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Introduction: Irritable Bowel Syndrome (IBS) is a gastrointestinal (GI) disorder that exhibits different GI and neurological symptoms such as abdominal pain, diarrhoea, constipation and mood disorders. The gut microbiota play an important role in modulating the communication between the Central Nervous System (CNS) and the GI tract, the so-called microbiota-gut-brain axis. In this context, IBS is a clear example of the alteration of the fine equilibrium between the gut microbiota and the CNS. Furthermore it has been observed that probiotic treatments of IBS results in the relief of GI and neurological symptoms, thus we could hypothesize the direct impact of the gut microbiota on IBS physiopathology. Since the IBS bacterial gut microbiota has been extensively studied we focused our attention also on the fungal counterpart of the gut microbiota, "the mycobiota". Methods and aims. The aim of the present work was to evaluate biodiversity in bacterial and fungal microbiota of a cohort of 20 IBS subject and 21 healthy subject (HS) through culture-based and metagenomics approaches. The bacterial diversity that relies on DNA polymorphism has been performed by means of cluster analysis of ARDRA profiles. Moreover the absolute abundances of bifidobacteria and enterobacteria has been evaluated by qPCR. For IBS gut mycobiota characterization, fungal strains from IBS stool samples were isolated and identified by mean of ITS1-4 sequencing. Furthermore, isolates were phenotypically characterized to evaluate their resistance to GI tract stresses (temperature, low pH and oxbile resistance), while Candida isolates were further clustered by means of their RAPD profiles. Results: The cluster analysis of ARDRA profiles showed that IBS bacterial microbiota clusters apart from HS microbiota. In addition we observed a 2.8-fold increase in the absolute abundance of enterobacteria in IBS subjects vs HS. The analysis of the gut mycobiota revealed significant differences in fungal isolates abundance in IBS subjects vs HS even if we did not find any significant difference in species richness. In particular, results showed an abundance of fungi (61.8% C. albicans), with a higher number of colony count 4.7 log10 CFU/g faeces in IBS subjects respect to HS faecal samples with 1.9 log10 CFU/g (48,75% C. albicans). RAPD prolfile analysis of C. albicans and C. parapsilosis showed that IBS isolates clustered apart from HS isolates, suggesting their different genotypical background. Finally, we observed that IBS fungal isolates showed different phenotypical features with an increased ability to growth to high temperatures and low pH respect to isolates from HS. Conclusions: Our results showed the presence of alterations in the microbial community structure of IBS subjects, both at bacterial and fungal level. The absolute abundance of enterobacteria in IBS subjects suggests their connection with putative inflammatory phenomena as previously observed in IBS and other pathologies (Lee and Park, 2014). The phenotypical characterization of IBS fungal isolates revealed that such isolates showed a different response in respect GI-like stresses. Moreover we observed that C. albicans and C. parapsilosis isolates from IBS are both phenotypically and genotypically different from HS Candida isolates suggesting their putative different ecological distribution. Finally we could hypotesize that dysbiosis of the gut microbiota in IBS could be one of the driving factor in IBS pathophysiology that could be responsible of intestinal fungal population overgrowth. Lee JY, Park KS. 2014. World J Gastroenterol 20:2456-2469

Irritable Bowel Syndrome (IBS) is a psychosomatic gastrointestinal disorder which aetiology is not well clear, although several factors indicate the involvement of the gut microbiota, remarking the importance of the microbiota-gut-brain axis in IBS pathophysiology. We therefore characterized the gut microbiota in a cohort of 20 IBS subjects and 21 healthy subjects (HS) focusing also on the characterization of the fungal gut microbiota.

