Integrating culture-based and molecular methods provides an improved assessment of microbial quality in a coastal lagoon.

Faecal pollution in aquatic environments is a worldwide public health concern, yet the reliability and comprehensiveness of the methods used to assess faecal contamination are still debated. We compared three approaches, namely a culture-based method to enumerate Faecal Indicator Bacteria (FIB), a FIB-targeting qPCR assay, and High-Throughput Sequencing (HTS) to detect faeces- and sewage-associated taxa in water and sediment samples of an impacted model lagoon and its adjacent sea across one year. Despite at different levels, all approaches agreed in showing a higher contamination in the lagoon than in the sea, and higher in sediments than water. FIB significantly correlated when considering separately sediment and water, and when using both cultivation and qPCR. Similarly, FIB correlated between cultivation and qPCR, but qPCR provided consistently higher estimates of FIB. Faeces-associated bacteria positively correlated with cultivated FIB in both compartments, whereas sewage-associated bacteria did only in water. Considering their benefits and limitations, we conclude that, in our study site, improved quali-quantitative information on contamination is provided when at least two approaches are combined (e.g., cultivation and qPCR or HTS data). Our results provide insights to move beyond the use of FIB to improve faecal pollution management in aquatic environments and to incorporate HTS analysis into routine monitoring.

Identification of Marine Biotechnology Value Chains with High Potential in the Northern Mediterranean Region.

Marine (blue) biotechnology is an emerging field enabling the valorization of new products and processes with massive potential for innovation and economic growth. In the Mediterranean region, this innovation potential is not exploited as well as in other European regions due to a lack of a clear identification of the different value chains and the high fragmentation of business innovation initiatives. As a result, several opportunities to create an innovative society are being missed. To address this problem, eight Northern Mediterranean countries (Croatia, France, Greece, Italy, Montenegro, Portugal, Slovenia and Spain) established five national blue biotechnology hubs to identify and address the bottlenecks that prevent the development of marine biotechnology in the region. Following a three-step approach (1. Analysis: setting the scene; 2. Transfer: identification of promising value chains; 3. Capitalization: community creation), we identified the three value chains that are most promising for the Northern Mediterranean region: algae production for added-value compounds, integrated multi-trophic aquaculture (IMTA) and valorization aquaculture/fisheries/processing by-products, unavoidable/unwanted catches and discards. The potential for the development and the technical and non-technical skills that are necessary to advance in this exciting field were identified through several stakeholder events which provided valuable insight and feedback that should be addressed for marine biotechnology in the Northern Mediterranean region to reach its full potential.

Host-associated and Environmental Microbiomes in an Open-Sea Mediterranean Gilthead Sea Bream Fish Farm.

Gilthead seabream is among the most important farmed fish species in the Mediterranean Sea. Several approaches are currently applied to assure a lower impact of diseases and higher productivity, including the exploration of the fish microbiome and its manipulation as a sustainable alternative to improve aquaculture practices. Here, using 16S rRNA gene high-throughput sequencing, we explored the microbiome of farmed seabream to assess similarities and differences among microbial assemblages associated to different tissues and compare them with those in the surrounding environment. Seabream had distinct associated microbiomes according to the tissue and compared to the marine environment. The gut hosted the most diverse microbiome; different sets of dominant ASVs characterized the environmental and fish samples. The similarity between fish and environmental microbiomes was higher in seawater than sediment (up to 7.8 times), and the highest similarity (3.9%) was observed between gill and seawater, suggesting that gills are more closely interacting with the environment. We finally analyzed the potential connections occurring among microbiomes. These connections were relatively low among the host's tissues and, in particular, between the gut and the others fish-related microbiomes; other tissues, including skin and gills, were found to be the most connected microbiomes. Our results suggest that, in mariculture, seabream microbiomes reflect only partially those in their surrounding environment and that the host is the primary driver shaping the seabream microbiome. These data provide a step forward to understand the role of the microbiome in farmed fish and farming environments, useful to enhance disease control, fish health, and environmental sustainability.

Time to integrate biotechnological approaches into fish gut microbiome research.

Like for other vertebrates, the fish microbiome is critical to the health of its host and has complex and dynamic interactions with the surrounding environment. Thus, the study of the fish microbiome can benefit from the new prospects gained by innovative biotechnological applications in human and other animals, that include manipulation of the associated microbial communities (to improve the health, productivity, and sustainability of fish production), in vitro gut simulators, synthetic microbial communities, and others. Here, we summarize the current state of knowledge on such biotechnological approaches to better understand and engineer the fish microbiome, as well as to advance our knowledge on host-microbes interactions. A particular focus is given to the most recent strategies for fish microbiome manipulation to improve fish health, food safety and environmental sustainability.

