Environmental conditions strongly influence interactions between bacteria and bacteriophages (phages). Here, we examined how osmolality (solute concentration) shapes the in vitro co-evolution of T4 phage and its host Escherichia coli during serial passage. When evolved independently, we observed substantial fitness gains in both bacteria and phages, particularly in high-osmotic conditions. During co-evolution, however, fitness gains were limited, bacterial populations consistently evolved phage resistance, and several phage populations went extinct. Furthermore, the resistance mechanisms varied by osmolality. In lower osmolalities, mutations disrupted phage-binding sites, conferring strong resistance. In higher osmolalities, mutations led to increased colonic acid production, producing a mucoid phenotype with weaker resistance. Because mucoidy has been associated with increased bacterial virulence, these findings suggest that gut-relevant osmotic conditions may constrain evolutionary trajectories, favoring resistance strategies that are less effective against phage but potentially more virulent, with important implications for phage therapy design.Phages offer a promising alternative to antibiotics, but their safety and efficacy strongly depend on the environmental conditions where the bacteria and phages interact. In the human gut, for instance, solute concentrations can vary widely due to factors such as food intolerances or laxative use. In this study, we show that such variations significantly impact how bacteria and phages co-evolve. In particular, we find that in higher osmolalities, bacteria evolve phage resistance through mucoidy-a phenotype linked with increased bacterial virulence-rather than receptor loss. This highlights the need to consider environmental factors when developing phage therapies.

The gastrointestinal tract is a critical interface for microplastic and nanoplastic exposure, yet causal links to health outcomes remain uncertain. We outline current methodological challenges and provide a list of minimum standards for rigorous study design, standardized reporting and careful attention to interpretations to distinguish mechanism from signals.

Abstract While environmental gradients are known to result in heterogeneous distributions of bacterial species along the gastrointestinal tract, the spatial distribution of genetic diversity within these species remains poorly understood. Because bacterial genetic variants influence host traits like inflammation and metabolism, understanding their distribution is critical. Here, we analyze ~30 common gut commensals in germ-free mice colonized with the same healthy human stool. Unexpectedly, we find that while species composition varied significantly across gut regions, genetic diversity within species remained remarkably uniform. This uniformity is driven by similar strain frequencies along the gut lumen, indicating that genetically divergent strains can coexist without spatial segregation. Furthermore, ~60 evolutionary adaptations arising within the mice tend to sweep globally throughout the gut, showing little region-specificity. We observe similar dynamics in conventional mice and humans, suggesting that uniform bacterial genetic diversity is a conserved, robust feature of mammalian gut ecosystems.

It has become increasingly appreciated that gut microbes influence host stress hormone responses through direct and indirect mechanisms. These relationships may have broad implications on hormone bioavailability, receptor signaling, and stress resilience. In this review, we summarize current evidence for microbe-stress factor interactions and their consequences for host physiology. We further examine how microbiota-stress crosstalk may contribute to inflammatory bowel disease, highlighting emerging mechanisms and potential microbiota-targeted therapies.

The human gut is a dynamic environment, where changes in pH, oxygen, and osmolality influence microbiota composition and disease. Monitoring these environmental shifts is crucial for advancing gut health diagnostics and therapeutics, yet non-invasive monitoring tools remain limited. Genetically tractable commensals, including Bacteroides thetaiotaomicron, offer promising chassis for engineering biosensors but lack modular systems for precise sensing and reporting. Here, we developed genetic tools for B. thetaiotaomicron, including (1) repressible promoters for tunable fluorescent protein expression, (2) a DNA-based system to modulate repressor activity, (3) a modular, fluorescence-based transcriptional reporter circuit, and (4) an alternative plasmid integration mode. Using these components, we engineered biosensors to detect increased gut osmolality caused by malabsorption and validated them in vitro and in a murine model of laxative-induced osmotic diarrhea. These biosensors enabled long-term, non-invasive reporting of gut osmolality from single-cell fluorescence, demonstrating the potential of gut bacteria as monitoring platforms in gut health applications.