During the last 25 years of activity, the Physical Oceanography Research Group, previously known as the PO-Unit, and currently established within the Department of Geosciences of the University of Malta, has been promoting the downscaling of broad scope marine core services to higher resolution local scale domains for the Maltese Islands. Several services are delivered either by an intrinsic data elaboration or by making use of and integrating the COPERNICUS Marine Environment Monitoring Service (CMEMS) data to local marine data streams. Local observations are also integrated with higher resolution forecasts for the preparation and provision of dedicated services that address real specific needs of sub-regional and coastal users. This effort has yielded valuable climatological datasets covering the Maltese coastal waters and spanning over several years. This work focusses on the climatologies derived from numerical models and satellites, and compiled within the Interreg MED programme AMAre (Actions for Marine Protected Areas) project., peer-reviewed

The human gut microbiota directly affect human health. Alterations in the composition of commensal bacteria can lead to chronic inflammation also predisposing individuals to fungal infections. Commensal fungi are important in human health and changes in commensal fungal populations have been shown to deeply affect pathologies not directly related to fungi, such as Inflammatory Bowel Diseases. Rett syndrome (RTT) is a neurodegenerative disorder caused by a mutation in MECP2. High proportion of RTT subjects suffer of gastrointestinal disorders, implying a link between RTT and the gut microbiota. We studied the bacterial and fungal gut microbiota of patients affected by RTT by a multilevel approach to understand whether changes in gut microbiota of RTT patients could be associated with GI abnormalities, inflammatory status and cytokine dysregulation previously observed. Our findings revealed that RTT patients harbor a distinct and less diverse bacterial and fungal microbiota compared to healthy subjects in which Bifidobacterium and Candida are the most abundant genera. The immunological responses exerted by RTT yeast isolates in our in vitro cell model suggest that fungi could contribute to the gastrointestinal abnormalities and intestinal inflammation observed in our RTT study cohort.

Rett syndrome (RTT) is a neurodegenerative disorder caused by a mutation in MECP2. To date, the mechanism that trigger the disease have yet to be explained. Gut microbiome controls CNS activities through endocrine, metabolic and immune pathways and yeast infections shift IDO’s activity leading to a reduction of kineurenine that act as a neuroprotective agent. In addition, kineurenine is affected by the immune system, dysfunction of which has been implicated in the etiology of ASD. The hypothesis we want to test is if alterations in microbiota-mediated immunomodulation are reflected in alterations in microbiota-mediated neuromodulation. We characterized the gut microbiome/mycobiome of RTT patients through culture based and metagenomics approaches. Stool sample from 50 RTT patient and 30 HS have been collected. DNA has been extracted and submitted to metagenomic analysis in order to analyze bacterial and fungal communities’ structure. We discovered C. albicans as significantly more represented in heathy controls with respect to RTT patients. On the contrary, the highly emerging potentially invasive species, Trichosporon spp. and S. cerevisiae have been isolated solely in RTT patients.

Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi—belonging to Aspergillus, Quambalaria and Bjerkandera genera—and bacteria—belonging to Staphylococcus genus—were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

In the present work, culture-based and culture-independent investigations were performed to determine the microbiota structure of the coelomic fluid of Mediterranean sea urchin Paracentrotus lividus individuals collected from two distinct geographical sites neighboring a high-density population bay and a nature reserve, respectively. Next Generation Sequencing analysis of 16S rRNA gene (rDNA) showed that members of the Proteobacteria, Bacteroidetes and Fusobacteria phyla, which have been previously reported to be commonly retrieved from marine invertebrates, dominate the overall population of microorganisms colonizing this liquid tissue, with minority bacterial genera exhibiting remarkable differences among individuals. Our results showed that there is a correlation between microbiota structure and geographical location of the echinoderm collection site, highlighting over-representation of metagenomic functions related to amino acid and bioactive peptides metabolism in specimens inhabiting the nature reserve. Finally, we also described the developmental delay and aberrations exhibited by sea urchin embryos exposed to distinct bacterial isolates, and showed that these defects rely upon hydrophilic compound(s) synthesized by the bacterial strains assayed. Altogether, our findings lay the groundwork to decipher the relationships of bacteria with sea urchins in their aquatic environment, also providing an additional layer of information to understand the biological roles of the coelomic fluid.

