Understanding the dynamic relationship between mucus-associated microbiota and host health is critical. However, studies predominantly using stool samples may not accurately represent these bacterial communities. Here, we investigated the mucus-associated microbiota in the gastrointestinal tract of mice and the terminal ileum of humans using different sample types: mucosal washes, brushes, scrapings, and intestinal contents in mice and biopsies, brushes and mucosal washes in humans. We used DNA quantification and 16S rRNA amplicon sequencing to evaluate the comparability of the information yielded from the different sample types under a controlled benchmark. In mice, mucosal washes and brushes had comparative bacterial DNA and host DNA contamination than scraping samples. Similarly, in humans, washes outperformed biopsies in bacterial DNA content. Read counts and microbiota alpha diversity remained remarkably similar in mice and between brushes and washes in humans. The composition of the microbiota varied based on the subsegment and sample type in mice and sample type in humans. We conclude that washes and brushes reduce host contamination without inducing substantial compositional bias when sampling mucosal microbiota. Our findings suggest that mucosal washes and brushes are a viable alternative to biopsies in humans and scrapings in mice, thereby improving the transferability of results across hosts. Our study highlights the importance of focusing on mucus-associated microbiota to better capture host-microbiome interactions at their closer interface.

The gut-brain axis (GBA) denotes the dynamic and bidirectional communication system that connects the gastrointestinal tract and the central nervous system (CNS). This review explored this axis, focusing on the role of microbial diversity and fitness in maintaining gastrointestinal health and preventing neurodegeneration, particularly in Alzheimer's disease (AD). Gut dysbiosis, characterized by the imbalance in populations of beneficial and harmful bacteria, has been associated with increased systemic inflammation, neuroinflammation, and the progression of AD through pathogenic mechanisms involving amyloid deposition, tauopathy, and increased blood-brain barrier (BBB) permeability. Emerging evidence highlighted the therapeutic potential of probiotics, dietary interventions, and intermittent fasting in restoring microbial balance, reducing inflammation, and minimizing neurodegenerative risks. Probiotics and synbiotics are promising in helping improve cognitive function and metabolic health, while dietary patterns like the Mediterranean diet were linked to decreased neuroinflammation and enhanced gut-brain communication. Despite significant advancement, further research is needed to elucidate the specific microbial strains, metabolites, and mechanisms influencing brain health. Future studies employing longitudinal designs and advanced omics technologies are essential to developing targeted microbiome-based therapies for managing AD-related disorders.

In situ imaging diagnosis and precise treatment of deep tumor tissues are hotspots in life sciences and medical research. In recent years, using focused ultrasound to remotely control engineered bacteria for drug release has become one of the methods for precise in vivo drug delivery. However, non-visualized engineered bacteria pose challenges for precise control within the body. Therefore, there is an urgent need for an engineered bacterial vector capable of deep tissue imaging to precisely locate bacteria in vivo. Acoustic reporter genes (ARGs) are biological elements used for deep tissue imaging, with gene clusters over 8 kb. However, ARGs are often tested on plasmids, which hinders stable expression in vivo and limits the space for inserting components that regulate drug release. Therefore, we used the attenuated Salmonella typhimurium VNP20009, known for its tumor-targeting ability, as the chassis bacteria. By using CRISPR-Cas9 technology, we inserted ARGs into the genome and optimized the promoter strength and copy number for ARG expression, constructing ultrasound-visible engineered bacteria expressing gas vesicles on the genome. Additionally, by knocking out the stress protein gene htrA in VNP20009, we increased the maximum injection dose by tenfold and the tumor specificity by a hundredfold. The constructed ultrasound-visible engineered bacteria can stably synthesize gas vesicles and output ultrasound signals while directly carrying drug plasmids for tumor therapy. Our research provides an effective vector for diagnosis and precise treatment.

Immune checkpoint blockade therapies have markedly advanced cancer treatment by invigorating antitumor immunity and extending patient survival. However, therapeutic resistance and immune-related toxicities remain major concerns. Emerging evidence indicates that microbial dysbiosis diminishes therapeutic response rates, while a diverse gut ecology and key beneficial taxa correlate with improved treatment outcomes. Therefore, there is a growing understanding that manipulating the gut microbiota could boost therapy efficacy. This review examines burgeoning methods that target the gut microbiome to optimize therapy and innovative diagnostic tools to detect dysbiosis, and highlights challenges that remain to be addressed in the field.

Probiotic microbes such as Lactobacillus may reduce serum total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol. The objective of this study was to assess the effect of Lactobacillus plantarum strains CECT7527, CECT7528, and CECT7529 (LP) on the serum lipids, cardiovascular parameters, and fecal gut microbiota composition in patients with mild hypercholesterolemia. A randomized, double-blinded, placebo-controlled clinical trial with 86 healthy adult participants with untreated elevated LDL cholesterol ≥ 160 mg/dl was conducted. Participants were randomly allocated to either placebo or LP (1.2 × 109 CFU/d) for 12 weeks. LDL, HDL, TC, and triglycerides (TG), cardiovascular parameters (blood pressure, arterial stiffness), and fecal gut microbiota composition (16S rRNA gene sequencing) were assessed at baseline and after 12 weeks. Both groups were comparable regarding age, sex, and LDL-C at baseline. LDL-C decreased (mean decrease - 6.6 mg/dl ±  - 14.0 mg/dl, Ptime*group = 0.006) in the LP group but not in the placebo group. No effects were observed on HDL, TG, or cardiovascular parameters or overall gut microbiota composition. Responders to LP intervention (> 5% LDL-C reduction) were characterized by higher BMI, pronounced TC reduction, higher abundance of fecal Roseburia, and lower abundance of Oscillibacter. In conclusion, 12 weeks of L. plantarum intake moderately reduced LDL-C and TC as compared to placebo. LDL-C-lowering efficacy of L. plantarum strains may potentially be dependent on individual difference in the gut microbiota. Trial registration: DRKS00020384, dated 07/01/2020.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has become a major health risk and a serious worldwide issue. MAFLD typically arises from aberrant lipid metabolism, insulin resistance, oxidative stress, and inflammation. However, subjacent causes are multifactorial. The gut has been proposed as a major factor in health and disease, and over the last decade, bacterial strains with potentially beneficial effects on the host have been identified. In vitro cell models have been commonly used as an early step before in vivo drug assessment and can confer complementary advantages in gut and liver health research. In this study, several selected strains of the order Bacteroidales were used in a three-cell line in vitro analysis (HT-29, Caco-2, and HepG2 cell lines) to investigate their potential as new-generation probiotics and microbiota therapeutics. Antimicrobial activity, a potentially useful trait, was studied, and the results showed that Bacteroidales can be a source of either wide- or narrow-spectrum antimicrobials targeting other closely related strains. Moreover, Bacteroides sp. 4_1_36 induced a significant decrease in gut permeability, as evidenced by the high TEER values in the Caco-2 monolayer assay, as well as a reduction in free fatty acid accumulation and improved fatty acid clearance in a steatosis HepG2 model. These results suggest that Bacteroidales may spearhead the next generation of probiotics to prevent or diminish MAFLD.

