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To date several studies address the important role of gut microbiome and its interplay with the human host in the health and disease status. However, the selection of a universal sampling matrix representative of the microbial biodiversity associated with the gastrointestinal (GI) tract, is still challenging. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colon washing feces (CWF), in order to evaluate their relative effectiveness and accuracy in representing the complexity of the human gut microbiome. A cohort of 30 volunteers was recruited and paired F and CWF samples were collected from each subject. Alpha diversity analysis confirmed a slightly higher biodiversity of CWF compared to F matched samples. Likewise, beta diversity analysis proved that paired F and CWF microbiomes were quite similar in the same individual, but remarkable inter-individual variability occurred among the microbiomes of all participants. Taxonomic analysis in matched samples was carried out to investigate the intra and inter individual/s variability. Firmicutes, Bacteroidota, Proteobacteria and Actinobacteriota were the main phyla in both F and CWF samples. At genus level, Bacteirodetes was the most abundant in F and CWF samples, followed by Faecalibacterium, Blautia and Escherichia-Shigella. Our study highlights an inter-individual variability greater than intra-individual variability for paired F and CWF samples. Indeed, an overall higher similarity was observed across matched F and CWF samples, suggesting, as expected, a remarkable overlap between the microbiomes inferred using the matched F and CWF samples. Notably, absolute quantification of total 16S rDNA by droplet digital PCR (ddPCR) revealed comparable overall microbial load between paired F and CWF samples. We report here the first comparative study on fecal and colon washing fecal samples for investigating the human gut microbiome and show that both types of samples may be used equally for the study of the gut microbiome. The presented results suggest that the combined use of both types of sampling matrices could represent a suitable choice to obtain a more complete overview of the human gut microbiota for addressing different biological and clinical questions.

Carcinogenesis; Microbiome; Tumor evolution Carcinogènesi; Microbioma; Evolució del tumor Carcinogénesis; Microbioma; Evolución del tumor Preclinical forms of gastrointestinal stromal tumor (GIST), small asymptomatic lesions, called microGIST, are detected in approximately 30% of the general population. Gastrointestinal stromal tumor driver mutation can be already detected in microGISTs, even if they do not progress into malignant cancer; these mutations are necessary, but insufficient events to foster tumor progression. Here we profiled the tissue microbiota of 60 gastrointestinal specimens in three different patient cohorts—micro, low-risk, and high-risk or metastatic GIST—exploring the compositional structure, predicted function, and microbial networks, with the aim of providing a complete overview of microbial ecology in GIST and its preclinical form. Comparing microGISTs and GISTs, both weighted and unweighted UniFrac and Bray–Curtis dissimilarities showed significant community-level separation between them and a pronounced difference in Proteobacteria, Firmicutes, and Bacteroidota was observed. Through the LEfSe tool, potential microbial biomarkers associated with a specific type of lesion were identified. In particular, GIST samples were significantly enriched in the phylum Proteobacteria compared to microGISTs. Several pathways involved in sugar metabolism were also highlighted in GISTs; this was expected as cancer usually displays high aerobic glycolysis in place of oxidative phosphorylation and rise of glucose flux to promote anabolic request. Our results highlight that specific differences do exist in the tissue microbiome community between GIST and benign lesions and that microbiome restructuration can drive the carcinogenesis process. Francesca Goriini and Federica Zanotti have been supported by Fondazione Cassa di Risparmio di Bologna.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigen- esis. We identified two anti-tumourigenic strains of the microbiota--Faecalibaculum rodentium and its human homologue, Holdemanella biformis--that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymeth- ane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endog- enous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

Bronchiolitis due to respiratory syncytial virus (RSV) or non-RSV agents is a health-menacing lower respiratory tract (LRT) disease of infants. Whereas RSV causes more severe disease than other viral agents may, genus-dominant fecal microbiota profiles have been identified in US hospitalized infants with bronchiolitis. We investigated the fecal microbiota composition of infants admitted to an Italian hospital with acute RSV (25/37 [67.6%]; group I) or non-RSV (12/37 [32.4%]; group II) bronchiolitis, and the relationship of fecal microbiota characteristics with the clinical characteristics of infants. Group I and group II infants differed significantly (24/25 [96.0%] versus 5/12 [41.7%]; P = 0.001) regarding 90% oxygen saturation (SpO2), which is an increased respiratory effort hallmark. Accordingly, impaired feeding in infants from group I was significantly more frequent than in infants from group II (19/25 [76.0%] versus 4/12 [33.3%]; P = 0.04). Conversely, the median (IQR) length of stay was not significantly different between the two groups (seven [3–14] for group I versus five [5–10] for group II; P = 0.11). The 16S ribosomal RNA V3–V4 region amplification of infants’ fecal samples resulted in 299 annotated amplicon sequence variants. Based on alpha- and beta-diversity microbiota downstream analyses, group I and group II infants had similar bacterial communities in their samples. Additionally, comparing infants having 2 (n = 29) with infants having ≥90% SpO2 (n = 8) showed that well-known dominant genera (Bacteroides, Bifidobacterium, Escherichia/Shigella, and Enterobacter/Veillonella) were differently, but not significantly (P = 0.44, P = 0.71, P = 0.98, and P = 0.41, respectively) abundant between the two subgroups. Overall, we showed that, regardless of RSV or non-RSV bronchiolitis etiology, no fecal microbiota-composing bacteria could be associated with the severity of acute bronchiolitis in infants. Larger and longitudinally conducted studies will be necessary to confirm these findings.

