Unexplored microbial diversity from 2,500 food metagenomes and links with the human microbiome.

Complex microbiomes are part of the food we eat and influence our own microbiome, but their diversity remains largely unexplored. Here, we generated the open access curatedFoodMetagenomicData (cFMD) resource by integrating 1,950 newly sequenced and 583 public food metagenomes. We produced 10,899 metagenome-assembled genomes spanning 1,036 prokaryotic and 108 eukaryotic species-level genome bins (SGBs), including 320 previously undescribed taxa. Food SGBs displayed significant microbial diversity within and between food categories. Extension to >20,000 human metagenomes revealed that food SGBs accounted on average for 3% of the adult gut microbiome. Strain-level analysis highlighted potential instances of food-to-gut transmission and intestinal colonization (e.g., Lacticaseibacillus paracasei) as well as SGBs with divergent genomic structures in food and humans (e.g., Streptococcus gallolyticus and Limosilactobabillus mucosae). The cFMD expands our knowledge on food microbiomes, their role in shaping the human microbiome, and supports future uses of metagenomics for food quality, safety, and authentication.

The person-to-person transmission landscape of the gut and oral microbiomes.

The human microbiome is an integral component of the human body and a co-determinant of several health conditions1,2. However, the extent to which interpersonal relations shape the individual genetic makeup of the microbiome and its transmission within and across populations remains largely unknown3,4. Here, capitalizing on more than 9,700 human metagenomes and computational strain-level profiling, we detected extensive bacterial strain sharing across individuals (more than 10 million instances) with distinct mother-to-infant, intra-household and intra-population transmission patterns. Mother-to-infant gut microbiome transmission was considerable and stable during infancy (around 50% of the same strains among shared species (strain-sharing rate)) and remained detectable at older ages. By contrast, the transmission of the oral microbiome occurred largely horizontally and was enhanced by the duration of cohabitation. There was substantial strain sharing among cohabiting individuals, with 12% and 32% median strain-sharing rates for the gut and oral microbiomes, and time since cohabitation affected strain sharing more than age or genetics did. Bacterial strain sharing additionally recapitulated host population structures better than species-level profiles did. Finally, distinct taxa appeared as efficient spreaders across transmission modes and were associated with different predicted bacterial phenotypes linked with out-of-host survival capabilities. The extent of microorganism transmission that we describe underscores its relevance in human microbiome studies5, especially those on non-infectious, microbiome-associated diseases.

Are Immunohistochemical Markers Useful in Phenotypic Gastric Cancer Classification?

To identify useful markers for prognostic and therapeutic purposes, The Cancer Genome Atlas (TCGA) provided a molecular classification of gastric cancers (GCs). Previous studies have used immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) to define immunophenotypic surrogate markers of the molecular alterations. Some critical issues concerning the correct definition of immunophenotypic groups have emerged in these studies that employed tissue microarrays (TMAs). We performed an immunophenotypic classification by evaluating MLH1, p53, HER2, E-cadherin, and Epstein-Barr virus (EBV) on the whole section of the surgical GC samples compared to most of the studies conducted on TMAs. We also investigated the immunohistochemical expression of PD-L1, a known therapeutic target. We identified the following immunophenotypic groups: EBV (2.9%); mismatch repair deficient (MMR-D) (7.2%); overexpressed p53 and/or HER2+ (61.4%); aberrant E-cadherin (11.4%); and normal pattern (17.1%). The use of surgical samples emphasized that some immunohistochemical markers were not useful for properly classifying the GC specimens. We can state that EBV (significantly correlated to PD-L1 expression) and MMR-D GCs are well-defined groups, mutually exclusive, and easily assessable with IHC and CISH, and could be candidates for immunotherapy with PD-1/PD-L1 inhibitors. As regards p53, our findings suggest that IHC assessment may be responsible for a misclassification of GC groups. Immunohistochemical evaluation of E-cadherin needs to be standardized, particularly in terms of the heterogeneous cytoplasmic/membranous staining pattern. Whether to consider the normal-pattern group as a separate category remains to be clarified. Because GC specimens with known therapeutic targets account for only 40%, we suggest reviewing the immunophenotypic classification to find new therapeutic targets, such as PD-L1, MLH1, and HER2.