Abstract Inferring bacterial growth rates is fundamental to understanding microbial interactions and community dynamics, but remains difficult in natural settings where timepoints are limited or organisms are unculturable. In these cases, a widely used method is the origin-to-terminus ratio, or peak-to-trough ratio (PTR), which estimates DNA replication activity by comparing the copy number of DNA at the replication origin and terminus. While PTR correlates well with cellular growth in uniform, idealized environments, it measures replication rather than net growth rate, and thus reflects growth only when there is no cell death. Despite this, PTR is widely applied across a range of laboratory and environmental contexts, where microbial populations frequently experience fluctuating stress, mortality, and subpopulation heterogeneity. Given its widespread use in such settings, we developed a stochastic, cell-based model that explicitly tracks DNA replication and cell death to quantify how different patterns and levels of mortality affect the relationship between PTR and net growth rate. We found that PTR and net growth rate are tightly correlated in idealized conditions; however, systematic deviations emerge when death rates vary over time or across subpopulations. We experimentally validated these predictions by exposing Escherichia coli to osmotic shock or antibiotics, and measuring net growth rate (by spot plating and observing the change in colony counts over time) and DNA replication activity (from qPCR with primers for the origin and terminus). Consistent with the predictions from our model, PTR correlated strongly with net growth rate in standard rich media, but not under stress. Together, these results provide a mechanistic and quantitative framework that clarifies the biological conditions under which PTR can be interpreted as a proxy for net growth rate.

Biofilms are structured microbial communities, known for their electron transfer properties, which are essential for metabolic processes and microbial survival. Here, we investigated the electrogenic properties of Bacillus subtilis, a bacterial producer of electron-donating biofilms. Interdigitated gold electrodes were utilized to continuously measure the electrochemical activity of biofilm-forming B. subtilis cells and genetic mutants unable to create them (biofilm-deficient). The formation of extracellular polymeric substances (EPS) and filamentous appendages was monitored via scanning electron microscopy (SEM). Chronoamperometry was used to assess electrochemical activity, which showed fluctuations in electrical current at specific time points in biofilm-forming cells. Cyclic voltammetry (CV) revealed significant differences between the voltammograms of biofilm-forming and biofilm-deficient cells, hypothesized to be a result of the reduction of secreted flavodoxin. Electrochemical impedance spectroscopy (EIS) was also performed at various intervals and analyzed using an equivalent circuit. We identified the presence of a charge transfer resistance (Rct) exclusively in biofilm which correlated to the time of increased electrochemical activity measured using chronoamperometry. Finally, through confocal microscopy, we found that the expression of a gene involved in biofilm matrix formation, tasA, was correlated with the time when charge transfer was measured. These results indicate that electrochemical activity is primarily present in biofilm-forming cells rather than in biofilm-deficient mutants. By combining electrochemical and microscopic methods, we developed a methodology to continuously monitor the stages of biofilm formation and showed that electrochemical activities within biofilms vary over time and there is a temporal relationship between these processes and the expression of genes responsible for biofilm development.

The microbiome has critical roles in human health and disease. Advances in high-throughput sequencing and metabolomics have revolutionized our understanding of human gut microbial communities and identified plausible associations with a variety of disorders. However, microbiome research remains constrained by challenges in establishing causality, an over-reliance on correlative studies, and methodological and analytical limitations. Artificial intelligence (AI) has emerged as a powerful tool to address these challenges; however, the seamless integration of preclinical models and clinical trials is crucial to maximizing the translational impact of microbiome studies. This manuscript critically evaluates best methodological practices and limitations in the field, focusing on how emerging AI tools can bridge the gap between microbial insights and clinical applications. Specifically, we emphasize the necessity of rigorous, reproducible methodologies that integrate multiomics approaches, preclinical models and clinical trials in the AI-driven era. We propose a practical framework for applying AI to microbiome studies, alongside strategic recommendations for clinical trial design, regulatory pathways, and best practices for microbiome-based informed diagnostics, AI training and clinical interventions. By establishing these guidelines, we aim to accelerate the translation of microbiome research into clinical practice, enabling precision medicine approaches informed by the human microbiome. Artificial intelligence (AI) is a powerful tool that could be applied to microbiome research. This Perspective discusses best practices and current limitations with the application of AI in microbiome data research, giving insights into future use and practical advice and recommendations on its use.

Wrinkled patterns in biofilms arise from buckling instabilities triggered by stresses that accumulate as growth is constrained by a stationary substrate. While nutrient availability, friction, and adhesion each influence wrinkling, their combined effects remain poorly understood. Here, we address this gap using a lattice-network model of biofilm morphogenesis. Under constant nutrient supply, wrinkles initiate at the center, where stresses are highest and isotropic, regardless of the level of friction or adhesion. Stronger adhesion delays wrinkling and decouples the length scale governing the buckling instability from the overall biofilm size. Heterogeneous adhesion lowers the critical stress by triggering wrinkles in weakly adhered regions, with the effect modulated by friction and the average adhesion. Under nonuniform nutrient supply, our model predicts that wrinkle initiation shifts from the center to the edge as initial nutrient availability decreases, a transition we experimentally validate using E. coli biofilms.

