The potential importance of the built-environment microbiome and its impact on human health.

There is increasing evidence that interactions between microbes and their hosts not only play a role in determining health and disease but also in emotions, thought, and behavior. Built environments greatly influence microbiome exposures because of their built-in highly specific microbiomes coproduced with myriad metaorganisms including humans, pets, plants, rodents, and insects. Seemingly static built structures host complex ecologies of microorganisms that are only starting to be mapped. These microbial ecologies of built environments are directly and interdependently affected by social, spatial, and technological norms. Advances in technology have made these organisms visible and forced the scientific community and architects to rethink gene-environment and microbe interactions respectively. Thus, built environment design must consider the microbiome, and research involving host-microbiome interaction must consider the built-environment. This paradigm shift becomes increasingly important as evidence grows that contemporary built environments are steadily reducing the microbial diversity essential for human health, well-being, and resilience while accelerating the symptoms of human chronic diseases including environmental allergies, and other more life-altering diseases. New models of design are required to balance maximizing exposure to microbial diversity while minimizing exposure to human-associated diseases. Sustained trans-disciplinary research across time (evolutionary, historical, and generational) and space (cultural and geographical) is needed to develop experimental design protocols that address multigenerational multispecies health and health equity in built environments.

Microbiome changes under enteral deprivation are dynamic and dependent on intestinal location.

BACKGROUND: The microbiome has a pivotal role in intestinal health, and nutrition has a major role shaping its structure. Enteral deprivation, in which no oral/enteral nutrition is administered, is common in hospitalized/gastrointestinal patients. The dynamics that enteral deprivation exerts on the microbial community, specifically in the small intestine, are not well understood. METHODS: Enteral deprivation was modeled with exclusive parenteral nutrition (EPN) mice. Mice were allocated to receive either EPN or saline and chow (control) and euthanized after 0, 2, 4, or 6 days. DNA was extracted from jejunum, ileum, and colon content. 16S sequencing was used to compare changes in microbial communities between groups. Functional pathways were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States. RESULTS: EPN-treated mice showed community changes throughout the intestine. Beta diversity in colon showed clear separation between the groups (Bray-Curtis, P < 0.001). Time-dependent dynamics were seen in ileal but not jejunal samples. Alpha diversity was lower in the colon of EPN mice compared with control/baseline mice (Chao1, P < 0.01) but not in ileum/jejunum. Progressive loss of single-taxon domination was seen, most notably in the small intestine. This was accompanied by increases/decreases in specific taxa. A clear separation was seen in the functional capacity of the community between fed and enterally deprived mice at the ileum and colon, which was observed early on. CONCLUSIONS: Enteral deprivation disturbs the microbial community in a spatial and dynamic manner. There should be further focus on studying the effect of these changes on the host.

A Murine Model of Maternal Micronutrient Deficiencies and Gut Inflammatory Host-microbe Interactions in the Offspring.

BACKGROUND & AIMS: Micronutrient deficiency (MND) (ie, lack of vitamins and minerals) during pregnancy is a major public health concern. Historically, studies have considered micronutrients in isolation; however, MNDs rarely occur alone. The impact of co-occurring MNDs on public health, mainly in shaping mucosal colonization by pathobionts from the Enterobacteriaceae family, remains undetermined due to lack of relevant animal models. METHODS: To establish a maternal murine model of multiple MND (MMND), we customized a diet deficient in vitamins (A, B12, and B9) and minerals (iron and zinc) that most commonly affect children and women of reproductive age. Thereafter, mucosal adherence by Enterobacteriaceae, the associated inflammatory markers, and proteomic profile of intestines were determined in the offspring of MMND mothers (hereafter, low micronutrient [LM] pups) via bacterial plating, flow cytometry, and mass spectrometry, respectively. For human validation, Enterobacteriaceae abundance, assessed via 16s sequencing of 3-month-old infant fecal samples (n = 100), was correlated with micronutrient metabolites using Spearman's correlation in meconium of children from the CHILD birth cohort. RESULTS: We developed an MMND model and reported an increase in colonic abundance of Enterobacteriaceae in LM pups at weaning. Findings from CHILD cohort confirmed a negative correlation between Enterobacteriaceae and micronutrient availability. Furthermore, pro-inflammatory cytokines and increased infiltration of lymphocyte antigen 6 complex high monocytes and M1-like macrophages were evident in the colons of LM pups. Mechanistically, mitochondrial dysfunction marked by reduced expression of nicotinamide adenine dinucleotide (NAD)H dehydrogenase and increased expression of NAD phosphate oxidase (Nox) 1 contributed to the Enterobacteriaceae bloom. CONCLUSION: This study establishes an early life MMND link to intestinal pathobiont colonization and mucosal inflammation via damaged mitochondria in the offspring.