Proteomics analysis of human serum of patients with non-small-cell lung cancer reveals proteins as diagnostic biomarker candidates.

Non-small-cell lung carcinomas (NSCLC) is the most common type of lung cancer and it has a poor prognosis, because overall survival after 5 years is 20-25% for all stages. Thus, it is extremely important to increase the survival rate in the early stages NSCLC by focusing on novel screening tests of cancer identifying specific biomarkers expression associated with a more accurate tumor staging and patient prognosis. In this study, we focused our attention on quantitative proteomics of three heavily glycosylated serum proteins: AMBP, α2 macroglobulin, and SERPINA1. In particular, we analyzed serum samples from 20 NSCLC lung adenocarcinoma cancer patients in early and advanced stages, and 10 healthy donors to obtain a relative quantification through the MRM analysis of these proteins that have shown to be markers of cancer development and progression. AMBP, α2 macroglobulin, and SERPINA1 were chosen because all of them possess endopeptidase inhibitor activity and play key roles in cancer. We observe a variation in the expression of these proteins linked to the stage of the disease. Therefore, we believe that proteins like α2 macroglobulin, αmicroglobulin/bikunin, and SERPINA1 could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.

The enigmatic role of matrix metalloproteinases in epithelial-to-mesenchymal transition of oral squamous cell carcinoma: Implications and nutraceutical aspects.

The most prevalent malignancy in the oral cavity is represented by oral squamous cell carcinoma, an aggressive disease mostly detected in low-income communities. This neoplasia is mostly diffused in older men particularly exposed to risk factors such as tobacco, alcohol, and a diet rich in fatty foods and poor in vegetables. In oral squamous cell carcinoma, a wide range of matrix-cleaving proteinases are involved in extracellular matrix remodeling of cancer microenvironment. In particular, matrix metalloproteinases (MMPs) represent the major and most investigated protagonists. Owing to their strong involvement in malignant pathologies, MMPs are considered the most promising new biomarkers in cancer diagnosis and prognosis. The interest in studying MMPs in oral cancer biology is also owing to their prominent role in epithelial-to-mesenchymal transition (EMT). EMT is an intricate process involving different complex pathways. EMT-related proteins are attractive diagnostic biomarkers that characterize the activation of biological events that promote cancer's aggressive expansion. Different antioncogenic natural compounds have been investigated to counteract oral carcinogenesis, with the scope of obtaining better clinical results and lower morbidity. In particular, we describe the role of different nutraceuticals used for the regulation of MMP-related invasion and proliferation of oral cancer cells.

Isolation, characterization and analysis of pro-inflammatory potential of Klebsiella pneumoniae outer membrane vesicles.

Outer membrane vesicles (OMVs) are potent virulence factors, naturally secreted by gram-negative bacteria. Since Klebsiella pneumoniae has emerged as an important nosocomial pathogen, because of resistance to a wide spectrum of antibiotics, it is crucial to investigate its pathogenetic mechanism microorganism secretes outer membrane vesicles (OMVs), but the pathogenesis of Klebsiella pneumoniae as it relates to OMVs has not been well elucidated. In this study we focused on the isolation, characterization and evaluation of the virulence potential of OMVs obtained from Klebsiella pneumoniae. Our data demonstrate that Klebsiella pneumoniae OMVs are important secretory nanocomplexes that elicit a potent inflammatory response. Since OMVs are clearly involved in the pathogenesis of this bacterium during infection, further studies are required to determine whether they could be future targets for novel therapy and potential vaccine against Klebsiella pneumoniae.

Draft genome sequences of multiple bacterial strains isolated from human feces.

