HOLOBIOMICS - Use of microbiomics for the exploration of microbial communities in holobionts.

Introducing more than a decade ago the High-Throughput Sequencing techniques we have exponentially increased our opportunities of shedding light on complex microbial communities. This revolution opened a ‘golden era’ in the new-born field of microbiomics, avoiding the culturing step that always represented a limiting factor in the characterization of particular and fastidious groups of microorganisms. Furthermore, it is clear the advantage of retrieving all the taxonomic and functional information encoded within a microbiome directly by sequencing a sample deriving from an environment of interest. The huge amount of information produced in studies relying on NGS represents a challenging task, constituting the main driver for the creation of the computational microbiologist: a new figure alongside the molecular microbiologist and classic microbiologist. This researcher’s work starts when the laboratory work ends and the sequencing process is completed: the aim of a computational microbiologist work is to deal with the vast amount of data generated by the sequencing process, producing biologically meaningful data. During my PhD I have focused on these latter tasks, dealing with the characterization at different levels of various holobionts, ranging from wild animals to humans, giving attention at the bacterial, fungal and viral fractions in ecosystems. In the present work I report the main achievements of my research work, whose common denominator is the bioinformatic approach to microbiome data. In the cases I studied, I observed a mutualistic microbiome that may follows adaptive strategies aimed at the conservation of the homeostasis of the total ecosystem. This work contributes to enrich the overall knowledge on the holobiont, also exploring some peculiar ecosystems for the first time. The data presented here may form the basis for future developments in the field, in order to obtain a more comprehensive profiling of bacterial, viral and fungal fractions within complex ecosystems

Effects of rearing density on growth, digestive conditions, welfare indicators and gut bacterial community of gilthead sea bream (Sparus aurata, L. 1758) fed different fishmeal and fish oil dietary levels

In Mediterranean aquaculture, significant advances have been made towards a reduction of marine-derived ingredients in aquafeed formulation, as well as in defining the effect on how environmental factors such as rearing density interact with fish health. Little research, however, has examined the interaction between rearing density and dietary composition on main key performance indicators, physiological processes and gut bacterial community. A study was undertaken, therefore to assess growth response, digestive enzyme activity, humoral immunity on skin mucus, plasma biochemistry and gut microbiota of gilthead sea bream (Sparus aurata, L. 1758) reared at high (HD, 36–44 kg m−3) and low (LD, 12–15 kg m−3) final stocking densities and fed high (FM30/FO15, 30% fishmeal FM, 15% fish oil, FO) and low (FM10/FO3; 10% FM and 3% FO) FM and FO levels. Isonitrogenous and isolipidic extruded diets were fed to triplicate fish groups (initial weight: 96.2 g) to overfeeding over 98 days. The densities tested had no major effects on overall growth and feed efficiency of sea bream reared at high or low FM and FO dietary level. However, HD seems to reduce feed intake compared to LD mainly in fish fed FM30/FO15. Results of digestive enzyme activity indicated a comparable digestive efficiency among rearing densities and within each dietary treatment even if intestinal brush border enzymes appeared to be more influenced by stocking density compared to gastric and pancreatic enzymes. Plasma parameters related to nutritional and physiological conditions were not affected by rearing densities under both nutritional conditions a similar observation was also achieved through the study of lysozyme, protease, antiprotease and total protein determination in skin mucus, however; in this case lysozyme was slightly reduced at HD. For the first time on this species, the effect of rearing density on gut bacterial community was studied. Different response in relation to dietary treatment under HD and LD were detected. Low FM-FO diet maintained steady the biodiversity of the gut bacterial community between LD and HD conditions while fish fed high FM-FO level showed a reduced biodiversity at HD. According to the results, it seems feasible to rear gilthead sea bream at the on-growing phase at a density up to 36–44 kg m−3 with low or high FM-FO diet without negatively affecting growth, feed efficiency, welfare condition and gut bacterial community.info:eu-repo/semantics/acceptedVersio

