A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

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.

Resources and tools for the high-throughput, multi-omic study of intestinal microbiota.

The human gut microbiome impacts several aspects of human health and disease, including digestion, drug metabolism and the propensity to develop various inflammatory, autoimmune and metabolic diseases. Many of the molecular processes that play a role in the activity and dynamics of the microbiota go beyond species and genic composition and thus, their understanding requires advanced bioinformatics support. This article aims to provide an up-to-date view of the resources and software tools that are being developed and used in human gut microbiome research, in particular data integration and systems-level analysis efforts. These efforts demonstrate the power of standardized and reproducible computational workflows for integrating and analysing varied omics data and gaining deeper insights into microbe community structure and function as well as host-microbe interactions.

Computational prediction of the bioactivity potential of proteomes based on expert knowledge.

Advances in the field of genome sequencing have enabled a comprehensive analysis and annotation of the dynamics of the protein inventory of cells. This has been proven particularly rewarding for microbial cells, for which the majority of proteins are already accessible to analysis through automatic metagenome annotation. The large-scale in silico screening of proteomes and metaproteomes is key to uncover bioactivities of translational, clinical and biotechnological interest, and to help assign functions to certain proteins, such as those predicted as hypothetical. This work introduces a new method for the prediction of the bioactivity potential of proteomes/metaproteomes, supporting the discovery of functionally relevant proteins based on prior knowledge. This methodology complements functional annotation enrichment methods by allowing the assignment of functions to proteins annotated as hypothetical/putative/uncharacterised, as well as and enabling the detection of specific bioactivities and the recovery of proteins from defined taxa. This work shows how the new method can be applied to screen proteome and metaproteome sets to obtain predictions of clinical or biotechnological interest based on reference datasets. Notably, with this methodology, the large information files obtained after DNA sequencing or protein identification experiments can be associated for translational purposes that, in cases such as antibiotic-resistance pathogens or foodborne diseases, may represent changes in how these important and global health burdens are approached in the clinical practice. Finally, the Sequence-based Expert-driven pRoteome bioactivity Prediction EnvironmENT, a public Web service implemented in Scala functional programming style, is introduced as means to ensure broad access to the method as well as to discuss main implementation issues, such as modularity, extensibility and interoperability.

P4P: a peptidome-based strain-level genome comparison web tool.

Peptidome similarity analysis enables researchers to gain insights into differential peptide profiles, providing a robust tool to discriminate strain-specific peptides, true intra-species differences among biological replicates or even microorganism-phenotype variations. However, no in silico peptide fingerprinting software existed to facilitate such phylogeny inference. Hence, we developed the Peptidomes for Phylogenies (P4P) web tool, which enables the survey of similarities between microbial proteomes and simplifies the process of obtaining new biological insights into their phylogeny. P4P can be used to analyze different peptide datasets, i.e. bacteria, viruses, eukaryotic species or even metaproteomes. Also, it is able to work with whole proteome datasets and experimental mass-to-charge lists originated from mass spectrometers. The ultimate aim is to generate a valid and manageable list of peptides that have phylogenetic signal and are potentially sample-specific. Sample-to-sample comparison is based on a consensus peak set matrix, which can be further submitted to phylogenetic analysis. P4P holds great potential for improving phylogenetic analyses in challenging taxonomic groups, biomarker identification or epidemiologic studies. Notably, P4P can be of interest for applications handling large proteomic datasets, which it is able to reduce to small matrices while maintaining high phylogenetic resolution. The web server is available at http://sing-group.org/p4p.

Improving Phylogeny Reconstruction at the Strain Level Using Peptidome Datasets.