The study of the microbiota is a subject of considerable and growing interest since it is drawing new important perspectives in the life sciences concerning the functional relationships between metazoans and microbial cells. In fact, it has already shown that the endogenous microbial community affects various physiological activities of multicellular organisms. The coelomic cavity of echinoderms contains a fluid in which coelomocytes are reported to exert immune functions like phagocytosis, opsonization and production of antimicrobial agents against marine bacteria [1, 2]. However, up to day nothing is known about the endogenous bacterial population of coelomic fluid. We focused on this issue, and, to this aim, both bacterial culture-dependent and –independent approaches were adopted. By the former approach, we isolated 8 distinct Gram-negative marine bacterial strains identified for their 16S rDNA sequence. Interestingly, almost all isolated strains show a considerable extracellular hydrolytic activity. Moreover, one of them exerts antimicrobial effect against Gram-negative bacteria, including most of the other strains isolated from the coelomic fluid. Finally, molecular investigation on metagenomic DNA composition is currently ongoing using Next Generation Sequence Technology. This study not only suggests insights on functional interaction between sea urchin and marine microorganisms, but also could provide a novel source of biochemical diversity for the production of bioactive compounds and enzymes that can find biotechnological applications. 1. Remziye Deveci et al. (2015). Journal of Morphology 276(5):583-8 2. Stabili L et al. (1996). Comp Biochem Physiol B Biochem Mol Biol 113(3):639-44

iNKT cells account for a relevant fraction of effector T-cells in the intestine and are considered an attractive platform for cancer immunotherapy. Although iNKT cells are cytotoxic lymphocytes, their functional role in colorectal cancer (CRC) is still controversial, limiting their therapeutic use. Thus, we examined the immune cell composition and iNKT cell phenotype of CRC lesions in patients (n = 118) and different murine models. High-dimensional single-cell flow-cytometry, metagenomics, and RNA sequencing experiments revealed that iNKT cells are enriched in tumor lesions. The tumor-associated pathobiont Fusobacterium nucleatum induces IL-17 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in iNKT cells without affecting their cytotoxic capability but promoting iNKT-mediated recruitment of neutrophils with polymorphonuclear myeloid-derived suppressor cells-like phenotype and functions. The lack of iNKT cells reduced the tumor burden and recruitment of immune suppressive neutrophils. iNKT cells in-vivo activation with α-galactosylceramide restored their anti-tumor function, suggesting that iNKT cells can be modulated to overcome CRC-associated immune evasion. Tumor co-infiltration by iNKT cells and neutrophils correlates with negative clinical outcomes, highlighting the importance of iNKT cells in the pathophysiology of CRC. Our results reveal a functional plasticity of iNKT cells in CRC, suggesting a pivotal role of iNKT cells in shaping the tumor microenvironment, with relevant implications for treatment.

Background The gut microbiota plays a central role in host physiology and in several pathological mechanisms in humans. Antibiotics compromise the composition and functions of the gut microbiota inducing long-lasting detrimental effects on the host. Recent studies suggest that the efficacy of different clinical therapies depends on the action of the gut microbiota. Here, we investigated how different antibiotic treatments affect the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in an experimental colitis model and in ex vivo experiments with human intestinal biopsies. Results Murine fecal donors were pre-treated with different antibiotics, i.e., vancomycin, streptomycin, and metronidazole before FMT administration to colitic animals. The analysis of the gut microbiome, fecal metabolome, and the immunophenotyping of colonic lamina propria immune cells revealed that antibiotic pre-treatment significantly influences the capability of the microbiota to control intestinal inflammation. Streptomycin and vancomycin-treated microbiota failed to control intestinal inflammation and were characterized by the blooming of pathobionts previously associated with IBD as well as with metabolites related to the presence of oxidative stress and metabolism of simple sugars. On the contrary, the metronidazole-treated microbiota retained its ability to control inflammation co-occurring with the enrichment of Lactobacillus and of innate immune responses involving iNKT cells. Furthermore, ex vivo cultures of human intestinal lamina propria mononuclear cells and iNKT cell clones from IBD patients with vancomycin pre-treated sterile fecal water showed a Th1/Th17 skewing in CD4+ T-cell populations; metronidazole, on the other hand, induced the polarization of iNKT cells toward the production of IL10. Conclusions Diverse antibiotic regimens affect the ability of the gut microbiota to control intestinal inflammation in experimental colitis by altering the microbial community structure and microbiota-derived metabolites.