Next-generation sequencing characterization of the gut bacterial community of gilthead sea bream (Sparus aurata, L.) fed low fishmeal based diets with increasing soybean meal levels

The present study was carried out to evaluate growth, gut histology and gut bacterial community of gilthead sea bream (Sparus aurata) fed with increasing dietary soybean meal (SBM) levels in a low fishmeal (FM) based diet, in comparison with a control diet. Five isoproteic and isolipidic experimental diets were formulated to contain increasing levels of SBM (0, 100, 200, and 300 g kg 121 named S0, S10, S20 and S30, respectively) with 150 g kg 121 of FM, and one control diet (C) without SBM and containing 350 g kg 121 of FM. Sixty sea bream (initial body weight 75.9 \ub1 1.9 g, n = 900) per tank were reared in a recirculation system at 23.0 \ub1 1.0 \ub0C and fed to satiation. The trial was run in triplicate and lasted 100 days. At the end of the trial fish fed the S30 diet showed a higher (P 64 0.05) specific growth rate (SGR) compared to S0 (SGR, 1.17 \ub1 0.03, 1.20 \ub1 0.01, 1.22 \ub1 0.01, 1.25 \ub1 0.01 and 1.21 \ub1 0.04 for S0, S10, S20, S30 and C, respectively), and a higher feed intake (FI) compared to S0, S10 and S20. Sea bream fed the C diet had a higher (P 64 0.05) FI compared to S0 (FI, 1.40 \ub1 0.01, 1.45 \ub1 0.01, 1.44 \ub1 0.03, 1.51 \ub1 0.03 and 1.46 \ub1 0.02 for S0, S10, S20, S30 and C, respectively). No significant differences in feed conversion rate, protein efficiency ratio, gross protein efficiency and gross lipid efficiency among the treatments were detected. No specific histopathological changes indicative of soy-induced enteritis were observed in the intestine of any fish examined. Gut bacterial community of the distal intestine content was analyzed by Next-Generation Sequencing. At the phylum level, the gut bacterial community was dominated by Firmicutes (relative abundance 71%), while the most represented family was Lactobacillaceae (26%). Even if no significant differences (P 64 0.05) in the gut bacterial community \u3b1 and \u3b2-diversity according to the different diets were detected, Cyanobacteria and Lactobacillaceae progressively increased from diet C to diet S30. In conclusion results of growth, nutrient utilization, gut histology and gut bacterial community indicate that SBM can be successfully incorporated up to a level of 300 g kg 121 with the inclusion of 150 g kg 121 of FM, without any deleterious effects on growth, protein utilization and gut health during the on-growing of sea bream

Effects of calcium carbonate inclusion in low fishmeal diets on growth, gastrointestinal pH, digestive enzyme activity and gut bacterial community of European sea bass (Dicentrarchus labrax L.) juveniles