Industrialized lifestyles are associated with increased risk of chronic inflammatory disorders and reduced microbiota diversity compared to traditional populations. Laxative treatments (LT) reduce microbial diversity, yet the effects of LT on autoimmune disease remain unknown. We hypothesized that LT may exacerbate experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We found that the microbiota of adult offspring of laxative treated (LTO) parents was significantly altered, including loss of Muribaculaceae intestinale (Mb) and several Clostridia species. Further, the severity and incidence of EAE was reduced in LTO mice compared to offspring from parents with no LT (NTO). At peak disease, CD4+ T cells from the central nervous system (CNS) of NTO mice expressed significantly more IFNγ, IL-17A, and GM-CSF compared to LTO mice and CNS microglia (CD45midCD11b+P2RY12+) from NTO mice had higher expression of the proliferation marker Ki67 and the damage response marker Clec7a. These data indicate that recruitment and activation of CNS immune cells is impaired in LTO mice. Adding Mb isolates to adult LTO mice prior to EAE induction increased EAE disease severity compared to LTO mice. Finally, LT in NTO prior to EAE does not ameliorate disease, suggesting that immune education in the presence of Mb and Clostridia species, and the physical presence of Mb in adult mice, prime inflammatory pathways that enhance susceptibility to autoimmune disease. Research in the Osborne lab is supported by the Canadian Institutes of Health Research, Multiple Sclerosis Canada, the Weston and Praespero Foundations. SP is supported through an endMS Doctoral Award. Neuroimmunology (NEUR)

The landscape of clinical microbiome research has dramatically evolved over the past decade. By leveraging in vivo and in vitro experimentation, multiomic approaches and computational biology, we have uncovered mechanisms of action and microbial metrics of association and identified effective ways to modify the microbiome in many diseases and treatment modalities. This Review explores recent advances in the clinical application of microbiome research over the past 5 years, while acknowledging existing barriers and highlighting opportunities. We focus on the translation of microbiome research into clinical practice, spearheaded by Food and Drug Administration (FDA)-approved microbiome therapies for recurrent Clostridioides difficile infections and the emerging fields of microbiome-based diagnostics and therapeutics. We highlight key examples of studies demonstrating how microbiome mechanisms, metrics and modifiers can advance clinical practice. We also discuss forward-looking perspectives on key challenges and opportunities toward integrating microbiome data into routine clinical practice, precision medicine and personalized healthcare and nutrition.

Wrinkling is a striking emergent behavior that occurs in microbial biofilms across many species. The phenomenon originates from an intricate interplay between environmental factors, cell-to-cell phenotypic heterogeneity, and mechanical forces, thus requiring insights from multiple disciplines, from biology through chemistry to physics, to be fully understood. We critically review current knowledge about wrinkle formation in biofilms, starting with an analysis of the shared and distinct features that characterize this morphology across different species and the potential evolutionary advantages associated with it. Leveraging the vast literature on Bacillus subtilis, we then focus on its biofilms to discuss in detail the molecular mechanisms and regulation of wrinkle formation, along with the environmental factors that impact this phenotype. We follow by summarizing the insights gained from theoretical and modeling work on the mechanical origin of wrinkle formation, and the related experimental studies that have attempted to measure the material properties of different biofilms. We conclude with a synthesis of the many physical, chemical, and biological factors at play and a discussion of the remaining open questions around complex architectures in biofilms.

The human gut is a highly dynamic physical environment where perturbations - including factors such as acidification, oxygenation, and particle concentration (osmolality) - can influence microbiota composition and contribute to disease states. Understanding gut environmental changes is essential for advancing diagnostic and therapeutic strategies for gut health. However, non-invasive methods for continuous monitoring remain limited. The bacterial gut microbiota represents a powerful platform for continuous, non-invasive biosensing technologies for the gut environment, with genetically tractable commensal species like Bacteroides thetaiotaomicron (B. theta) emerging as promising hosts for engineered biosensors. However, the availability of genetic tools for precise, modular environmental sensing and reporting control in B. theta remains limited. Here, we present an expanded genetic engineering toolkit for B. theta that enables precise, fluorescence-based environmental sensing of the gut environment. This toolkit includes (i) three libraries of orthogonally inducible promoters capable of driving fluorescence expression, (ii) a DNA-based system to tune repressor activity in B. theta, (iii) a resulting modular transcriptional reporter circuit that integrates native promoter activation with fluorescent outputs, and (iv) characterization of a novel plasmid integration mode in B. theta. To demonstrate the utility of these tools, we engineered biosensors for gut malabsorption, a condition characterized by increased luminal osmolality. Using identified osmolality-responsive native promoters from B. theta, we made biosensors capable of detecting changes in gut physiology through graded fluorescent outputs. These biosensors were validated both in vitro and in vivo using a murine model of laxative-induced malabsorption, where they enabled near real-time, non-invasive monitoring of single-cell response from fecal samples with sensitivity to subclinical malabsorption levels. By expanding the genetic toolkit for B. theta and demonstrating its use in a physiologically relevant context, this approach highlights the potential of engineered gut bacteria as a monitoring platform for diverse gut health applications. This work advances strategies for microbial biosensing and positions gut commensals as key players in next-generation diagnostic methods. ### Competing Interest Statement The authors have declared no competing interest.

