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Lack of reproducibility is a prominent problem in biomedical research. An important source of variation in animal experiments is the microbiome, but little is known about specific changes in the microbiota composition that cause phenotypic differences. Here, we show that genetically similar laboratory mice obtained from four different commercial vendors exhibited marked phenotypic variation in their susceptibility to Salmonella infection. Faecal microbiota transplant into germ-free mice replicated donor susceptibility, revealing that variability was due to changes in the gut microbiota composition. Co-housing of mice only partially transferred protection against Salmonella infection, suggesting that minority species within the gut microbiota might confer this trait. Consistent with this idea, we identified endogenous Enterobacteriaceae, a low-abundance taxon, as a keystone species responsible for variation in the susceptibility to Salmonella infection. Protection conferred by endogenous Enterobacteriaceae could be modelled by inoculating mice with probiotic Escherichia coli, which conferred resistance by using its aerobic metabolism to compete with Salmonella for resources. We conclude that a mechanistic understanding of phenotypic variation can accelerate development of strategies for enhancing the reproducibility of animal experiments. Variable susceptibility to Salmonella infection across genetically similar mice from commercial vendors is due to differential colonization of the gut microbiome by endogenous Enterobacteriaceae.

An imbalance in our microbiota may contribute to many human diseases, but the mechanistic underpinnings of dysbiosis remain poorly understood. We argue that dysbiosis is secondary to a defect in microbiota-nourishing immunity, a part of our immune system that balances the microbiota to attain colonization resistance against environmental exposure to microorganisms. We discuss this new hypothesis and its implications for ulcerative colitis, an inflammatory bowel disease of the large intestine.

Sorbara et al. report a critical function of the healthy intestinal microbiota in preventing the expansion of antibiotic-resistant Enterobacteriaceae that depends on the production of high levels of short chain fatty acids and an acidic environment to trigger intracellular acidification of Enterobacteriaceae., Klebsiella pneumoniae, Escherichia coli, and other members of the Enterobacteriaceae family are common human pathogens that have acquired broad antibiotic resistance, rendering infection by some strains virtually untreatable. Enterobacteriaceae are intestinal residents, but generally represent, Graphical Abstract

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E.coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E.coli. In contrast, E.coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species.

Summary The colonic microbiota exhibits cross-sectional heterogeneity, but the mechanisms that govern its spatial organization remain incompletely understood. Here we used Citrobacter rodentium, a pathogen that colonizes the colonic surface, to identify microbial traits that license growth and survival in this spatial niche. Previous work showed that during colonic crypt hyperplasia, type III secretion system (T3SS)-mediated intimate epithelial attachment provides C. rodentium with oxygen for aerobic respiration. However, we find that prior to the development of colonic crypt hyperplasia, T3SS-mediated intimate attachment is not required for aerobic respiration but for hydrogen peroxide (H2O2) respiration using cytochrome c peroxidase (Ccp). The epithelial NADPH oxidase NOX1 is the primary source of luminal H2O2 early after C. rodentium infection and is required for Ccp-dependent growth. Our results suggest that NOX1-derived H2O2 is a resource that governs bacterial growth and survival in close proximity to the mucosal surface during gut homeostasis.

A balanced gut microbiota is important for health, but the mechanisms maintaining homeostasis are incompletely understood. Anaerobiosis of the healthy colon drives the composition of the gut microbiota towards a dominance of obligate anaerobes, while dysbiosis is often associated with a sustained increase in the abundance of facultative anaerobic Proteobacteria, indicative of a disruption in anaerobiosis. The colonic epithelium is hypoxic, but intestinal inflammation or antibiotic treatment increases epithelial oxygenation in the colon, thereby disrupting anaerobiosis to drive a dysbiotic expansion of facultative anaerobic Proteobacteria through aerobic respiration. These observations suggest a dysbiotic expansion of Proteobacteria is a potential diagnostic microbial signature of epithelial dysfunction, a hypothesis that could spawn novel preventative or therapeutic strategies for a broad spectrum of human diseases.

