In Vitro Modelling of Oral Microbial Invasion in the Human Colon.

Recent advances in the human microbiome characterization have revealed significant oral microbial detection in stools of dysbiotic patients. However, little is known about the potential interactions of these invasive oral microorganisms with commensal intestinal microbiota and the host. In this proof-of-concept study, we proposed a new model of oral-to-gut invasion by the combined use of an in vitro model simulating both the physicochemical and microbial (lumen- and mucus-associated microbes) parameters of the human colon (M-ARCOL), a salivary enrichment protocol, and whole-metagenome shotgun sequencing. Oral invasion of the intestinal microbiota was simulated by injection of enriched saliva in the in vitro colon model inoculated with a fecal sample from the same healthy adult donor. The mucosal compartment of M-ARCOL was able to retain the highest species richness levels over time, while species richness levels decreased in the luminal compartment. This study also showed that oral microorganisms preferably colonized the mucosal microenvironment, suggesting potential oral-to-intestinal mucosal competitions. This new model of oral-to-gut invasion can provide useful mechanistic insights into the role of oral microbiome in various disease processes. IMPORTANCE Here, we propose a new model of oral-to-gut invasion by the combined use of an in vitro model simulating both the physicochemical and microbial (lumen- and mucus-associated microbes) parameters of the human colon (M-ARCOL), a salivary enrichment protocol, and whole-metagenome shotgun sequencing. Our study revealed the importance of integrating the mucus compartment, which retained higher microbial richness during fermentation, showed the preference of oral microbial invaders for the mucosal resources, and indicated potential oral-to-intestinal mucosal competitions. It also underlined promising opportunities to further understand mechanisms of oral invasion into the human gut microbiome, define microbe-microbe and mucus-microbe interactions in a compartmentalized fashion, and help to better characterize the potential of oral microbial invasion and their persistence in the gut.

Exposure to polyethylene microplastics alters immature gut microbiome in an infant in vitro gut model.

Infants are characterized by an immaturity of the gut ecosystem and a high exposure to microplastics (MPs) through diet, dust and suckling. However, the bidirectional interactions between MPs and the immature infant intestinal microbiota remain unknown. Our study aims to investigate the impact of chronic exposure to polyethylene (PE) MPs on the gut microbiota and intestinal barrier of infants, using the new Toddler mucosal Artificial Colon coupled with a co-culture of epithelial and mucus-secreting cells. Gut microbiota composition was determined by 16S metabarcoding and microbial activities were evaluated by gas, short chain fatty acid and volatolomics analyses. Gut barrier integrity was assessed via evaluation of intestinal permeability, inflammation and mucus synthesis. Exposure to PE MPs induced gut microbial shifts increasing α-diversity and abundance of potentially harmful pathobionts, such as Dethiosulfovibrionaceae and Enterobacteriaceae. Those changes were associated to butyrate production decrease and major changes in volatile organic compounds profiles. In contrast, no significant impact of PE MPs on the gut barrier, as mediated by microbial metabolites, was reported. For the first time, this study indicates that ingestion of PE MPs can induce perturbations in the gut microbiome of infants. Next step would be to further investigate the potential vector effect of MPs.

Microplastics: What happens in the human digestive tract? First evidences in adults using in vitro gut models.

Microplastics (MPs) are ubiquitous in the environment and humans are inevitably exposed to them. However, the effects of MPs in the human digestive environment are largely unknown. The aim of our study was to investigate the impact of repeated exposure to polyethylene (PE) MPs on the human gut microbiota and intestinal barrier using, under adult conditions, the Mucosal Artificial Colon (M-ARCOL) model, coupled with a co-culture of intestinal epithelial and mucus-secreting cells. The composition of the luminal and mucosal gut microbiota was determined by 16S metabarcoding and microbial activities were characterized by gas, short chain fatty acid, volatolomic and AhR activity analyses. Gut barrier integrity was assessed via intestinal permeability, inflammation and mucin synthesis. First, exposure to PE MPs induced donor-dependent effects. Second, an increase in abundances of potentially harmful pathobionts, Desulfovibrionaceae and Enterobacteriaceae, and a decrease in beneficial bacteria such as Christensenellaceae and Akkermansiaceae were observed. These bacterial shifts were associated with changes in volatile organic compounds profiles, notably characterized by increased indole 3-methyl- production. Finally, no significant impact of PE MPs mediated by changes in gut microbial metabolites was reported on the intestinal barrier. Given these adverse effects of repeated ingestion of PE MPs on the human gut microbiota, studying at-risk populations like infants would be a valuable advance.