The secretory immunoglobulin A (SIgA) in mammalian gut protects the organism from infections and contributes to host physiology by shaping microbiota composition. The mechanisms regulating the adaptive SIgA response towards gut microbes are poorly defined. Deletion of P2rx7, encoding for the ATP-gated ionotropic P2X7 receptor, leads to T follicular helper (Tfh) cells expansion in the Peyer’s patches (PPs) of the small intestine, enhanced germinal centre (GC) reaction and IgA secretion; the resulting alterations of the gut microbiota in turn affects host metabolism. Here, we define gut microbiota modifications that correlate with deregulated SIgA secretion and metabolic alterations in P2rx7−/− mice. In particular, Lactobacillus shows enhanced SIgA coating in P2rx7−/− with respect to wild-type (WT) mice. The abundance of SIgA-coated lactobacilli positively correlates with Tfh cells number and body weight, suggesting Lactobacillus-specific SIgA response conditions host metabolism. Accordingly, oral administration of intestinal Lactobacillus isolates from P2rx7−/− mice to WT animals results in altered glucose homeostasis and fat deposition. Thus, enhanced SIgA production by P2X7 insufficiency promotes Lactobacillus colonization that interferes with systemic metabolic homeostasis. These data indicate that P2X7 receptor-mediated regulation of commensals coating by SIgA is important in tuning the selection of bacterial taxa, which condition host metabolism.

Campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. Currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. Secretory immunoglobulin A (SIgA) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. In this study, we describe the prophylactic activity of orally delivered recombinant SIgA generated from two human monoclonal antibodies (CAA1 and CCG4) isolated for their reactivity against the flagellar-capping protein FliD, which is essential for bacteria motility and highly conserved across Campylobacter species associated with severe enteritis. In an immunocompetent weaned mouse model, a single oral administration of FliD-reactive SIgA CAA1 or CCG4 at 2 h before infection significantly enhances Campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (PMN) cells infiltration in the cecum lamina propria. Our data indicate that the prophylactic activity of CAA1 and CCG4 is not only dependent on the specificity to FliD but also on the use of the SIgA format, as the immunoglobulin G (IgG) versions of the same antibodies did not confer a comparable protective effect. Our work emphasizes the potential of FliD as a target for the development of vaccines and supports the concept that orally administered FliD-reactive SIgA can be developed to prevent or mitigate the severity of Campylobacter infections as well as the development of post-infection syndromes.

T cell dependent secretory IgA (SIgA) generated in the Peyer’s patches (PPs) of the small intestine shapes a broadly diverse microbiota that is crucial for host physiology. The mutualistic co-evolution of host and microbes led to the relative tolerance of host’s immune system towards commensal microorganisms. The ATP-gated ionotropic P2X7 receptor limits T follicular helper (Tfh) cells expansion and germinal center (GC) reaction in the PPs. Here we show that transient depletion of intestinal ATP can dramatically improve high-affinity IgA response against both live and inactivated oral vaccines. Ectopic expression of Shigella flexneri periplasmic ATP-diphosphohydrolase (apyrase) abolishes ATP release by bacteria and improves the specific IgA response against live oral vaccines. Antibody responses primed in the absence of intestinal extracellular ATP (eATP) also provide superior protection from enteropathogenic infection. Thus, modulation of eATP in the small intestine can affect high-affinity IgA response against gut colonizing bacteria., Nature Communications, 10, ISSN:2041-1723

