Meta-analysis of the Cetacea gut microbiome: Diversity, co-evolution, and interaction with the anthropogenic pathobiome.

Despite their critical roles in marine ecosystems, only few studies have addressed the gut microbiome (GM) of cetaceans in a comprehensive way. Being long-living apex predators with a carnivorous diet but evolved from herbivorous ancestors, cetaceans are an ideal model for studying GM-host evolutionary drivers of symbiosis and represent a valuable proxy of overall marine ecosystem health. Here, we investigated the GM of eight different cetacean species, including both Odontocetes (toothed whales) and Mysticetes (baleen whales), by means of 16S rRNA-targeted amplicon sequencing. We collected faecal samples from free-ranging cetaceans circulating within the Pelagos Sanctuary (North-western Mediterranean Sea) and we also included publicly available cetacean gut microbiome sequences. Overall, we show a clear GM trajectory related to host phylogeny and taxonomy (i.e., phylosymbiosis), with remarkable GM variations which may reflect adaptations to different diets between baleen and toothed whales. While most samples were found to be infected by protozoan parasites of potential anthropic origin, we report that this phenomenon did not lead to severe GM dysbiosis. This study underlines the importance of both host phylogeny and diet in shaping the GM of cetaceans, highlighting the role of neutral processes as well as environmental factors in the establishment of this GM-host symbiosis. Furthermore, the presence of potentially human-derived protozoan parasites in faeces of free-ranging cetaceans emphasizes the importance of these animals as bioindicators of anthropic impact on marine ecosystems.

Dispersion of antimicrobial resistant bacteria in pig farms and in the surrounding environment.

BACKGROUND: Antimicrobial resistance has been identified as a major threat to global health. The pig food chain is considered an important source of antimicrobial resistance genes (ARGs). However, there is still a lack of knowledge on the dispersion of ARGs in pig production system, including the external environment. RESULTS: In the present study, we longitudinally followed one swine farm located in Italy from the weaning phase to the slaughterhouse to comprehensively assess the diversity of ARGs, their diffusion, and the bacteria associated with them. We obtained shotgun metagenomic sequences from 294 samples, including pig feces, farm environment, soil around the farm, wastewater, and slaughterhouse environment. We identified a total of 530 species-level genome bins (SGBs), which allowed us to assess the dispersion of microorganisms and their associated ARGs in the farm system. We identified 309 SGBs being shared between the animals gut microbiome, the internal and external farm environments. Specifically, these SGBs were characterized by a diverse and complex resistome, with ARGs active against 18 different classes of antibiotic compounds, well matching antibiotic use in the pig food chain in Europe. CONCLUSIONS: Collectively, our results highlight the urgency to implement more effective countermeasures to limit the dispersion of ARGs in the pig food systems and the relevance of metagenomics-based approaches to monitor the spread of ARGs for the safety of the farm working environment and the surrounding ecosystems.

Routes of dispersion of antibiotic resistance genes from the poultry farm system.

Poultry farms are hotspots for the development and spread of antibiotic resistance genes (ARGs), due to high stocking densities and extensive use of antibiotics, posing a threat of spread and contagion to workers and the external environment. Here, we applied shotgun metagenome sequencing to characterize the gut microbiome and resistome of poultry, workers and their households - also including microbiomes from the internal and external farm environment - in three different farms in Italy during a complete rearing cycle. Our results highlighted a relevant overlap among the microbiomes of poultry, workers, and their families (gut and skin), with clinically relevant ARGs and associated mobile elements shared in both poultry and human samples. On a finer scale, the reconstruction of species-level genome bins (SGBs) allowed us to delineate the dynamics of microorganism and ARGs dispersion from farm systems. We found the associations with worker microbiomes representing the main route of ARGs dispersion from poultry to human populations. Collectively, our findings clearly demonstrate the urgent need to implement more effective procedures to counteract ARGs dispersion from poultry food systems and the relevance of metagenomics-based metacommunity approaches to monitor the ARGs dispersion process for the safety of the working environment on farms.

Microbiome variation at the clam-sediment interface may explain changes in local productivity of Chamelea gallina in the North Adriatic sea.

