and sequenced for the V3-V4 region of the bacterial 16S rRNA. Only 3.9% of the total 181 operational taxonomic units (OTUs) were shared among all fish. Between 5 and 74 OTUs were unique to each fish species. Each of the investigated fish species had a distinct profile of dominant OTUs, i.e. cumulative relative abundance of ≥ 80%. Co-occurrence network analysis for each fish species showed that all networks were strongly dominated by positive correlations between the abundances of their OTUs. However, each fish species had different network characteristics suggesting the differential significance of the OTUs in each of the five fish species midgut. The results of the present study may provide evidence that adult fish farmed in the Mediterranean Sea have a rather divergent and species-specific gut microbiota profile, which are shaped independently of the similar environmental conditions under which they grow.

Everything is not everywhere: can marine compartments shape phytoplankton assemblages?

The idea that 'everything is everywhere, but the environment selects' has been seminal in microbial biogeography, and marine phytoplankton is one of the prototypical groups used to illustrate this. The typical argument has been that phytoplankton is ubiquitous, but that distinct assemblages form under environmental selection. It is well established that phytoplankton assemblages vary considerably between coastal ecosystems. However, the relative roles of compartmentalization of regional seas and site-specific environmental conditions in shaping assemblage structures have not been specifically examined. We collected data from coastal embayments that fall within two different water compartments within the same regional sea and are characterized by highly localized environmental pressures. We used principal coordinates of neighbour matrices (PCNM) and asymmetric eigenvector maps (AEM) models to partition the effects that spatial structures, environmental conditions and their overlap had on the variation in assemblage composition. Our models explained a high percentage of variation in assemblage composition (59-65%) and showed that spatial structure consistent with marine compartmentalization played a more important role than local environmental conditions. At least during the study period, surface currents connecting sites within the two compartments failed to generate sufficient dispersal to offset the impact of differences due to compartmentalization. In other words, our findings suggest that, even for a prototypical cosmopolitan group, everything is not everywhere.

Host-Associated Bacterial Succession during the Early Embryonic Stages and First Feeding in Farmed Gilthead Sea Bream (Sparusaurata).

One of the most widely reared fish in the Mediterranean Sea is Sparus aurata. The succession of S. aurata whole-body microbiota in fertilized eggs, five, 15, 21 and 71 days post hatch (dph) larvae and the contribution of the rearing water and the provided feed (rotifers, Artemia sp. and commercial diet) to the host's microbiota was investigated by 454 pyrosequencing of the 16S rRNA gene diversity. In total, 1917 bacterial operational taxonomic units (OTUs) were found in all samples. On average, between 93 ± 2.1 and 366 ± 9.2 bacterial OTUs per sample were found, with most of them belonging to Proteobacteria and Bacteroidetes. Ten OTUs were shared between all S. aurata stages and were also detected in the rearing water or diet. The highest OTU richness occurred at the egg stage and the lowest at the yolk sac stage (5 dph). The rearing water and diet microbial communities contributed in S. aurata microbiota without overlaps in their microbial composition and structure. The commercial diet showed higher contribution to the S. aurata microbiota than the rearing water. After stage D71 the observed microbiota showed similarities with that of adult S. aurata as indicated by the increased number of OTUs associated with γ-Proteobacteria and Firmicutes.

Ecogenomic characterization of widespread, closely-related SAR11 clades of the freshwater genus "Candidatus Fonsibacter" and proposal of Ca. Fonsibacter lacus sp. nov.

The ubiquitous alpha-proteobacteria of the order "Candidatus Pelagibacterales" (SAR11) are highly abundant in aquatic environments, and among them, members of the monophyletic lineage LD12 (also known as SAR11 clade IIIb) are specifically found in lacustrine ecosystems. Clade IIIb bacteria are some of the most prominent members of freshwater environments, but little is known about their biology due to the lack of genome representatives. Only recently, the first non-marine isolate was cultured and described as "Candidatus Fonsibacter ubiquis". Here, we expand the collection of freshwater IIIb representatives and describe a new IIIb species of the genus "Ca. Fonsibacter". Specifically, we assembled a collection of 67 freshwater metagenomic datasets from the interconnected lakes of the Chattahoochee River basin (GA, USA) and obtained nearly complete metagenome-assembled genomes (MAGs) representing 5 distinct IIIb subclades, roughly equivalent to species based on genomic standards, including the previously described "Ca. F. ubiquis". Genomic comparisons between members of the IIIb species revealed high similarity in gene content. However, when comparing their abundance profiles in the Chattahoochee basin and various aquatic environments, differences in temporal and spatial distributions among the distinct species were observed implying niche differentiation might be underlying the coexistence of the highly functionally similar representatives. The name Ca. Fonsibacter lacus sp. nov. is proposed for the most abundant and widespread species in the Chattahoochee River basin and various freshwater ecosystems.