Diabetes mellitus, a most common endocrine disorder of glucose metabolism, has become a global epidemic and poses a serious public health threat with an increased socio-economic burden. Escalating incidence of diabetes is correlated with changes in lifestyle and food habits that cause gut microbiome dysbiosis and β-cells damage, which can be addressed with dietary interventions containing probiotics. Hence, the search for probiotics of human origin with anti-diabetic, anti-AGE, and anti-ACE potentials has gained renewed interest for the effective management of diabetes and its associated complications. The present study used an alloxan (AXN)-induced diabetic rat model to investigate the effects of potential probiotic Lacticaseibacillus casei MKU1, Lactiplantibacillus pentosus MKU3, and Lactiplantibacillus plantarum MKU7 administration individually on physiochemical parameters related to diabetic pathogenesis. Experimental animals were randomly allotted into six groups viz. NCG (control), DCG (AXN), DGM (metformin), DGP1 (MKU1), DGP2 (MKU3), and DGP3 (MKU7), and biochemical data like serum glucose, insulin, AngII, ACE, HbA1c, and TNF-α levels were measured until 90 days. Our results suggest that oral administration with MKU1, MKU3, or MKU7 significantly improved serum insulin levels, glycemic control, glucose tolerance, and body weight. Additionally, β-cell mass was increased by preserving islet integrity in Lactobacillus-treated diabetic rats, whereas TNF-α (~40%), AngII (~30%), and ACE levels (~50%) were strongly inhibited and enhanced sIgA production (5.8 folds) abundantly. Furthermore, Lactobacillus administration positively influenced the gut microbiome with a significant increase in the abundance of Lactobacillus species and the beneficial Bacteroides uniformis and Bacteroides fragilis, while decreased the pathogenic Proteus vulgaris and Parabacteroides distasonis. Among the probiotic treatment groups, L. pentosus MKU3 performed greatly in almost all parameters, indicating its potential use for alleviating diabetes-associated complications.

The gastrointestinal tract is a dynamic biomechanical environment where physical forces, cellular processes, and microbial interactions converge to shape the gut health and disease. In this review, we examine the unique mechanical properties of the gut, including peristalsis, viscoelasticity, shear stress, and tissue stiffness, and their roles in modulating host mechanosignaling and microbial behavior under physiological and pathological conditions. We discuss how these mechanical forces regulate gut epithelial integrity, immune responses, and microbial colonization, leading to distinct ecological niches across different intestinal segments. Furthermore, we highlight recent advancements in 3D culture systems and gut-on-a-chip models that accurately recapitulate the complex interplay between biomechanics and gut microbiota. By elucidating the intricate relationship between mechanobiology and gut function, this review underscores the potential for mechanotherapeutic strategies to modulate host-microbe interactions, offering promising avenues for the prevention and treatment of disorders such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer.

Klebsiella pneumoniae (Kp) is an opportunistic pathogen that poses a major threat in healthcare settings. The gut is a primary Kp reservoir in hospitalized patients, and colonization is a major risk factor for Kp infection. The stability of virulence determinants such as capsule and lipopolysaccharide during gut colonization is largely unexplored. In a murine gut colonization model, we demonstrated that spontaneous capsule loss occurs rapidly but varies by Kp pathotype. A classical Kp strain and a carbapenem-resistant strain of the epidemic sequence type 258 lineage had significant levels (median of 25% and 9.5%, respectively) of capsule loss. In contrast, a hypervirulent strain did not lose capsule to a significant degree (median 0.1%), despite readily losing capsule during in vitro passage. Insertion sequences (ISs) or mutations were found disrupting capsule operon genes of all isolates and in O-antigen encoding genes in a subset of isolates. Mouse-derived acapsular isolates from two pathotypes had significant fitness defects in a murine pneumonia model. Removal of the IS in the capsule operon in a mouse-derived acapsular classical isolate restored capsule production to wild-type levels. Genomic analysis of Klebsiella rectal isolates from hospitalized patients found that 18 of 245 strains (7%) had at least one IS disrupting the capsule operon. Combined, these data indicate that Kp capsule loss can occur during gut colonization in a strain-dependent manner, not only impacting strain virulence but also potentially altering patient infection risk.

In hospitalized patients, gut colonization by the bacterial pathogen Klebsiella pneumoniae (Kp) is a major risk factor for the development of infections. The genome of Kp varies across isolates, and the presence of certain virulence genes is associated with the ability to progress from colonization to infection. Here, we identified that virulence genes encoding capsule and lipopolysaccharide, which normally protect bacteria from the immune system, are disrupted by mutations during murine gut colonization. These mutations occurred frequently in some isolates of Kp but not others, and these virulence gene mutants from the gut were defective in causing infections. An analysis of 245 human gut isolates demonstrated that this capsule loss also occurred in patients. This work highlights that mutations that decrease virulence occur during gut colonization, the propensity for these mutations differs by isolate, and that stability of virulence genes is important to consider when assessing infection risk in patients.