Partitioning and sources of microbial pollution in the Venice Lagoon.

Microbial pollutants are a serious threat to human and environmental health in coastal areas. Based on the hypothesis that pollution from multiple sources may produce a distinct microbial signature and that microbial pollutants seem to distribute between a free-living and a particle-attached fraction, we investigated the occurrence, partitioning and sources of microbial pollutants in water samples collected in the Venice Lagoon (Italy). The area was taken as a case study of an environment characterized by a long history of industrial pollution and by growing human pressure. We found a variety of pollutants from several sources, with sewage-associated and faecal bacteria accounting for up to 5.98% of microbial communities. Sewage-associated pollutants were most abundant close to the city centre. Faecal pollution was highest in the area of the industrial port and was dominated by human inputs, whereas contamination from animal faeces was mainly detected at the interface with the mainland. Microbial pollutants were almost exclusively associated with the particle-attached fraction. The samples also contained other potential pathogens. Our findings stress the need for monitoring and managing microbial pollution in highly urbanized lagoon and semi-enclosed systems and suggest that management plans to reduce microbial inputs to the waterways should include measures to reduce particulate matter inputs to the lagoon. Finally, High-Throughput Sequencing combined with computational approaches proved critical to assess water quality and appears to be a valuable tool to support the monitoring of waterborne diseases.

Occurrence and distribution of microbial pollutants in coastal areas of the Adriatic Sea influenced by river discharge.

The transport of a variety of pollutants from agricultural, industrial and urbanised areas makes rivers major contributors to the contamination of coastal marine environments. Too little is known of their role in carrying pathogens to the coast. We used DNA-based metabarcoding data to describe the microbial community composition in seawater and sediment collected in front of the estuary of the Tronto, the Chienti and the Esino, three Italian rivers with different pollution levels that empty into the north-central Adriatic Sea, and to detect and measure within these communities the relative abundance of microbial pollutants, including traditional faecal indicators and alternative faecal and sewage-associated pollutants. We then applied the FORENSIC algorithm to distinguish human from non-human sources of microbial pollution and FAPROTAX to map prokaryotic clades to established metabolic or other ecologically relevant functions. Finally, we searched the dataset for other common pathogenic taxa. Seawater and sediment contained numerous potentially pathogenic bacteria, mainly faecal and sewage-associated. The samples collected in front of the Tronto estuary showed the highest level of contamination, likely sewage-associated. The pathogenic signature showed a weak but positive correlation with some nutrients and strong correlations with some polycyclic aromatic hydrocarbons. This study confirms that rivers transport pathogenic bacteria to the coastal sea and highlights the value of expanding the use of HTS data, source tracking and functional identification tools to detect microbial pollutants and identify their sources with a view to gaining a better understanding of the pathways of sewage-associated discharges to the sea.

Plastics occurrence in juveniles of Engraulis encrasicolus and Sardina pilchardus in the Southern Tyrrhenian Sea.

We report the presence of microplastics on the external surface and in the gastrointestinal tract of white late-larval and juvenile stages (fry) of clupeid fishes caught in the Southern Tyrrhenian Sea. The average highest number of plastics debris was recorded on Sardina pilchardus (0.53 items/specimen); a lower average number of items was observed for Engraulis encrasicolus (0.26 items/specimen). The plastics were characterized by fibers that differed in shape, colour and composition. Polyester, polypropylene, polyacrylonitrile, polyethylene, polyamide, nylon, rayon and polyurethane segments were detected by Raman and FTIR spectroscopies. Traces of organic components and dyes, compounds that are generally included in the polymer matrix to modify its base properties, were also identified on microplastics. Our results raise concerns for the potential transfer of synthetic materials through the marine food web and into humans, given the prominent role of S. pilchardus and E. encrasicolus within the food web as main food source for many marine species.

Inorganic and Organic Carbon Uptake Processes and Their Connection to Microbial Diversity in Meso- and Bathypelagic Arctic Waters (Eastern Fram Strait).

