Cell Host & Microbe

The mechanisms by which maturation of the infant gut microbiota promotes resistance to pathogen colonization remain poorly understood. Infant botulism, a potentially fatal disease caused by intestinal colonization by Clostridium botulinum, provides a striking example of age-dependent susceptibility to infection1. Although the gut microbiota has long been implicated in protection against C. botulinum2-6, the responsible bacterial species and underlying mechanisms have yet to be elucidated. Here, we show that acquisition of Stickland-metabolizing Clostridia during infant gut microbiota maturation confers resistance to C. botulinum colonization through competition for shared amino-acid-dependent nutritional niches. In human fecal microbiota-transplanted mice, longitudinally collected infant microbiotas exhibited a clear transition from susceptibility to resistance. Intestinal metabolomic analysis identified 5-aminovalerate as a hallmark metabolite of the resistant microbiota, implicating Stickland metabolism, an amino acid metabolic pathway also utilized by C. botulinum. Guided by this finding, metagenomic analysis revealed enrichment of Stickland-metabolizing Clostridia in resistant microbiotas, including Clostridioides difficile, a bacterium frequently carried by healthy infants. Intestinal metabolic signatures of C. botulinum and infant-derived Stickland-metabolizing Clostridia suggested competition for shared amino-acid-dependent nutritional niches. Consistent with this model, C. difficile suppressed C. botulinum expansion through nutrient competition. Together, these findings identify nutritional niche competition as a mechanism by which microbiota maturation promotes resistance to C. botulinum colonization. This work demonstrates how acquisition of specific microbial metabolic functions during early life can shift the gut microbiota from a susceptible to a resistant state.

Clostridioides difficile is an urgent threat to human health. Current treatments for C. difficile infections (CDIs) are antibiotics and microbiome restoration therapy (MRT) for recurrent cases. However, antibiotics contribute to antibiotic resistance and recurrent CDIs and the long-term sustainability and accessibility of MRTs remains to be determined. Since a dysbiotic gut microbiome is the primary risk factor for CDI, a better understanding of the interactions between C. difficile, the microbiome, and the host will aid development of treatments with improved precision. Emerging evidence supports that butyrate, a prominent end product of gut microbiome metabolism, is a key determinant of C. difficile pathogenesis. Notably, C. difficile releases more of its toxins TcdA and TcdB in butyrate-rich environments. Here, we demonstrate that butyrate-dependent toxin release is not driven by two previously characterized modes of toxin release (e.g., TcdE-dependent secretion or Cwp19-dependent autolysis). Instead, butyrate enhances the expression of a broadly conserved endolysin (EndD), which is responsible for butyrate-dependent toxin release. We additionally demonstrate that endD-dependent toxin release does not universally occur under all growth conditions and that its expression is dependent on the late-stage sporulation sigma factor SigK. Overall, our findings provide deeper insight into butyrate-dependent effects on C. difficile pathogenesis and set the stage for future work to better understand the molecular and genetic underpinnings of endD regulation.

Infancy is a critical developmental window during which the gut ecosystem assembles and helps train the immune system, thereby setting trajectories for lifelong health. Bacteria and viruses are equally numerous in this early ecosystem, yet the gut viromes composition, dynamics, and health relevance remain poorly understood. Here, we show that the infant gut virome is diverse, dynamic, and linked to health outcomes. We performed comprehensive virome profiling of 1,110 longitudinal fecal samples from 314 mother-infant pairs from the Dutch birth cohort Lifelines NEXT using both virus-like particle enrichment (VLP) and total metagenomic sequencing (MGS). We find only 18.9% compositional overlap between the VLP- and MGS-metaviromes, with VLP recovering the active virome and most novel species and MGS predominantly capturing temperate phages. By combining both methods, we identified 8,348 novel virus species spanning diverse hosts, from bacteria to humans, and all major viral genome types (dsDNA, ssDNA, and RNA). We find that bacteriophages frequently encode metabolic functions, including genes related to B vitamin metabolism. We further observe that the development of the infant gut virome is shaped by both host factors, including delivery mode and feeding practices, and continuous switching of temperate phage lifecycles. Notably, the relative abundance of induced temperate phages is also associated with eczema development within the first year of life. Together, these findings establish the infant gut virome as a dynamic and clinically relevant component of early-life microbial development and highlight how comprehensive dual-method profiling is a necessary framework for future virome research.