Bacterial isolation is necessary for functional and mechanistic analyses, and the increased human microbiome diversity revealed by metagenomic sequencing is expanding the relevant cultivation targets. Here, we report 46 draft genome sequences of bacterial isolates obtained from fecal samples of healthy adults in Trento and Milan (Italy), including strains from seven taxonomically uncharacterized species.

In-depth characterization of food and environmental microbiomes across different meat processing plants.

BACKGROUND: Processing environments can be an important source of pathogenic and spoilage microorganisms that cross contaminate meat and meat products. The aim of this study was to characterize the microbiome of raw materials, processing environments and end products from 19 facilities producing different meat products. RESULTS: The taxonomic profiles of the microbial communities evolved along processing, from raw materials to end products, suggesting that food contact (FC) surfaces play an important role in modulating the microbiome of final products. Some species persisted with the highest relative abundance in raw materials, food processing environments and/or in the final product, including species from the genera Pseudomonas, Staphylococcus, Brochothrix, Acinetobacter and Psychrobacter. Processing environments showed a very diverse core microbiota, partially shared with the products. Pseudomonas fragi and Pseudomonas sp. Lz4W (in all sample and facility types) and Brochothrix thermosphacta, Psychrobacter sp. and Psychrobacter sp. P11F6 (in raw materials, FC surfaces and end products) were prominent members of the core microbiota for all facilities, while Latilactobacillus sakei was found as a dominant species exclusively in end products from the facilities producing fermented sausages. Processing environments showed a higher amount of antimicrobial resistance genes and virulence factors than raw materials and end products. One thousand four hundred twenty-one medium/high-quality metagenome-assembled genomes (MAGs) were reconstructed. Of these, 274 high-quality MAGs (completeness > 90%) corresponded to 210 putative new species, mostly found in processing environments. For two relevant taxa in meat curing and fermentation processes (S. equorum and L. sakei, respectively), phylogenetic variation was observed associated with the specific processing facility under study, which suggests that specific strains of these taxa may be selected in different meat processing plants, likely contributing to the peculiar sensorial traits of the end products produced in them. CONCLUSIONS: Overall, our findings provide the most detailed metagenomics-based perspective up to now of the microbes that thrive in meat, meat products and associated environments and open avenues for future research activities to better understand the microbiome functionality and potential contribution to meat quality and safety. Video Abstract.

Neuroblastoma is associated with alterations in gut microbiome composition subsequent to maternal microbial seeding.

BACKGROUND: Neuroblastoma is the most frequent extracranial solid tumour in children, accounting for ∼15% of deaths due to cancer in childhood. The most common clinical presentation are abdominal tumours. An altered gut microbiome composition has been linked to multiple cancer types, and reported in murine models of neuroblastoma. Whether children with neuroblastoma display alterations in gut microbiome composition remains unexplored. METHODS: We assessed gut microbiome composition by shotgun metagenomic profiling in an observational cross-sectional study on 288 individuals, consisting of patients with a diagnosis of neuroblastoma at disease onset (N = 63), healthy controls matching the patients on the main covariates of microbiome composition (N = 94), healthy siblings of the patients (N = 13), mothers of patients (N = 59), and mothers of the controls (N = 59). We examined taxonomic and functional microbiome composition and mother-infant strain transmission patterns. FINDINGS: Patients with neuroblastoma displayed alterations in gut microbiome composition characterised by reduced microbiome richness, decreased relative abundances of 18 species (including Phocaeicola dorei and Bifidobacterium bifidum), enriched protein fermentation and reduced carbohydrate fermentation potential. Using machine learning, we could successfully discriminate patients from controls (AUC = 82%). Healthy siblings did not display such alterations but resembled the healthy control group. No significant differences in maternal microbiome composition nor mother-to-offspring transmission were detected. INTERPRETATION: Patients with neuroblastoma display alterations in taxonomic and functional gut microbiome composition, which cannot be traced to differential maternal seeding. Follow-up research should include investigating potential causal links. FUNDING: Italian Ministry of Health Ricerca Corrente and Ricerca Finalizzata 5 per mille (to MPonzoni); Fondazione Italiana Neuroblastoma (to MPonzoni); European Research Council (ERC-StG project MetaPG-716575 and ERC-CoG microTOUCH-101045015 to NS); the European H2020 program ONCOBIOME-825410 project (to NS); the National Cancer Institute of the National Institutes of Health 1U01CA230551 (to NS); the Premio Internazionale Lombardia e Ricerca 2019 (to NS); the MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN) Bando 2017 Grant 2017J3E2W2 (to NS); EMBO ALTF 593-2020 and Knowledge Generation Project from the Spanish Ministry of Science and Innovation (PID2022-139328OA-I00) (to MV-C).