Gut bugs and systemic disease risk What people eat has an immediate selective effect on the microbial populations resident in the gut. A high-fat diet is associated with the occurrence of microbes that catabolize choline and the accumulation of trimethylamine N -oxide (TMAO) in the bloodstream, a contributing factor for heart disease. Yoo et al . explored the microbial organisms and pathways that convert choline into TMAO in mice. Although gene clusters for choline metabolism are found widely among the microbiota, it is only the facultative anaerobes that become abundant in hosts on a high-fat diet. A high-fat diet impairs mitochondrial uptake of oxygen into host enterocytes and elevates nitrate in the mucus, which in turn weakens healthy anaerobic gut function. Facultative anaerobes such as the pathobiont Escherichia coli become dominant, which leads to an overall increase in the amount of choline catabolized into the precursor for TMAO. Whether this pathway plays a role in heart disease remains unclear. —CA

In ecological terms, the microbiome is defined as the microbiota and its environment, a definition that encompasses the human host. The size, species composition, and biogeography of microbial communities is shaped by host interactions, and, in turn, the microbiota influences many aspects of human health. Here we discuss the concept of microbiota-nourishing immunity, a host-microbe chimera composed of the microbiota and host factors that shape the microbial ecosystem, which functions in conferring colonization resistance against pathogens. We propose that dysbiosis is a biomarker of a weakening in microbiota-nourishing immunity and that homeostasis can be defined as a state of immune competence. Microbiota-nourishing immunity thus provides a conceptual framework to further examine the mechanisms that preserve a healthy microbiome and the drivers and consequences of dysbiosis.

Our skin and mucosal surfaces are colonized by diverse microbial communities, collectively known as the microbiota [1]. The microbiota provides benefits as microbial metabolites contribute to host nutrition and immune education, although the viability of germ-free animals conjectures that these two functions are not essential for life. However, environmental exposure makes germ-free animals prone to lethal infection, illustrating that the microbiota confers a third function that is often vital, namely, the ability to confer colonization resistance against pathogens [2]. Colonization resistance is an acquired trait, because the microbiota is assembled after birth by attaining maternal and environmental microbes [3]. To coexist, each species within the microbial community needs to be able to utilize a critical resource better than any other member of the microbiota, and the abundance of this growth-limiting resource determines the abundance of the species, a concept known as the nutrient-niche hypothesis [4]. The conceptual framework of the nutrient-niche hypothesis suggests that the neonate microbiota will mature until all discrete nutrient-niches have been filled with a suitable occupant, thereby reaching an equilibrium state [5]. Assuming the same anatomical location in different individuals exposes similar nutrient-niches, the nutrient-niche hypothesis further predicts that the metabolic pathways that enable each member within the microbial community to utilize its growth-limiting nutrient must be conserved between different individuals. Consistent with this prediction, metabolic pathways encoded by the microbiota are very similar between individuals [1]. However, carriage of microbial taxa varies greatly within a healthy population [1], an observation that is not explained by the nutrient-niche hypothesis and remains poorly understood. Priority effects generate variation in taxa carriage Host genetic variation explains only a small fraction of taxonomic microbiota variation between individuals, whereas environmental influences dominate this trait [6]. An important environmental influence in the gastrointestinal tract is the diet, which determines the availability of a subset of growth-limiting nutrients, thereby adding or subtracting nutrient-niches [7, 8]. For example, microbiota-accessible carbohydrates found in dietary fiber determine the abundance of fiber-consuming saccharolytic bacteria in the gut microbiota, and prolonged dietary fiber starvation can lead to an irreversible extinction of species specialized in devouring this critical resource by eliminating their nutrient-niche [8]. Although diet can generate statistically significant changes in the taxonomic composition of the gut microbiota, these changes are small compared to the variation observed between individuals. Furthermore, diet does not provide a plausible explanation for the taxonomic diversity observed in microbial communities outside the gastrointestinal tract [1]. Instead, a critical factor generating taxonomic microbiota diversity between individuals is the order of species arrival and timing by which host surfaces are colonized early in life [9]. The colonization order influences both the outcome of microbial community assembly and the ecological success of individual microbes [3, 9]. These priority effects are preserved in mice lacking adaptive immunity, suggesting that acquired host responses are not a major source of taxonomic diversity in the microbiota composition [9]. Priority effects are mediated through niche preemption or niche modification and can involve the genetic adaptation of microbes to a niche [9, 12], but the underlying mechanisms are incompletely understood. Mechanistic insights into this “first come, first serve” phenomenon suggest that the microbe that initially occupies a nutrient-niche in a neonate gains priority access to the growth-limiting nutrient that defines its nutrient-niche [10]. A growth-limiting resource that determines the abundance of facultative anaerobic Enterobacteriaceae (phylum Proteobacteria) within the microbiota of the large intestine is the availability of respiratory electron acceptors, such as oxygen [11]. Escherichia coli (family Enterobacteriaceae) has access to oxygen in the ceca of neonate chicks when it is inoculated one day prior to challenge with Salmonella enterica (family Enterobacteriaceae) but not when neonate chicks receive both species at the same time [10], suggesting that order and timing of gut colonization determine whether growth-limiting resources are accessible to a microbe. Henceforth we will refer to the concept that the founding occupant gains priority access to the growth-limiting resource that defines its nutrient-niche as the “founder hypothesis.” The founder hypothesis suggests that stochastic effects that govern the initial exposure of neonates to microbes that become founding occupants of each nutrient-niche are a prominent source of taxonomic variation in the microbiota composition between individuals (Fig 1) [3]. Open in a separate window Fig 1 The founder hypothesis. The principles of the founder hypothesis are shown schematically for a single nutrient-niche. Stochastic effects governing microbial exposure during infancy determine which microbial species (red or blue rods) establishes residency in the nutrient-niche, thereby generating diversity in taxa carriage between individuals. The founding occupant gains priority access to the growth-limiting resource that defines its nutrient-niche. These priority effects enable the occupant to confer colonization resistance against environmental exposure to microorganisms that are suitable contenders for the same nutrient-niche. The resulting resistance to stress imposed through environmental exposure to microorganisms produces microbiota resistance.

