Cancers doi: 10.20517/mrr.2025.69

Authors: Anastasia V. Poznyak,Aleksey A. Vatlin,Vsevolod V. Pavshintsev,Nikita A. Mitkin,Olga N. Maltseva,Aleksandra S. Utkina,Alexander N. Orekhov

Rheumatoid arthritis (RA) is a chronic autoimmune disease preceded by a prolonged preclinical phase marked by the emergence of autoantibodies and mucosal immune dysregulation. Evidence from human studies and animal models consistently demonstrates that gut microbiota dysbiosis contributes to this transition, particularly through impaired intestinal barrier function, activation of pro-inflammatory pathways, and molecular mimicry. Specific taxa - including Prevotella copri, Collinsella aerofaciens, and reductions in butyrate-producing bacteria - have been linked to heightened systemic inflammation, increased T helper 17 responses, and the generation of RA-associated autoantibodies. Current research also indicates that anti-rheumatic medications such as methotrexate, sulfasalazine, and minocycline produce measurable shifts in gut microbial composition, suggesting that microbiota-drug interactions may influence treatment response. Therapeutic approaches aimed at modifying gut ecology - including dietary interventions, prebiotics, probiotics, and fecal microbiota transplantation - show early potential in restoring microbial balance, improving intestinal barrier integrity, and reducing inflammatory markers, although evidence in the preclinical RA stage remains limited. Additionally, emerging data highlight the importance of intestinal autophagy and microRNA networks in regulating epithelial integrity and systemic immune activation. Taken together, the literature supports a mechanistic link between gut dysbiosis and the onset of RA. It points to microbiota-targeted strategies as promising avenues for delaying or preventing disease progression. Future studies should prioritize longitudinal analyses and interventional trials focusing specifically on individuals at risk for RA.

Cancers doi: 10.20517/mrr.2025.96

Authors: Simen Hyll Hansen,Niharika Bhattacharjee,Chang Hu,Maria Gjerstad Maseng,Olle Grannö,Corinna Bang,Christine Olbjørn,Gøri Perminow,Jørgen Valeur,May-Bente Bengtson,Svein Oskar Frigstad,Svend Andersen,Tone Bergene Aabrekk,Trond Espen Detlie,Andre Franke,Vendel A. Kristensen,Jonas Halfvarson,Marte Lie Høivik,Ravishankar K. Iyer,Johannes Hov

Background: Inflammatory bowel diseases (IBDs) are complex conditions marked by chronic inflammation in the gastrointestinal tract. Traditional classification separates IBD into Crohn’s disease and ulcerative colitis, but this division may not fully capture disease heterogeneity. Here, we examine whether microbiome-driven subtyping can describe novel clinical IBD phenotypes. To achieve this, we applied unsupervised clustering to fecal microbiota profiles from the population-based Inflammatory Bowel Disease in South-Eastern Norway III (IBSEN III) cohort.

Methods: A Gaussian Mixture Model (GMM) was used to cluster participants with IBD based on microbiome composition and examine associations between clusters and clinical outcomes, including inflammatory markers and disease severity during the first year after inclusion.

Results: Three microbiome-based clusters were identified: CLO (dominated by Clostridia UCG-014), ALF (Agathobacter, Lachnoclostridium, and Faecalibacterium), and RUM (Ruminococcus gnavus). Participants in the RUM cluster had a higher risk of future severe disease than those in the CLO cluster, even among participants with remission-to-mild disease at inclusion (21% vs. 6%, P < 0.00001). This association could not be explained by antibiotic use or baseline disease severity. Cluster membership alone performed comparably to fecal calprotectin in distinguishing severe disease, and a combined model significantly improved accuracy (P < 0.0001).

Conclusion: Our findings demonstrate a connection between microbiome composition and the risk of severe disease development, which is partly independent of inflammation levels at the time of sampling. Microbiome-informed subgrouping could lead to more personalized treatment strategies. Further validation is needed to determine the clinical utility of these clusters.