Obesity and diet are associated with colorectal cancer (CRC) risk, and microbiome could mediate this risk factor. To investigate this interaction, we performed a case-control study (34 CRC cases and 32 controls) and analyzed fecal microbiota composition using 16S rRNA metabarcoding and sub-sequential shotgun analyses of genomic bacterial DNA to evaluate the role of microbiome and diet in CRC etiology, taking into account vitamin D and other risk biomarkers. Dietary habits were evaluated using a short questionnaire. Multivariate methods for data integration and mediation analysis models were used to investigate causal relationships. CRC cases were significantly more often deficient in vitamin D than controls (p = 0.04); FokI and CYP24A1 polymorphism frequency were different between cases and controls (p = 0.03 and p = 0.02, respectively). A diet poor in fatty fish and rich in carbohydrates was found to be significantly associated with CRC risk (p = 0.011). The mediation analysis confirmed the significant role of the microbiome in mediating CRC risk-increasing levels of Bifidobacteria/Escherichia genera ratio, an indicator of “healthy” intestinal microbiome, can overcome the effect of diet on CRC risk (p = 0.03). This study suggests that microbiome mediates the diet effect on CRC risk, and that vitamin D, markers of inflammation, and adipokines are other factors to consider in order to achieve a better knowledge of the whole carcinogenic process., SCOPUS: ar.j, info:eu-repo/semantics/published

Gastric cancer (GC) is the fifth most prevalent cancer worldwide and the third leading cause of global cancer mortality. With the advances of the omic studies, a heterogeneous GC landscape has been revealed, with significant molecular diversity. Given the multifaceted nature of GC, identification of different patient subsets with prognostic and/or predictive outcomes is a key aspect to allow tailoring of specific treatments. Recently, the involvement of the microbiota in gastric carcinogenesis has been described. To deepen this aspect, we compared microbiota composition in signet-ring cell carcinoma (SRCC) and adenocarcinoma (ADC), two distinct GC subtypes. To this purpose, 10 ADC and 10 SRCC and their paired non-tumor (PNT) counterparts were evaluated for microbiota composition through 16S rRNA analysis. Weighted and unweighted UniFrac and Bray&ndash, Curtis dissimilarity showed significant community-level separation between ADC and SRCC. Through the LEfSe (linear discriminant analysis coupled with effect size) tool, we identified potential microbial biomarkers associated with GC subtypes. In particular, SRCCs were significantly enriched in the phyla Fusobacteria, Bacteroidetes, Patescibacteria, whereas in the ADC type, Proteobacteria and Acidobacteria phyla were found. Overall, our data add new insights into GC heterogeneity and may contribute to deepening the GC classification.

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9-36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9-14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1-36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial "Candidatus Aquiluna" (~19% at 14.5% salinity). Interestingly, "Candidatus Aquiluna" had not been identified before in thalassohaline waters.