Role of mucus-bacteria interactions in Enterotoxigenic Escherichia coli (ETEC) H10407 virulence and interplay with human microbiome.

The intestinal mucus layer has a dual role in human health constituting a well-known microbial niche that supports gut microbiota maintenance but also acting as a physical barrier against enteric pathogens. Enterotoxigenic Escherichia coli (ETEC), the major agent responsible for traveler's diarrhea, is able to bind and degrade intestinal mucins, representing an important but understudied virulent trait of the pathogen. Using a set of complementary in vitro approaches simulating the human digestive environment, this study aimed to describe how the mucus microenvironment could shape different aspects of the human ETEC strain H10407 pathophysiology, namely its survival, adhesion, virulence gene expression, interleukin-8 induction and interactions with human fecal microbiota. Using the TNO gastrointestinal model (TIM-1) simulating the physicochemical conditions of the human upper gastrointestinal (GI) tract, we reported that mucus secretion and physical surface sustained ETEC survival, probably by helping it to face GI stresses. When integrating the host part in Caco2/HT29-MTX co-culture model, we demonstrated that mucus secreting-cells favored ETEC adhesion and virulence gene expression, but did not impede ETEC Interleukin-8 (IL-8) induction. Furthermore, we proved that mucosal surface did not favor ETEC colonization in a complex gut microbial background simulated in batch fecal experiments. However, the mucus-specific microbiota was widely modified upon the ETEC challenge suggesting its role in the pathogen infectious cycle. Using multi-targeted in vitro approaches, this study supports the major role played by mucus in ETEC pathophysiology, opening avenues in the design of new treatment strategies.

A child is not an adult: development of a new in vitro model of the toddler colon.

Early life is a critical period where gut ecosystem and functions are being established with significant impact on health. For regulatory, technical, and cost reasons, in vitro gut models can be used as a relevant alternative to in vivo assays. An exhaustive literature review was conducted to adapt the Mucosal Artificial Colon (M-ARCOL) to specific physicochemical (pH, transit time, and nutritional composition of ileal effluents) and microbial parameters from toddlers in the age range of 6 months-3 years, resulting in the Tm-ARCOL. In vitro fermentations were performed to validate this newly developed colonic model compared to in vivo toddler data. Results were also compared to those obtained with the classical adult configuration. Fecal samples from 5 toddlers and 4 adults were used to inoculate bioreactors, and continuous fermentations were performed for 8 days. Gut microbiota structure (lumen and mucus-associated microbiota) and functions (gas and short-chain fatty acids) were monitored. Clearly distinct microbial signatures were obtained between the two in vitro conditions, with lower α-diversity indices and higher abundances of infant-related microbial populations (e.g., Bifidobacteriaceae, Enterobacteriaceae) in toddler versus adult conditions. In accordance with in vivo data, methane was found only in adult bioreactors, while higher percentage of acetate but lower proportions of propionate and butyrate was measured in toddlers compared to adults. This new in vitro model will provide a powerful platform for gut microbiome mechanistic studies in a pediatric context, both in nutritional- (e.g., nutrients, probiotics, prebiotics) and health-related (e.g., drugs, enteric pathogens) studies. KEY POINTS: • Development of a novel in vitro colonic model recapitulating the toddler environment. • Specific toddler versus adult digestive conditions are preserved in vitro. • The new model provides a powerful platform for microbiome mechanistic studies.