BACKGROUND: The clam Chamelea gallina is an ecologically and economically important marine species in the Northwestern Adriatic Sea, which currently suffers from occasional, and still unexplained, widespread mortality events. In order to provide some glimpses in this direction, this study explores the connections between microbiome variations at the clam-sediment interface and the nutritional status of clams collected at four Italian production sites along the Emilia Romagna coast, with different mortality incidence, higher in the Northern sites and lower in the Southern sites. RESULTS: According to our findings, each production site showed a peculiar microbiome arrangement at the clam-sediment interface, with features that clearly differentiate the Northern and Southern sites, with the latter also being associated with a better nutritional status of the animal. Interestingly, the C. gallina digestive gland microbiome from the Southern sites was enriched in some health-promoting microbiome components, capable of supplying the host with essential nutrients and defensive molecules. Furthermore, in experiments conducted under controlled conditions in aquaria, we provided preliminary evidence of the prebiotic action of sediments from the Southern sites, allowing to boost the acquisition of previously identified health-promoting components of the digestive gland microbiome by clams from the Northern sites. CONCLUSIONS: Taken together, our findings may help define innovative microbiome-based management strategies for the preservation of the productivity of C. gallina clams in the Adriatic Sea, through the identification and maintenance of a probiotic niche at the animal-sediment interface.

Microbiome study of a coupled aquaponic system: unveiling the independency of bacterial communities and their beneficial influences among different compartments.

To understand the microbiome composition and interplay among bacterial communities in different compartments of a coupled freshwater aquaponics system growing flathead grey mullet (Mugil cephalus) and lettuces (Lactuca sativa), 16S rRNA gene amplicon sequencing of the V3-V4 region was analysed from each compartment (fish intestine, water from the sedimentation tank, bioballs from the biological filter, water and biofilm from the hydroponic unit, and lettuce roots). The bacterial communities of each sample group showed a stable diversity during all the trial, except for the fish gut microbiota, which displayed lower alpha diversity values. Regarding beta diversity, the structure of bacterial communities belonging to the biofilm adhering to the hydroponic tank walls, bioballs, and lettuce roots resembled each other (weighted and unweighted UniFrac distances), while bacteria from water samples also clustered together. However, both of the above-mentioned bacterial communities did not resemble those of fish gut. We found a low or almost null number of shared Amplicon Sequence Variants (ASVs) among sampled groups which indicated that each compartment worked as an independent microbiome. Regarding fish health and food safety, the microbiome profile did not reveal neither fish pathogens nor bacterial species potentially pathogenic for food health, highlighting the safety of this sustainable food production system.

Extraction method for the multiresidue analysis of legacy and emerging pollutants in marine mussels from the Adriatic Sea.

The release of hazardous chemicals into aquatic environments has long been a known problem, but its full impact has only recently been realized. This study presents a validated liquid chromatography-mass spectrometry (HPLC-MS/MS) method for detecting pharmaceutical and pesticide residues in mussels (Mytilus galloprovincialis). An innovative MS-compatible extraction method was developed and validated, demonstrating successful recovery rates for analytes at three different concentration levels (25-95%). The method detected the target analytes at ng/g concentrations with high accuracy (-7% to 11%) and low relative standard deviation (<10%) for both intra-day and inter-day analyses. After validation, the method was applied to mussel samples collected from a commercial farm near Senigallia, Adriatic Sea, detecting different contaminants in the range of 2-40 ng/g (dry weight). The study provides a valuable tool for investigating the potential threats posed by diverse contaminant classes with high annual tonnage, including analytes with known persistence and/or illegal status.

Seasonal dynamics of the microbiome-host response to pharmaceuticals and pesticides in Mytilus galloprovincialis farmed in the Northwestern Adriatic Sea.