Microbial transmission in the social microbiome and host health and disease.

Although social interactions are known to drive pathogen transmission, the contributions of socially transmissible host-associated mutualists and commensals to host health and disease remain poorly explored. We use the concept of the social microbiome-the microbial metacommunity of a social network of hosts-to analyze the implications of social microbial transmission for host health and disease. We investigate the contributions of socially transmissible microbes to both eco-evolutionary microbiome community processes (colonization resistance, the evolution of virulence, and reactions to ecological disturbance) and microbial transmission-based processes (transmission of microbes with metabolic and immune effects, inter-specific transmission, transmission of antibiotic-resistant microbes, and transmission of viruses). We consider the implications of social microbial transmission for communicable and non-communicable diseases and evaluate the importance of a socially transmissible component underlying canonically non-communicable diseases. The social transmission of mutualists and commensals may play a significant, under-appreciated role in the social determinants of health and may act as a hidden force in social evolution.

Microbiota-mediated effects of Parkinson's disease medications on Parkinsonian non-motor symptoms in male transgenic mice.

Parkinson's disease (PD) is characterized by motor symptoms and a loss of dopaminergic neurons, as well as a variety of non-motor symptoms, including constipation, depression, and anxiety. Recently, evidence has also accumulated for a link between gut microbiota and PD. Most PD patients are on dopamine replacement therapy, primarily a combination of L-DOPA and carbidopa; however, the effect of these medications on the microbiota and non-motor symptoms in PD is still unclear. In this study, we explored the effects of chronic oral treatment with L-DOPA plus carbidopa (LDCD) on the gut microbiota and non-motor symptoms in males of a transgenic mouse model of PD (dbl-PAC-Tg(SNCAA53T);Snca-/-). To further test whether the effects of these PD medications were mediated by the gut microbiota, oral antibiotic treatment (Abx; vancomycin and neomycin) was included both with and without concurrent LDCD treatment. Post-treatment, the gastrointestinal, motor, and behavioral phenotypes were profiled, and fecal, ileal, and jejunal samples were analyzed for gut microbiota composition by 16S sequencing. LDCD treatment was found to improve symptoms of constipation and depression in this model, concurrent with increases in Turicibacter abundance in the ileum. Abx treatment worsened the symptoms of constipation, possibly through decreased levels of short-chain fatty acids and disrupted gut barrier function. LDCD + Abx treatment showed an interaction effect on behavioral symptoms that was also associated with ileal Turicibacter levels. This study demonstrates that, in a mouse model, PD medications and antibiotics affect PD-related non-motor symptoms potentially via the gut microbiota.IMPORTANCEThe motor symptoms of Parkinson's disease (PD) are caused by a loss of dopamine-producing neurons and are commonly treated with dopamine replacement therapy (L-DOPA plus carbidopa). PD has also been associated with altered gut microbiota composition. However, the effects of these PD medications on PD-related non-motor symptoms and the gut microbiota have not been well characterized. This study uses a transgenic mouse model of PD to help resolve medication-induced microbiota alterations from those that are potentially disease relevant within a PD context, and explores how long-term treatment may interact with the gut microbiota to impact non-motor symptoms.

The Effect of Nutrient Deprivation on Early Life Small Intestinal Mucosal Microbiome and Host Proteome.

BACKGROUND: Nutrition plays a vital role in shaping the intestinal microbiome. However, many hospitalized children undergo periods of fasting during medical treatment. Changes to the small intestinal microbiota in early life in the setting of enteral deprivation have not been well described. OBJECTIVE: The aim of this study was to investigate the impact of enteral deprivation on the small intestinal mucosal microbiome and to identify factors that shape this interaction in infancy. METHODS: Intestinal biopsies were collected from proximal (fed) and distal (unfed) small bowel at the time of ostomy closure in children with a small intestinal enterostomy. Mucosal and luminal microbiome comparisons were performed including ß-diversity and differential abundance and correlations with clinical factors were analyzed. Host proteomics were compared between fed and unfed samples and correlated with microbiome parameters. Finally, microbial results were validated in another cohort of pediatric patients. RESULTS: Samples from 13 children (median age 84 d) were collected. Mucosal microbiome communities in the fed and unfed segments were strikingly similar [paired UniFrac distance (ß-diversity)], whereas luminal effluent differed significantly from fed samples (PERMANOVA, P = 0.003). Multivariate analysis revealed patient as the strongest predictor of the UniFrac distance. Environmental variables did not influence the intrapatient microbial dissimilarity. Host proteomics were similar intrapatient (paired fed-unfed Euclidian distance) and showed a correlation with the UniFrac distance (Spearman rho = 0.71, P < 0.001). Specific proteins and functional clusters were significantly different between paired samples, including lipid metabolism and intracellular trafficking, whereas no difference was seen in innate immune proteins. The microbiome results were validated in a different cohort with similar characteristics. CONCLUSION: We found the host to be the most dominant factor in the structure of the early life small intestinal mucosal microbiome. Nutrient deprivation was associated with specific changes in the host proteome. Further research is needed to better understand this host-microbe-nutrition interaction.