© 2017, American Association for the Advancement of Science. All rights reserved. Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator–activated receptor g (PPAR-g). Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-g signaling. Microbiota-induced PPAR-g signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward b-oxidation. Therefore, microbiota-activated PPAR-g signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.

Summary Neonates are highly susceptible to infection with enteric pathogens, but the underlying mechanisms are not resolved. We show that neonatal chick colonization with Salmonella enterica serovar Enteritidis requires a virulence-factor-dependent increase in epithelial oxygenation, which drives pathogen expansion by aerobic respiration. Co-infection experiments with an Escherichia coli strain carrying an oxygen-sensitive reporter suggest that S. Enteritidis competes with commensal Enterobacteriaceae for oxygen. A combination of Enterobacteriaceae and spore-forming bacteria, but not colonization with either community alone, confers colonization resistance against S. Enteritidis in neonatal chicks, phenocopying germ-free mice associated with adult chicken microbiota. Combining spore-forming bacteria with a probiotic E. coli isolate protects germ-free mice from pathogen colonization, but the protection is lost when the ability to respire oxygen under micro-aerophilic conditions is genetically ablated in E. coli. These results suggest that commensal Enterobacteriaceae contribute to colonization resistance by competing with S. Enteritidis for oxygen, a resource critical for pathogen expansion.

An imbalance in the colonic microbiota might underlie many human diseases, but the mechanisms that maintain homeostasis remain elusive. Recent insights suggest that colonocyte metabolism functions as a control switch, mediating a shift between homeostatic and dysbiotic communities. During homeostasis, colonocyte metabolism is directed toward oxidative phosphorylation, resulting in high epithelial oxygen consumption. The consequent epithelial hypoxia helps to maintain a microbial community dominated by obligate anaerobic bacteria, which provide benefit by converting fiber into fermentation products absorbed by the host. Conditions that alter the metabolism of the colonic epithelium increase epithelial oxygenation, thereby driving an expansion of facultative anaerobic bacteria, a hallmark of dysbiosis in the colon. Enteric pathogens subvert colonocyte metabolism to escape niche protection conferred by the gut microbiota. The reverse strategy, a metabolic reprogramming to restore colonocyte hypoxia, represents a promising new therapeutic approach for rebalancing the colonic microbiota in a broad spectrum of human diseases.