Many commensal bacteria that peacefully reside in the human microbiome are also able to cause acute opportunistic infections. Emerging evidence suggests that within-host evolution contributes to infection, but the genetic mechanisms facilitating the progression of opportunistic pathogens from carriage to acute infection remain unknown. Here, we prospectively collected native samples from four microbiome niches of 13 critically ill patients to assess the evolutionary dynamics leading up to infection. Among three patients we have observed eleven healthcare-associated infections (HAI) caused by nine pathogen species. Leveraging a culture-based approach, we demonstrate that the microbiome is frequently (73%) colonized by the pathogen lineage already before or at the time of diagnosis. Moreover, we identify a short-lived, non-synonymous mutation (F126L) within the fimbriae regulator gene fimZ of Enterobacter hormaechei, first detectable within the gut and subsequently associated with HAI before becoming replaced by body-wide sweeps of independent treatment-associated mutations. Despite fimZ [F126L] being globally undetected, we can show in vitro and in vivo that the F126L mutation leads to elevated biofilm formation, cell adhesion and virulence, suggesting a role during HAI. Our work highlights the power of prospective, population-wide investigation of pathogens to elucidate rapid evolution linked to disease.

HLA-DQ2/8 haplotypes are established genetic risk factors for autoimmune diseases and are known to influence gut microbiota assembly in early life. However, their impact on the adult microbiome and functional consequences for host physiology remain unclear. Here, we performed a genotype-stratified multi-omics analysis of 60 healthy adults, including 28 HLA-DQ2/8 carriers and 32 non-carriers. We found that host HLA-DQ2/8 genotype was significantly associated with gut microbiome composition, with an effect size exceeding that of sex and BMI. HLA-DQ2/8 carriers exhibited higher gut microbial alpha-diversity, lower virulence factor abundance, and a distinct species profile enriched in butyrate-producing taxa. We identified pervasive intra-species phylogenetic and functional divergence linked to the DQ2/8 genotype. This diversification reflects predicted HLA-restricted microbial peptide-binding specificities, suggesting a possible role for antigen presentation-mediated immune selection, a mechanism further supported by AlphaFold3 structural modeling. We found an enrichment of microbial pathways for pantothenate and coenzyme A (CoA) biosynthesis in carriers, primarily driven by functionally divergent Blautia obeum strains. This functional shift paralleled lower levels of serum pantothenate and HDL-cholesterol in the host. Our findings suggest a potential genotype-microbiome-host axis where antigen presentation-mediated immune selection may modulate microbial adaptation, with possible implications for the host availability of essential cofactor precursors and lipid metabolism.

Bdellovibrio and like organisms (BALOs) are obligate bacterial predators that shape microbial communities by promoting species diversity, yet they have long been considered irrelevant to the human gut due to their presumed obligate aerobic lifestyle. Here, we challenge this view through a combined meta-analytic, experimental, and conceptual investigation of BALOs in human microbiomes. Reanalyzing 168,000 consistently processed samples from the Human Microbiome Compendium spanning 482 studies, we detected BALOs in more than 80 studies and across multiple body sites worldwide, with a gut prevalence of 2.4%, a finding confirmed by reanalysis of the PRIME database for 16S rRNA microbiome data. Strikingly, BALO presence was consistently associated with higher microbial alpha-diversity across body sites and disease contexts. Biopsy-derived samples showed a substantially higher prevalence than fecal samples, suggesting a mucosa-proximal niche. Our laboratory experiments showed that multiple Bdellovibrio strains can delay the loss of microbial diversity in vitro and remain active under gut-relevant conditions, including 37C, pH 6.5, and in the presence of mucus. Genomic analyses further revealed terminal reductases, including nitrite reductases, in several BALO genomes, indicating the capacity for anaerobic or microaerobic respiration, consistent with persistence in mucosal microenvironments. Notably, the metabolic and ecological profiles of cultured BALOs closely match those of facultative anaerobes, which constitute their preferred prey and are central drivers of dysbiosis in inflammatory bowel disease, diabetes, colorectal cancer, and chronic kidney disease. Building on these findings, we propose a conceptual framework in which BALOs contribute to gut homeostasis by controlling the expansion of facultative anaerobes under inflammatory conditions, thereby facilitating the restoration of fermentative, butyrate-producing communities. Together, our results establish BALOs as consistent, functionally relevant members of the human microbiome and a promising natural candidate for therapeutic strategies targeting chronic gut disease.