Time to rethink academic publishing: the peer reviewer crisis.

There is concern that the time taken to publish academic papers in microbiological science has significantly increased in recent years. While the data do not specifically support this, evidence suggests that editors are having to invite more and more reviewers to identify those willing to perform peer review.

Multiple micronutrient deficiencies in early life cause multi-kingdom alterations in the gut microbiome and intrinsic antibiotic resistance genes in mice.

Globally, ~340 million children suffer from multiple micronutrient deficiencies, accompanied by high pathogenic burden and death due to multidrug-resistant bacteria. The microbiome is a reservoir of antimicrobial resistance (AMR), but the implications of undernutrition on the resistome is unclear. Here we used a postnatal mouse model that is deficient in multiple micronutrients (that is, zinc, folate, iron, vitamin A and vitamin B12 deficient) and shotgun metagenomic sequencing of faecal samples to characterize gut microbiome structure and functional potential, and the resistome. Enterobacteriaceae were enriched in micronutrient-deficient mice compared with mice fed an isocaloric experimental control diet. The mycobiome and virome were also altered with multiple micronutrient deficiencies including increased fungal pathogens such as Candida dubliniensis and bacteriophages. Despite being antibiotic naïve, micronutrient deficiency was associated with increased enrichment of genes and gene networks encoded by pathogenic bacteria that are directly or indirectly associated with intrinsic antibiotic resistance. Bacterial oxidative stress was associated with intrinsic antibiotic resistance in these mice. This analysis reveals multi-kingdom alterations in the gut microbiome as a result of co-occurring multiple micronutrient deficiencies and the implications for antibiotic resistance.

Quorum sensing modulates bacterial virulence and colonization dynamics of the gastrointestinal pathogen Citrobacter rodentium.

Quorum Sensing (QS) is a form of cell-to-cell communication that enables bacteria to modify behavior according to their population density. While QS has been proposed as a potential intervention against pathogen infection, QS-mediated communication within the mammalian digestive tract remains understudied. Using an LC-MS/MS approach, we discovered that Citrobacter rodentium, a natural murine pathogen used to model human infection by pathogenic Escherichia coli, utilizes the CroIR system to produce three QS-molecules. We then profiled their accumulation both in vitro and across different gastrointestinal sites over the course of infection. Importantly, we found that in the absence of QS capabilities the virulence of C. rodentium is enhanced. This highlights the role of QS as an effective mechanism to regulate virulence according to the pathogen's spatio-temporal context to optimize colonization and transmission success. These results also demonstrate that inhibiting QS may not always be an effective strategy for the control of virulence.

Early Life Exposure to Human Milk Oligosaccharides Reduces Allergic Response in a Murine Asthma Model.

Background: Studies suggest that early-life gut microbiota composition and intestinal short-chain fatty acids (SCFAs) are linked to future asthma susceptibility. Furthermore, infancy offers a critical time window to modulate the microbiota and associated metabolites through diet-microbe interactions to promote infant health. Human milk oligosaccharides (HMOs), nondigestible carbohydrates abundant in breast milk, are prebiotics selectively metabolized by gut microbiota that consequently modify microbiome composition and SCFA production. Methods: Using a house dust mite mouse model of allergy, we investigated the impacts of early oral treatment of pups with biologically relevant doses of 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL), two of the most abundant HMOs in human milk, in amelioration of allergic airway disease severity. Results: We found that administration of 2'-FL and 6'-SL during early life reduced lung histopathology scores, circulating IgE, cytokine levels, and inflammatory cell infiltration, all hallmark symptoms of allergic asthma. HMO supplementation also increased the relative abundance of intestinal Bacteroidetes and Clostridia, known SCFA producers within the gut. Indeed, we detected increased SCFA concentrations in both the intestine and blood of adult mice who received HMOs prior to weaning. Conclusion: We propose a model in which orally administered HMOs delivered during early life shift the microbiota toward increased production of SCFAs, which dampens the allergic immune responses behind allergy and asthma. Overall, these data suggest the potential for HMO supplementation to protect infants against asthma development later in life, with possible benefits against additional atopic diseases such as eczema and food allergies.