Marine mussels, especially Mytilus galloprovincialis, are well-established sentinel species, being naturally resistant to the exposure to multiple xenobiotics of natural and anthropogenic origin. Even if the response to multiple xenobiotic exposure is well known at the host level, the role of the mussel-associated microbiome in the animal response to environmental pollution is poorly explored, despite its potential in xenobiotic detoxification and its important role in host development, protection, and adaptation. Here, we characterized the microbiome-host integrative response of M. galloprovincialis in a real-world setting, involving exposure to a complex pattern of emerging pollutants, as occurs in the Northwestern Adriatic Sea. A total of 387 mussel individuals from 3 commercial farms, spanning about 200 km along the Northwestern Adriatic coast, and in 3 different seasons, were collected. Multiresidue analysis (for quantitative xenobiotic determination), transcriptomics (for host physiological response), and metagenomics (for host-associated microbial taxonomical and functional features) analyses were performed on the digestive glands. According to our findings, M. galloprovincialis responds to the presence of the complex pattern of multiple emerging pollutants - including the antibiotics sulfamethoxazole, erythromycin, and tetracycline, the herbicides atrazine and metolachlor, and the insecticide N,N-diethyl-m-toluamide - integrating host defense mechanisms, e.g., through upregulation of transcripts involved in animal metabolic activity, and microbiome-mediated detoxification functions, including microbial functionalities involved in multidrug or tetracycline resistance. Overall, our data highlight the importance of the mussel-associated microbiome as a strategic player for the orchestration of resistance to the multixenobiotic exposure at the holobiont level, providing strategic functionalities for the detoxification of multiple xenobiotic substances, as occurring in real world exposure settings. Complementing the host with microbiome-dependent xenobiotic degradative and resistance genes, the M. galloprovincialis digestive gland associated microbiome can have an important role in the detoxification of emerging pollutants in a context of high anthropogenic pressure, supporting the relevance of mussel systems as potential animal-based bioremediation tool.

Sorbed environmental contaminants increase the harmful effects of microplastics in adult zebrafish, Danio rerio.

Aquatic animals ingest Microplastics (MPs) which have the potential to affect the uptake and bioavailability of sorbed co-contaminants. However, the effects on living organisms still need to be properly understood. The present study was designed to assess the combined effects of MPs and environmental contaminants on zebrafish (Danio rerio) health and behavior. Adult specimens were fed according to three different protocols: 1) untreated food (Control group); 2) food supplemented with 0.4 mg/L pristine polyethylene-MPs (PE-MPs; 0.1-0.3 mm diameter) (PEv group); 3) food supplemented with 0.4 mg/L PE-MPs previously incubated (PEi group) for 2 months in seawater. Analysis of contaminants in PEi detected trace elements, such as lead and copper. After 15 days of exposure, zebrafish underwent behavioral analysis and were then dissected to sample gills and intestine for histology, and the latter also for microbiome analysis. Occurrence of PEv and PEi in the intestine and contaminants in the fish carcass were analyzed. Both PEv- and PEi-administered fish differed from controls in the assays performed, but PEi produced more harmful effects in most instances. Overall, MPs after environmental exposure revealed higher potential to alter fish health through combined effects (e.g. proportion of microplastics, pollutants and/or microorganisms).

Composition and biodiversity of soil and root-associated microbiome in Vitis vinifera cultivar Lambrusco distinguish the microbial terroir of the Lambrusco DOC protected designation of origin area on a local scale.

Introduction: Wines produced from the same grape cultivars but in different locations possess distinctive qualities leading to different consumer's appreciation, preferences, and thus purchase choices. Here, we explore the possible importance of microbiomes at the soil-plant interface as a determinant of the terroir properties in grapevine production, which confer specific growth performances and wine chemo-sensory properties at the local scale. Methods: In particular, we investigated the variation in microbial communities associated with the roots of Vitis vinifera cultivar Lambrusco, as well as with surrounding bulk soils, in different vineyards across the "Consorzio Tutela Lambrusco DOC" protected designation of origin area (PDO, Emilia Romagna, Italy), considering viticultural sites located both inside and outside the consortium in two different seasons (June and November 2021). Results: According to our findings, rhizospheric and soil microbiomes show significant structural differences in relation to the sampling site, regardless of seasonality, while endophytic microbiomes seem to be completely unaffected by such variables. Furthermore, a deeper insight into the microbial terroir of PDO areas highlighted the presence of some rhizospheric microorganisms enriched inside the consortium and characterizing the PDO regardless of both sampling season and farming strategy. These include Bacillus, Paenibacillus, and Azospirillum, which are all well-known plant growth-promoting bacteria. Discussion: Taken together, our results suggest a connection between soil and root microbiomes of V. vinifera cultivar Lambrusco and the local designation of origin, emphasizing the potential role of PDO-enriched plant growth-promoting bacteria in vine growing and final quality of the Lambrusco DOC wine.

Functional characterization and taxonomic classification of novel gammaproteobacterial diversity in sponges.