COVID-19 vaccines markedly reduce disease severity, but their ability to block infection and transmission remains limited and variable.1 A better understanding of early mucosal immune programs that constrain viral replication at susceptible upper respiratory sites is needed to develop more effective antiviral strategies. However, temporal and anatomical antiviral dynamics are difficult to resolve without longitudinal, paired-site sampling beginning before infection onset. We quantified longitudinal viral load by RT-qPCR and human gene expression by mRNA sequencing in 1,237 samples prospectively collected daily from the nasal cavity, oral cavity, and oropharynx of 16 individuals starting from the onset of naturally acquired SARS-CoV-2 infection, and 16 age-, sex-, and vaccination-matched uninfected individuals. Here we show that Type I interferon (IFN) responses are initiated concurrently across these upper respiratory sites, even before local viral detection. In contrast, Type II IFN initiation is more spatially variable, and earlier nasal Type II IFN initiation is associated with reduced viral replication, prior COVID-19 vaccination, and higher tissue-resident memory T cell (TRM) signature expression. These findings demonstrate that in addition to humoral immunity, prior vaccination primes rapid, inducible mucosal Type II IFN responses, likely mediated by rare TRM upon viral encounter, to limit viral replication and spread.

Commensal microbes in the gastrointestinal tract are central to host health, yet they must adapt to frequent perturbations such as intestinal inflammation that challenges microbial homeostasis. A major challenge during inflammation is exposure to host-derived reactive nitrogen species (RNS), which damage macromolecules and impair microbial fitness, but how commensals orchestrate defense against nitrosative stress remains poorly defined. Here, we show that Bacteroides thetaiotaomicron mounts a protective RNS-defense program centered on the hybrid cluster protein Hcp, which is required for fitness under nitrosative stress. We identify a nitrite-responsive SnoA locus (Stress-responsive Nitric Oxide regulator A) that promotes HcpR-dependent hcp expression. In vivo, this pathway promotes commensal resilience in both an antibiotic-perturbed, Nos2-dependent model of intestinal nitrosative stress and during Salmonella-induced gut inflammation. Together, our findings identify a regulatory pathway that enables a dominant gut commensal to withstand host-derived nitrosative stress and persist during intestinal inflammation.

Antibiotics (ABXs) represent the current standard of care for treating Clostridioides difficile infection (CDI). Paradoxically, ABX-induced dysbiosis is the primary risk factor for CDI, as disruption of the colonic microbial ecosystem creates an opportunity for C. difficile colonization. Given that ABXs can also alter immune responses, we investigated whether ABXs prime the colonic immune milieu for CDI susceptibility. Here, we implicate ABXs in driving CDI severity through the emergence of pathogenic CCR5-reliant immune populations in the mouse colon. High-throughput immune cell profiling revealed that ABXs shift the colonic immune compartment toward a CCR5-associated type I immunity signature, marked by an expansion of CCR5+ ILC1s and CCR5+ Th1 cells. A partial genetic deletion of CCR5 reversed CDI severity, alleviating colonic inflammation and improving survival. Pharmacological inhibition of the CCL3/4/5-CCR5 circuit also recapitulated these favorable disease outcomes, which we attribute to reduced colonic CCR5+ ILC1, CCR5+ Th1, and CCR5+ CD8 T cell populations during CDI. Together, our findings extend beyond dysbiosis as the canonical CDI risk factor and establish ABX-induced immune imbalance as an underappreciated determinant of CDI susceptibility.