Cancers doi: 10.20517/mrr.2025.100

Authors: Jung Yeol Sung,Hyunjun Choi,Alexa R. Gormley,Sung Woo Kim

The objective was to quantify the absolute abundance of the jejunal mucosa-associated microbiota in pigs and develop a standardized spike-in control protocol. The spike-in control, containing bacteria that are Gram-negative (Imtechella halotolerans) and Gram-positive (Allobacillus halotolerans), was diluted 100 times and 10 μL (100,000 bacterial cells of each species) was added to 100 mg of porcine jejunal mucosa samples. The relative abundance of the jejunal mucosa-associated microbiota differed between the diet groups, whereas no difference was observed in their absolute abundance. In conclusion, the spike-in control containing 100,000 cells of Imtechella halotolerans and Allobacillus halotolerans can be used to quantify the absolute abundance of microbiota in 100 mg of pig jejunal mucosa.

Cancers doi: 10.20517/mrr.2025.119

Authors: Gabriele Andrea Lugli,Emanuele Selleri,Chiara Tarracchini,Aryanna Muscò,Silvia Petraro,Giulia Longhi,Leonardo Mancabelli,Christian Milani,Francesca Turroni,Marco Ventura

Background: Bifidobacteria are recognized as one of the most influential bacterial groups inhabiting the human gut, capable of modulating the host’s health. To understand how these bacteria interact with their hosts, it is essential to investigate their extracellular structures, such as pili. While the presence of sortase-dependent pili has already been investigated in the Bifidobacterium genus, limited information is available on Type IV pili (T4P), which have previously been identified in a few species as tight adherence (Tad) loci.

Methods: Here, we explored the T4P distribution across the currently 117 (sub)species representing all described taxa of the Bifidobacterium genus, revealing two distinct loci unevenly distributed within the genus through in silico genomic analyses supported by in vitro validation.

Results: Our analysis identified a conserved Type IVc pili (T4cP) structure across all bifidobacterial taxa, with minor predicted structural variations in members of the Bifidobacterium longum and Bifidobacterium boum phylogenetic groups. This T4cP structure, also known as Tad, exhibited an ancestral, non-retractile architecture typically associated with stable colonization and long-term persistence. In addition, a secondary Type IVa pili (T4aP) structure was detected in 13 bifidobacterial species. These species are associated with specific ecological niches, including primate, bovine, and porcine hosts, suggesting a link between this locus and host-associated adaptation.

Conclusion: Notably, twitching motility assays demonstrated that Bifidobacterium adolescentis strains harboring the T4aP locus exhibit motility in response to specific environmental signals, observed upon starch supplementation of the growth medium, thereby challenging the traditional view of bifidobacteria as a strictly non-motile bacterial genus.

Cancers doi: 10.20517/mrr.2025.85

Authors: Kayo Ikuta,Akihiro Kunisawa,Itaru Dekio,Arisa Ito,Qiuyi Wang,Kazuhiro Kawamura,Masaki Yamada,Sumi Nakamura,Yoshihiro Hayakawa,Takuma Higurashi,Junko Iida,Eiichiro Fukusaki,Toru Suzuki,Mitsuharu Matsumoto

Objectives: Targeted metabolomic analysis of faecal samples has been limited by narrow chemical coverage. Here, we established a multiplexed, triple quadrupole mass spectrometry (TQMS)-based targeted metabolomics workflow. This workflow allows accurate detection and semi-quantification of diverse faecal metabolites and provides a methodological platform for studying host-microbiome metabolic interactions.

Methods: Faecal metabolomes from germ-free (GF) mice, ex-germ-free (Ex-GF) mice, and human participants were analysed using TQMS-based targeted metabolomics. The analysis comprised multiple methods targeting amino acids and their derivatives, carbohydrates, short-chain fatty acids, bile acids, lipid mediators, and phospholipids.

Results: In total, 607 low-molecular-weight metabolites in 44 chemical categories were detected and semi-quantified. Faecal metabolomes of GF and Ex-GF mice were analysed, uncovering 341 intestinal microbiome-dependent metabolites. A proof-of-concept analysis using faecal samples from five patients with colorectal cancer demonstrated the successful application of this platform to human clinical material, highlighting its strong potential for future disease-oriented metabolomic investigations.

Conclusion: We developed a multi-targeted faecal metabolomics platform that substantially expands the chemical space accessible to targeted analysis. This workflow provides a methodological foundation for future large-scale and translational studies.