A mutualistic co-evolution exists between the host and its associated microbiota in the human body. Bacteria establish ecological niches in various tissues of the body, locally influencing their physiology and functions, but also contributing to the well-being of the whole organism through systemic communication with other distant niches (axis). Emerging evidence indicates that when the composition of the microbiota inhabiting the niche changes toward a pathogenic state (dysbiosis) and interactions with the host become unbalanced, diseases may present. In addition, imbalances within a single niche can cause dysbiosis in distant organs. Current research efforts are focused on elucidating the mechanisms leading to dysbiosis, with the goal of restoring tissue homeostasis. In vitro models can provide critical experimental platforms to address this need, by reproducing the niche cyto-architecture and physiology with high fidelity. This review surveys current in in vitro host-microbiota research strategies and provides a roadmap that can guide the field in further developing physiologically relevant in vitro models of ecological niches, thus enabling investigation of the role of the microbiota in human health and diseases. Lastly, given the Food and Drug Administration Modernization Act 2.0, this review highlights emerging in vitro strategies to support the development and validation of new therapies on the market.

The role of gut microbiota in human health and disease is becoming increasingly recognized. Historically, the impact of human gut microbiota on health has been studied using clinical trials and animal models. However, clinical studies often struggle with controlling variables and pinpointing disease-causing factors, while animal models fall short of accurately replicating the human gut environment. Additionally, continuous sample collection for gut microbiota analysis in vivo presents significant ethical and technical challenges. To address these limitations, in vitro fermentation models have emerged as promising alternatives. These models aim to simulate the structural and functional characteristics of the human gut in a controlled setting, offering valuable insights into microbial behavior. This review highlights current knowledge and technological advances in in vitro cultivation systems for human gut microbiota, focusing on key elements such as three-dimensional scaffolds, culture media, fermentation systems, and analytical techniques. By examining these components, the review establishes a framework for improving methods to cultivate and study human gut microbiota, enhancing research methodologies for better understanding microbial interactions, behavior, and adaptation in diverse environments.

We developed a two-tiered strategy aiming to identify gut bacteria functionally linked to the development of multiple sclerosis (MS). First, we compared gut microbial profiles in a cohort of 81 monozygotic twins discordant for MS. This approach allowed to minimize confounding effects by genetic and early environmental factors and identified over 50 differently abundant taxa with the majority of increased taxa within the Firmicutes. These included taxa previously described to be associated with MS (Anaerotruncus colihominis and Eisenbergiella tayi), along with newly identified taxa, such as Copromonas and Acutalibacter. Second, we interrogated the intestinal habitat and functional impact of individual taxa on the development of MS-like disease. In an exploratory approach, we enteroscopically sampled microbiota from different gut segments of selected twin pairs and compared their compositional profiles. To assess their functional potential, samples were orally transferred into germfree transgenic mice prone to develop spontaneous MS-like experimental autoimmune encephalomyelitis (EAE) upon bacterial colonization. We found that MS-derived ileal microbiota induced EAE at substantially higher rates than analogous material from healthy twin donors. Furthermore, female mice were more susceptible to disease development than males. The likely active organisms were identified as Eisenbergiella tayi and Lachnoclostridium, members of the Lachnospiraceae family. Our results identify potentially disease-facilitating bacteria sampled from the ileum of MS affected twins. The experimental strategy may pave the way to functionally understand the role of gut microbiota in initiation of MS.

Preterm Premature Rupture of Membrane (PPROM) constitutes a prevalent obstetric complication that markedly heightens the risk of neonatal mortality and low birth weight, while also potentially impacting the psychological well-being of the mother. Despite established associations between PPROM and various underlying medical conditions or lifestyle factor, a definitive treatment strategy continues to remain unattainable. Contemporary research indicates that dysbiosis of the gut microbiome may play a significant role in the pathogenesis of PPROM. Consequently, this study endeavors to gather recent findings related to the mechanisms underlying intestinal dysbiosis in relation to PPROM. It aims to offer novel insights into this critical issue. An increasing amount of evidence suggests that specific intestinal bacteria have the capacity to translocate into the vascular system and the amniotic cavity during pregnancy. This happens as a consequence of imbalances or dysbiosis within the gut microbiota. This translocation may be facilitated by the presence of bacteria within the amniotic cavity, modifications in the vaginal microbiota, and activation of the Hypothalamus-Pituitary-Adrenal (HPA) axis, which initiates a physiological cascade that accelerates the progression of PPROM. In light of these findings, the preservation of gut microbial homeostasis, particularly through the application of probiotics or dietary modifications, may serve to alleviate the detrimental effects of dysbiosis on PPROM.

This study explored the conversion of citrus juice side streams into fermentable oligosaccharides for potential gut health benefits. Alcohol washed, insoluble lemon peel waste was enzymatically treated using technical pectinolytic enzyme preparations, yielding mixtures of galactose- arabinose- and either methyl-esterified or non-methyl-esterified galacturonic acid oligosaccharides (OS) with a Δ4,5-unsaturated non-reducing end resulting in mixtures of pectin-derived OS: POS and POSNME. Both mixtures were completely fermented during in vitro batch fermentation by proximal and distal microbiota of three healthy adult donors. Fermentation by distal and proximal microbiota resulted in similar methyl-ester-dependent mechanisms of POS utilization, yielding health beneficial acetate, propionate and butyrate in significant amounts. Arabinose-, galactose- and non-methyl-esterified Δ4,5-unsaturated galacturonic acid OS were utilized significantly faster by the distal and proximal microbiota of donors 1 and 2 compared to methyl-esterified Δ4,5-unsaturated galacturonic acid OS, suggesting methyl-esterification of Δ4,5-unsaturated galacturonic acid oligosaccharides as a substantial regulator of POS fermentability. The findings presented in this manuscript suggest that carbohydrate molecular structure and availability, rather than microbiota composition, determine carbohydrate fermentation patterns along the colon, emphasizing that the consumption of differently fermentable fiber is essential to promote gut health along the colon.