Typical bacterial strain differentiation methods are often challenged by high genetic similarity between strains. To address this problem, we introduce a novel in silico peptide fingerprinting method based on conventional wet-lab protocols that enables the identification of potential strain-specific peptides. These can be further investigated using in vitro approaches, laying a foundation for the development of biomarker detection and application-specific methods. This novel method aims at reducing large amounts of comparative peptide data to binary matrices while maintaining a high phylogenetic resolution. The underlying case study concerns the Bacillus cereus group, namely the differentiation of Bacillus thuringiensis, Bacillus anthracis and Bacillus cereus strains. Results show that trees based on cytoplasmic and extracellular peptidomes are only marginally in conflict with those based on whole proteomes, as inferred by the established Genome-BLAST Distance Phylogeny (GBDP) method. Hence, these results indicate that the two approaches can most likely be used complementarily even in other organismal groups. The obtained results confirm previous reports about the misclassification of many strains within the B. cereus group. Moreover, our method was able to separate the B. anthracis strains with high resolution, similarly to the GBDP results as benchmarked via Bayesian inference and both Maximum Likelihood and Maximum Parsimony. In addition to the presented phylogenomic applications, whole-peptide fingerprinting might also become a valuable complementary technique to digital DNA-DNA hybridization, notably for bacterial classification at the species and subspecies level in the future.

A peptidome-based phylogeny pipeline reveals differential peptides at the strain level within Bifidobacterium animalis subsp. lactis.

Bifidobacteria are gut commensal microorganisms belonging to the Actinobacteria group. Some specific strains of Bifidobacterium animalis subsp. lactis are used in functional foods as they are able to exert health-promoting effects in the human host. Due to the limited genetic variability within this subspecies, it is sometimes difficult for a manufacturer to properly track its strain once included in dairy products or functional foods. In this paper, we present a peptidome-based analysis in which the proteomes of a set of B. animalis subsp. lactis strains were digested in silico with human gut endopeptidases. The molecular masses were compared along all the strains to detect strain-specific peptides. These peptides may be interesting towards the development of methodologies for strain identification in the final product.

Favorable subgingival plaque microbiome shifts are associated with clinical treatment for peri-implant diseases.

We performed a longitudinal shotgun metagenomic investigation of the plaque microbiome associated with peri-implant diseases in a cohort of 91 subjects with 320 quality-controlled metagenomes. Through recently improved taxonomic profiling methods, we identified the most discriminative species between healthy and diseased subjects at baseline, evaluated their change over time, and provided evidence that clinical treatment had a positive effect on plaque microbiome composition in patients affected by mucositis and peri-implantitis.

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).

Establishment of a non-Westernized gut microbiota in men who have sex with men is associated with sexual practices.

The human gut microbiota is influenced by various factors, including health status and environmental conditions, yet considerable inter-individual differences remain unexplained. Previous studies identified that the gut microbiota of men who have sex with men (MSM) is distinct from that of non-MSM. Here, we reveal through species-level microbiota analysis using shotgun metagenomics that the gut microbiota of many MSM with Western origin resembles gut microbial communities of non-Westernized populations. Specifically, MSM gut microbiomes are frequently dominated by members of the Prevotellaceae family, including co-colonization of species from the Segatella copri complex and unknown Prevotellaceae members. Questionnaire-based analysis exploring inter-individual differences in MSM links specific sexual practices to microbiota composition. Moreover, machine learning identifies microbial features associated with sexual activities in MSM. Together, this study shows associations of sexual activities with gut microbiome alterations in MSM, which may have a large impact on population-based microbiota studies.

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.

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.

Towards the isolation of more robust next generation probiotics: The first aerotolerant Bifidobacterium bifidum strain.

This work reports on the first described aerotolerant Bifidobacterium bifidum strain, Bifidobacterium bifidum IPLA60003, which has the ability to form colonies on the surface of agar plates under aerobic conditions, a weird phenotype that to our knowledge has never been observed in B. bifidum. The strain IPLA60003 was generated after random UV mutagenesis from an intestinal isolate. It incorporates 26 single nucleotide polymorphisms that activate the expression of native oxidative-defense mechanisms such as the alkyl hydroxyperoxide reductase, the glycolytic pathway and several genes coding for enzymes involved in redox reactions. In the present work, we discuss the molecular mechanisms underlying the aerotolerance phenotype of B. bifidum IPLA60003, which will open new strategies for the selection and inclusion of probiotic gut strains and next generation probiotics into functional foods.

Targeted depletion of pks+ bacteria from a fecal microbiota using specific antibodies.