Cancers doi: 10.20517/mrr.2025.104

Authors: Shahna Fathima,Tina Sarkar,Navneet Sharma,Paul E Kilgore,Huan H Nguyen

Aim: This study investigated the effects of Muno-IgY®, a multi-pathogen-specific immunoglobulin Y (IgY), on microbial growth, adhesion, fermentation activity, and immune signaling using a multi-tiered in vitro approach.

Methods: IgY activity was first evaluated in Caco-2 adhesion and invasion assays using adherent-invasive Escherichia coli (AIEC) at optimized concentrations, followed by assessment in a SHIME® in vitro gut model inoculated with human fecal microbiota enriched in Enterobacteriaceae. Microbial composition, fermentation markers, and metabolite production were analyzed, and downstream effects on epithelial barrier integrity and immune signaling were evaluated using a Caco-2/peripheral blood mononuclear cell (PBMC) co-culture model exposed to SHIME® effluents.

Results: Muno-IgY® significantly reduced AIEC adhesion/invasion from 39.76% in controls to 13.08% and 9.13% at 3 and 6 mg/mL, respectively (P < 0.05). In the SHIME® model, IgY significantly increased acetate and propionate production (P < 0.05), alongside a marked increase in ammonium concentration (P < 0.01). Microbial biomass increased modestly, while alpha- and beta-diversity indices were not significantly altered. The compositional shifts indicated enrichment of beneficial and mucin-associated taxa and reduction of opportunistic or pathogenic species in the Muno-IgY® group. In Caco-2/PBMC co-cultures, IgY-treated effluent decreased transepithelial electrical resistance (TEER) indicating reduced barrier integrity (P < 0.05) but significantly decreased pro-inflammatory cytokines Interferon-γ (IFN-γ) and Interleukin-22 (IL-22) (P < 0.05).

Conclusions: Muno-IgY® demonstrates the ability to inhibit pathogen adhesion and modulate microbial composition and immune responses in vitro. These findings support its potential as a non-antibiotic approach for microbiome-targeted interventions, although further validation in vivo is required.

Cancers doi: 10.20517/mrr.2025.112

Authors: Taylor D. Ticer,Pramita Suresh,Subhomitra Ghoshal,Anna M. Tingler,Rachel Stubler,Adelaide E. Horvath,Janiece S. Glover,Terri N. Ellis,Melinda A. Engevik

Background: Klebsiella pneumoniae can colonize the gastrointestinal tract, yet its interactions with intestinal mucus remain poorly defined. In this study, we examined the capacity of Klebsiella pneumoniae (K. pneumoniae) to adhere and use intestinal mucus and its associated glycans.

Methods: Multiple commercial and clinical K. pneumoniae isolates were tested for adhesion to porcine and human Mucin 2 (MUC2) using fluorescence-based assays and microscopy. In vivo mucus localization was examined in colonized mice by fluorescent in situ hybridization (FISH). Genomic analyses of K. pneumoniae genomes were performed to identify glycosyl hydrolases and sugar utilization pathways. Growth on mucin-derived monosaccharides or intact mucus was assessed in minimal media. Biofilm formation and aminoglycoside susceptibility were measured in the presence or absence of mucus.

Results: All K. pneumoniae strains adhered robustly to porcine and human MUC2 in vitro and we found K. pneumoniae localized to the murine mucus layer in vivo. Genomic analysis of over 1,000 K. pneumoniae isolates revealed that most strains possess glycosyl hydrolases targeting internal galactose, N-acetyl-glucosamine (GlcNAc), and N-acetyl-galactosamine (GalNAc) glycan sugars, though they lack enzymes to cleave terminal fucose or N-acetyl-neuraminic acid. Consistent with this finding, we found that K. pneumoniae alone could not grow in minimal media with intact mucus as a sole carbon source. However, we found that K. pneumoniae could grow with free mucus glycan-derived sugars galactose, GlcNAc, GalNAc fucose and N-acetyl-neuraminic acid. Mucus did not alter biofilm formation, but it significantly increased sensitivity to gentamicin, kanamycin and streptomycin.

Conclusion: These findings identify mucus as an important modulator of K. pneumoniae colonization and antibiotic responsiveness.