Negative experiences during adolescence, such as social isolation (SI), bullying, and abuse, increase the risk of psychiatric diseases in adulthood. However, the pathogenesis of psychiatric diseases induced by these factors remain poorly understood. In adolescents, stress affects the intestinal homeostasis in the gut-brain axis. This study determined whether adolescent SI induces behavioral abnormalities by disrupting colonic function. Adolescent mice exposed to SI exhibit spatial cognitive deficits and microglial activation in the hippocampus (HIP). SI decreased the differentiation of mucin-producing goblet cells, which was accompanied by alterations in the composition of the gut microbiota, particularly the depletion of mucin-feeding bacteria. Treatment with rebamipide, which promotes goblet cell differentiation in the colon, attenuated SI-induced spatial cognitive deficits and microglial activation in the HIP and decreased cystine, a downstream metabolite of homocysteine. Treatment with cystine ameliorated SI-induced spatial cognitive deficits and increased microglial C-C motif chemokine ligand 7 (CCL7) levels in the HIP. Inhibition of CCL7 receptors by antagonists of CC motif chemokine receptors 2 (CCR2) and 3 (CCR3) in the HIP prevented spatial cognitive deficits induced by SI. Infusion of CCL7 into the HIP following microglial ablation with clodronate liposome induced spatial cognitive deficits. These findings suggest that adolescent SI decreases serum cystine levels by damaging the colonic goblet cells, resulting in spatial cognitive deficits by triggering microglial activation in the HIP. Our results indicate that increased CCL7 expression in hippocampal microglia may contribute to spatial cognitive deficits by activating CCR2 and CCR3.

Everyday actions such as eating, tooth brushing or applying cosmetics inherently modulate our microbiome. Advances in sequencing technologies now facilitate detailed microbial profiling, driving intentional microbiome-targeted product development. Inspired by an academic-industry workshop held in January 2024, this review explores the oral, skin and gut microbiomes, focussing on the potential long-term implications of perturbations. Key challenges in microbiome safety assessment include confounding factors (ecological variability, host influences and external conditions like geography and diet) and biases from experimental measurements and bioinformatics analyses. The taxonomic composition of the microbiome has been associated with both health and disease, and perturbations like regular disruption of the dental biofilm are essential for preventing caries and inflammatory gum disease. However, further research is required to understand the potential long-term impacts of microbiome disturbances, particularly in vulnerable populations including infants. We propose that emerging technologies, such as omics technologies to characterize microbiome functions rather than taxa, leveraging artificial intelligence to interpret clinical study data and in vitro models to characterize and measure host-microbiome interaction endpoints, could all enhance the risk assessments. The workshop emphasized the importance of detailed documentation, transparency and openness in computational models to reduce uncertainties. Harmonisation of methods could help bridge regulatory gaps and streamline safety assessments but should remain flexible enough to allow innovation and technological advancements. Continued scientific collaboration and public engagement are critical for long-term microbiome monitoring, which is essential to advancing safety assessments of microbiome perturbations.

Dysbiosis, which refers to an imbalance in the composition of the gut microbiome, has been associated with a range of metabolic disorders, including type 2 diabetes, obesity, and metabolic syndrome. Although the exact mechanisms connecting gut dysbiosis to these conditions are not fully understood, various lines of evidence strongly suggest a substantial role for the interaction between the gut microbiome, mitochondria, and epigenetics. Current studies suggest that the gut microbiome has the potential to affect mitochondrial function and biogenesis through the production of metabolites. A well-balanced microbiota plays a pivotal role in supporting normal mitochondrial and cellular functions by providing metabolites that are essential for mitochondrial bioenergetics and signaling pathways. Conversely, in the context of illnesses, an unbalanced microbiota can impact mitochondrial function, leading to increased aerobic glycolysis, reduced oxidative phosphorylation and fatty acid oxidation, alterations in mitochondrial membrane permeability, and heightened resistance to cellular apoptosis. Mitochondrial activity can also influence the composition and function of the gut microbiota. Because of the intricate interplay between nuclear and mitochondrial communication, the nuclear epigenome can regulate mitochondrial function, and conversely, mitochondria can produce metabolic signals that initiate epigenetic changes within the nucleus. Given the epigenetic modifications triggered by metabolic signals from mitochondria in response to stress or damage, targeting an imbalanced microbiota through interventions could offer a promising strategy to alleviate the epigenetic alterations arising from disrupted mitochondrial signaling.

Butylated hydroxyanisole (BHA) continues to raise consumer concerns. All previous evaluations of this additive have failed to consider its effect on the gut microbiome, even though it enters the colon. An ex vivo model was used to assess the effect of BHA on microbial communities from 24 donors, aged infants to older adults. A dose of 0.35 g/L BHA elicited no statistically significant changes in the functional outputs or community structure for any age group. Although not large enough to affect community diversity, there were some significant decreases at the phylum level. Among the genes most significantly affected by treatment with BHA across age groups are those involved in lipopolysaccharide synthesis and bacterial electron transport encoded by Bacteroidota, Proteobacteria, and Verrucomicrobiota. Given what is known about the intracellular activity of BHA, these genes may hint at a mechanism behind BHA's evident, but minimally detrimental effect on the gut microbiota.

With growing recognition of the pivotal role of gut microbiota in human health, probiotics have gained widespread attention for their potential to restore microbial homeostasis. However, a critical challenge persists: limited colonization efficiency among most probiotic strains compromises their therapeutic efficacy. This overview synthesizes ecological principles with cutting-edge microbiome research to elucidate the dynamic interplay between dietary components and probiotic colonization within the intestinal niche. This overview systematically analyzes: (1) stage-specific colonization mechanisms spanning microbial introduction, establishment, and proliferation; (2) nutrient-driven modulation of gut microbiota composition and function; and (3) the dual role of common dietary patterns as both facilitators and disruptors of probiotic persistence. Notably, this overview identifies key dietary strategies, including precision delivery of prebiotic fibers and polyphenol-microbiota crosstalk, that enhance niche adaptation through pH optimization, adhesion potentiation, and competitive exclusion of pathogens. Furthermore, this overview critically evaluates current limitations in probiotic research, particularly strain-specific variability and methodological constraints in simulating host-microbe-diet tripartite interactions. To bridge these gaps, this overview proposes an interdisciplinary framework integrating omics-driven strain selection, engineered delivery systems, and personalized nutrition models. Collectively, this work advances a mechanistic understanding of diet-microbiota interactions while providing actionable insights for developing targeted probiotic therapies and evidence-based dietary interventions to optimize gut ecosystem resilience.