Jellyfish blooms are frequent and widespread in coastal areas worldwide, often associated with significant ecological and socio-economic consequences. Recent studies have also suggested cnidarian jellyfish may act as vectors of bacterial pathogens. The scyphomedusa Rhizostoma pulmo is an outbreak-forming jellyfish widely occurring across the Mediterranean basin. Using combination of culture-based approaches and a high-throughput amplicon sequencing (HTS), and based on available knowledge on a warm-affinity jellyfish-associated microbiome, we compared the microbial community associated with R. pulmo adult jellyfish in the Gulf of Taranto (Ionian Sea) between summer (July 2016) and winter (February 2017) sampling periods. The jellyfish-associated microbiota was investigated in three distinct compartments, namely umbrella, oral arms, and the mucus secretion. Actinobacteria, Bacteroidetes, Chlamydiae, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Fusobacteria, Planctomycetes, Proteobacteria, Rhodothermaeota, Spirochaetes, Tenericutes, and Thaumarchaeota were the phyla isolated from all the three R. pulmo compartments in the sampling times. In particular, the main genera Mycoplasma and Spiroplasma, belonging to the class Mollicutes (phylum Tenericutes), have been identified in all the three jellyfish compartments. The taxonomic microbial data were coupled with metabolic profiles resulting from the utilization of 31 different carbon sources by the BIOLOG Eco-Plate system. Microorganisms associated with mucus are characterized by great diversity. The counts of culturable heterotrophic bacteria and potential metabolic activities are also remarkable. Results are discussed in terms of R. pulmo ecology, the potential health hazard for marine and human life as well as the potential biotechnological applications related to the associated microbiome.

Summary The ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh) cell abundance in the Peyer’s patches (PPs) of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help., Graphical Abstract, Highlights • P2X7 receptor activity in Tfh cells is important for shaping the gut microbiota • Control of secretory IgA by Tfh cells promotes a healthy gut ecosystem • Lack of P2X7 in Tfh cells results in selection of an obesogenic microbiota • Sensing of extracellular ATP by P2X7 in Tfh cells promotes host metabolic balance, Gut commensals contribute to host metabolic homeostasis. ATP released by bacteria limits IgA secretion in the small intestine via the P2X7 receptor expressed in T follicular helper cells. Perruzza et al. show that this regulatory mechanism is important for shaping a diverse microbiota that promote host metabolic homeostasis.

The endometrium is a challenging site for metagenomic analysis due to difficulties in obtaining uncontaminated samples and the limited abundance of the bacterial population. Indeed, solid correlations between endometrial physio-pathologic conditions and bacteria compositions have not yet been firmly established. Nevertheless, the study of the endometrial microbiota is of great interest due to the close correlations between microbiota profiles, women&rsquo, s health, and successful pregnancies. In this study, we decided to tackle the study of the endometrial microbiota through analysis of bacterial population in women subjected to elective caesarean delivery. As a pilot study, a cohort of 19 Caucasian women at full term of normal pregnancy and with a prospection of elective caesarean delivery was enrolled for endometrium sampling at the time of caesarean section. Sampling was carried out by endometrial biopsy soon after the delivery of the newborn and the discharge of the placenta and fetal membranes from the uterus. Bacterial composition was established by a deep metabarcoding next generation sequencing (NGS) procedure addressing the V5&ndash, V6 hypervariable region of the 16S rRNA gene. Amplicon sequences were analysed by bioinformatic procedures for denoising and taxonomic classification. The RDP database was used as 16S rRNA reference collection. Metabarcoding analysis showed the presence of a common bacterial composition, including six genera classifiable within the human microbiota (Cutibacterium, Escherichia, Staphylococcus, Acinetobacter, Streptococcus, Corynebacterium), that could be part of the core endometrial microbiota under the specific conditions examined. These results can provide useful information for future studies on the correlations between bacteria and successful pregnancies.

The possible existence of yet undiscovered human tumorigenic viruses is still under scrutiny. The development of large-scale sequencing technologies, coupled with bioinformatics techniques for the characterization of metagenomic sequences, have provided an invaluable tool for the detection of unknown, infectious, tumorigenic agents, as demonstrated by several recent studies. However, discoveries of novel viruses possibly associated with tumorigenesis are scarce at best. Here, we apply a rigorous bioinformatics workflow to investigate in depth tumor metagenomes from a small but carefully selected cohort of immunosuppressed patients. While a variegated bacterial microbiome was associated with each tumor, no evidence of the presence of putative oncoviruses was found. These results are consistent with the major findings of several recent papers and suggest that new human tumorigenic viruses are not common even in immunosuppressed populations.