Quantifying the changes in genetic diversity within sequence-discrete bacterial populations across a spatial and temporal riverine gradient.

Recent diversity studies have revealed that microbial communities of natural environments are dominated by species-like, sequence-discrete populations. However, how stable the sequence and gene-content diversity are within these populations and especially in highly dynamic lotic habitats remain unclear. Here we quantified the dynamics of intra-population diversity in samples spanning two years and five sites in the Kalamas River (Northwest Greece). A significant positive correlation was observed between higher intra-population sequence diversity and longer persistence over time, revealing that more diverse populations tended to represent more autochthonous (vs. allochthonous) community members. Assessment of intra-population gene-content changes caused by strain replacement or gene loss over time revealed different profiles with the majority of populations exhibiting gene-content changes close to 10% of the total genes, while one population exhibited ~21% change. The variable genes were enriched in hypothetical proteins and mobile elements, and thus, were probably functionally neutral or attributable to phage predation. A few notable exceptions to this pattern were also noted such as phototrophy-related proteins in summer vs. winter populations. Taken together, these results revealed that some freshwater genomes are remarkably dynamic, even across short time and spatial scales, and have implications for the bacterial species concept and microbial source tracking.

Gut Bacterial Communities in Geographically Distant Populations of Farmed Sea Bream (Sparus aurata) and Sea Bass (Dicentrarchus labrax).

This study investigated the profile of the autochthonous gut bacterial communities in adult individuals of Sparus aurata and Dicentrarchus labrax reared in sea cages in five distantly located aquaculture farms in Greece and determine the impact of geographic location on them in order to detect the core gut microbiota of these commercially important fish species. Data analyses resulted in no significant geographic impact in the gut microbial communities within the two host species, while strong similarities between them were also present. Our survey revealed the existence of a core gut microbiota within and between the two host species independent of diet and geographic location consisting of the Delftia, Pseudomonas, Pelomonas, Propionibacterium, and Atopostipes genera.

Assessment of the Virological Quality of Marine and Running Surface Waters in NW Greece: A Case Study.

The virological quality of surface marine and running water samples collected from Igoumenitsa gulf and Kalamas river (NW Greece) was assessed from October 2012 to September 2013. Sampling sites were exposed to different land and/or anthropogenic effects. Seawater samples were collected monthly from five sampling stations (new harbor, old harbor, wastewater treatment plant outlet, protected Natura area, Drepano beach). Viral targets included human adenoviruses (hAdVs), as index human viruses, while noroviruses (NoVs) and hepatitis A virus (HAV) were also studied. Kalamas river samples were collected seasonally, from three sampling stations (Soulopoulo, Dam, Sagiada-estuaries), while viral targets included also porcine adenoviruses (pAdVs) and bovine polyoma viruses (bPyVs), as additional index viruses. All water samples were analyzed for standard bacterial indicators, as well. Physicochemical and meteorological data were also collected. Based on the standard bacterial indices, both sea and river water samples did not exceed the limits set according to Directive 2006/7/EU. However, positive samples for hAdVs were found occasionally in all sampling sites in Igoumenitsa gulf (23.3%, 14/60) showing fecal contamination of human origin. Moreover, HAV was detected once, in the sampling site of the old port (at 510 GC/L). Most of the Kalamas water samples were found positive for hAdVs (58.3%, 7/12), while human noroviruses GI (NoVGI) (8.3%, 1/12) and GII (NoVGII) (16.7%, 2/12) were also detected. HAV, pAdVs, and bovine polyomaviruses (bPyVs) were not detected in any of the analyzed samples. No statistically significant correlations were found between classic bacterial indicators and viral targets, nor between viruses and meteorological data. Overall, the present study contributed to the collection of useful data for the biomonitoring of the region, and the assessment of the overall impact of anthropogenic activities. It provided also valuable information for the evaluation of the risk of waterborne viral infections and the protection of public health. It was the first virological study in the area and one of the few in Greece.

Changes in Microbial (Bacteria and Archaea) Plankton Community Structure after Artificial Dispersal in Grazer-Free Microcosms.