Inter-facility characterization of bacteria in seafood processing plants: Exploring potential reservoirs of spoilage organisms and the resistome.

A study was conducted in fish processing facilities to investigate the microbial composition, microbial metabolic potential, and distribution of antibiotic resistance genes. Whole metagenomic sequencing was used to analyze microbial communities from different processing rooms, operators and fish products. Taxonomic analyses identified the genera Pseudomonas and Psychrobacter as the most prevalent bacteria. A Principal Component Analysis revealed a distinct separation between fish product and environmental samples, as well as differences between fish product samples from companies processing either Gadidae or Salmonidae fish. Some particular bacterial genera and species were associated with specific processing rooms and operators. Metabolic analysis of metagenome assembled genomes demonstrated variations in microbiota metabolic profiles of microbiota across rooms and fish products. The study also examined the presence of antibiotic-resistance genes in fish processing environments, contributing to the understanding of microbial dynamics, metabolic potential, and implications for fish spoilage.

Discovering and exploring the hidden diversity of human gut viruses using highly enriched virome samples.

Viruses are an abundant and crucial component of the human microbiome, but accurately discovering them via metagenomics is still challenging. Currently, the available viral reference genomes poorly represent the diversity in microbiome samples, and expanding such a set of viral references is difficult. As a result, many viruses are still undetectable through metagenomics even when considering the power of de novo metagenomic assembly and binning, as viruses lack universal markers. Here, we describe a novel approach to catalog new viral members of the human gut microbiome and show how the resulting resource improves metagenomic analyses. We retrieved >3,000 viral-like particles (VLP) enriched metagenomic samples (viromes), evaluated the efficiency of the enrichment in each sample to leverage the viromes of highest purity, and applied multiple analysis steps involving assembly and comparison with hundreds of thousands of metagenome-assembled genomes to discover new viral genomes. We reported over 162,000 viral sequences passing quality control from thousands of gut metagenomes and viromes. The great majority of the retrieved viral sequences (~94.4%) were of unknown origin, most had a CRISPR spacer matching host bacteria, and four of them could be detected in >50% of a set of 18,756 gut metagenomes we surveyed. We included the obtained collection of sequences in a new MetaPhlAn 4.1 release, which can quantify reads within a metagenome matching the known and newly uncovered viral diversity. Additionally, we released the viral database for further virome and metagenomic studies of the human microbiome.

Microbial composition associated with biliary stents in patients undergoing pancreatic resection for cancer.

Malignant bile duct obstruction is typically treated by biliary stenting, which however increases the risk of bacterial infections. Here, we analyzed the microbial content of the biliary stents from 56 patients finding widespread microbial colonization. Seventeen of 36 prevalent stent species are common oral microbiome members, associate with disease conditions when present in the gut, and include dozens of biofilm- and antimicrobial resistance-related genes. This work provides an overview of the microbial communities populating the stents.

Influence of microbiota-associated metabolic reprogramming on clinical outcome in patients with melanoma from the randomized adjuvant dendritic cell-based MIND-DC trial.