Cancers doi: 10.20517/mrr.2026.04

Authors: Evangelia N. Kerezoudi,Sue McKay,Seta Kurt,Maaike De Kreek,Jelle De Medts,Lynn Verstrepen,Jonas Ghyselinck,Lieven Van Meulebroek,Wim Calame,Ruud Albers,Annick Mercenier,Robert J. Brummer,Ignacio Rangel

Background: Pectic rhamnogalacturonan-I (RG-I) is a dietary fiber that modulates the gut-immune axis. This study evaluates a novel variant of RG-I from chicory root (chRG-I).

Methods: In a randomized, double-blind, placebo-controlled trial, 55 healthy adults were stratified by habitual fiber intake and baseline Bifidobacterium levels before receiving 500 mg/day of chRG-I or placebo for four weeks. Primary endpoints included fecal Bifidobacterium counts. Secondary outcomes assessed fecal metabolites, systemic immune cell activation markers, and gastrointestinal symptoms. To provide mechanistic insights, donor-matched fecal samples were used in in vitro fermentation and Caco-2/peripheral blood mononuclear cell co-culture gut barrier models.

Results: Supplementation with chRG-I induced a statistically significant bifidogenic effect, with absolute levels peaking at week three, and lower levels of some fecal short-chain fatty acids (SCFA) compared to placebo. However, donor-matched in vitro fermentations with chRG-I confirmed robust production of SCFA and reduction of branched-chain fatty acids levels (BCFA). Systemically, chRG-I upregulated HLA-DR expression on myeloid dendritic cells. Clinically, chRG-I was well-tolerated and slightly improved stool consistency compared to placebo. In an intestinal barrier challenge model, chRG-I fermentates (a pool of metabolites including SCFA and fragments of chRG-I) protected barrier integrity, modulated the cytokine milieu away from a predominantly pro-inflammatory response, as characterized by increased IL4 and IL22 and reduced IL9, IL17A, and IL21.

Conclusion: Supplementation with a low dose of chRG-I is well-tolerated, beneficially modulates the gut microbiome - which can protect the intestinal barrier, and subtly enhances systemic immune readiness, suggesting that chRG-I may have benefits as a functional food ingredient.

Cancers doi: 10.20517/mrr.2026.63

Authors: Chanon Kunasol,Chaloemwut Piyaphattachai,Jirawat Saengsin,Nipon Chattipakorn,Siriporn C. Chattipakorn

Osteomyelitis is an inflammatory bone disorder that includes both infectious and noninfectious forms and remains a major cause of bone destruction, impaired skeletal repair, and long-term disability. Although bacterial infection is the predominant cause, disease progression is influenced by interactions among microorganisms, host immunity, inflammation, and bone remodeling. This review critically evaluates current evidence from genome-wide association and Mendelian randomization studies, clinical investigations, and experimental animal models. Clinical studies demonstrate that osteomyelitic bone contains polymicrobial communities enriched with anaerobic and biofilm-forming bacteria, whereas oral microbial dysbiosis differs between chronic bacterial and chronic nonbacterial osteomyelitis. Experimental studies indicate that gut microbial dysbiosis is associated with impaired intestinal barrier function, altered microbial metabolites, systemic inflammation, and delayed bone repair. Current evidence therefore suggests that microbial communities detected at local sites are associated with infection, biofilm formation, and local inflammatory responses, whereas the gut microbiome may influence systemic immunity, inflammatory tone, and bone remodeling. Although microbiome-targeted interventions have shown beneficial effects in experimental models, current evidence remains largely associative, and mechanistic and longitudinal studies are limited. A better understanding of microbiome-host interactions may facilitate the development of microbiome-based strategies for the prevention and treatment of osteomyelitis.

Cancers doi: 10.20517/mrr.2026.08

Authors: Wanyu Yang,Shuigen Bian,Huizi Tan

Background: Irritable bowel syndrome (IBS) is a highly prevalent disorder that significantly impairs quality of life, yet effective treatments remain limited. While prior work from our laboratory linked the Lachnospiraceae family to IBS severity, conflicting sequencing data necessitated a deeper investigation into this taxonomic group.

Methods: We evaluated the therapeutic potential of four representative Lachnospiraceae strains in IBS mice with standardized gut microbiota baseline (SynCom-23). Core symptoms were assessed via body weight, fecal status, and visceral hypersensitivity. Intestinal inflammation, oxidative stress, and barrier function were evaluated by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (RT-qPCR), and gut microbiota and metabolites were analyzed by 16S rRNA sequencing, gas chromatography-flame ionization detector (GC-FID), and liquid chromatography-mass spectrometry (LC-MS), together with brain neurotransmitter systems.