The gut microbiome produces numerous metabolites that influence mammalian health. While microbiome composition and diet influence metabolite concentrations, how these factors interact remains incompletely defined. Here we find production of imidazole propionate (ImP), a microbial metabolite associated with cardiometabolic and neurodegenerative diseases, is determined by the balance of competing metabolic pathways that catabolize histidine to ImP or short-chain fatty acids (SCFAs). We show glutamate serves as a preferred substrate that selectively inhibits histidine conversion to SCFAs, redirecting flux to increased ImP production across mouse- and human-derived microbial communities. We find dietary monosodium glutamate (MSG) acting via this mechanism boosts ImP production in the mouse gut, transiently impairing glucose tolerance and increasing systemic ImP. These findings show that predictable interactions between dietary substrate and microbial competition control systemic ImP levels, providing a mechanistic framework for understanding microbiome metabolite production more broadly.

The intestinal tract is a reservoir for Extended-Spectrum {beta}-Lactamase (ESBL)-producing Escherichia coli. Asymptomatic gut colonization by these pathobionts represents a major risk for extraintestinal infections. Despite clinical relevance, the genetic basis of gut colonization by ESBL E. coli remains poorly understood. Here, we determined how the microbiota shapes the fitness landscape of diverse ESBL E. coli strains, defining the functional requirements for intestinal colonization. In microbiota-depleted hosts, colonization relies mostly on metabolic functions. In contrast, in mice harbouring a microbiota, pathoadaptive functions associated with adhesion and biofilm formation are dominant determinants of E. coli fitness, together with accessory virulence functions. Consistent with these observations, experimental evolution in mice reveals convergent adaptation of ESBL E. coli to the presence of a complex microbiota through enhanced adhesion. These findings establish the microbiota as a major ecological driver of pathoadaptation in antibiotic-resistant pathobionts.

Molecular hydrogen (H2) and hydrogen sulfide (H2S) are central gut metabolites that shape microbial metabolism and affect host health. In Crohns disease (CD), the shift in microbiota composition ( dysbiosis) is associated with intestinal accumulation of these gases, but the responsible microbes remain poorly resolved. Here, we analysed 4,644 bacterial and archaeal species-level genomes from the Unified Human Gastrointestinal Genome Collection to identify H2-cycling microbes, assessed their prevalence in ca. 1,700 stool metagenomes from healthy and diseased individuals, and validated their activity using culture-based incubations of stool isolates and biopsy samples. Approximately half of all species encoded H2-producing abilities, with acetate- and propionate-forming fermenters such as Phocaeicola and Bacteroides dominating healthy cohorts, whereas comparatively few taxa, including Escherichia and Megamonas, encoded H2 consuming abilities. In CD, H2 producers became more abundant but less diverse, favouring species with multiple H2-evolving hydrogenases and more fermentation routes, especially Clostridium and Enterocloster species. Consistently, isolates enriched in CD produced H2 faster and at higher concentrations than health-associated isolates. Increased H2S-producing capacity in CD was driven mainly by these H2-producing fermenters carrying anaerobic sulfite reductases (Asr), rather than sulfate-reducing bacteria, and was supported by elevated H2S production in Asr-positive isolates, likely providing an additional electron sink. These findings provide a species-resolved view of gut gas metabolism and implicate metabolically flexible fermenters in excessive gas and sulfide production in gut disorders.

Pneumonia is a leading cause of death globally and disproportionately affects children in lower- and middle-income countries. To explore microbial and immune correlates of disease and death, we performed metagenomic sequencing of upper respiratory tract (URT) microbiome in 309 children in Mali with pneumonia and 150 age- and season- and site-matched controls. We show that the URT microbiome matures throughout early life and is influenced by breastfeeding. URT microbiome maturation was disrupted during pneumonia resulting in loss of commensal species and expansion of pathobionts, which was linked to disease severity and death. Analysis of serum antibody levels revealed that low levels of passively acquired antibody from mothers, deficient antibody responses to RSV, and persistent autoantibody to cytokines were associated with pneumonia mortality in an age-dependent manner. These findings underscore the complex nature of pneumonia and identify microbial and immune factors for risk stratification and therapeutic interventions in pediatric pneumonia.