Tumor immunosurveillance plays a major role in melanoma, prompting the development of immunotherapy strategies. The gut microbiota composition, influencing peripheral and tumoral immune tonus, earned its credentials among predictors of survival in melanoma. The MIND-DC phase III trial (NCT02993315) randomized (2:1 ratio) 148 patients with stage IIIB/C melanoma to adjuvant treatment with autologous natural dendritic cell (nDC) or placebo (PL). Overall, 144 patients collected serum and stool samples before and after 2 bimonthly injections to perform metabolomics (MB) and metagenomics (MG) as prespecified exploratory analysis. Clinical outcomes are reported separately. Here we show that different microbes were associated with prognosis, with the health-related Faecalibacterium prausnitzii standing out as the main beneficial taxon for no recurrence at 2 years (p = 0.008 at baseline, nDC arm). Therapy coincided with major MB perturbations (acylcarnitines, carboxylic and fatty acids). Despite randomization, nDC arm exhibited MG and MB bias at baseline: relative under-representation of F. prausnitzii, and perturbations of primary biliary acids (BA). F. prausnitzii anticorrelated with BA, medium- and long-chain acylcarnitines. Combined, these MG and MB biomarkers markedly determined prognosis. Altogether, the host-microbial interaction may play a role in localized melanoma. We value systematic MG and MB profiling in randomized trials to avoid baseline differences attributed to host-microbe interactions.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Longitudinal gut microbiome changes in immune checkpoint blockade-treated advanced melanoma.

Multiple clinical trials targeting the gut microbiome are being conducted to optimize treatment outcomes for immune checkpoint blockade (ICB). To improve the success of these interventions, understanding gut microbiome changes during ICB is urgently needed. Here through longitudinal microbiome profiling of 175 patients treated with ICB for advanced melanoma, we show that several microbial species-level genome bins (SGBs) and pathways exhibit distinct patterns from baseline in patients achieving progression-free survival (PFS) of 12 months or longer (PFS ≥12) versus patients with PFS shorter than 12 months (PFS <12). Out of 99 SGBs that could discriminate between these two groups, 20 were differentially abundant only at baseline, while 42 were differentially abundant only after treatment initiation. We identify five and four SGBs that had consistently higher abundances in patients with PFS ≥12 and <12 months, respectively. Constructing a log ratio of these SGBs, we find an association with overall survival. Finally, we find different microbial dynamics in different clinical contexts including the type of ICB regimen, development of immune-related adverse events and concomitant medication use. Insights into the longitudinal dynamics of the gut microbiome in association with host factors and treatment regimens will be critical for guiding rational microbiome-targeted therapies aimed at enhancing ICB efficacy.

Draft genome sequence of the anatoxin-a producing cyanobacterium Tychonema bourrellyi B0820 isolated from the epilimnion of the deep Alpine Lake Garda.

We report the draft genome sequence of strain B0820 of the cyanobacterium Tychonema bourrellyi isolated from the epilimnion of Lake Garda and assembled from a metagenome of a non-axenic culture. The strain analyzed was shown to produce anatoxin-a, a potent neurotoxin that can cause fatal intoxication in exposed organisms.

Draft genome sequence of a representative strain of the Catenibacterium genus isolated from human feces.

A strain from a previously undescribed species belonging to the Catenibacterium genus was isolated from the stool of a healthy volunteer. The strain is strictly anaerobic, and the genome encodes a CRISPR-Cas system and genes related to trimethylamine production.

Maternal and food microbial sources shape the infant microbiome of a rural Ethiopian population.