Results: We found that all four strains effectively alleviated IBS symptoms, reducing weight loss (P < 0.001) and visceral hypersensitivity markers calcitonin gene-related peptide (CGRP) and transient receptor potential vanilloid 1 (TRPV1) (P < 0.0001), potentially through distinct pathways: Blautia wexlerae MW-022 produces substantial acetic acid, stimulates Lactobacillaceae proliferation, and targets the γ-aminobutyric acid/glutamate (GABA/Glu) system. Roseburia faecis MW-024 produces butyrate, affecting the GABA/Glu system. Dorea longicatena MW-023 enhances the serotonin (5-HT) system at synthesis and reception levels; and Coprococcus eutactus DSM107541 modulates the 5-HT system via the “clearance-reception” mode. Combined strains achieved more comprehensive gut micro-ecology restoration.

Conclusion: This study systematically elucidates the heterogeneity in the mechanisms by which Lachnospiraceae strains treat IBS at the strain level. Different strains achieve precise regulation through their specific gut-brain axes. These findings offer strain-level insights and a foundation for developing Lachnospiraceae-based microbial therapies.

Cancers doi: 10.20517/mrr.2026.03

Authors: Raphaela Joos,Aonghus Lavelle,Eugene Dempsey,Catherine Stanton,R. Paul Ross

The early-life gut microbiome is tightly linked to different aspects of infant development. Microbial colonisation patterns have been repeatedly shown to play a role in a variety of paediatric outcomes, ranging from metabolism and immune function to neurodevelopment. Concomitantly, the identification of early-life biomarkers is crucial, especially considering that for various conditions, reliable diagnostic tools only emerge in early childhood. As such, microbiome data collected in the first two years of life may offer valuable prospects for early detection, prevention, quantification or even correction of adverse health trajectories. With the increasing availability of high-resolution microbiome data, researchers are leveraging both traditional statistical approaches and machine learning (ML) methods to analyse the evolution of these complex microbial communities. While statistical models are well-suited for identifying associations between microbiome features and health states, ML methods allow for predicting health outcomes from those features. This review explores the role of the early-life gut microbiome in infant health and development, with a focus on how data acquisition and analytical methods can shape current knowledge. We contrast statistical approaches with ML methods, summarising key findings on microbial succession and factors influencing it. By addressing current challenges and identifying areas for methodological refinement, we aim to discuss the potential of the microbiome in the assessment of current and future health states of an individual and aid in the development of more robust, clinically-relevant models for paediatric care.

Cancers doi: 10.20517/mrr.2026.19

Authors: Emanuele Selleri,Giulia Longhi,Chiara Tarracchini,Erica de Carolis,Silvia Petraro,Leonardo Mancabelli,Christian Milani,Francesca Turroni,Marco Ventura,Gabriele Andrea Lugli

Background: Bifidobacterium bifidum (B. bifidum) is an infant gut symbiont specialized in degrading host-derived glycans. Despite its relevance in early life, the species’ genomic diversity has not yet been comprehensively surveyed, and current reference collections capture only a fraction of the global B. bifidum pangenome.

Methods: In this study, we reconstructed the first comprehensive pangenome of B. bifidum using 1,351 high-quality genomes, including metagenome-assembled genomes. This dataset was used for in silico comparative genomics analyses to identify species-specific genetic and functional features. In vitro transcriptomics analyses were further performed to validate and functionally characterize selected species-specific traits.

Results: Comparative genomic analysis with other human-associated bifidobacteria species identified 667 B. bifidum-specific clusters of orthologous genes mostly involved in carbohydrate utilization, osmotic regulation, and host interaction. Notably, B. bifidum displays the most extensive enzymatic repertoire for host-glycan degradation, dedicating 43% of its conserved glycoside hydrolases to these substrates. We identified significant gain-of-function events, including two unique phosphotransferase systems (PTS) for disaccharide uptake. Transcriptomic profiling corroborated the functional relevance of these PTS clusters, which were significantly up-regulated during growth on human milk oligosaccharides, mucin, and N-acetylglucosamine. While the species exhibits high genomic stability, a localized divergence (average nucleotide identitiy, ANI < 98.5%) was identified in rural, non-Westernized populations, reflecting niche-specific adaptations.