Fishmeal (FM) possesses one of the highest buffering capacities in comparison to most alternative vegetable aquafeed ingredients and its decreasing content in current formulations might affect the ideal gastrointestinal environment for digestive enzyme action and gut bacterial community of carnivorous fish species. A study was undertaken, therefore, to assess growth response, gastrointestinal pH, digestive enzyme activity and gut bacterial community of European sea bass (Dicentrarchus labrax L.) juveniles fed low FM diets (10% FM) with or without a feed buffering additive, calcium carbonate (FM10 + B and FM10, respectively) in comparison to a standard FM diet (20% FM, FM20). Three isonitrogenous and isolipidic extruded diets were fed to triplicate fish groups of 80 individuals (initial weight: 23 g) to overfeeding over 64 days. No significant differences due to low FM dietary levels were observed in final body weight, specific growth rate, feed intake, feed and protein efficiency. Low FM diet did not affect gastrointestinal pH in the stomach, anterior intestine, mid-intestine and hindgut at 0, 4, 8 and 12 hours post meal (hpm) while the inclusion of calcium carbonate in low FM diet seems to slightly increase the pH in the hindgut at 12 hpm. The absence of significant differences of pepsin, chymotrypsin, amylase and lipase activity suggests a comparable digestive efficiency among treatments although trypsin activity was slightly reduced in low FM diets after 4 hpm. Decreasing FM content seems to exert an effect on the overall gut bacterial community analysed by next-generation sequencing even if no significant effects on specific bacterial component were detected. The gut bacterial community in all the treatments was particularly rich in lactic acid bacteria such as Lactobacillus spp. which may provide important beneficial functions for the host and be associated with a healthy intestinal epithelium. According to the results, increasing the feed buffering capacity does not seem to improve digestive conditions while it is feasible to include 10% FM dietary level in practical formulation for European sea bass juveniles without negatively affecting growth, feed efficiency and digestive luminal conditions.info:eu-repo/semantics/publishedVersio

Microbial Community Dynamics in Mother’s Milk and Infant’s Mouth and Gut in Moderately Preterm Infants

Mother’s own milk represents the optimal source for preterm infant nutrition, as it promotes immune defenses and gastrointestinal function, protects against necrotizing enterocolitis, improves long-term clinical outcome and is hypothesized to drive gut microbiota assembly. Preterm infants at birth usually do not receive their mother’s milk directly from the breast, because active suckling and coordination between suckling, swallowing and breathing do not develop until 32–34 weeks gestational age, but actual breastfeeding is usually possible as they grow older. Here, we enrolled moderately preterm infants (gestational age 32–34 weeks) to longitudinally characterize mothers’ milk and infants’ gut and oral microbiomes, up to more than 200 days after birth, through 16S rRNA sequencing. This peculiar population offers the chance to disentangle the differential contribution of human milk feeding per se vs. actual breastfeeding in the development of infant microbiomes, that have both been acknowledged as crucial contributors to short and long-term infant health status. In this cohort, the milk microbiome composition seemed to change following the infant’s latching to the mother’s breast, shifting toward a more diverse microbial community dominated by typical oral microbes, i.e., Streptococcus and Rothia. Even if all infants in the present study were fed human milk, features typical of healthy, full term, exclusively breastfed infants, i.e., high percentages of Bifidobacterium and low abundances of Pseudomonas in fecal and oral samples, respectively, were detected in samples taken after actual breastfeeding started. These findings underline the importance of encouraging not only human milk feeding, but also an early start of actual breastfeeding in preterm infants, since the infant’s latching to the mother’s breast might constitute an independent factor helping the health-promoting assembly of the infant gut microbiome

Modulation of gut microbiota dysbioses in type 2 diabetic patients by macrobiotic Ma-Pi 2 diet

The study was carried out with internal funding

ViromeScan: a new tool for metagenomic viral community profiling

BACKGROUND: Bioinformatics tools available for metagenomic sequencing analysis are principally devoted to the identification of microorganisms populating an ecological niche, but they usually do not consider viruses. Only some software have been designed to profile the viral sequences, however they are not efficient in the characterization of viruses in the context of complex communities, like the intestinal microbiota, containing bacteria, archeabacteria, eukaryotic microorganisms and viruses. In any case, a comprehensive description of the host-microbiota interactions can not ignore the profile of eukaryotic viruses within the virome, as viruses are definitely critical for the regulation of the host immunophenotype. RESULTS: ViromeScan is an innovative metagenomic analysis tool that characterizes the taxonomy of the virome directly from raw data of next-generation sequencing. The tool uses hierarchical databases for eukaryotic viruses to unambiguously assign reads to viral species more accurately and >1000 fold faster than other existing approaches. We validated ViromeScan on synthetic microbial communities and applied it on metagenomic samples of the Human Microbiome Project, providing a sensitive eukaryotic virome profiling of different human body sites. CONCLUSIONS: ViromeScan allows the user to explore and taxonomically characterize the virome from metagenomic reads, efficiently denoising samples from reads of other microorganisms. This implies that users can fully characterize the microbiome, including bacteria and viruses, by shotgun metagenomic sequencing followed by different bioinformatic pipelines. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2446-3) contains supplementary material, which is available to authorized users