Sponges harbour exceptionally diverse microbial communities, whose members are largely uncultured. The class Gammaproteobacteria often dominates the microbial communities of various sponge species, but most of its diversity remains functional and taxonomically uncharacterised. Here we reconstructed and characterised 32 metagenome-assembled genomes (MAGs) derived from three sponge species. These MAGs represent ten novel species and belong to seven orders, of which one is new. We propose nomenclature for all these taxa. These new species comprise sponge-specific bacteria with varying levels of host specificity. Functional gene profiling highlights significant differences in metabolic capabilities across the ten species, though each also often exhibited a large degree of metabolic diversity involving various nitrogen- and sulfur-based compounds. The genomic features of the ten species suggest they have evolved to form symbiotic interaction with their hosts or are well-adapted to survive within the sponge environment. These Gammaproteobacteria are proposed to scavenge substrates from the host environment, including metabolites or cellular components of the sponge. Their diverse metabolic capabilities may allow for efficient cycling of organic matter in the sponge environment, potentially to the benefit of the host and other symbionts.

Microbiome network in the pelagic and benthic offshore systems of the northern Adriatic Sea (Mediterranean Sea).

Because of their recognized global importance, there is now the urgent need to map diversity and distribution patterns of marine microbial communities. Even if available studies provided some advances in the understanding the biogeographical patterns of marine microbiomes at the global scale, their degree of plasticity at the local scale it is still underexplored, and functional implications still need to be dissected. In this scenario here we provide a synoptical study on the microbiomes of the water column and surface sediments from 19 sites in a 130 km2 area located 13.5 km afar from the coast in the North-Western Adriatic Sea (Italy), providing the finest-scale mapping of marine microbiomes in the Mediterranean Sea. Pelagic and benthic microbiomes in the study area showed sector specific-patterns and distinct assemblage structures, corresponding to specific variations in the microbiome network structure. While maintaining a balanced structure in terms of potential ecosystem services (e.g., hydrocarbon degradation and nutrient cycling), sector-specific patterns of over-abundant modules-and taxa-were defined, with the South sector (the closest to the coast) characterized by microbial groups of terrestrial origins, both in the pelagic and the benthic realms. By the granular assessment of the marine microbiome changes at the local scale, we have been able to describe, to our knowledge at the first time, the integration of terrestrial microorganisms in the marine microbiome networks, as a possible natural process characterizing eutrophic coastal area. This raises the question about the biological threshold for terrestrial microorganisms to be admitted in the marine microbiome networks, without altering the ecological balance.

Metagenomic shifts in mucus, tissue and skeleton of the coral Balanophyllia europaea living along a natural CO2 gradient.

Using the Mediterranean coral Balanophyllia europaea naturally growing along a pH gradient close to Panarea island (Italy) as a model, we explored the role of host-associated microbiomes in coral acclimatization to ocean acidification (OA). Coral samples were collected at three sites along the gradient, mimicking seawater conditions projected for 2100 under different IPCC (The Intergovernmental Panel on Climate Change) scenarios, and mucus, soft tissue and skeleton associated microbiomes were characterized by shotgun metagenomics. According to our findings, OA induced functional changes in the microbiomes genetic potential that could mitigate the sub-optimal environmental conditions at three levels: i. selection of bacteria genetically equipped with functions related to stress resistance; ii. shifts in microbial carbohydrate metabolism from energy production to maintenance of cell membranes and walls integrity; iii. gain of functions able to respond to variations in nitrogen needs at the holobiont level, such as genes devoted to organic nitrogen mobilization. We hence provided hypotheses about the functional role of the coral associated microbiome in favoring host acclimatation to OA, remarking on the importance of considering the crosstalk among all the components of the holobiont to unveil how and to what extent corals will maintain their functionality under forthcoming ocean conditions.

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.

Particulate matter emission sources and meteorological parameters combine to shape the airborne bacteria communities in the Ligurian coast, Italy.

Aim of the present study is to explore how the chemical composition of particulate matter (PM) and meteorological conditions combine in shaping the air microbiome in Savona (Italy), a medium-size, heavily inhabited urban settlement, hosting a wide range of industrial activities. In particular, the air microbiome and PM10 were monitored over six months in 2012. During that time, the air microbiome was highly dynamic, fluctuating between different compositional states, likely resulting from the aerosolization of different microbiomes emission sources. According to our findings, this dynamic process depends on the combination of local meteorological parameters and particle emission sources, which may affect the prevalent aerosolized microbiomes, thus representing further fundamental tools for source apportionment in a holistic approach encompassing chemical as well as microbiological pollution. In particular, we showed that, in the investigated area, industrial emissions and winds blowing from the inlands combine with an airborne microbiome which include faecal microbiomes components, suggesting multiple citizens' exposure to both chemicals and microorganisms of faecal origin, as related to landscape exploitation and population density. In conclusion, our findings support the need to include monitoring of the air microbiome compositional structure as a relevant factor for the final assessment of local air quality.