Conclusion: The identified genomic framework highlighted a distinct evolutionary path of B. bifidum, placing this taxon as a metabolic cornerstone in the neonatal gut via extensive metabolic specialization toward glycan hosts.

Cancers doi: 10.20517/mrr.2026.25

Authors: Paerhande Dilinuer,Jehangir Khan,Ming Li,Yu Wu,Xiaoying Zheng,Zhongdao Wu,Datao Lin,Hanano Yamada,Jeremy Bouyer,Kostas Bourtzis,Dongjing Zhang

Aim: The Sterile Insect Technique is a promising method for Aedes albopictus control, but its effectiveness hinges on male quality, which may be compromised during mass-rearing and irradiation. Reliable biological indicators capable of reflecting irradiation-induced quality loss remain limited. Gut microbiota regulates mosquito development, immunity, and metabolism and may therefore provide potential indicators of sterile male quality. This study explored whether gut microbiota composition can reflect irradiation-induced quality impairment in males of a long-term (over 3 years) mass-reared Ae. albopictus GT strain.

Methods: Male pupae were exposed to X-ray irradiation without filtration at an estimated absorbed dose of 78 Gy. Emergence rate, flight ability, longevity, metabolic capacity, and gut microbiota (via 16S rRNA gene sequencing, culture-based methods, and qPCR) were compared between irradiated and non-irradiated males.

Results: Irradiation significantly reduced emergence rate, flight ability, and longevity. It did not alter gut microbiota diversity, but reshaped its composition, including the emergence of Sphingobacterium, increased abundance of Stenotrophomonas and Comamonas, and decreased levels of Delftia and Achromobacter. Similarity was observed in the culturable gut bacterial community, confirmed by qPCR with eight selected key taxa. Metabolic changes included reduced glutamine synthetase activity and elevated glucose levels in irradiated males.

Conclusion: These results show that irradiation-induced quality loss is accompanied by reproducible shifts in gut microbiota composition and metabolic status. These findings suggest that gut microbiota profiles may serve as potential indicators of irradiation-associated quality changes in sterile males and provide preliminary evidence for microbiota-informed quality assessment approaches in Ae. albopictus SIT programs.

Cancers doi: 10.20517/mrr.2025.91

Authors: Erin Chard,Adelaide E. Horvath,Makenna Grozis,Renata Rocha do Nascimento,Paul R. S. Baker,Santosh Kapil Kumar Gorti,Robert Proos,Thomas D. Horvath,Melinda A. Engevik

Background: The intestinal mucus layer is comprised of heavily glycosylated mucins, including mucin 2 (MUC2), that serve as a nutrient source for certain bacterial members of the gut microbiota. Only a subset of gut commensals encode the glycoside hydrolases required to degrade mucin glycans. However, mucin-degrading microbes can release glycans and generate compounds that can cross-fed non-mucin degrading microbes, creating complex microbial networks. While pairwise studies have shown that mucin degradation drives cross-feeding and metabolite exchange, the broader impact of mucins on community structure and metabolic output remains poorly understood.

Objective: In this study, we sought to identify how a defined microbial consortium of human commensals with varied mucin-degrading capacities responds to MUC2 to shape community composition and metabolic output.

Methods: A defined consortium of human gut commensals with varied mucin-degrading capacities was cultivated in anaerobic bioreactors in the presence or absence of porcine MUC2. Community composition was assessed, and extracellular metabolites were quantified using targeted and untargeted metabolomic profiling.

Results: MUC2 supplementation significantly altered community structure, promoting the expansion of Akkermansia muciniphila while reducing Prevotella. MUC2 also reshaped microbial metabolism, decreasing acetate levels while increasing propionate, butyrate, and formate. In addition, MUC2 supplementation altered amino acid utilization and vitamin metabolism and reduced several neuroactive compounds, including glutamate, γ-aminobutyric acid (GABA), and anthranilic acid, while increasing tryptamine levels.

Conclusion: These findings demonstrate that mucins exert broad effects on microbial community structure and metabolic output. Collectively, this work highlights the central role of bacterial cross-feeding in shaping gut ecosystem function.