Background Rett syndrome (RTT) is a neurological disorder mainly caused by mutations in MeCP2 gene. It has been shown that MeCP2 impairments can lead to cytokine dysregulation due to MeCP2 regulatory role in T-helper and T-reg mediated responses, thus contributing to the pro-inflammatory status associated with RTT. Furthermore, RTT subjects suffer from an intestinal dysbiosis characterized by an abnormal expansion of the Candida population, a known factor responsible for the hyper-activation of pro-inflammatory immune responses. Therefore, we asked whether the intestinal fungal population of RTT subjects might contribute the sub-inflammatory status triggered by MeCP2 deficiency. Methods We evaluated the cultivable gut mycobiota from a cohort of 50 RTT patients and 29 healthy controls characterizing the faecal fungal isolates for their virulence-related traits, antifungal resistance and immune reactivity in order to elucidate the role of fungi in RTT’s intestinal dysbiosis and gastrointestinal physiology. Results Candida parapsilosis, the most abundant yeast species in RTT subjects, showed distinct genotypic profiles if compared to healthy controls’ isolates as measured by hierarchical clustering analysis from RAPD genotyping. Their phenotypical analysis revealed that RTT’s isolates produced more biofilm and were significantly more resistant to azole antifungals compared to the isolates from the healthy controls. In addition, the high levels of IL-1β and IL-10 produced by peripheral blood mononuclear cells and the mixed Th1/Th17 cells population induced by RTT C. parapsilosis isolates suggest the capacity of these intestinal fungi to persist within the host, being potentially involved in chronic, pro-inflammatory responses. Conclusions Here we demonstrated that intestinal C. parapsilosis isolates from RTT subjects hold phenotypic traits that might favour the previously observed low-grade intestinal inflammatory status associated with RTT. Therefore, the presence of putative virulent, pro-inflammatory C. parapsilosis strains in RTT could represent an additional factor in RTT’s gastrointestinal pathophysiology, whose mechanisms are not yet clearly understood. Electronic supplementary material The online version of this article (10.1186/s12876-018-0785-z) contains supplementary material, which is available to authorized users.

Nucleotide-binding Oligomerization Domain-2 (NOD2) mutations are associated with an increased risk to develop Crohn’s Disease. In previous studies, we have shown that Nod2−/− mice manifest increased proportion of Lamina Propria (LP) CD4+ LAP+ Foxp3− regulatory cells, when compared with Nod2+/+ mice, while CD4+ Foxp3 + regulatory cells were not affected. Here, we investigated the Nod2 gut microbiota, by 16S rRNA pyrosequencing, at steady state and after TNBS-colitis induction in mice reared separately or in cohousing, correlating the microbial profiles with LP regulatory T cells proportion and tissue cytokines content. We found that enrichment of Rikenella and Alistipes (Rikenellaceae) in Nod2−/− mice at 8 weeks of age reared separately was associated with increased proportion of CD4+ LAP+ Foxp3− cells and less severe TNBS-colitis. In co-housed mice the acquisition of Rickenellaceae by Nod2+/+ mice was associated with increased CD4+ LAP+ Foxp3− proportion and less severe colitis. Severe colitis was associated with enrichment of gram-negative pathobionts (Escherichia and Enterococcus), while less severe colitis with protective bacteria (Barnesiella, Odoribacter and Clostridium IV). Environmental factors acting on genetic background with different outcomes according to their impact on microbiota, predispose in different ways to inflammation. These results open a new scenario for therapeutic attempt to re-establish eubiosis in Inflammatory Bowel Disease patients with NOD2 polymorphisms.

Background Autism spectrum disorders (ASDs) are neurodevelopmental conditions characterized by social and behavioural impairments. In addition to neurological symptoms, ASD subjects frequently suffer from gastrointestinal abnormalities, thus implying a role of the gut microbiota in ASD gastrointestinal pathophysiology. Results Here, we characterized the bacterial and fungal gut microbiota in a cohort of autistic individuals demonstrating the presence of an altered microbial community structure. A fraction of 90% of the autistic subjects were classified as severe ASDs. We found a significant increase in the Firmicutes/Bacteroidetes ratio in autistic subjects due to a reduction of the Bacteroidetes relative abundance. At the genus level, we observed a decrease in the relative abundance of Alistipes, Bilophila, Dialister, Parabacteroides, and Veillonella in the ASD cohort, while Collinsella, Corynebacterium, Dorea, and Lactobacillus were significantly increased. Constipation has been then associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by high levels of bacterial taxa belonging to Escherichia/Shigella and Clostridium cluster XVIII. We also observed that the relative abundance of the fungal genus Candida was more than double in the autistic than neurotypical subjects, yet due to a larger dispersion of values, this difference was only partially significant. Conclusions The finding that, besides the bacterial gut microbiota, also the gut mycobiota contributes to the alteration of the intestinal microbial community structure in ASDs opens the possibility for new potential intervention strategies aimed at the relief of gastrointestinal symptoms in ASDs. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0242-1) contains supplementary material, which is available to authorized users.