Microbes are considered to have a global distribution due to their high dispersal capabilities. However, our knowledge of the way geographically distant microbial communities assemble after dispersal in a new environment is limited. In this study, we examined whether communities would converge because similar taxa would be selected under the same environmental conditions, or would diverge because of initial community composition, after artificial dispersal. To this aim, a microcosm experiment was performed, in which the temporal changes in the composition and diversity of different prokaryoplankton assemblages from three distant geographic coastal areas (Banyuls-sur-Mer in northwest Mediterranean Sea, Pagasitikos Gulf in northeast Mediterranean and Woods Hole, MA, USA in the northwest Atlantic), were studied. Diversity was investigated using amplicon pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA. The three assemblages were grown separately in particle free and autoclaved Banyuls-sur-mer seawater at 18 °C in the dark. We found that the variability of prokaryoplankton community diversity (expressed as richness, evenness and dominance) as well as the composition were driven by patterns observed in Bacteria. Regarding community composition, similarities were found between treatments at family level. However, at the OTU level microbial communities from the three different original locations diverge rather than converge during incubation. It is suggested that slight differences in the composition of the initial prokaryoplankton communities, resulted in separate clusters the following days even when growth took place under identical abiotic conditions.

Anthropogenic effects on bacterial diversity and function along a river-to-estuary gradient in Northwest Greece revealed by metagenomics.

Studies assessing the effects of anthropogenic inputs on the taxonomic and functional diversity of bacterioplankton communities in lotic ecosystems are limited. Here, we applied 16S rRNA gene amplicon and whole-genome shotgun sequencing to examine the microbial diversity in samples from the Kalamas River (Northwest Greece), a mid-size river that runs through agricultural and NATURA-protected areas, but also receives urban sewage from a large city through a manmade ditch. Samples from three different locations between the exit of the ditch and the estuary, during three different months showed that temporal differences of taxonomic and functional diversity were more pronounced than spatial ones, and <1% of total taxa were shared among all samples, revealing a highly dynamic ecosystem. Comparisons of gene diversity with other aquatic habitats showed that only the high flow winter samples resembled more to freshwater environments while samples during the decreased water flow months were dominated by sewage inputs and soil-related organisms. Notably, microbial human gut signals were detectable over background freshwater and soil/runoff related signals, even at tens of kilometers downstream the city. These findings revealed the significance of allochthonous inputs on the composition and dynamics of river bacterial communities, and highlighted the potential of metagenomics for source tracking purposes.

Spatially uniform but temporally variable bacterioplankton in a semi-enclosed coastal area.

Studies focusing on the temporal and spatial dynamics of bacterioplankton communities within littoral areas undergoing direct influences from the coast are quite limited. In addition, they are more complicated to resolve compared to communities in the open ocean. In order to elucidate the effects of spatial vs. temporal variability on bacterial communities in a highly land-influenced semi-enclosed gulf, surface bacterioplankton communities from five coastal sites in Igoumenitsa Gulf (Ionian Sea, Greece) were analyzed over a nine-month period using 16S rDNA 454-pyrosequencing. Temporal differences were more pronounced than spatial ones, with lower diversity indices observed during the summer months. During winter and early spring, bacterial communities were dominated by SAR11 representatives, while this pattern changed in May when they were abruptly replaced by members of Flavobacteriales, Pseudomonadales, and Alteromonadales. Additionally, correlation analysis showed high negative correlations between the presence of SAR11 OTUs in relation to temperature and sunlight that might have driven, directly or indirectly, the disappearance of these OTUs in the summer months. The dominance of SAR11 during the winter months further supported the global distribution of the clade, not only in the open-sea, but also in coastal systems. This study revealed that specific bacteria exhibited distinct succession patterns in an anthropogenic-impacted coastal system. The major bacterioplankton component was represented by commonly found marine bacteria exhibiting seasonal dynamics, while freshwater and terrestrial-related phylotypes were absent.

Dietary differences are reflected on the gut prokaryotic community structure of wild and commercially reared sea bream (Sparus aurata).

We compared the gut prokaryotic communities in wild, organically-, and conventionally reared sea bream (Sparus aurata) individuals. Gut microbial communities were identified using tag pyrosequencing of the 16S rRNA genes. There were distinct prokaryotic communities in the three different fish nutritional treatments, with the bacteria dominating over the Archaea. Most of the Bacteria belonged to the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. The number of bacterial operational taxonomic units (OTUs) was reduced from the wild to the conventionally reared fish, implying a response of the gut microorganisms to the supplied food and possibly alterations in food assimilation. The dominant bacterial OTU in all examined fish was closely related to the genus Diaphorobacter. This is the first time that a member of the ß-Proteobacteria, which dominate in freshwaters, are so important in a marine fish gut. In total the majority of the few Archaea OTUs found, were related to methane metabolism. The inferred physiological roles of the dominant prokaryotes are related to the metabolism of carbohydrates and nitrogenous compounds. This study showed the responsive feature of the sea bream gut prokaryotic communities to their diets and also the differences of the conventional in comparison to the organic and wild sea bream gut microbiota.