Antimicrobial-resistant Enterobacteriaceae are increasingly a clinical challenge. In particular, extraintestinal pathogenic Escherichia coli and Klebsiella pneumoniae threaten public health. Vaccination presents a long-term strategy to reduce both drug-susceptible and resistant infections while maintaining current clinical therapies. The review aims to emphasize the need for vaccines targeting extraintestinal pathogenic E. coli and K. pneumoniae by providing an overview of disease burden, antimicrobial resistance, therapeutics, and vaccine development.

The human gut contains diverse communities of bacteriophage, whose interactions with the broader microbiome and potential roles in human health are only beginning to be uncovered. Here, we combine multiple types of data to quantitatively estimate gut phage population dynamics and lifestyle characteristics in human subjects. Unifying results from previous studies, we show that an average human gut contains a low ratio of phage particles to bacterial cells (∼1:100) but a much larger ratio of phage genomes to bacterial genomes (∼4:1), implying that most gut phage are effectively temperate (e.g., integrated prophage and phage-plasmids). By integrating imaging and sequencing data with a generalized model of temperate phage dynamics, we estimate that phage induction and lysis occur at a low average rate (∼0.001-0.01 per bacterium per day), imposing only a modest fitness burden on their bacterial hosts. Consistent with these estimates, we find that the phage composition of a diverse synthetic community in gnotobiotic mice can be quantitatively predicted from bacterial abundances alone while still exhibiting phage diversity comparable to native human microbiomes. These results provide a foundation for interpreting existing and future studies on links between the gut virome and human health.

Many plants and animals house symbiotic microorganisms in specialized tissues or organs. Here, we used multidimensional in situ imaging techniques to illuminate how host organ structure and bacterial microbiogeography contribute to the symbiotic function of an organ in the Hawaiian bobtail squid, Euprymna scolopes. Along with the well-studied light organ, female E. scolopes harbor a community of bacteria in the accessory nidamental gland (ANG). The ANG is a dense network of epithelium-lined tubules, some of which are dominated by a single bacterial taxon. These bacteria are deposited into squid eggs, where they defend the developing embryos from harmful biofouling. This study used a combination of imaging techniques to visualize different dimensions of the ANG and its bacterial communities. Imaging entire organs with light sheet microscopy revealed that the ANG is a composite tissue of individual, non-intersecting tubules that each harbor their own bacterial population. The organ is bisected, with tubules converging toward two points at the posterior end. At these points, tubules empty into a space where bacteria can mix with squid jelly to be deposited onto eggs. Observations of the symbiotic community correlated bacterial taxa with cell morphology and revealed that tubule populations varied: some tubules contained populations of mixed taxa, whereas others contained only one bacterial genus. Together, these data shed light on how bacterial populations interact within the ANG and how the host uses physical structure to maintain and employ a symbiotic bacterial population in a defensive context.IMPORTANCESequence-based microbiome studies have revealed much about how hosts interact with communities of symbiotic microbiota but often lack a spatial understanding of how microbes relate to each other and the host in which they reside. This study uses a combination of microscopy techniques to reveal how the structure of a symbiotic organ in the female bobtail squid, Euprymna scolopes, houses diverse, beneficial bacterial populations and deploys them for egg defense. These findings suggest that spatial partitioning may be key to harboring a diverse population of antimicrobial-producing bacteria and establishing a foundation for further understanding how host structures mediate symbiotic interactions.

A chronic autoimmune illness that causes joint destruction and inflammation, rheumatoid arthritis (RA) often results in disability. Genetic, environmental, and immune system variables all have a role in the pathophysiology of RA. The complex community of bacteria that live in the gastrointestinal system, known as the gut microbiota, has been implicated in the onset and progression of RA in recent years, according to mounting data. An imbalance in the gut microbiota's composition, known as dysbiosis, has been noted in RA patients. This imbalance may impact inflammatory pathways and immunological responses, which in turn may contribute to the development and severity of the illness. Research has shown that some bacterial species, including Firmicutes, Bacteroidetes, and Proteobacteria, are either more abundant or less prevalent in RA patients than in healthy people. The gut-immune system axis may be modulated, immunological tolerance may be affected, and pro-inflammatory cytokine production may be enhanced by these microbial changes, all of which may lead to systemic inflammation linked to RA. Moreover, changes in intestinal permeability and a rise in microbial metabolite translocation may make autoimmune reactions worse. Probiotics, antibiotics, and dietary changes have also been investigated as possible treatment approaches to help RA patients regain the balance of their gut microbiota. Still up for debate, however, are the precise ways in which the gut microbiome affects RA. Comprehending the complex connection between gut microbiota and RA may give new perspectives on managing and preventing the condition, as well as future prospects for medicines that target the microbiome.

Metabolic fluxes between cells, organisms, or communities drive ecosystem assembly and functioning and explain higher-level biological organization. Exometabolite-mediated inter-organismal interactions, however, remain poorly described due to technical challenges in measuring these interactions. Here, we present MetaFlowTrain, an easy-to-assemble, cheap, semi-high-throughput, and modular fluidic system in which multiple media can be flushed at adjustable flow rates into gnotobiotic microchambers accommodating diverse micro-organisms, ranging from bacteria to small eukaryotes. These microchambers can be used alone or connected in series to create microchamber trains within which metabolites, but not organisms, directionally travel between microchambers to modulate organismal growth. Using MetaFlowTrain, we uncover soil conditioning effects on synthetic community structure and plant growth, and reveal microbial antagonism mediated by exometabolite production. Our study highlights MetaFlowTrain as a versatile system for investigating plant-microbe-microbe metabolic interactions. We also discuss the system´s potential to discover metabolites that function as signaling molecules, drugs, or antimicrobials across various systems.