Delayed gut microbiota maturation in the first year of life is a hallmark of pediatric allergic disease.

Allergic diseases affect millions of people worldwide. An increase in their prevalence has been associated with alterations in the gut microbiome, i.e., the microorganisms and their genes within the gastrointestinal tract. Maturation of the infant immune system and gut microbiota occur in parallel; thus, the conformation of the microbiome may determine if tolerant immune programming arises within the infant. Here we show, using deeply phenotyped participants in the CHILD birth cohort (n = 1115), that there are early-life influences and microbiome features which are uniformly associated with four distinct allergic diagnoses at 5 years: atopic dermatitis (AD, n = 367), asthma (As, n = 165), food allergy (FA, n = 136), and allergic rhinitis (AR, n = 187). In a subset with shotgun metagenomic and metabolomic profiling (n = 589), we discover that impaired 1-year microbiota maturation may be universal to pediatric allergies (AD p = 0.000014; As p = 0.0073; FA p = 0.00083; and AR p = 0.0021). Extending this, we find a core set of functional and metabolic imbalances characterized by compromised mucous integrity, elevated oxidative activity, decreased secondary fermentation, and elevated trace amines, to be a significant mediator between microbiota maturation at age 1 year and allergic diagnoses at age 5 years (ßindirect = -2.28; p = 0.0020). Microbiota maturation thus provides a focal point to identify deviations from normative development to predict and prevent allergic disease.

Multiple micronutrient deficiencies alter energy metabolism in host and gut microbiome in an early-life murine model.

Introduction: Micronutrients perform a wide range of physiological functions essential for growth and development. However, most people still need to meet the estimated average requirement worldwide. Globally, 2 billion people suffer from micronutrient deficiency, most of which are co-occurring deficiencies in children under age five. Despite decades of research, animal models studying multiple micronutrient deficiencies within the early-life period are lacking, which hinders our complete understanding of the long-term health implications and may contribute to the inefficacy of some nutritional interventions. Evidence supporting the Developmental Origins of Health and Disease (DOHaD) theory demonstrates that early-life nutritional deficiencies carry life-long consequences mediated through various mechanisms such as abnormal metabolic programming, stunting, altered body composition, and the gut microbiome. However, this is largely unexplored in the multiple micronutrient deficient host. Methods: we developed a preclinical model to examine undernutrition's metabolic and functional impact on the host and gut microbiome early in life. Three-week-old weanling C57BL/6N male mice were fed a low-micronutrient diet deficient in zinc, folate, iron, vitamin A, and vitamin B12 or a control diet for 4-weeks. Results: Our results showed that early-life multiple micronutrient deficiencies induced stunting, altered body composition, impaired glucose and insulin tolerance, and altered the levels of other micronutrients not depleted in the diet within the host. In addition, functional metagenomics profiling and a carbohydrate fermentation assay showed an increased microbial preference for simple sugars rather than complex ones, suggestive of a less developed microbiome in the low-micronutrient-fed mice. Moreover, we found that a zinc-only deficient diet was not sufficient to induce these phenotypes, further supporting the importance of studying co-occurring deficiencies. Discussion: Together, these findings highlight a previously unappreciated role of early-life multiple micronutrient deficiencies in shaping the metabolic phenome of the host and gut microbiome through altered glucose energy metabolism, which may have implications for metabolic disease later in life in micronutrient-deficient survivors.

Human milk oligosaccharides: potential therapeutic aids for allergic diseases.

Childhood allergy, including asthma, eczema, and food allergies, is a major global health burden, with prevalence increasing dramatically and novel interventions needed. Emerging research suggests that human milk oligosaccharides (HMOs), complex glycans found in breastmilk, have allergy-protective properties, indicating exciting therapeutic potential. This review evaluates current literature on the role of HMOs in allergy, assesses underlying immunological mechanisms, and discusses future research needed to translate findings into clinical implications. HMOs may mediate allergy risk through multiple structure-specific mechanisms, including microbiome modification, intestinal barrier maturation, immunomodulation, and gene regulation. Findings emphasize the importance of breastfeeding encouragement and HMO-supplemented formula milk for high allergy-risk infants. Although further investigation is necessary to determine the most efficacious structures against varying allergy phenotypes and their long-term efficacy, HMOs may represent a promising complementary tool for childhood allergy prevention.

Gut microbiota, malnutrition, and immunity: COVID-19's confounding triad.

