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.

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.

Rice protein concentrate as a fish meal substitute in Oreochromis niloticus: Effects on immune response, intestinal cytokines, Aeromonas veronii resistance, and gut microbiota composition.

The potential of rice protein concentrate (RPC) to substitute fishmeal (FM) protein in the diet of Oreochromis niloticus was assessed in a five-month-long feeding trial. Fishmeal protein was replaced by RPC at rates of 0% (control), 25%, 50%, and 75% (RPC0, RPC25, RPC50, and RPC75, respectively). RPC25 had no significant effect on antioxidant capacity (total antioxidant capacity; superoxide dismutase, catalase, and glutathione peroxidase activities) and immune indices (lysozyme, nitric oxide, antiprotease, and bactericidal activities) after one, two, and five months of feeding, while the values for these parameters were significantly lower in the RPC75 group compared to those in the RPC0 group. The RPC25 group showed higher mRNA levels of the intestinal cytokines IL-1ß, IL-10ß, TGF-ß, and TNF-α than the control group. In fish affected by Aeromonas veronii, the highest significant cumulative mortality was recorded in the RPC75 group, followed by the RPC50, RPC25, and control groups. Gut microbiome analyses showed a reduction in microbial diversity in response to the addition of RPC, regardless of the RPC content, and the composition of the community of the RPC samples differed from that of the control. RPC-enriched diets resulted in higher relative abundances of Bacteroidetes and Fusobacteria in the gut compared to that in the gut of the control fish. In summary, RPC can be used to replace up to 25% of the FM protein in the diet of O. niloticus, while improving the antioxidant capacity, immunocompetence, and disease resistance of the fish.

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.

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.

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.

Microbiological, physico-chemical, nutritional and sensory characterization of traditional Matsoni: selection and use of autochthonous multiple strain cultures to extend its shelf-life.

Matsoni, a traditional Georgian fermented milk, has variable quality and stability besides a short shelf-life (72-120 h at 6 °C) due to inadequate production and storage conditions. To individuate its typical traits as well as select and exploit autochthonous starter cultures to standardize its overall quality without altering its typicality, we carried out a thorough physico-chemical, sensorial and microbial characterization of traditional Matsoni. A polyphasic approach, including a culture-independent (PCR-DGGE) and two PCR culture-dependent methods, was employed to study the ecology of Matsoni. Overall, the microbial ecosystem of Matsoni resulted largely dominated by Streptococcus (S.) thermophilus and Lactobacillus (Lb.) delbrueckii subsp. bulgaricus. High loads of other lactic acid bacteria species, including Lb. helveticus, Lb. paracasei and Leuconostoc (Leuc.) lactis were found to occur as well. A selected autochthonous multiple strain culture (AMSC) composed of one Lb. bulgaricus, one Lb. paracasei and one S. thermophilus strain, applied for the pilot-scale production of traditional Matsoni, resulted the best in terms of enhanced shelf-life (one month), sensorial and nutritional quality without altering its overall typical quality. This AMSC is at disposal of SMEs who need to exploit and standardize the overall quality of this traditional fermented milk, preserving its typicality.

Culture-Dependent and Culture-Independent Nucleic-Acid-Based Methods Used in the Microbial Safety Assessment of Milk and Dairy Products.

Despite great advances in the diagnostics and better awareness for food safety and security worldwide, significant numbers of foodborne outbreaks have been traced back to the consumption of milk and dairy products contaminated with pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Campylobacter spp., and pathogenic Escherichia coli. Several culture-dependent and culture-independent nucleic acid-based methods have been proposed to identify, detect, and type milk- and dairyborne pathogenic bacteria. In our review, we will provide an overview on why it is of utmost importance to ascertain the presence of pathogenic microorganisms in milk and milk products; thereafter, we will describe the most commonly used culture-dependent and culture-independent methods, as well as the most attractive ones with regard to their future exploitation, providing the reader with new insights into how and when they can be exploited to ensure the enumeration, and accurate detection at both species and strain level of the most important milk- and dairyborne pathogenic bacteria, even if in a viable but nonculturable state.

Increasing trends in faecal pollution revealed over a decade in the central Adriatic Sea (Italy).

Faecal contamination of the coastal sea poses widespread hazard to human and environmental health and is predicted to rise in response to global change and human pressure. For better management and risk reduction it is thus imperative to clarify and predict trends of faecal pollution over spatial and temporal scales, and to assess links with climate and other variables. Here, we investigated the spatio-temporal variation in the Faecal Indicator Bacteria (FIB) Escherichia coli and enterococci, over a time frame spanning 11 years (2011-2021) along a coastal area covering approximately 40 km and 59 bathing sites in the Marche region (Adriatic Sea, Italy), characterized by intense beach tourism, high riverine inputs, resident population, maritime traffic and industrial activities. Our analysis, that considers 5,183 measurements during the bathing season (April to October), shows that FIB abundance varied significantly among years. A general, although not significant, increase over time of both FIB was observed, mainly due to a general reduction of structural zeros (i.e., zeros originated from the actual absence of the response variable) over the examined time period. FIB abundances displayed their maxima and minima in different years according to the municipality, with overall peaks recorded in different months (May-June or September), whereas the lowest values were always observed in October. FIB levels were not significantly related neither to rainfalls nor to river discharge, but the activation of combined sewer overflows (CSOs), typically occurring after intense rainfall events, appeared as a necessary condition for the high faecal contamination levels. Considering climate change scenarios predicting significant increases in extreme weather events, our findings support the usefulness of analysing long-term trends to identify pollution sources, and the prioritization of control strategies to better manage the release of microbial pollutants from combined sewer overflows in coastal waters to reduce human risks.