Acalypha australis L. (AAL), a traditional medicinal herb from the Euphorbiaceae family, has been widely used in Chinese medicine for its heat-clearing, detoxifying, and diuretic properties, as well as for treating gastrointestinal disorders such as diarrhea and dysentery. Its reported anti-inflammatory and hemostatic effects are closely linked to inflammatory pathways. While previous studies have demonstrated AAL's efficacy in acute colitis, its therapeutic potential in chronic colitis and the underlying mechanisms remain largely unexplored.

This study aims to investigate the therapeutic efficacy of AAL in dextran sulfate sodium (DSS)-induced chronic colitis and elucidate its anti-inflammatory and barrier-protective mechanisms, with a specific focus on the FABP4/PPARγ/NF-κB signaling pathway.

The chemical composition of AAL was characterized using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Chronic colitis was induced in mice through three cycles of DSS administration, and the therapeutic effects of AAL were evaluated by assessing body weight, Disease Activity Index (DAI), colon length, and pathological alterations. Enzyme-linked immunosorbent assay (ELISA) was used to quantify inflammatory cytokine levels. Immunohistochemistry and Western blotting were performed to assess mucosal barrier proteins, including Mucin 2 (MUC2), zonula occludens-1 (ZO-1), and Occludin, as well as key signaling proteins such as fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activated receptor gamma (PPARγ), and phosphorylated P65 (p-P65). Proteomic analysis combined with Gene Set Enrichment Analysis (GSEA) was conducted to identify differentially expressed proteins and enriched pathways. The role of the FABP4/PPARγ/NF-κB axis was further validated using the PPARγ antagonist GW9662. Additionally, molecular docking and molecular dynamics simulations were employed to identify bioactive components in AAL and their interactions with FABP4 and PPARγ.

UPLC-QTOF-MS analysis identified 47 compounds in AAL, including flavonoids, terpenoids, and polyphenols. Bergaptol and corilagin were identified as major constituents with potential anti-inflammatory properties. AAL treatment significantly alleviated chronic colitis symptoms, as evidenced by reduced DAI scores, restoration of body weight, and improved colon length. Pathological and immunohistochemical analyses demonstrated that AAL preserved intestinal mucosal integrity by upregulating MUC2, ZO-1, and Occludin expression. Proteomic and GSEA analyses identified the FABP4/PPARγ/NF-κB pathway as a key target of AAL. Western blotting confirmed that AAL suppressed FABP4 expression, enhanced PPARγ levels, and reduced p-P65 expression, indicating inhibition of NF-κB activation. Notably, the therapeutic effects of AAL were abolished by GW9662, further validating the involvement of PPARγ signaling. Molecular docking and molecular dynamics simulations demonstrated strong binding affinities of bergaptol and corilagin to FABP4 and PPARγ, suggesting their role as active compounds responsible for AAL's therapeutic effects.

AAL effectively mitigates chronic colitis by preserving intestinal barrier integrity, suppressing inflammatory responses, and modulating the FABP4/PPARγ/NF-κB pathway. The bioactive compounds bergaptol and corilagin may contribute to these therapeutic effects, highlighting AAL as a promising natural therapeutic agent for ulcerative colitis.

Microbial alterations in different tumor locations of colorectal cancer (CRC) remain unclear. Here, 1,375 fecal metagenomes from six in-house and published datasets were analyzed, including 128 right-sided CRC (rCRC), 168 left-sided CRC (lCRC), 250 rectal cancer (RC), and 829 controls. Firmicutes progressively increase from rCRC, lCRC, to RC, in contrast to the gradual decrease of Bacteroidetes. Tumor location-associated fecal microbes are identified, including Veillonella parvula for rCRC, Streptococcus angionosus for lCRC, and Peptostreptococcus anaerobius for RC, while Fusobacterium nucleatum is enriched in all tumor locations. Tumor location-associated bacteria correlate with patient survival. Clinically, we establish a microbial biomarker panel for each tumor location that accurately diagnoses rCRC (area under the receiver operating characteristic curve [AUC] = 91.59%), lCRC (AUC = 91.69%), or RC (AUC = 90.53%) from controls. Tumor location-specific biomarkers also have higher diagnostic accuracy (AUC = 91.38%) than location-non-specific biomarkers (AUC = 82.92%). Overall, we characterize fecal microbes associated with different CRC tumor locations, highlighting that tumor location should be considered in non-invasive diagnosis.

With advances in sequencing techniques, microbiota dysbiosis and pathogenic microbes that accelerate colorectal cancer progression have been identified and widely reported. However, few studies have focused on the microbiota taxa of rectal mucus in rectal cancer (RC) patients. Here, we analyzed the composition and characteristics of the rectal mucosa microbiota of RC patients from Wenzhou city, China, and compared the results with those of healthy controls.

To explore the changes in the characteristics of the rectal mucosal flora associated with RC, and identify biomarkers of microbe taxa for RC.

Rectal mucosa samples from a Chinese cohort of 72 recently diagnosed RC patients and 71 healthy controls were obtained. A validation cohort, which included 22 RC patients and 60 healthy controls, was also established. Changes in the rectal mucosal flora were observed by cultivation, 16S ribosomal DNA gene sequencing analysis and quantitative polymerase chain reaction analysis.