ViromeScan: a new tool for metagenomic viral community profiling

Bioinformatics tools available for metagenomic sequencing analysis are principally devoted to the identification of microorganisms populating an ecological niche, but they usually do not consider viruses. Only some software have been designed to profile the viral sequences, however they are not efficient in the characterization of viruses in the context of complex communities, like the intestinal microbiota, containing bacteria, archeabacteria, eukaryotic microorganisms and viruses. In any case, a comprehensive description of the host-microbiota interactions can not ignore the profile of eukaryotic viruses within the virome, as viruses are definitely critical for the regulation of the host immunophenotype

Variation of Carbohydrate-Active Enzyme Patterns in the Gut Microbiota of Italian Healthy Subjects and Type 2 Diabetes Patients

The human gut microbiota (GM) has been associated, to date, with various complex functions, essentials for the host health. Among these, it is certainly worth noting the degradation of the so-called microbiota-accessible carbohydrates (MACs), which the GM breaks down through specific enzymes, referred to as carbohydrate-active enzymes (CAZymes). This degradation constitutes the first step in the production of short-chain fatty acids (SCFAs), key microbial small molecules having multiple health-promoting effects for the host organism. The decline in MAC dietary intake in urban Western populations forced the shrinkage of CAZyme repertoire in the GM, as shown by the literature comparing the microbiome layout between Western urban citizens and traditional rural populations. Even if this reduction in GM functional complexity has been associated with the onset of the so-called “diseases of civilization,” only few information regarding the CAZyme variation within Western populations has been provided to date, and its connections with diet and health are still unexplored. In this scenario, here we explore the GM-encoded CAZyme repertoire across two Italian adult cohorts, including healthy lean subjects consuming a Mediterranean diet and obese patients affected by type 2 diabetes, consuming a high-fat diet. In order to impute the CAZyme panel, a pipeline consisting of publicly available software – QIIME, FragGeneScan and HMMER – was specifically implemented. Our study highlighted the existence of robust clusters of bacterial species sharing a common MAC degradation profile in the Italian GM, allowing the stratification of the individual GM into different steady states according to the carbohydrate degradation profile, with possible connections with diet and health

Temporal dynamics of the gut microbiota in people sharing a confined environment, a 520-day ground-based space simulation, MARS500

Background: The intestinal microbial communities and their temporal dynamics are gaining increasing interest due to the significant implications for human health. Recent studies have shown the dynamic behavior of the gut microbiota in free-living, healthy persons. To date, it is not known whether these dynamics are applicable during prolonged life sharing in a confined and controlled environment. Results: The MARS500 project, the longest ground-based space simulation ever, provided us with a unique opportunity to trace the crew microbiota over 520 days of isolated confinement, such as that faced by astronauts in real long-term interplanetary space flights, and after returning to regular life, for a total of 2 years. According to our data, even under the strictly controlled conditions of an enclosed environment, the human gut microbiota is inherently dynamic, capable of shifting between different steady states, typically with rearrangements of autochthonous members. Notwithstanding a strong individuality in the overall gut microbiota trajectory, some key microbial components showed conserved temporal dynamics, with potential implications for the maintenance of a health-promoting, mutualistic microbiota configuration. Conclusions: Sharing life in a confined habitat does not affect the resilience of the individual gut microbial ecosystem, even in the long term. However, the temporal dynamics of certain microbiota components should be monitored when programming future mission simulations and real space flights, to prevent breakdowns in the metabolic and immunological homeostasis of the crewmembers