Background Plant seeds possess a complex microbiota which may play a crucial rolein a plethora of mechanisms, such as preservation, germination, seedling development, plant growth and health. In particular, seed endophytic microbiota is gaining more and more consideration due to the fact that it may be vertically transmitted to ensure individual competitive advantages. The Citrusspecies, including C.limon,areone of the most economically important evergreen fruit crops in the world. Objectives i)Identification and characterization ofmicrobial isolates from Citrus limon L. Burm. F. seed endosphere. ii) Structure of Citrus limon L. Burm. F. seed endosphytic microbiota. Methods -Surface-sterilization of Citrus limon L. Burm. F. seeds. - Strain isolation and phylogenetic characterization by 16S rDNA sequence. - Next Generation Sequence Technology analysis (NGS) of metagenomic DNA by pyrosequencing of 16S rDNA. - Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy (FISH-CLSM). Conclusions Culture-dependent analysis allowed the isolation of several Staphylococcus spp. and fungi belonging to genera Aspergillus, Quambalaria and Efibula from surface-sterilized seeds. These results weresupported by the detection of bacterial cells and micro–colonies in seed cryosections by FISH-CLSM. In fact, this finding highlighted the presence of Firmicutes and other bacteria colonizing intercellular spaces. NGS-basedcharacterization using metagenomic DNA from surface-sterilized seeds is being carried out in order to elucidatethe microbiota structure. Culture-dependent approaches allowed the isolation of several bacterial strains belonging to the genus Staphylococcus and several fungal strains belonging to the genera Aspergillus, Quambalaria and Efibulafrom seed endosphere. These results were supported by the detection of bacterial cells and micro–colonies in seed cryosections by FISH-CLSM. In particular, this analysis highlighted the presence of Firmicutes and other bacteria colonizing intercellular spaces. NGS-based characterization using metagenomic DNA from surface-sterilized seeds is being carried out in order to elucidate the microbiota structure.

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley.

The fungal component of the human gut microbiota has been neglected for long time due to the low relative abundance of fungi with respect to bacteria, and only recently few reports have explored its composition and dynamics in health or disease. The application of metagenomics methods to the full understanding of fungal communities is currently limited by the under representation of fungal DNA with respect to the bacterial one, as well as by the limited ability to discriminate passengers from colonizers. Here, we investigated the gut mycobiota of a cohort of healthy subjects in order to reduce the gap of knowledge concerning fungal intestinal communities in the healthy status further screening for phenotypical traits that could reflect fungi adaptation to the host. We studied the fecal fungal populations of 111 healthy subjects by means of cultivation on fungal selective media and by amplicon-based ITS1 metagenomics analysis on a subset of 57 individuals. We then characterized the isolated fungi for their tolerance to gastrointestinal (GI) tract-like challenges and their susceptibility to antifungals. A total of 34 different fungal species were isolated showing several phenotypic characteristics associated with intestinal environment such as tolerance to body temperature (37°C), to acidic and oxidative stress, and to bile salts exposure. We found a high frequency of azoles resistance in fungal isolates, with potential and significant clinical impact. Analyses of fungal communities revealed that the human gut mycobiota differs in function of individuals' life stage in a gender-related fashion. The combination of metagenomics and fungal cultivation allowed an in-depth understanding of the fungal intestinal community structure associated to the healthy status and the commensalism-related traits of isolated fungi. We further discussed comparatively the results of sequencing and cultivation to critically evaluate the application of metagenomics-based approaches to fungal gut populations.