The 16S ribosomal DNA results demonstrated that RC patients presented increased bacterial community richness and alpha diversity as well as an altered rectal mucosal microbiota, with depletion of Proteobacteria and Thermi and enrichment of Bacteroidetes and Fusobacteria in cancerous mucosal tissues (CM) and enrichment of Firmicutes and Cyanobacteria in adjacent noncancerous mucosal tissues (AM). The culture results showed that the mean loads of Escherichia coli, Bifidobacterium, Enterococcus, and Lactobacillus were significantly reduced in RC patients. The ratios of Prevotella to Ruminococcus [areas under the receiver operating curve: 0.795 in AM vs normal control mucosa (NM), 0.77 in CM vs NM] and of Prevotella stercorea to Propionibacterium acnes (areas under the receiver operating curve: 0.808 in AM vs NM, 0.843 in CM vs NM) exhibited excellent abilities to differentiate between healthy controls and RC patients.

RC patients have an altered rectal mucosal microbiota, and the ratio of Prevotella to Ruminococcus or the ratio of Prevotella stercorea to Propionibacterium acnes may serve as a marker for RC diagnosis.

Dry eye disease (DED) is a multifactorial condition with complex and incompletely understood molecular mechanisms. Advances in multi-omics technologies, including genomics, transcriptomics, proteomics, metabolomics, and microbiomics, have provided new insights into the pathophysiology of DED. Genomic analyses have identified key genetic variants linked to immune regulation and lacrimal gland function. Transcriptomic studies reveal upregulated inflammatory pathways in ocular surface tissues, implicating these as core drivers of chronic inflammation. Proteomic research highlights significant alterations in tear protein composition, especially proteins involved in inflammation and tissue repair. Metabolomics studies focus on disrupted lipid metabolism and oxidative stress, which are crucial in maintaining tear film stability. Furthermore, microbiome research has demonstrated reduced microbial diversity and increased pathogenic bacteria, exacerbating inflammatory responses. The integration of multi-omics data allows for the identification of novel biomarkers and therapeutic targets, enabling precision diagnostics and personalized treatments. Therefore, this review highlights the critical importance of multi-omics approaches in deepening our understanding of DED's complex molecular mechanisms and their potential to transform clinical management and therapeutic innovations in this challenging field.

Bacterial populations often have complex spatial structures, which can impact their evolution. Here, we study how spatial structure affects the evolution of antibiotic resistance in a bacterial population. We consider a minimal model of spatially structured populations where all demes (i.e., subpopulations) are identical and connected to each other by identical migration rates. We show that spatial structure can facilitate the survival of a bacterial population to antibiotic treatment, starting from a sensitive inoculum. Specifically, the bacterial population can be rescued if antibiotic resistant mutants appear and are present when drug is added, and spatial structure can impact the fate of these mutants and the probability that they are present. Indeed, the probability of fixation of neutral or deleterious mutations providing drug resistance is increased in smaller populations. This promotes local fixation of resistant mutants in the structured population, which facilitates evolutionary rescue by drug resistance in the rare mutation regime. Once the population is rescued by resistance, migrations allow resistant mutants to spread in all demes. Our main result that spatial structure facilitates evolutionary rescue by antibiotic resistance extends to more complex spatial structures, and to the case where there are resistant mutants in the inoculum.

Mapping the spatial locations of gut bacteria in their native environment enhances our understanding of bacteria-host interactions and the physiological and pathological roles these microbes play. However, the intricate composition of bacterial communities in millimeter-scale intestinal tissues presents a great challenge for in situ imaging of their spatial distributions. To address this, we introduce a three-dimensional (3D) imaging strategy that combines a fluorescent tetrapeptide (TetraAA-AcLys) metabolic labeling probe with a tissue clearing protocol. This method enables high-resolution visualization of the microbiota within intact colon, allowing for clear observation of the 3D distribution of gut bacteria across various sections, without interference from host tissues. Moreover, 3D quantitative analysis of the labeled bacteria in a enteritis model reveals their penetration into the mucus layer in colon, highlighting the technique's potential for studying gut microbiota biogeography in health and disease. This 3D imaging method offers valuable spatial insights into the dynamic relationship between the microbial community and its host.

Probiotics are pivotal in maintaining or restoring the balance of human intestinal microbiota, a crucial factor in mitigating diseases and preserving the host's health. Exploration into Bacteroides spp. reveals substantial promise in their development as next-generation probiotics due to their profound interaction with host immune cells and capability to regulate the microbiome's metabolism by significantly impacting metabolite production. These beneficial bacteria exhibit potential in ameliorating various health issues such as intestinal disorders, cardiovascular diseases, behavioral disorders, and even cancer. Though it's important to note that a high percentage of them are as well opportunistic pathogens, posing risks under certain conditions. Studies highlight their role in modifying immune responses and improving health conditions by regulating lymphocytes, controlling metabolism, and preventing inflammation and cancer. The safety and efficacy of Bacteroides strains are currently under scrutiny by the European Commission for authorization in food processing, marking a significant step towards their commercialization. The recent advancements in bacterial isolation and sequencing methodologies, coupled with the integration of Metagenome-Assembled Genomes (MAGs) binning from metagenomics data, continue to unveil the potential of Bacteroides spp., aiding in the broader understanding and application of these novel probiotics in health and disease management.

The translocation of bacteria from intestinal tracts into blood vessels and distal organs plays pivotal roles in the pathogenesis of numerous severe diseases. Intravital monitoring of bacterial translocation, however, is not yet feasible, which greatly hinders us from comprehending this spatially and temporally dynamic process. Here we report an in vivo fluorogenic labeling method, which enables in situ imaging of mouse gut microbiota and real-time tracking of the translocated bacteria. By mimicking the peptidoglycan stem peptide in bacteria, a tetrapeptide probe composed of alternating D- and L-amino acids and separately equipped with a fluorophore and a quencher on the N- and C-terminal amino acid, is designed. Because of its resistance to host proteases, it can be directly used in gavage and achieves fluorogenic labeling of the microbiota in the gut via the functioning of the L,D-transpeptidases of the labeled bacteria. Using intravital two-photon microscopy, we then successfully visualize the translocation of gut bacteria into the bloodstream and liver in obesity mouse models. This technique can help further exploration into the spatiotemporal activities of gut microbiota in vivo, and be valuable in investigating the less understood pathogenicity of bacterial translocation in many severe diseases.