The human microbiome seeding starts at birth, when pioneer microbes are acquired mainly from the mother. Mode of delivery, antibiotic prophylaxis, and feeding method have been studied as modulators of mother-to-infant microbiome transmission, but other key influencing factors like modern westernized lifestyles with high hygienization, high-calorie diets, and urban settings, compared with non-westernized lifestyles have not been investigated yet. In this study, we explored the mother-infant sharing of characterized and uncharacterized microbiome members via strain-resolved metagenomics in a cohort of Ethiopian mothers and infants, and we compared them with four other cohorts with different lifestyles. The westernized and non-westernized newborns' microbiomes composition overlapped during the first months of life more than later in life, likely reflecting similar initial breast-milk-based diets. Ethiopian and other non-westernized infants shared a smaller fraction of the microbiome with their mothers than did most westernized populations, despite showing a higher microbiome diversity, and uncharacterized species represented a substantial fraction of those shared in the Ethiopian cohort. Moreover, we identified uncharacterized species belonging to the Selenomonadaceae and Prevotellaceae families specifically present and shared only in the Ethiopian cohort, and we showed that a locally produced fermented food, injera, can contribute to the higher diversity observed in the Ethiopian infants' gut with bacteria that are not part of the human microbiome but are acquired through fermented food consumption. Taken together, these findings highlight the fact that lifestyle can impact the gut microbiome composition not only through differences in diet, drug consumption, and environmental factors but also through its effect on mother-infant strain-sharing patterns.

Fecal microbiota transplantation plus anti-PD-1 immunotherapy in advanced melanoma: a phase I trial.

Fecal microbiota transplantation (FMT) represents a potential strategy to overcome resistance to immune checkpoint inhibitors in patients with refractory melanoma; however, the role of FMT in first-line treatment settings has not been evaluated. We conducted a multicenter phase I trial combining healthy donor FMT with the PD-1 inhibitors nivolumab or pembrolizumab in 20 previously untreated patients with advanced melanoma. The primary end point was safety. No grade 3 adverse events were reported from FMT alone. Five patients (25%) experienced grade 3 immune-related adverse events from combination therapy. Key secondary end points were objective response rate, changes in gut microbiome composition and systemic immune and metabolomics analyses. The objective response rate was 65% (13 of 20), including four (20%) complete responses. Longitudinal microbiome profiling revealed that all patients engrafted strains from their respective donors; however, the acquired similarity between donor and patient microbiomes only increased over time in responders. Responders experienced an enrichment of immunogenic and a loss of deleterious bacteria following FMT. Avatar mouse models confirmed the role of healthy donor feces in increasing anti-PD-1 efficacy. Our results show that FMT from healthy donors is safe in the first-line setting and warrants further investigation in combination with immune checkpoint inhibitors. ClinicalTrials.gov identifier NCT03772899 .

Variability of strain engraftment and predictability of microbiome composition after fecal microbiota transplantation across different diseases.

Fecal microbiota transplantation (FMT) is highly effective against recurrent Clostridioides difficile infection and is considered a promising treatment for other microbiome-related disorders, but a comprehensive understanding of microbial engraftment dynamics is lacking, which prevents informed applications of this therapeutic approach. Here, we performed an integrated shotgun metagenomic systematic meta-analysis of new and publicly available stool microbiomes collected from 226 triads of donors, pre-FMT recipients and post-FMT recipients across eight different disease types. By leveraging improved metagenomic strain-profiling to infer strain sharing, we found that recipients with higher donor strain engraftment were more likely to experience clinical success after FMT (P = 0.017) when evaluated across studies. Considering all cohorts, increased engraftment was noted in individuals receiving FMT from multiple routes (for example, both via capsules and colonoscopy during the same treatment) as well as in antibiotic-treated recipients with infectious diseases compared with antibiotic-naïve patients with noncommunicable diseases. Bacteroidetes and Actinobacteria species (including Bifidobacteria) displayed higher engraftment than Firmicutes except for six under-characterized Firmicutes species. Cross-dataset machine learning predicted the presence or absence of species in the post-FMT recipient at 0.77 average AUROC in leave-one-dataset-out evaluation, and highlighted the relevance of microbial abundance, prevalence and taxonomy to infer post-FMT species presence. By exploring the dynamics of microbiome engraftment after FMT and their association with clinical variables, our study uncovered species-specific engraftment patterns and presented machine learning models able to predict donors that might optimize post-FMT specific microbiome characteristics for disease-targeted FMT protocols.