International audience; Evolution of Bordetella pertussis is driven by natural and vaccine pressures. Isolates circulating in regions with high vaccination coverage present multiple allelic and antigenic variations as compared to isolates collected before introduction of vaccination. Furthermore, during the last epidemics reported in regions using pertussis acellular vaccines, isolates deficient for vaccine antigens, such as pertactin (PRN), were reported to reach high proportions of circulating isolates. More sporadic filamentous hemagglutinin (FHA) or pertussis toxin (PT) deficient isolates were also collected. The whole genome of some recent French isolates, deficient or non-deficient in vaccine antigens, were analyzed. Transcription profiles of the expression of the main virulence factors were also compared. The invasive phenotype in an in vitro human tracheal epithelial (HTE) cell model of infection was evaluated. Our genomic analysis focused on SNPs related to virulence genes known to be more likely to present allelic polymorphism. Transcriptomic data indicated that isolates circulating since the introduction of pertussis vaccines present lower transcription levels of the main virulence genes than the isolates of the pre-vaccine era. Furthermore, isolates not producing FHA present significantly higher expression levels of the entire set of genes tested. Finally, we observed that recent isolates are more invasive in HTE cells when compared to the reference strain, but no multiplication occurs within cells.

The Yaghnob Valley in Tajikistan is inhabited by a small isolated human population. The area is geographically isolated, causing the inhabitant’s culture, food and lifestyle to remain uninfluenced by the rest of the world. Traditional methods of fermenting milk involve the use of indigenous microorganisms, leading to the production of a variety of fermented milk products. The people in Yaghnobi produce a fermented milk from goat, which constitutes one of their main foods. The aim of the current work was to determine which yeasts were present in the original Yaghnobi fermented milk as well as in the same product reproduced at home for three years with monthly reculturing in cow milk. Thirty yeasts have been isolated using different lab media such as M17, MRS, WL,YPD and YPD plus chloramphenicol, and colonies were re-cultivated until pure. Twenty isolates have been obtained from the original product and 10 from fermented milk reproduced at home. They were identified by Sanger sequencing of the amplified ribosomal Internal Transcribed Spacers (ITS1-4). Furthermore, RFLP analyses of the ITS1-4 region were performed when quality, coverage or similarity of ITS1-4sequences did not permitted the unambiguously identification of the yeast isolates. Only for Saccharomyces cerevisiae, identity was also confirmed with GTG5Rep PCR. The results showed the presence of Kluyveromycesmarxianus (11), Pichia fermentans (7) and Saccharomyces cerevisiae (10) in both products. However, Kluyveromyceslactis (1) was found in the original fermented milk sample and Kazachstaniaunispora(1) in home maintained fermented milk. Great attention has nowadays been devoted to microbial resources and the study of traditional fermented products can be a very interesting source of microbial biodiversity. This study provides for the first time data on yeast composition and characteristics in naturally Yaghnobi fermented milk, showing an unexpected richness in yeast communities leading to fermented milk production.

Introduction In the Yaghnob Valley in Tajikistan lives an isolated human population, who still utilize ancient fermentation methods to produce fermented milk as one of their main foods. The area is very isolated, so theinhabitants and their diet have remained protected without influence or contamination from the surroundingworld. Thus, it is interesting to study the microbial biodiversity of their fermented food.The aim of the present work is thephenotypic characterization of yeasts isolated from Yaghnobi fermented milk. Materials and Methods Thirty yeast isolates were obtained from the original Yaghnobi fermented milk and from the sameone reproduced at home for three years.The isolation was performed on M17, MRS, WL,YPD and YPD plus chloramphenicol. Phenotypic characterization were assessed by growth i) on different carbon sources, ii) in presence of ox bile, iii) at high temperatures, iv) in acidic condition and v) in presence of hydrogen peroxide. Also the invasiveness in YPD and hyphae formation was studied. Results The 30 yeast strains, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae plus one Kazachstania unispora and one Kluyveromyces lactis. revealed different phenotypes within the same species; e.g. invasiveness from 0 to 3 among the isolates of both S. cerevisiae and P. fermentans, the latter species also showed large variations in thermo-tolerance (inhibition from 40°Cto 46°C), and varying tolerance to acidic conditions. Only the strain of K. lactis and about 50%of K .marxianus strains grew at 46°C. All strains of K. marxianus grew at ox bile up to 2%. Of the S. cerevisiae strains, 50% grew in ox bile up to 2%, while two strains were inhibited already at 0.5%. Conclusions The data obtained suggest the presenceof yeasts with interesting probiotic properties in Yaghnobi fermented milk, such as the ability to survive in harsh gastrointestinal environment. The complete probiotic characterization of these yeasts will be investigated further.