The deep Arctic Ocean is increasingly vulnerable to climate change effects, yet our understanding of its microbial processes is limited. We collected samples from shelf waters, mesopelagic Atlantic Waters (AW) and bathypelagic Norwegian Sea Deep Waters (NSDW) in the eastern Fram Strait, along coast-to-offshore transects off Svalbard during boreal summer. We measured community respiration, heterotrophic carbon production (HCP), and dissolved inorganic carbon utilization (DICu) together with prokaryotic abundance, diversity, and metagenomic predictions. In deep samples, HCP was significantly faster in AW than in NSDW, while we observed no differences in DICu rates. Organic carbon uptake was higher than its inorganic counterpart, suggesting a major reliance of deep microbial Arctic communities on heterotrophic metabolism. Community structure and spatial distribution followed the hydrography of water masses. Distinct from other oceans, the most abundant OTU in our deep samples was represented by the archaeal MG-II. To address the potential biogeochemical role of each water mass-specific microbial community, as well as their link with the measured rates, PICRUSt-based predicted metagenomes were built. The results showed that pathways of auto- and heterotrophic carbon utilization differed between the deep water masses, although this was not reflected in measured DICu rates. Our findings provide new insights to understand microbial processes and diversity in the dark Arctic Ocean and to progress toward a better comprehension of the biogeochemical cycles and their trends in light of climate changes.

Plastics occurrence in the gastrointestinal tract of Zeus faber and Lepidopus caudatus from the Tyrrhenian Sea.

The present study investigates the occurrence of plastic pollution in two commercially important marine teleosts (Zeus faber and Lepidopus caudatus) from the northern coasts of Sicily (Tyrrhenian Sea). Plastics occurrence in the gastrointestinal tract was higher in Lepidopus caudatus (78.1%) than Zeus faber (51.4%). Debris characterization, carried out by micro-Raman spectroscopy, allowed identified the main types of found polymers as: polypropylene (PP), polyamide (PA), nylon and, to a lesser extent, polyethylene (PE). Of the two fish species studied, the silver scabbardfish appeared to be the more vulnerable to plastic ingestion. Our study represents a starting point that may pave the way for future investigation of the fate, accumulation and transfer of plastic debris to upper trophic levels, to verify their potential toxicity and to better understand strategies to mitigate this phenomenon.

Biostimulation of in situ microbial degradation processes in organically-enriched sediments mitigates the impact of aquaculture.

Fish farm deposition, resulting in organic matter accumulation on bottom sediments, has been identified as among the main phenomena causing negative environmental impacts in aquaculture. An in situ bioremediation treatment was carried out in order to reduce the organic matter accumulation in the fish farm sediments by promoting the natural microbial biodegradation processes. To assess the effect of the treatment, the concentration of organic matter in the sediment and its microbial degradation, as well as the response of the benthic prokaryotic community, were investigated. The results showed a significant effect of the treatment in stimulating microbial degradation rates, and the consequent decrease in the concentration of biochemical components beneath the cages during the treatment. During the bioremediation process, the prokaryotic community in the fish farm sediment responded to the overall improvement of the sediment conditions by showing the decrease of certain anaerobic taxa (e.g. Clostridiales, Acidaminobacteraceae and Caldilinaceae). This suggested that the bioactivator was effective in promoting a shift from an anaerobic to an aerobic metabolism in the prokaryotic community. However, the larger importance of Lachnospiraceae (members of the gut and faecal microbiota of the farmed fishes) in treated compared to non-treated sediments suggested that the bioactivator was not efficient in reducing the accumulation of faecal bacteria from the farmed fishes. Our results indicate that bioremediation is a promising tool to mitigate the aquaculture impact in fish farm sediments, and that further research needs to be oriented to identifying more successful interventions able to specifically target also fish-faeces related microbes.

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.

Status of faecal pollution in ports: A basin-wide investigation in the Adriatic Sea.

Ports are subject to a variety of anthropogenic impacts, and there is mounting evidence of faecal contamination through several routes. Yet, little is known about pollution in ports by faecal indicator bacteria (FIB). FIB spatio-temporal dynamics were assessed in 12 ports of the Adriatic Sea, a semi-enclosed basin under strong anthropogenic pressure, and their relationships with environmental variables were explored to gain insight into pollution sources. FIB were abundant in ports, often more so than in adjacent areas; their abundance patterns were related to salinity, oxygen, and nutrient levels. In addition, a molecular method, quantitative (q)PCR, was used to quantify FIB. qPCR enabled faster FIB determination and water quality monitoring that culture-based methods. These data provide robust baseline evidence of faecal contamination in ports and can be used to improve the management of routine port activities (dredging and ballast water exchange), having potential to spread pathogens in the sea.

Massive shelf dense water flow influences plankton community structure and particle transport over long distance.