Comparison of the Norway lobster (Nephrops norvegicus) gut bacterial communities using 16S rDNA clone libraries and pyrosequencing.

By comparing 16S rDNA cloning and 454 pyrosequencing in the Nephrops norvegicus midgut, several common bacterial OTUs were detected. However, when only one method is to be selected, it needs to be considered whether the revealing of rare OTUs or their accurate phylogenetic relationships is mostly preferred.

Changes of bacterioplankton apparent species richness in two ornamental fish aquaria.

We analysed the 16S rRNA gene diversity within the bacterioplankton community in the water column of the ornamental fish Pterophyllum scalare and Archocentrus nigrofasciatus aquaria during a 60-day growth experiment in order to detect any dominant bacterial species and their possible association with the rearing organisms. The basic physical and chemical parameters remained stable but the bacterial community at 0, 30 and 60 days showed marked differences in bacterial cell abundance and diversity. We found high species richness but no dominant phylotypes were detected. Only few of the phylotypes were found in more than one time point per treatment and always with low relative abundance. The majority of the common phylotypes belonged to the Proteobacteria phylum and were closely related to Acinetobacter junii, Pseudomonas sp., Nevskia ramosa, Vogesella perlucida, Chitinomonas taiwanensis, Acidovorax sp., Pelomonas saccharophila and the rest belonged to the α-Proteobacteria, Bacteroidetes, Actinobacteria, candidate division OP11 and one unaffiliated group. Several of these phylotypes were closely related to known taxa including Sphingopyxis chilensis, Flexibacter aurantiacus subsp. excathedrus and Mycobacterium sp. Despite the high phylogenetic diversity most of the inferred ecophysiological roles of the found phylotypes are related to nitrogen metabolism, a key process for fish aquaria.

Gut bacteria associated with different diets in reared Nephrops norvegicus.

The impact of different diets on the gut microbiota of reared Nephrops norvegicus was investigated based on bacterial 16S rRNA gene diversity. Specimens were collected from Pagasitikos Gulf (Greece) and kept in experimental rearing tanks, under in situ conditions, for 6months. Treatments included three diets: frozen natural (mussel) food (M), dry formulated pellet (P) and starvation (S). Gut samples were collected at the initiation of the experiment, and after 3 and 6months. Tank water and diet samples were also analyzed for bacterial 16S rRNA gene diversity. Statistical analysis separated the two groups fed or starved (M and P vs. S samples). Most gut bacteria were not related to the water or diet bacteria, while bacterial diversity was higher in the starvation samples. M and P samples were dominated by Gammaproteobacteria, Epsilonproteobacteria and Tenericutes. Phylotypes clustering in Photobacterium leiognathi, Shewanella sp. and Entomoplasmatales had high frequencies in the M and P samples but low sequence frequencies in S samples. The study showed that feeding resulted in the selection of specific species, which also occurs in the natural population, and might be associated with the animal's nutrition.

Temporal shifts of the Norway lobster (Nephrops norvegicus) gut bacterial communities.

The aim of this study was to investigate the gut bacterial communities of Nephrops norvegicus individuals, using a suite of molecular tools consisting of automated ribosomal intergenic spacer analysis, 16S rRNA gene-internal transcribed spacer clone libraries and FISH. The animals were collected from Pagasitikos Gulf, Greece, during different months of the year. The diversity of the gut bacterial communities was found to mostly vary with sampling time, which could be related to temporal variations in food supply. The 16S rRNA gene diversity analysis showed dominance of specific phylotypes for each month studied. February, May, July, August and October samples were rich in sequences related to the gammaproteobacterial genera Pseudoalteromonas, Psychrobacter and Photobacterium. September and December samples were dominated by phylotypes affiliated with uncultured representatives of Mollicutes, which are generally associated with the intestinal tracts of various animals. The presence of Gammaproteobacteria and uncultured Mollicutes in August and September samples, respectively, was further confirmed by FISH. None of the morphometric parameters considered was related to the temporal pattern of dominant bacterial communities.