Strains of Bifidobacterium animalis subsp. lactis are extensively exploited by the food industry as health-promoting bacteria, although the genetic variability of members belonging to this taxon has so far not received much scientific attention. In this article, we describe the complete genetic makeup of the B. animalis subsp. lactis Bl12 genome and discuss the genetic relatedness of this strain with other sequenced strains belonging to this taxon. Moreover, a detailed comparative genomic analysis of B. animalis subsp. lactis genomes was performed, which revealed a closely related and isogenic nature of all currently available B. animalis subsp. lactis strains, thus strongly suggesting a closed pan-genome structure of this bacterial group.

The international Muon Ionization Cooling Experiment (MICE), which is under construction at the Rutherford Appleton Laboratory (RAL), will demonstrate the principle of ionization cooling as a technique for the reduction of the phase-space volume occupied by a muon beam. Ionization cooling channels are required for the Neutrino Factory and the Muon Collider. MICE will evaluate in detail the performance of a single lattice cell of the Feasibility Study 2 cooling channel. The MICE Muon Beam has been constructed at the ISIS synchrotron at RAL, and in MICE Step I, it has been characterized using the MICE beam-instrumentation system. In this paper, the MICE Muon Beam and beam-line instrumentation are described. The muon rate is presented as a function of the beam loss generated by the MICE target dipping into the ISIS proton beam. For a 1 V signal from the ISIS beam-loss monitors downstream of our target we obtain a 30 KHz muon rate, with a neglible pion contamination in the beam. The international Muon Ionization Cooling Experiment (MICE), which is under construction at the Rutherford Appleton Laboratory (RAL), will demonstrate the principle of ionization cooling as a technique for the reduction of the phase-space volume occupied by a muon beam. Ionization cooling channels are required for the Neutrino Factory and the Muon Collider. MICE will evaluate in detail the performance of a single lattice cell of the Feasibility Study 2 cooling channel. The MICE Muon Beam has been constructed at the ISIS synchrotron at RAL, and in MICE Step I, it has been characterized using the MICE beam-instrumentation system. In this paper, the MICE Muon Beam and beam-line instrumentation are described. The muon rate is presented as a function of the beam loss generated by the MICE target dipping into the ISIS proton beam. For a 1 V signal from the ISIS beam-loss monitors downstream of our target we obtain a 30 KHz instantaneous muon rate, with a neglible pion contamination in the beam.

The Bifidobacterium bifidum PRL2010 genome encodes a relatively small set of predicted carbohydrate transporters. Growth experiments and transcriptome analyses of B. bifidum PRL2010 revealed that carbohydrate utilization in this microorganism appears to be restricted to a relatively low number of carbohydrates.

Botaccini, Francesca et al., Bifidobacteria are known as anaerobic/microaerophilic and fermentative microorganisms, which commonly inhabit the gastrointestinal tract of various animals and insects. Analysis of the 2,167,301 bp genome of Bifidobacterium asteroides PRL2011, a strain isolated from the hindgut of Apis mellifera var. ligustica, commonly known as the honey bee, revealed its predicted capability for respiratory metabolism. Conservation of the latter gene clusters in various B. asteroides strains enforces the notion that respiration is a common metabolic feature of this ancient bifidobacterial species, which has been lost in currently known mammal-derived Bifidobacterium species. In fact, phylogenomic based analyses suggested an ancient origin of B. asteroides and indicates it as an ancestor of the genus Bifidobacterium. Furthermore, the B. asteroides PRL2011 genome encodes various enzymes for coping with toxic products that arise as a result of oxygen-mediated respiration. © 2012 Bottacini et al., This work was financially supported by the Cariparma Bank Foundation to MV. This work was also financially supported by a Federation European Microbiology society (FEMS) Advanced Fellowship 2011 and an Irish Research Council for Science, Engineering & Technology (IRCSET) Embark postdoctoral fellowship to FT. DV is a member of The Alimentary Pharmabiotic Centre and the Alimentary Glycoscience Research Cluster, both funded by Science Foundation Ireland (SFI), through the Irish Government's National Development Plan (grant nos. 07/CE/B1368 and 08/SRC/B1393, respectively).