Lentils and Yeast Fibers: A New Strategy to Mitigate Enterotoxigenic Escherichia coli (ETEC) Strain H10407 Virulence?

Dietary fibers exhibit well-known beneficial effects on human health, but their anti-infectious properties against enteric pathogens have been poorly investigated. Enterotoxigenic Escherichia coli (ETEC) is a major food-borne pathogen that causes acute traveler's diarrhea. Its virulence traits mainly rely on adhesion to an epithelial surface, mucus degradation, and the secretion of two enterotoxins associated with intestinal inflammation. With the increasing burden of antibiotic resistance worldwide, there is an imperious need to develop novel alternative strategies to control ETEC infections. This study aimed to investigate, using complementary in vitro approaches, the inhibitory potential of two dietary-fiber-containing products (a lentil extract and yeast cell walls) against the human ETEC reference strain H10407. We showed that the lentil extract decreased toxin production in a dose-dependent manner, reduced pro-inflammatory interleukin-8 production, and modulated mucus-related gene induction in ETEC-infected mucus-secreting intestinal cells. We also report that the yeast product reduced ETEC adhesion to mucin and Caco-2/HT29-MTX cells. Both fiber-containing products strengthened intestinal barrier function and modulated toxin-related gene expression. In a complex human gut microbial background, both products did not elicit a significant effect on ETEC colonization. These pioneering data demonstrate the promising role of dietary fibers in controlling different stages of the ETEC infection process.

In vitro models of gut digestion across childhood: current developments, challenges and future trends.

The human digestion is a multi-step and multi-compartment process essential for human health, at the heart of many issues raised by academics, the medical world and industrials from the food, nutrition and pharma fields. In the first years of life, major dietary changes occur and are concomitant with an evolution of the whole child digestive tract anatomy and physiology, including colonization of gut microbiota. All these phenomena are influenced by child exposure to environmental compounds, such as drugs (especially antibiotics) and food pollutants, but also childhood infections. Due to obvious ethical, regulatory and technical limitations, in vivo approaches in animal and human are more and more restricted to favor complementary in vitro approaches. This review summarizes current knowledge on the evolution of child gut physiology from birth to 3 years old regarding physicochemical, mechanical and microbial parameters. Then, all the available in vitro models of the child digestive tract are described, ranging from the simplest static mono-compartmental systems to the most sophisticated dynamic and multi-compartmental models, and mimicking from the oral phase to the colon compartment. Lastly, we detail the main applications of child gut models in nutritional, pharmaceutical and microbiological studies and discuss the limitations and challenges facing this field of research.

Nitric Oxide Impacts Human Gut Microbiota Diversity and Functionalities.

The disruption of gut microbiota homeostasis has been associated with numerous diseases and with a disproportionate inflammatory response, including overproduction of nitric oxide (NO) in the intestinal lumen. However, the influence of NO on the human gut microbiota has not been well characterized yet. We used in vitro fermentation systems inoculated with human fecal samples to monitor the effect of repetitive NO pulses on the gut microbiota. NO exposure increased the redox potential and modified the fermentation profile and gas production. The overall metabolome was modified, reflecting less strict anaerobic conditions and shifts in amino acid and nitrogen metabolism. NO exposure led to a microbial shift in diversity with a decrease in Clostridium leptum group and Faecalibacterium prausnitzii biomass and an increased abundance of the Dialister genus. Escherichia coli, Enterococcus faecalis, and Proteus mirabilis operational taxonomic unit abundance increased, and strains from those species isolated after NO stress showed resistance to high NO concentrations. As a whole, NO quickly changed microbial fermentations, functions, and composition in a pulse- and dose-dependent manner. NO could shift, over time, the trophic chain to conditions that are unfavorable for strict anaerobic microbial processes, implying that a prolonged or uncontrolled inflammation has detrimental and irreversible consequences on the human microbiome. IMPORTANCE Gut microbiota dysbiosis has been associated with inflammatory diseases. The human inflammatory response leads to an overproduction of nitric oxide (NO) in the gut. However, so far, the influence of NO on the human gut microbiota has not been characterized. In this study, we used in vitro fermentation systems with human fecal samples to understand the effect of NO on the microbiota: NO modified the microbial composition and its functionality. High NO concentration depleted the microbiota of beneficial butyrate-producing species and favored potentially deleterious species (E. coli, E. faecalis, and P. mirabilis), which we showed can sustain high NO concentrations. Our work shows that NO may participate in the vicious circle of inflammation, leading to detrimental and irreversible consequences on human health.