The pks island is one of the most prevalent pathogenicity islands among the Escherichia coli strains that colonize the colon of colorectal carcinoma (CRC) patients. This pathogenic island encodes the production of a nonribosomal polyketide-peptide named colibactin, which induces double-strand breaks in DNA molecules. Detection or even depletion of this pks-producing bacteria could help to understand the role of these strains in the context of CRC. In this work, we performed a large-scale in silico screening of the pks cluster in more than 6,000 isolates of E. coli. The results obtained reveal that not all the pks-detected strains could produce a functional genotoxin and, using antibodies against pks-specific peptides from surface cell proteins, a methodology for detection and depletion of pks+ bacteria in gut microbiotas was proposed. With our method, we were able to deplete a human gut microbiota of this pks+ strains, opening the door to strain-directed microbiota modification and intervention studies that allow us to understand the relation between these genotoxic strains and some gastrointestinal diseases. IMPORTANCE The human gut microbiome has also been hypothesized to play a crucial role in the development and progression of colorectal carcinoma (CRC). Between the microorganisms of this community, the Escherichia coli strains carrying the pks genomic island were shown to be capable of promoting colon tumorigenesis in a colorectal cancer mouse model, and their presence seems to be directly related to a distinct mutational signature in patients suffering CRC. This work proposes a novel method for the detection and depletion of pks-carrying bacteria in human gut microbiotas. In contrast to methods based on probes, this methodology allows the depletion of low-abundance bacterial strains maintaining the viability of both targeted and non-targeted fractions of the microbiota, allowing the study of the contribution of these pks-carrying strains to different diseases, such as CRC, and their role in other physiological, metabolic or immune processes.

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.

Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4.

Metagenomic assembly enables new organism discovery from microbial communities, but it can only capture few abundant organisms from most metagenomes. Here we present MetaPhlAn 4, which integrates information from metagenome assemblies and microbial isolate genomes for more comprehensive metagenomic taxonomic profiling. From a curated collection of 1.01 M prokaryotic reference and metagenome-assembled genomes, we define unique marker genes for 26,970 species-level genome bins, 4,992 of them taxonomically unidentified at the species level. MetaPhlAn 4 explains ~20% more reads in most international human gut microbiomes and >40% in less-characterized environments such as the rumen microbiome and proves more accurate than available alternatives on synthetic evaluations while also reliably quantifying organisms with no cultured isolates. Application of the method to >24,500 metagenomes highlights previously undetected species to be strong biomarkers for host conditions and lifestyles in human and mouse microbiomes and shows that even previously uncharacterized species can be genetically profiled at the resolution of single microbial strains.

MetaPhlAn 4 profiling of unknown species-level genome bins improves the characterization of diet-associated microbiome changes in mice.

Mouse models are key tools for investigating host-microbiome interactions. However, shotgun metagenomics can only profile a limited fraction of the mouse gut microbiome. Here, we employ a metagenomic profiling method, MetaPhlAn 4, which exploits a large catalog of metagenome-assembled genomes (including 22,718 metagenome-assembled genomes from mice) to improve the profiling of the mouse gut microbiome. We combine 622 samples from eight public datasets and an additional cohort of 97 mouse microbiomes, and we assess the potential of MetaPhlAn 4 to better identify diet-related changes in the host microbiome using a meta-analysis approach. We find multiple, strong, and reproducible diet-related microbial biomarkers, largely increasing those identifiable by other available methods relying only on reference information. The strongest drivers of the diet-induced changes are uncharacterized and previously undetected taxa, confirming the importance of adopting metagenomic methods integrating metagenomic assemblies for comprehensive profiling.

Extension of the Segatella copri complex to 13 species with distinct large extrachromosomal elements and associations with host conditions.

The Segatella copri (formerly Prevotella copri) complex (ScC) comprises taxa that are key members of the human gut microbiome. It was previously described to contain four distinct phylogenetic clades. Combining targeted isolation with large-scale metagenomic analysis, we defined 13 distinct Segatella copri-related species, expanding the ScC complex beyond four clades. Complete genome reconstruction of thirteen strains from seven species unveiled the presence of genetically diverse large circular extrachromosomal elements. These elements are consistently present in most ScC species, contributing to intra- and inter-species diversities. The nine species-level clades present in humans display striking differences in prevalence and intra-species genetic makeup across human populations. Based on a meta-analysis, we found reproducible associations between members of ScC and the male sex and positive correlations with lower visceral fat and favorable markers of cardiometabolic health. Our work uncovers genomic diversity within ScC, facilitating a better characterization of the human microbiome.