Depression is a debilitating and poorly understood mental disorder. There is an urgency to explore new potential biological mechanisms of depression and the gut microbiota is a promising research area.

Our study was aim to understand regional heterogeneity and potential molecular mechanisms underlying depression induced by dysbiosis of mucus-associated microbiota.

Here, we only selected female macaques because they are more likely to form a natural social hierarchy in a harem-like environment. Because high-ranking macaques rarely displayed depressive-like behaviors, we selected seven monkeys from high-ranking individuals as control group (HC) and the same number of low-ranking ones as depressive-like group (DL), which displayed significant depressive-like behaviors. Then, we collected mucus from the duodenum, jejunum, ileum, cecum and colon of DL and HC monkeys for shotgun metagenomic sequencing, to profile the biogeography of mucus-associated microbiota along duodenum to colon.

Compared with HC, DL macaques displayed noticeable depressive-like behaviors such as longer duration of huddle and sit alone behaviors (negative emotion behaviors), and fewer duration of locomotion, amicable and ingestion activities (positive emotion behaviors). Moreover, the alpha diversity index (Chao) could predict aforementioned depressive-like behaviors along duodenum to colon. Further, we identified that genus Pseudomonas was consistently decreased in DL group throughout the entire intestinal tract except for the jejunum. Specifically, there were 10, 18 and 28 decreased Pseudomonas spp. identified in ileum, cecum and colon, respectively. Moreover, a bacterial module mainly composed of Pseudomonas spp. was positively associated with three positive emotion behaviors. Functionally, Pseudomonaswas mainly involved in microbiota derived lipid metabolisms such as PPAR signaling pathway, cholesterol metabolism, and fat digestion and absorption.

Different regions of intestinal mucus-associated microbiota revealed that depletion of genus Pseudomonas is associated with depressive-like behaviors in female macaques, which might induce depressive phenotypes through regulating lipid metabolism.

Growing evidence suggests that cancer should not be viewed solely as a genetic disease but also as the result of functional defects in the metaorganism, including disturbances in the gut microbiota (i.e., gut dysbiosis). The human microbiota plays a critical role in regulating epithelial barrier function in the gut, airways, and skin, along with host metabolism and systemic immune responses against microbes and cancer. Collaborative international networks, such as ONCOBIOME, are essential in advancing research equity and building microbiome resources to identify and validate microbiota-related biomarkers and therapies. In this review, we explore the intricate relationship between the microbiome, metabolism, and cancer immunity, and we propose microbiota-based strategies to improve outcomes for individuals at risk of developing cancer or living with the disease.

With the popularization of modern lifestyles, the spectrum of intestinal diseases has become increasingly diverse, presenting significant challenges in its management. Traditional pharmaceutical interventions have struggled to keep pace with these changes, leaving many patients refractory to conventional pharmaceutical treatments. Fecal microbiota transplantation (FMT) has emerged as a promising therapeutic approach for enterogenic diseases. Still, controversies persist regarding its active constituents, mechanism of action, scheme of treatment evaluation, indications, and contraindications. In this review, we investigated the efficacy of FMT in addressing gastrointestinal and extraintestinal conditions, drawing from follow-up data on over 8000 patients. We systematically addressed the controversies surrounding FMT's clinical application. We delved into key issues such as its technical nature, evaluation methods, microbial restoration mechanisms, and impact on the host-microbiota interactions. Additionally, we explored the potential colonization patterns of FMT-engrafted new microbiota throughout the entire intestine and elucidated the specific pathways through which the new microbiota modulates host immunity, metabolism, and genome.

SUMMARYThe development of multicellularity represents a key evolutionary transition that is crucial for the emergence of complex life forms. Although multicellularity has traditionally been studied in eukaryotes, it originates in prokaryotes. Coordinated aggregation of individual cells within the confines of a colony results in emerging, higher-level functions that benefit the population as a whole. During colony differentiation, an almost infinite number of ecological and physiological population-forming forces are at work, creating complex, intricate colony structures with divergent functions. Understanding the assembly and dynamics of such populations requires resolving individual cells or cell groups within such macroscopic structures. Addressing how each cell contributes to the collective action requires pushing the resolution boundaries of key technologies that will be presented in this review. In particular, single-cell techniques provide powerful tools for studying bacterial multicellularity with unprecedented spatial and temporal resolution. These advancements include novel microscopic techniques, mass spectrometry imaging, flow cytometry, spatial transcriptomics, single-bacteria RNA sequencing, and the integration of spatiotemporal transcriptomics with microscopy, alongside advanced microfluidic cultivation systems. This review encourages exploring the synergistic potential of the new technologies in the study of bacterial multicellularity, with a particular focus on individuals in differentiated bacterial biofilms (colonies). It highlights how resolving population structures at the single-cell level and understanding their respective functions can elucidate the overarching functions of bacterial multicellular populations.

The gut-liver axis plays a crucial role in maintaining metabolic balance and overall human health. It orchestrates various processes, such as blood flow, nutrient transfer, metabolite processing, and immune cell communication between the two organs. Traditional methods, such as animal models and two-dimensional (2D) cell cultures, are insufficient in fully replicating the intricate functions of the gut-liver axis. The emergence of microfluidic technology has revolutionized this field, facilitating the development of organ-on-a-chip (OOC) systems. These systems are capable of mimicking the complex structures and dynamic environments of the gut and liver in vitro and incorporating sensors for real-time monitoring. In this article, we review the latest progress in gut-on-a-chip (GOC) and liver-on-a-chip (LOC) systems, as well as the integrated gut-liver-on-a-chip (GLOC) models. Our focus lies in the simulation of physiological parameters, three-dimensional (3D) structural mimicry, microbiome integration, and multicellular co-culture. All these aspects are essential for constructing accurate in vitro models of the gut and liver. Furthermore, we explore the current applications of OOC technology in the study of the gut and liver, including its use in disease modeling, toxicity testing, and drug screening. Finally, we discuss the challenges that remain and outline potential future directions for advancing GOC and LOC development in vitro.