In Vitro Evaluation of Dietary Fiber Anti-Infectious Properties against Food-Borne Enterotoxigenic Escherichia coli.

Dietary fibers have well-known beneficial effects on human health, but their anti-infectious properties against human enteric pathogens have been poorly investigated. Enterotoxigenic Escherichia coli (ETEC) is the main agent of travelers' diarrhea, against which targeted preventive strategies are currently lacking. ETEC pathogenesis relies on multiple virulence factors allowing interactions with the intestinal mucosal layer and toxins triggering the onset of diarrheal symptoms. Here, we used complementary in vitro assays to study the antagonistic properties of eight fiber-containing products from cereals, legumes or microbes against the prototypical human ETEC strain H10407. Inhibitory effects of these products on the pathogen were tested through growth, toxin production and mucus/cell adhesion inhibition assays. None of the tested compounds inhibited ETEC strain H10407 growth, while lentil extract was able to decrease heat labile toxin (LT) concentration in culture media. Lentil extract and specific yeast cell walls also interfered with ETEC strain H10407 adhesion to mucin beads and human intestinal cells. These results constitute a first step in the use of dietary fibers as a nutritional strategy to prevent ETEC infection. Further work will be dedicated to the study of fiber/ETEC interactions within a complex gut microbial background.

[Irritable bowel syndrome: Role of gut microbiota]. / Syndrome de l'intestin irritable - Rôle du microbiote intestinal.

Irritable Bowel Syndrome (IBS) is a functional disorder of the gastrointestinal tract with high prevalence. IBS, in particular the diarrheic subtype, is associated with alterations in gut microbiota composition and functionality, called dysbiosis. However, the treatment of this disease mainly relies on the patient's symptoms without considering the gut microbiota perturbations. In this review, we present epidemiological data about IBS-D. Then, we describe the main pathophysiological mechanisms associated with this disease, by focusing on gut microbiota alterations. We end up discussing the current therapies now available.

TITLE: Syndrome de l'intestin irritable - Rôle du microbiote intestinal. ABSTRACT: Le syndrome de l'intestin irritable (SII) est un trouble fonctionnel digestif dont la prévalence est très élevée. Ce syndrome, et notamment son sous-type diarrhéique (SII-D), est associé à des perturbations de la composition et des fonctions du microbiote intestinal à l'origine d'une dysbiose. Pourtant, la maladie est principalement traitée en fonction des symptômes des patients atteints, sans que la perturbation de leur microbiote ne soit prise en compte. Dans cette revue, nous détaillerons les données épidémiologiques de la maladie. Nous traiterons ensuite des principaux mécanismes physiopathologiques, notamment de l'impact des perturbations du microbiote intestinal. Le sous-type diarrhéique (SII-D) étant le plus fréquent [1], nous nous concentrerons principalement sur celui-ci. Nous introduirons enfin les thérapies actuelles utilisées.

Use of the Dynamic TIM-1 Model for an In-Depth Understanding of the Survival and Virulence Gene Expression of Shiga Toxin-Producing Escherichia coli in the Human Stomach and Small Intestine.