The Pseudomonas syringae species complex includes major crop pathogens that use a type III secretion system (T3SS) to inject effectors into plant cells, suppressing immunity and promoting disease. The cherry canker pathogen Pseudomonas amygdali pv. morsprunorum (Pam) carries the effector gene hopAR1 on a prophage, PamPP1, which belongs to a novel Caudoviricetes family widespread across the P. syringae complex and likely acquired before pathovar divergence. Deletion of PamPP1 shows that this prophage enhances Pam virulence independently of hopAR1, instead it alters the T3SS operon expression both in vitro and in planta. These prophage-driven transcriptional changes likely reshape how Pam interacts with plant immunity, highlighting how bacteriophages rewire bacterial transcriptomes and contribute to the evolution and emergence of plant diseases.

Cholera causes severe diarrheal illness in young children, but the mechanisms underlying age-dependent susceptibility remain unclear. Experimental cholera in neonatal mice recapitulates age-dependent susceptibility: suckling mice are susceptible to Vibrio cholerae colonization and cholera toxin (CT)-dependent disease, whereas adult mice are not readily colonized and do not develop cholera-like disease. Here, we define a developmental window in which susceptibility declines sharply over the first two postnatal weeks. Maternal antibiotic exposure disrupted vertical transmission of maternal microbiota to offspring and altered distal small intestinal microbiota assembly, extending the window of susceptibility to CT-dependent V. cholerae colonization and disease. Pups born to antibiotic-treated dams exhibited reduced Lactobacillaceae and increased Enterobacteriaceae, and reintroduction of an endogenous Lactobacillus isolate restored offspring lactobacilli levels and reestablished resistance to experimental cholera at two weeks of age. Consistent with a direct protective role, increasing lactobacilli in susceptible neonatal mice reduced experimental cholera burden, and cultures of the endogenous Lactobacillus isolate as well as spent culture media acidified the in vitro growth environment and rapidly eliminated recoverable V. cholerae. Together, these findings identify vertical transmission of maternal microbiota to offspring and lactobacilli-associated antagonism as determinants of early-life resistance to cholera.

Intra-species genomic variation results from diversity-generating processes and supplies the raw material for subsequent natural selection. Environmental stress can be regarded as the ultimate accelerator of these processes, especially for microorganisms, which can alter their DNA if presented with nutrient limitation, toxins, or pathogen attack. Chronic intestinal inflammation, as in inflammatory bowel diseases (IBD), may be regarded as prolonged environmental stress for gut commensal bacteria, bringing a large number of enteric species down to undetectable levels. However, it remains unclear how the microbes that survive the IBD gut environment actually respond to IBD stress, and whether their stress response may leave a transient or permanent signature in their genomes. To investigate whether IBD stress induces and selects for certain genetic diversity, we performed metagenomic analyses on gut species in IBD patients and Controls. We focused on strain diversity within a single individual, which might be the result of more recent diversification processes under stress. We found measurable differences at the genome level between IBD and Controls, yet this was species-dependent. We then investigated gene-level diversity and found that certain functions were more likely to be enriched with either neutral divergence, functional divergence, or both. Functions that were enriched in IBD with both kinds of diversity were associated with motility and iron-scavenging, among others. These results may point towards functions that are under selection in the context of IBD stress, and could inform future mechanistic work, exploring previously unknown routes of bacterial diversification and adaptation to stress in the gut microbiome.

The vertebrate digestive tract harbors complex microbial communities whose influences on phenotypes and fitness in non-model organisms remain poorly understood. Here, we show that colonization with gut microbiota is critical for digestive development and function in a non-model songbird, the House sparrow (Passer domesticus). We raised nestlings under sterile (axenic) conditions and compared them to nestlings reconstituted (conventionalized) with microbiota from adult sparrows. Conventionalization drove significant increases in villus length, crypt depth, goblet cell density, and mucosal thickness in the small intestine. Conventionalized nestlings also exhibited increased growth of several digestive organs and elevated circulating bile acid levels, including bacterial metabolites known to promote growth and lipid metabolism in vertebrates. These results reveal functions of songbird microbiota and establish axenic methods for non-model oviparous vertebrates.