Dense waters (DW) formation in shelf areas and their cascading off the shelf break play a major role in ventilating deep waters, thus potentially affecting ecosystem functioning and biogeochemical cycles. However, whether DW flow across shelves may affect the composition and structure of plankton communities down to the seafloor and the particles transport over long distances has not been fully investigated. Following the 2012 north Adriatic Sea cold outbreak, DW masses were intercepted at ca. 460 km south the area of origin and compared to resident ones in term of plankton biomass partitioning (pico to micro size) and phytoplankton species composition. Results indicated a relatively higher contribution of heterotrophs in DW than in deep resident water masses, probably as result of DW-mediated advection of fresh organic matter available to consumers. DWs showed unusual high abundances of Skeletonema sp., a diatom that bloomed in the north Adriatic during DW formation. The Lagrangian numerical model set up on this diatom confirmed that DW flow could be an important mechanism for plankton/particles export to deep waters. We conclude that the predicted climate-induced variability in DW formation events could have the potential to affect the ecosystem functioning of the deeper part of the Mediterranean basin, even at significant distance from generation sites.

Seasonal rather than spatial variability drives planktonic and benthic bacterial diversity in a microtidal lagoon and the adjacent open sea.

Coastal lagoons are highly productive ecosystems, which are experiencing a variety of human disturbances at increasing frequency. Bacteria are key ecological players within lagoons, yet little is known about the magnitude, patterns and drivers of diversity in these transitional environments. We carried out a seasonal study in the Venice Lagoon (Italy) and the adjacent sea, to simultaneously explore diversity patterns in different domains (pelagic, benthic) and their spatio-temporal variability, and test the role of environmental gradients in structuring assemblages. Community composition differed between lagoon and open sea, and between domains. The dominant phyla varied temporally, with varying trends for the two domains, suggesting different environmental constraints on the assemblages. The percentage of freshwater taxa within the lagoon increased during higher river run-off, pointing at the lagoon as a dynamic mosaic of microbial taxa that generate the metacommunity across the whole hydrological continuum. Seasonality was more important than spatial variability in shaping assemblages. Network analyses indicated more interactions between several genera and environmental variables in the open sea than the lagoon. Our study provides evidences for a temporally dynamic nature of bacterial assemblages in lagoons and suggests that an interplay of seasonally influenced environmental drivers shape assemblages in these vulnerable ecosystems.

Unveiling the role and life strategies of viruses from the surface to the dark ocean.

Viruses are a key component of marine ecosystems, but the assessment of their global role in regulating microbial communities and the flux of carbon is precluded by a paucity of data, particularly in the deep ocean. We assessed patterns in viral abundance and production and the role of viral lysis as a driver of prokaryote mortality, from surface to bathypelagic layers, across the tropical and subtropical oceans. Viral abundance showed significant differences between oceans in the epipelagic and mesopelagic, but not in the bathypelagic, and decreased with depth, with an average power-law scaling exponent of -1.03 km-1 from an average of 7.76 × 106 viruses ml-1 in the epipelagic to 0.62 × 106 viruses ml-1 in the bathypelagic layer with an average integrated (0 to 4000 m) viral stock of about 0.004 to 0.044 g C m-2, half of which is found below 775 m. Lysogenic viral production was higher than lytic viral production in surface waters, whereas the opposite was found in the bathypelagic, where prokaryotic mortality due to viruses was estimated to be 60 times higher than grazing. Free viruses had turnover times of 0.1 days in the bathypelagic, revealing that viruses in the bathypelagic are highly dynamic. On the basis of the rates of lysed prokaryotic cells, we estimated that viruses release 145 Gt C year-1 in the global tropical and subtropical oceans. The active viral processes reported here demonstrate the importance of viruses in the production of dissolved organic carbon in the dark ocean, a major pathway in carbon cycling.

Dense water plumes modulate richness and productivity of deep sea microbes.

Growing evidence indicates that dense water formation and flow over the continental shelf is a globally relevant oceanographic process, potentially affecting microbial assemblages down to the deep ocean. However, the extent and consequences of this influence have yet to be investigated. Here it is shown that dense water propagation to the deep ocean increases the abundance of prokaryotic plankton, and stimulates carbon production and organic matter degradation rates. Dense waters spilling off the shelf modifies community composition of deep sea microbial assemblages, leading to the increased relevance of taxa likely originating from the sea surface and the seafloor. This phenomenon can be explained by a combination of factors that interplay during the dense waters propagation, such as the transport of surface microbes to the ocean floor (delivering in our site 0.1 megatons of C), the stimulation of microbial metabolism due to increased ventilation and nutrients availability, the sediment re-suspension, and the mixing with ambient waters along the path. Thus, these results highlight a hitherto unidentified role for dense currents flowing over continental shelves in influencing deep sea microbes. In light of climate projections, this process will affect significantly the microbial functioning and biogeochemical cycling of large sectors of the ocean interior.

Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs).

Prokaryotes in coastal sediments are fundamental players in the ecosystem functioning and regulate processes relevant in the global biogeochemical cycles. Nevertheless, knowledge on benthic microbial diversity patterns across spatial scales, or as function to anthropogenic influence, is still limited. We investigated the microbial diversity in two of the most chemically polluted sites along the coast of Italy. One site is the Po River Prodelta (Northern Adriatic Sea), which receives contaminant discharge from one of the largest rivers in Europe. The other site, the Mar Piccolo of Taranto (Ionian Sea), is a chronically polluted area due to steel production plants, oil refineries, and intense maritime traffic. We collected sediments from 30 stations along gradients of contamination, and studied prokaryotic diversity using Illumina sequencing of amplicons of a 16S rDNA gene fragment. The main sediment variables and the concentration of eleven metals, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were measured. Chemical analyses confirmed the high contamination in both sites, with concentrations of PCBs particularly high and often exceeding the sediment guidelines. The analysis of more than 3 millions 16S rDNA sequences showed that richness decreased with higher contamination levels. Multivariate analyses showed that contaminants significantly shaped community composition. Assemblages differed significantly between the two sites, but showed wide within-site variations related with spatial gradients in the chemical contamination, and the presence of a core set of OTUs shared by the two geographically distant sites. A larger importance of PCB-degrading taxa was observed in the Mar Piccolo, suggesting their potential selection in this historically polluted site. Our results indicate that sediment contamination by multiple contaminants significantly alter benthic prokaryotic diversity in coastal areas, and suggests considering the potential contribution of the resident microbes to contaminant bioremediation actions.

Understanding the association of Escherichia coli with diverse macroalgae in the lagoon of Venice.

Recent studies provided evidence that the macroalga Cladopohora in lakes hosts associated Escherichia coli, with consequences on the environmental and human health. We expanded these investigations to other macroalgae (Ulva spp., Sargassum muticum and Undaria pinnatifida) widespread in the lagoon of Venice (Italy). Attached E. coli were abundant, accounting up to 3,250 CFU gram(-1) of alga. Macroalgal-associated isolates belonged to all E. coli phylogroups, including pathogenic ones, and to Escherichia cryptic clades. Attached E. coli showed potential to grow even at in situ temperature on macroalgal extracts as only source of carbon and nutrients, and ability to produce biofilm in vitro. The genotypic diversity of the attached isolates was high, with significant differences between algae and the overlying water. Our evidences suggest that attached populations consist of both resident and transient strains, likely resulting from the heterogeneous input of fecal bacteria from the city. We report that cosmopolitan and invasive macroalgae may serve as source of E. coli, including pathogenic genotypes, and that this habitat can potentially support their growth. Considering the global diffusion of the macroalgae here studied, this phenomenon is likely occurring in other coastal cities worldwide and deserves further investigations from either the sanitary and ecological perspectives.

The ocean sampling day consortium.

Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world's oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits.

Adhesion of marine cryptic Escherichia isolates to human intestinal epithelial cells.

Five distinct cryptic lineages (clades I-V) have recently been recognized in the Escherichia genus. The five clades encompass strains that are phenotypically and taxonomically indistinguishable from Escherichia coli sensu stricto; however, scant data are available on their ecology, virulence and pathogenic properties. In this study 20 cryptic E. coli strains isolated from marine sediments were investigated to gain insights into their virulence characteristics and genetic traits. The ability to adhere to intestinal cells was highest among clade V strains, which also harbored the genes involved in gut colonization as well as the genes (pduC and eut operon) typically found in environmentally adapted E. coli strains. The pduC gene was significantly associated with clade V. Multilocus sequence typing of three representative clade V isolates revealed new sequence types (STs) and showed that the strains shared two allelic loci (adk 51 and recA 37). Our findings suggest that cryptic Escherichia lineages are common in coastal marine sediments and that this habitat may be suitable for their growth and persistence outside the host. On the other hand, detection in clade V strains of a gene repertoire and adhesion properties similar to those of intestinal pathogenic strains could indicate their potential virulence. It could be argued that there is a dual nature of cryptic clade V strains, where the ability to survive and persist in a secondary habitat does not involve the loss of the host-associated lifestyle. Clade V could be a group of closely related, environmentally adapted E. coli strains.