Due to obvious ethical and technical reasons, it remains very difficult to evaluate the survival and expression of virulence genes of food-borne pathogens, such as Shiga toxin-producing Escherichia coli (STEC) in the human gastrointestinal tract. Here, we describe the use of the dynamic TNO (Toegepast Natuurwetenschappelijk Onderzoek) gastrointestinal model (TIM-1) as a powerful in vitro tool to obtain the kinetics of STEC survival by plate counting, the regulation of major virulence genes by RT-qPCR, and the production of Shiga toxins by ELISA, in the human stomach and small intestine. The gut model was adapted in order that in vitro digestions were performed both under adult and child digestive conditions, specific at risk populations for STEC infections.

Microplastics in the human digestive environment: A focus on the potential and challenges facing in vitro gut model development.

Plastic pollution is a major issue worldwide, generating massive amounts of smaller plastic particles, including microplastics (MPs). Their ubiquitous nature in the environment but also in foodstuff and consumer packaged goods has revealed potential threats to humans who can be contaminated mainly through air, food and water consumption. In this review, the current literature on human exposure to MPs is summarized with a focus on the gastrointestinal tract as portal of entry. Then, we discuss the vector effect of MPs, in their pristine versus weathered forms, with well-known contaminants as heavy metals and chemicals, or more emerging ones as antibiotics or microbial pathogens, like Pseudomonas spp., Vibrio spp., Campylobacter spp. and Escherichia coli. Comprehensive knowledge on MP fate in the gastrointestinal tract and their potential impact on gut homeostasis disruption, including gut microbiota, mucus and epithelial barrier, is reported in vitro and in vivo in mammals. Special emphasis is given on the crucial need of developing robust in vitro gut models to adequately simulate human digestive physiology and absorption processes. Finally, this review points out future research directions on MPs in human intestinal health.

Tripartite relationship between gut microbiota, intestinal mucus and dietary fibers: towards preventive strategies against enteric infections.

The human gut is inhabited by a large variety of microorganims involved in many physiological processes and collectively referred as to gut microbiota. Disrupted microbiome has been associated with negative health outcomes and especially could promote the onset of enteric infections. To sustain their growth and persistence within the human digestive tract, gut microbes and enteric pathogens rely on two main polysaccharide compartments, namely dietary fibers and mucus carbohydrates. Several evidences suggest that the three-way relationship between gut microbiota, dietary fibers and mucus layer could unravel the capacity of enteric pathogens to colonise the human digestive tract and ultimately lead to infection. The review starts by shedding light on similarities and differences between dietary fibers and mucus carbohydrates structures and functions. Next, we provide an overview of the interactions of these two components with the third partner, namely, the gut microbiota, under health and disease situations. The review will then provide insights into the relevance of using dietary fibers interventions to prevent enteric infections with a focus on gut microbial imbalance and impaired-mucus integrity. Facing the numerous challenges in studying microbiota-pathogen-dietary fiber-mucus interactions, we lastly describe the characteristics and potentialities of currently available in vitro models of the human gut.

IL-22-induced cell extrusion and IL-18-induced cell death prevent and cure rotavirus infection.

Bacterial flagellin can elicit production of TLR5-mediated IL-22 and NLRC4-mediated IL-18 cytokines that act in concert to cure and prevent rotavirus (RV) infection. This study investigated the mechanism by which these cytokines act to impede RV. Although IL-18 and IL-22 induce each other's expression, we found that IL-18 and IL-22 both impeded RV independently of one another and did so by distinct mechanisms that involved activation of their cognate receptors in intestinal epithelial cells (IEC). IL-22 drove IEC proliferation and migration toward villus tips, which resulted in increased extrusion of highly differentiated IEC that serve as the site of RV replication. In contrast, IL-18 induced cell death of RV-infected IEC thus directly interrupting the RV replication cycle, resulting in spewing of incompetent virus into the intestinal lumen and causing a rapid drop in the number of RV-infected IEC. Together, these actions resulted in rapid and complete expulsion of RV, even in hosts with severely compromised immune systems. These results suggest that a cocktail of IL-18 and IL-22 might be a means of treating viral infections that preferentially target short-lived epithelial cells.

Comparative methods for fecal sample storage to preserve gut microbial structure and function in an in vitro model of the human colon.