Intestinal inflammation caused by Salmonella enterica serovar Typhimurium increases the availability of electron acceptors that fuel a respiratory growth of the pathogen in the intestinal lumen. Here we show that one of the carbon sources driving this respiratory expansion in the mouse model is 1,2-propanediol, a microbial fermentation product. 1,2-propanediol utilization required intestinal inflammation induced by virulence factors of the pathogen. S. Typhimurium used both aerobic and anaerobic respiration to consume 1,2-propanediol and expand in the murine large intestine. 1,2-propanediol-utilization did not confer a benefit in germ-free mice, but the pdu genes conferred a fitness advantage upon S. Typhimurium in mice mono-associated with Bacteroides fragilis or Bacteroides thetaiotaomicron. Collectively, our data suggest that intestinal inflammation enables S. Typhimurium to sidestep nutritional competition by respiring a microbiota-derived fermentation product., Author Summary Salmonella enterica serovar Typhimurium induces intestinal inflammation to induce the generation of host-derived respiratory electron acceptors, thereby driving a respiratory pathogen expansion, which aids infectious transmission by the fecal oral route. However, the identity of nutrients serving as electron donors to enable S. Typhimurium to edge out competing microbes in the competitive environment of the gut are just beginning to be worked out. Here we demonstrate that aerobic and anaerobic respiratory pathways cooperate to promote growth of Salmonella on the microbial fermentation product 1,2-propanediol. We propose that pathogen-induced intestinal inflammation enables Salmonella to sidestep nutritional competition with the largely anaerobic microbiota by respiring a microbe-derived metabolite that cannot be consumed by fermentation.

Enteropathogenic Escherichia coli (EPEC) is a major cause of food poisoning, leading to significant morbidity and mortality. EPEC virulence is dependent on a type III secretion system (T3SS), a molecular syringe employed by EPEC to inject effector proteins into host cells. The injected effector proteins subvert host cellular functions to the benefit of the infecting bacteria. The T3SS and related genes reside in several operons clustered in the locus of enterocyte effacement (LEE). We carried out simultaneous analysis of the expression dynamics of all the LEE promoters and the rate of maturation of the T3SS. The results showed that expression of the LEE1 operon is activated immediately upon shifting the culture to inducing conditions, while expression of other LEE promoters is activated only ∼70 min postinduction. Parallel analysis showed that the T3SS becomes functional around 100 min postinduction. The T3SS core proteins EscS, EscT, EscU, and EscR are predicted to be involved in the first step of T3SS assembly and are therefore included among the LEE1 genes. However, interfering with the temporal regulation of EscS, EscT, EscU, and EscR expression has only a marginal effect on the rate of the T3SS assembly. This study provides a comprehensive description of the transcription dynamics of all the LEE genes and correlates it to that of T3SS biogenesis.

Several major bacterial pathogens use the type III secretion system (TTSS) to deliver virulence factors into host cells. Bacterial Rho GTPase activating proteins (RhoGAPs) comprise a remarkable family of type III secreted toxins that modulate cytoskeletal dynamics and manipulate cellular signaling pathways. We show that the RhoGAP activity ofSalmonellaSptP andPseudomonasExoS toxins is resistant to variations in the concentration of NaCl or MgCl2, unlike the known salt dependant nature of the activity of some eukaryotic GAPs such as p190, RanGAP and p120GAP. Furthermore, SptP-GAP and ExoS-GAP display full activity after treatment at 80°C or with 6 murea, which suggests that these protein domains are capable of spontaneous folding into an active state following denaturing such as what might occur upon transit through the TTSS needle. We determined the catalytic activity of bacterial GAPs for Rac1, CDC42 and RhoA GTPases and found that ExoS, in addition toYersiniaYopE andAeromonasAexT toxins, display higher catalytic efficiencies for Rac1 and CDC42 than the known eukaryotic GAPs, making them the most catalytically efficient RhoGAPs known. This study expands our knowledge of the mechanism of action of GAPs and of the ways bacteria mimic host activities and promote catalysis of eukaryotic signaling proteins.

The N terminus of the Aeromonas salmonicida ADP-ribosylating toxin AexT displays in vitro GTPase-activating protein (GAP) activity for Rac1, CDC42, and RhoA. HeLa cells transfected with the AexT N terminus exhibit rounding and actin disordering. We propose that the Aeromonas salmonicida AexT toxin is a novel member of the growing family of bacterial RhoGAPs.

Bacterial GTPase-activating proteins (GAPs) subvert their host's eukaryotic Rho GTPases to their own advantage. Studies of bacterial GAPs extend our understanding of the action of eukaryotic GAPs, provide new tools for studies of cytoskeletal dynamics and offer new targets for anti-bacterial drugs.