Age and environment are the main drivers shaping the wild common sole (Solea solea) microbiota.

Microbiota plays an essential role in fish growth and health and may be influenced by the changing environmental conditions. Here, we explored the microbiota of wild common sole, one of the most important fishery resources in the Mediterranean Sea, collected from different areas in the North Adriatic Sea. Our results show that the sole microbiota differs from that of the surrounding environment and among the different body sites (gill, skin and gut). Gut microbiota composition showed to be strongly related to fish age, rather than maturity, sex or sampling site. Age-related shifts in gut microbial communities were identified, with increased abundances of Bacteroidia and Desulfobacteria, unveiling potential microbial proxies for age estimation crucial for fisheries management. Our results expand the limited knowledge of the wild common sole microbiota, also in the light of the potential usefulness of the fish microbiota as a tool for future stock identification and connectivity studies.

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.

Inter-comparison of marine microbiome sampling protocols.

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d'Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22-3 µm, 3-20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and "whole water" (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume.

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.

Nested interactions between chemosynthetic lucinid bivalves and seagrass promote ecosystem functioning in contaminated sediments.

In seagrass sediments, lucinid bivalves and their chemoautotrophic bacterial symbionts consume H2S, relying indirectly on the plant productivity for the presence of the reduced chemical. Additionally, the role of lucinid bivalves in N provisioning to the plant (through N2 fixation by the symbionts) was hypothesized. Thus, lucinids may contribute to sediment detoxification and plant fitness. Seagrasses are subject to ever-increasing human pressure in coastal environments. Here, disentangling nested interactions between chemosynthetic lucinid bivalves and seagrass exposed to pollution may help to understand seagrass ecosystem dynamics and to develop successful seagrass restoration programs that consider the roles of animal-microbe symbioses. We evaluated the capacity of lucinid bivalves (Loripes orbiculatus) to promote nutrient cycling and seagrass (Cymodocea nodosa) growth during a 6-week mesocosm experiment. A fully crossed design was used to test for the effect of sediment contamination (metals, nutrients, and hydrocarbons) on plant and bivalve (alone or interacting) fitness, assessed by mortality, growth, and photosynthetic efficiency, and for the effect of their nested interaction on sediment biogeochemistry. Plants performed better in the contaminated sediment, where a larger pool of dissolved nitrogen combined with the presence of other trace elements allowed for an improved photosynthetic efficiency. In fact, pore water nitrogen accumulated during the experiment in the controls, while it was consumed in the contaminated sediment. This trend was accentuated when lucinids were present. Concurrently, the interaction between clams and plants benefitted both organisms and promoted plant growth irrespective of the sediment type. In particular, the interaction with lucinid clams resulted in higher aboveground biomass of C. nodosa in terms of leaf growth, leaf surface, and leaf biomass. Our results consolidate the notion that nested interactions involving animal-microbe associations promote ecosystem functioning, and potentially help designing unconventional seagrass restoration strategies that exploit chemosynthetic symbioses.

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks.

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.

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.

Impact of Marine Aquaculture on the Microbiome Associated with Nearby Holobionts: The Case of Patella caerulea Living in Proximity of Sea Bream Aquaculture Cages.

Aquaculture plays a major role in the coastal economy of the Mediterranean Sea. This raises the issue of the impact of fish cages on the surrounding environment. Here, we explore the impact of aquaculture on the composition of the digestive gland microbiome of a representative locally dwelling wild holobiont, the grazer gastropod Patella caerulea, at an aquaculture facility located in Southern Sicily, Italy. The microbiome was assessed in individuals collected on sea bream aquaculture cages and on a rocky coastal tract located about 1.2 km from the cages, as the control site. Patella caerulea microbiome variations were explained in the broad marine metacommunity context, assessing the water and sediment microbiome composition at both sites, and characterizing the microbiome associated with the farmed sea bream. The P. caerulea digestive gland microbiome at the aquaculture site was characterized by a lower diversity, the loss of microorganisms sensitive to heavy metal contamination, and by the acquisition of fish pathogens and parasites. However, we also observed possible adaptive responses of the P. caerulea digestive gland microbiome at the aquaculture site, including the acquisition of putative bacteria able to deal with metal and sulfide accumulation, highlighting the inherent microbiome potential to drive the host acclimation to stressful conditions.

Autochthonous and Probiotic Lactic Acid Bacteria Employed for Production of "Advanced Traditional Cheeses".

Microbial characterization of two Italian traditional cheeses, Giuncata and Caciotta Leccese, was carried out, with the aim to isolate autochthonous bacterial strains to be used as starters to improve and standardize the quality of these cheeses. More than 400 bacterial isolates were found, using PCR-based identification, to belong to 12 species of the Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc genera. The dominant strains were screened for antagonistic activity against pathogenic and spoilage bacteria and exopolysaccharide production, acidification, and proteolytic activity. Since Streptococcus macedonicus was found to be the most prevalent lactic acid bacteria species present in milk and in both types of cheese, the best performing strain of this species was successfully used, alone or in combination with a selected autochthonous Lactococcus lactis strain, in pilot-scale productions of Giuncata and Caciotta Leccese cheeses, respectively. The combined inoculums of selected autochthonous strains positively influenced the sensory characteristics of both Giuncata and Caciotta cheeses. Finally, the selected autochthonous cultures were enriched with a potentially probiotic Lactobacillus rhamnosus strain and successfully used in pilot-scale productions of these traditional cheeses. To the best of our knowledge, this is the first study reporting the use of an autochthonous S. macedonicus strain as a starter for the production of cheeses with added probiotics. In addition, the identification of the probiotic strain in the feces of healthy volunteers fed with the advanced traditional cheese proved its effectiveness as a carrier for the delivery of probiotics to the human body.