In vitro gut models, such as the mucosal artificial colon (M-ARCOL), provide timely and cost-efficient alternatives to in vivo assays allowing mechanistic studies to better understand the role of human microbiome in health and disease. Using such models inoculated with human fecal samples may require a critical step of stool storage. The effects of preservation methods on microbial structure and function in in vitro gut models have been poorly investigated. This study aimed to assess the impact of three commonly used preserving methods, compared with fresh fecal samples used as a control, on the kinetics of lumen and mucus-associated microbiota colonization in the M-ARCOL model. Feces from two healthy donors were frozen 48 h at - 80 °C with or without cryoprotectant (10% glycerol) or lyophilized with maltodextrin and trehalose prior to inoculation of four parallel bioreactors (e.g., fresh stool, raw stool stored at - 80 °C, stool stored at - 80 °C with glycerol and lyophilized stool). Microbiota composition and diversity (qPCR and 16S metabarcoding) as well as metabolic activity (gases and short chain fatty acids) were monitored throughout the fermentation process (9 days). All the preservative treatments allowed the maintaining inside the M-ARCOL of a complex and functional microbiota, but considering stabilization time of microbial profiles and activities (and not technical constraints associated with the supply of frozen material), our results highlighted 48 h freezing at - 80 °C without cryoprotectant as the most efficient method. These results will help scientists to determine the most accurate method for fecal storage prior to inoculation of in vitro gut microbiome models. KEY POINTS: • In vitro ARCOL model reproduces luminal and mucosal human microbiome. • Short-term storage of fecal sample influences microbial stabilization and activity. • 48 h freezing at - 80°C: most efficient method to preserve microbial ecosystem. • Scientific and technical requirements: influencers of preservation method.

Differential miRNA-Gene Expression in M Cells in Response to Crohn's Disease-Associated AIEC.

Adherent-invasive Escherichia coli (AIEC), which abnormally colonize the ileal mucosa of Crohn's disease (CD) patients, are able to invade intestinal epithelial cells (IECs) and translocate through M cells overlying Peyer's patches. The levels of microRNA (miRNA) and gene expression in IECs and M cells upon AIEC infection have not been investigated. Here, we used human intestinal epithelial Caco-2 monolayers and an in vitro M-cell model of AIEC translocation to analyze comprehensive miRNA and gene profiling under basal condition and upon infection with the reference AIEC LF82 strain. Our results showed that AIEC LF82 translocated through M cells but not Caco-2 monolayers. Both differential gene expression and miRNA profile in M cells compared to Caco-2 cells were obtained. In addition, AIEC infection induces changes in gene and miRNA profiles in both Caco-2 and M cells. In silico analysis showed that certain genes dysregulated upon AIEC infection were potential targets of AIEC-dysregulated miRNAs, suggesting a miRNA-mediated regulation of gene expression during AIEC infection in Caco-2, as well as M cells. This study facilitates the discovery of M cell-specific and AIEC response-specific gene-miRNA signature and enhances the molecular understanding of M cell biology under basal condition and in response to infection with CD-associated AIEC.

Experimental models to study intestinal microbes-mucus interactions in health and disease.

A close symbiotic relationship exists between the intestinal microbiota and its host. A critical component of gut homeostasis is the presence of a mucus layer covering the gastrointestinal tract. Mucus is a viscoelastic gel at the interface between the luminal content and the host tissue that provides a habitat to the gut microbiota and protects the intestinal epithelium. The review starts by setting up the biological context underpinning the need for experimental models to study gut bacteria-mucus interactions in the digestive environment. We provide an overview of the structure and function of intestinal mucus and mucins, their interactions with intestinal bacteria (including commensal, probiotics and pathogenic microorganisms) and their role in modulating health and disease states. We then describe the characteristics and potentials of experimental models currently available to study the mechanisms underpinning the interaction of mucus with gut microbes, including in vitro, ex vivo and in vivo models. We then discuss the limitations and challenges facing this field of research.