Bifidobacterium longum (Bl.) is a key early-life gut symbiont, yet its evolutionary origin and mechanisms underlying the global biogeographic distribution of its subspecies remain poorly resolved. Here, we compiled a global genomic atlas of >7,000 MAGs/genomes from infants, domesticated animals, non-human primates, and ancient humans. High-resolution phylogenomic and functional analyses expanded infant-associated subspecies to five. Compared with non-human primates, B. longum was more prevalent in domesticated animals and ancient humans dating from 150 to 1,500 years ago. Moreover, human- and livestock-derived lineages from the same geographic regions clustered together, suggesting potential host-associated transmission. Ecologically, Bl. infantis and Bl. longum predominated in non-Western and Western infants, respectively, independent of breastfeeding, delivery mode, or antibiotic exposure. Instead, their distribution was associated with co-occurring microbes and HMO-driven cross-feeding interactions. These findings explain subspecies differentiation in the gut microbiota of Western and non-Western infants and provide a framework for community-mediated interventions in early life.

Understanding how zoonotic pathogens diversify across reservoir hosts remains a central question in evolutionary biology and infectious disease research. Here, we address this challenge using Leptospira interrogans, a globally distributed bacterial pathogen with an extremely wide range of animal reservoirs, as a model. After defining 13 distinct genogroups that largely align with serogroups, we selected the strongly host-adapted rodent-associated lineage, which is more frequently associated with fatal outcomes in patients, and the cattle-associated lineage for further analysis. A comprehensive approach integrating multi-omics analyses and host-specific infection assays showed that these two genogroups have followed distinct evolutionary trajectories associated with host specialization. Genomics demonstrated that specialized genogroups are genetically less diverse, characterized by divergence in membrane and signaling genes, and by the acquisition of host-adaptive functions. Changes in gene expression and protein production revealed distinct regulatory programs, predominantly affecting virulence pathways in rodent-borne lineage and stress responses in cattle-borne lineage. Consistently, rodent-borne lineage causes greater disruption of human epithelial barrier integrity and elicits an attenuated host-dependent macrophage inflammatory response relative to cattle-borne lineage. Collectively, these findings reveal distinct host-adaptive strategies and remarkable evolutionary plasticity in a major zoonotic bacterium, highlighting the central role of intraspecies heterogeneity in shaping host specialization.

Background Colonisation resistance provided by the gut microbiota is a critical barrier to pathogen invasion, yet its study in vivo is constrained by the complexity and cost of vertebrate models. Here, we developed a humanised Galleria mellonella infection model by inoculating wax moth larvae with complex human faecal microbiota. 16S rRNA gene sequencing confirmed stable, reproducible establishment of a diverse human associated community across larvae over four days. Results Humanised larvae exhibited colonisation resistance against Salmonella enterica serovar Typhimurium, with mortality reduced to 20% compared to 90% in non colonised controls. To test whether prophages could overcome this barrier, we infected larvae with isogenic S. Tm strains differing in the presence of prophage P22. Infection with the P22 carrying strain resulted in a threefold higher larval mortality (60% vs. 20%), increased pathogen load, and a significant reduction in the abundance of resident E. coli. Free P22 virions were detected early after infection, indicating extensive prophage activity. Notably, P22 can neither adsorb nor lyse resident E. coli, indicating that prophage mediated invasion success did not rely on direct lysis. Instead, using high throughput metabolic profiling paired with whole genome sequencing of three replicate lineages, we found that phage activation intensified resource partitioning, accelerating functional metabolic adaptations in E. coli that significantly reduced the niche overlap between the invading pathogen and the commensal E. coli. Conclusion Our findings establish the first humanised G. mellonella model supporting complex human microbiota and provide a novel non lytic mechanism by which prophages influence species interactions. This scalable, low cost model offers a new platform to dissect pathogen phage microbiota interactions relevant to human gut ecology.