Phylogeny resolved, metabolism revealed: functional radiation within a widespread and divergent clade of sponge symbionts.

The symbiosis between bacteria and sponges has arguably the longest evolutionary history for any extant metazoan lineage, yet little is known about bacterial evolution or adaptation in this process. An example of often dominant and widespread bacterial symbionts of sponges is a clade of uncultured and uncharacterised Proteobacteria. Here we set out to characterise this group using metagenomics, in-depth phylogenetic analyses, metatranscriptomics, and fluorescence in situ hybridisation microscopy. We obtained five metagenome-assembled-genomes (MAGs) from different sponge species that, together with a previously published MAG (AqS2), comprise two families within a new gammaproteobacterial order that we named UTethybacterales. Members of this order share a heterotrophic lifestyle but vary in their predicted ability to use various carbon, nitrogen and sulfur sources, including taurine, spermidine and dimethylsulfoniopropionate. The deep branching of the UTethybacterales within the Gammaproteobacteria and their almost exclusive presence in sponges suggests they have entered a symbiosis with their host relatively early in evolutionary time and have subsequently functionally radiated. This is reflected in quite distinct lifestyles of various species of UTethybacterales, most notably their diverse morphologies, predicted substrate preferences, and localisation within the sponge tissue. This study provides new insight into the evolution of metazoan-bacteria symbiosis.

Tissue-scale microbiota of the Mediterranean mussel (Mytilus galloprovincialis) and its relationship with the environment.

In this study, we characterize the structural variation of the microbiota of Mytilus galloprovincialis at the tissue scale, also exploring the connection with the microbial ecosystem of the surrounding water. Mussels were sampled within a farm located in the North-Western Adriatic Sea and microbiota composition was analyzed in gills, hemolymph, digestive glands, stomach and foot by Next Generation Sequencing marker gene approach. Mussels showed a distinctive microbiota structure, with specific declinations at the tissue level. Indeed, each tissue is characterized by a distinct pattern of dominant families, reflecting a peculiar adaptation to the respective tissue niche. For instance, the microbiota of the digestive gland is characterized by Ruminococcaceae and Lachnospiraceae, being shaped to ferment complex polysaccharides of dietary origin into short-chain fatty acids, well matching the general asset of the animal gut microbiota. Conversely, the gill and hemolymph ecosystems are dominated by marine microorganisms with aerobic oxidative metabolism, consistent with the role played by these tissues as an interface with the external environment. Our findings highlight the putative importance of mussel microbiota for different aspects of host physiology, with ultimate repercussions on mussel health and productivity.

Expressed protein profile of a Tectomicrobium and other microbial symbionts in the marine sponge Aplysina aerophoba as evidenced by metaproteomics.

Aplysina aerophoba is an emerging model marine sponge, with a well-characterized microbial community in terms of diversity and structure. However, little is known about the expressed functional capabilities of its associated microbes. Here, we present the first metaproteomics-based study of the microbiome of A. aerophoba. We found that transport and degradation of halogenated and chloroaromatic compounds are common active processes in the sponge microbiomes. Our data further reveal that the highest number of proteins were affiliated to a sponge-associated Tectomicrobium, presumably from the family Entotheonellaceae, as well as to the well-known symbiont "Candidatus Synechococcus spongiarium", suggesting a high metabolic activity of these two microorganisms in situ. Evidence for nitric oxide (NO) conversion to nitrous oxide was consistently observed for Tectomicrobia across replicates, by production of the NorQ protein. Moreover, we found a potential energy-yielding pathway through CO oxidation by putative Chloroflexi bacteria. Finally, we observed expression of enzymes that may be involved in the transformation of chitin, glycoproteins, glycolipids and glucans into smaller molecules, consistent with glycosyl hydrolases predicted from analyses of the genomes of Poribacteria sponge symbionts. Thus, this study provides crucial links between expressed proteins and specific members of the A. aerophoba microbiome.