The coronavirus disease has swept the world, bringing scientists from multiple disciplines together to work on a focused cause. In this forum, we discuss different roles that microbiota, malnutrition, and immunity have on severity of coronavirus disease and the importance of studying them from a gut-systemic perspective using multi-omics approaches.

Early-life interactions between the microbiota and immune system: impact on immune system development and atopic disease.

Prenatal and early postnatal life represent key periods of immune system development. In addition to genetics and host biology, environment has a large and irreversible role in the immune maturation and health of an infant. One key player in this process is the gut microbiota, a diverse community of microorganisms that colonizes the human intestine. The diet, environment and medical interventions experienced by an infant determine the establishment and progression of the intestinal microbiota, which interacts with and trains the developing immune system. Several chronic immune-mediated diseases have been linked to an altered gut microbiota during early infancy. The recent rise in allergic disease incidence has been explained by the 'hygiene hypothesis', which states that societal changes in developed countries have led to reduced early-life microbial exposures, negatively impacting immunity. Although human cohort studies across the globe have established a correlation between early-life microbiota composition and atopy, mechanistic links and specific host-microorganism interactions are still being uncovered. Here, we detail the progression of immune system and microbiota maturation in early life, highlight the mechanistic links between microbes and the immune system, and summarize the role of early-life host-microorganism interactions in allergic disease development.

GrlR, a negative regulator in enteropathogenic E. coli, also represses the expression of LEE virulence genes independently of its interaction with its cognate partner GrlA.

Introduction: Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood. Methods: To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis. Results and discussion: We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Enterohemorrhagic Escherichia coli responds to gut microbiota metabolites by altering metabolism and activating stress responses.

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

Breastfeeding enrichment of B. longum subsp. infantis mitigates the effect of antibiotics on the microbiota and childhood asthma risk.

BACKGROUND: Early antibiotic exposure is linked to persistent disruption of the infant gut microbiome and subsequent elevated pediatric asthma risk. Breastfeeding acts as a primary modulator of the gut microbiome during early life, but its effect on asthma development has remained unclear. METHODS: We harnessed the CHILD cohort to interrogate the influence of breastfeeding on antibiotic-associated asthma risk in a subset of children (n = 2,521). We then profiled the infant microbiomes in a subset of these children (n = 1,338) using shotgun metagenomic sequencing and compared human milk oligosaccharide and fatty acid composition from paired maternal human milk samples for 561 of these infants. FINDINGS: Children who took antibiotics without breastfeeding had 3-fold higher asthma odds, whereas there was no such association in children who received antibiotics while breastfeeding. This benefit was associated with widespread "re-balancing" of taxonomic and functional components of the infant microbiome. Functional changes associated with asthma protection were linked to enriched Bifidobacterium longum subsp. infantis colonization. Network analysis identified a selection of fucosylated human milk oligosaccharides in paired maternal samples that were positively associated with B. infantis and these broader functional changes. CONCLUSIONS: Our data suggest that breastfeeding and antibiotics have opposing effects on the infant microbiome and that breastfeeding enrichment of B. infantis is associated with reduced antibiotic-associated asthma risk. FUNDING: This work was supported in part by the Canadian Institutes of Health Research; the Allergy, Genes and Environment Network of Centres of Excellence; Genome Canada; and Genome British Columbia.

Gastric acid and escape to systemic circulation represent major bottlenecks to host infection by Citrobacter rodentium.

The gastrointestinal (GI) environment plays a critical role in shaping enteric infections. Host environmental factors create bottlenecks, restrictive events that reduce the genetic diversity of invading bacterial populations. However, the identity and impact of bottleneck events on bacterial infection are largely unknown. We used Citrobacter rodentium infection of mice, a model of human pathogenic Escherichia coli infections, to examine bacterial population dynamics and quantify bottlenecks to host colonization. Using Sequence Tag-based Analysis of Microbial Populations (STAMP) we characterized the founding population size (Nb') and relatedness of C. rodentium populations at relevant tissue sites during early- and peak-infection. We demonstrate that the GI environment severely restricts the colonizing population, with an average Nb' of only 12-43 lineages (of 2,000+ inoculated) identified regardless of time or biogeographic location. Passage through gastric acid and escape to the systemic circulation were identified as major bottlenecks during C. rodentium colonization. Manipulating such events by increasing gastric pH dramatically increased intestinal Nb'. Importantly, removal of the stomach acid barrier had downstream consequences on host systemic colonization, morbidity, and mortality. These findings highlight the capability of the host GI environment to limit early pathogen colonization, controlling the population of initial founders with consequences for downstream infection outcomes.