Jellyfish represent an important component of marine food webs characterized by large fluctuations of population density, with the ability to abruptly form outbreaks, followed by rarity periods. In spite of considerable efforts to investigate how jellyfish populations are responding globally to anthropogenic change, available evidence still remains unclear. In the last 50 years, jellyfish arc seemingly on the rise in a number of coastal areas, including the Mediterranean Sea, where jellyfish blooms periodically become an issue to marine and maritime human activities. Their impacts on marine organism welfare have been poorly quantified. The jellyfish, Rhizostoma pulmo, is an outbreak-forming scyphomedusa whose large populations spread across the Mediterranean, with increasing periodicity and variable abundance. Studies on cnidarian jellyfish suggested being important vectors of bacterial pathogens. In the present study, by combination of conventional culture-based methods and a high-throughput amplicon sequencing (HTS) approach, we characterized the diversity of the bacterial community associated with this jellyfish during their summer outbreak. Three distinct jellyfish compartments, namely umbrella, oral arms, and the mucus secretion obtained from whole specimens were screened for specifically associated microbiota. A total of 17 phyla, 30 classes, 73 orders, 146 families and 329 genera of microbial organisms were represented in R. pulmo samples with three major lades (ie. Spiroplasma, Mycoplasma and Wolinella) representing over 90% of the retrieved total sequences. The taxonomic microbial inventory was then combined with metabolic profiling data obtained from the Biolog Eco-Plate system. Significant differences among the jellyfish compartments were detected in terms of bacterial abundance, diversity and metabolic utilization of 31 different carbon sources with the highest value of abundance and metabolic potential in the mucus secretion compared to the umbrella and oral arms. Results are discussed in the framework of the species ecology as well as the potential health hazard for marine organisms and humans. (C) 2019 Elsevier B.V. All rights reserved.

The classification of reads from a metagenomic sample using a reference taxonomy is usually based on first mapping the reads to the reference sequences and then classifying each read at a node under the lowest common ancestor of the candidate sequences in the reference taxonomy with the least classification error. However, this taxonomic annotation can be biased by an imbalanced taxonomy and also by the presence of multiple nodes in the taxonomy with the least classification error for a given read. In this article, we show that the Rand index is a better indicator of classification error than the often used area under thereceiver operating characteristic (ROC) curve andF-measure for both balanced and imbalanced reference taxonomies, and we also address the second source of bias by reducing the taxonomic annotation problem for a whole metagenomic sample to a set cover problem, for which a logarithmic approximation can be obtained in linear time and an exact solution can be obtained by integer linear programming. Experimental results with a proof-of-concept implementation of the set cover approach to taxonomic annotation in a next release of the TANGO software show that the set cover approach further reduces ambiguity in the taxonomic annotation obtained with TANGO without distorting the relative abundance profile of the metagenomic sample.

Lactobacillus rossiae is an obligately hetero-fermentative lactic acid bacterium, which can be isolated from a broad range of environments including sourdoughs, vegetables, fermented meat and flour, as well as the gastrointestinal tract of both humans and animals. In order to unravel distinctive genomic features of this particular species and investigate the phylogenetic positioning within the genus Lactobacillus, comparative genomics and phylogenomic approaches, followed by functional analyses were performed on L. rossiae DSM 15814(T), showing how this type strain not only occupies an independent phylogenetic branch, but also possesses genomic features underscoring its biotechnological potential. This strain in fact represents one of a small number of bacteria known to encode a complete de novo biosynthetic pathway of vitamin B-12 (in addition to other B vitamins such as folate and riboflavin). In addition, it possesses the capacity to utilize an extensive set of carbon sources, a characteristic that may contribute to environmental adaptation, perhaps enabling the strain's ability to populate different niches.

Summary Shotgun metagenomics by high-throughput sequencing may allow deep and accurate characterization of host-associated total microbiomes, including bacteria, viruses, protists and fungi. However, the analysis of such sequencing data is still extremely challenging in terms of both overall accuracy and computational efficiency, and current methodologies show substantial variability in misclassification rate and resolution at lower taxonomic ranks or are limited to specific life domains (e.g. only bacteria). We present here MetaShot, a workflow for assessing the total microbiome composition from host-associated shotgun sequence data, and show its overall optimal accuracy performance by analyzing both simulated and real datasets. Availability and Implementation https://github.com/bfosso/MetaShot Supplementary information Supplementary data are available at Bioinformatics online.

Currently, there is very little information available regarding the microbiome associated with the wine production chain. Here, we used an amplicon sequencing approach based on high-throughput sequencing (HTS) to obtain a comprehensive assessment of the bacterial community associated with the production of three Apulian red wines, from grape to final product. The relationships among grape variety, the microbial community, and fermentation was investigated. Moreover, the winery microbiota was evaluated compared to the autochthonous species in vineyards that persist until the end of the winemaking process. The analysis highlighted the remarkable dynamics within the microbial communities during fermentation. A common microbial core shared among the examined wine varieties was observed, and the unique taxonomic signature of each wine appellation was revealed. New species belonging to the genus Halomonas were also reported. This study demonstrates the potential of this metagenomic approach, supported by optimized protocols, for identifying the biodiversity of the wine supply chain. The developed experimental pipeline offers new prospects for other research fields in which a comprehensive view of microbial community complexity and dynamics is desirable.