We discuss the opportunity for public health microbiome curricula to bridge the gaps in knowledge that exist between microbiome researchers and the lay public. We propose equipping public health professionals, important facilitators of public outreach and behaviour change, with three public health curriculum modules focused on breastfeeding, antibiotics and diet. These modules shift the focus from microbes as pathogens to potential partners in promoting health across the life course. Current public health messages cover only the 'tip of the iceberg' in exploring mechanisms, and this microbiome curriculum dives below the surface to provide fresh perspectives. These microbiome insights allow us to move beyond a focus on microbes as pathogens to understand the numerous collaborative roles played by the microbiome in producing health, and the upstream factors influencing the microbiome, thereby offering mechanistic insights that can be harnessed for public health education.

Environmental gradients exist throughout the digestive tract, driving spatial variation in the membership and abundance of bacterial species along the gut. However, less is known about the distribution of genetic diversity within bacterial species along the gut. Understanding this distribution is important because bacterial genetic variants confer traits important for the functioning of the microbiome and are also known to impart phenotypes to the hosts, including local inflammation along the gut and the ability to digest food. Thus, to be able to understand how the microbiome functions at a mechanistic level, it is essential to understand how genetic diversity is organized along the gut and the ecological and evolutionary processes that give rise to this organization. In this study, we analyzed bacterial genetic diversity of approximately 30 common gut commensals in five regions along the gut lumen in germ-free mice colonized with the same healthy human stool sample. While species membership and abundances varied considerably along the gut, genetic diversity within species was substantially more uniform. Driving this uniformity were similar strain frequencies along the gut, implying that multiple, genetically divergent strains of the same species can coexist within a host without spatially segregating. Additionally, the approximately 60 unique evolutionary adaptations arising within mice tended to sweep throughout the gut, showing little specificity for particular gut regions. Together, our findings show that genetic diversity may be more uniform along the gut than species diversity, which implies that species presence-absence may play a larger role than genetic variation in responding to varied environments along the gut.

In the United States, an estimated 14 million colonoscopies are performed yearly, each requiring patients to undergo bowel preparation, a laxative cleansing of the intestine’s luminal contents. Despite its widespread use, the effects of bowel preparation on gut physiology and susceptibility to pathogens remain poorly understood, particularly in individuals with compromised gut health. Using mouse and in vitro models, we find that bowel preparation with the laxative polyethylene glycol rapidly disrupts the gut, transiently increasing susceptibility to infection by Salmonella Typhimurium, including a non-motile mutant, and by gut pathobionts derived from ulcerative colitis microbiota. Bowel preparation also facilitates bacterial translocation to extraintestinal sites (mesenteric lymph nodes, liver, and spleen) and exacerbates inflammation in a chemically induced colitis model. Although these findings are preclinical, they suggest that bowel preparation may have underappreciated risks in vulnerable populations and warrant further clinical investigation.

Bacteriophages, or phages, are viruses that infect bacteria and offer a promising approach to combating antimicrobial resistance in the gut by selectively targeting harmful bacteria while preserving the broader microbiota. This study examines how variations in osmolality (solute concentration), which can occur in the gut due to factors such as food intolerances or laxative use, affect the in vitro co-evolution of T4 phage and its host Escherichia coli. When evolved independently, we observed substantial fitness gains in both bacteria (growth rate) and phages (productivity), especially at higher osmolalities. During co-evolution, phage resistance emerged in all co-evolved bacterial populations. However, the resistance mechanism varied by osmolality: in lower osmolalities, mutations disrupted phage binding sites, conferring strong resistance to phages. Conversely, in higher osmolalities mutations led to increased colonic acid production, resulting in a mucoid phenotype associated with weaker phage resistance. This bacterial phenotype has been linked with increased bacterial virulence, underscoring the need to consider environmental factors when designing phage therapies. ### Competing Interest Statement The authors have declared no competing interest.