Early-Life Microbiota Exposure Restricts Myeloid-Derived Suppressor Cell-Driven Colonic Tumorigenesis.

Gut microbiota and their metabolites are instrumental in regulating homeostasis at intestinal and extraintestinal sites. However, the complex effects of prenatal and early postnatal microbial exposure on adult health and disease outcomes remain incompletely understood. Here, we showed that mice raised under germ-free conditions until weaning and then transferred to specific pathogen-free (SPF) conditions harbored altered microbiota composition, augmented inflammatory cytokine and chemokine expression, and were hyper-susceptible to colitis-associated tumorigenesis later in adulthood. Increased number and size of colon tumors and intestinal epithelial cell proliferation in recolonized germ-free mice were associated with augmented intratumoral CXCL1, CXCL2, and CXCL5 expression and granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation. Consistent with these findings, CXCR2 neutralization in recolonized germ-free mice completely reversed the exacerbated susceptibility to colitis-associated tumorigenesis. Collectively, our findings highlight a crucial role for early-life microbial exposure in establishing intestinal homeostasis that restrains colon cancer in adulthood.

Modulation of Enterohaemorrhagic Escherichia coli Survival and Virulence in the Human Gastrointestinal Tract.

Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen responsible for human diseases ranging from diarrhoea to life-threatening complications. Survival of the pathogen and modulation of virulence gene expression along the human gastrointestinal tract (GIT) are key features in bacterial pathogenesis, but remain poorly described, due to a paucity of relevant model systems. This review will provide an overview of the in vitro and in vivo studies investigating the effect of abiotic (e.g., gastric acid, bile, low oxygen concentration or fluid shear) and biotic (e.g., gut microbiota, short chain fatty acids or host hormones) parameters of the human gut on EHEC survival and/or virulence (especially in relation with motility, adhesion and toxin production). Despite their relevance, these studies display important limitations considering the complexity of the human digestive environment. These include the evaluation of only one single digestive parameter at a time, lack of dynamic flux and compartmentalization, and the absence of a complex human gut microbiota. In a last part of the review, we will discuss how dynamic multi-compartmental in vitro models of the human gut represent a novel platform for elucidating spatial and temporal modulation of EHEC survival and virulence along the GIT, and provide new insights into EHEC pathogenesis.

Toxin-positive Clostridium difficile latently infect mouse colonies and protect against highly pathogenic C. difficile.

OBJECTIVE: Clostridium difficile is a toxin-producing bacterium and a leading cause of antibiotic-associated disease. The ability of C. difficile to form spores and infect antibiotic-treated persons at low multiplicity of infection (MOI) underlies its large disease burden. However, C. difficile-induced disease might also result from long-harboured C. difficile that blooms in individuals administered antibiotics. DESIGN: Mice purchased from multiple vendors and repeatedly testing negative for this pathogen by quantitative PCR bloomed C. difficile following antibiotic treatment. This endogenous C. difficile strain, herein termed LEM1, which formed spores and produced toxin, was compared with highly pathogenic C. difficile strain VPI10463. RESULTS: Whole-genome sequencing revealed that LEM1 and VPI10463 shared 95% of their genes, including all known virulence genes. In contrast to VPI10463, LEM1 did not induce overt disease when administered to antibiotic-treated or germ-free mice, even at high doses. Rather, blooms of LEM1 correlated with survival following VPI10463 inoculation, and exogenous administration of LEM1 before or shortly following VPI10463 inoculation prevented C. difficile-induced death. Accordingly, despite similar growth properties in vitro, LEM1 strongly outcompeted VPI10463 in mice even at 100-fold lower inocula. CONCLUSIONS: These results highlight the difficulty of determining whether individual cases of C. difficile infection resulted from a bloom of endogenous C. difficile or a new exposure to this pathogen. In addition to impacting the design of studies using mouse models of C. difficile-induced disease, this study identified, isolated and characterised an endogenous murine spore-forming C. difficile strain able to decrease colonisation, associated disease and death induced by a pathogenic C. difficile strain.