Lactococcus lactis CNCM I-5388 versus NCDO2118 by its GABA hyperproduction ability, counteracts faster stress-induced intestinal hypersensitivity in rats.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut-brain interactions and gut bacteria may alleviate IBS symptoms, including VH. γ-amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L-glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA-producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA-producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10-day oral treatment. Here, we identified the strain CNCM I-5388, with a four-fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and in vitro analyses revealed the same optimal pH for GAD activity; however, CNCM I-5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, in vivo experiments have demonstrated that CNCM I-5388 has faster anti-VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I-5388 anti-VH effects partially persisted after 5-day treatment interruption and after a single oral treatment. These findings highlight CNCM I-5388 as a potential therapeutic agent for managing VH in IBS patients.

Natural diversity of lactococci in γ-aminobutyric acid (GABA) production and genetic and phenotypic determinants.

BACKGROUND: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M). RESULTS: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the PgadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the PgadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production. CONCLUSIONS: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity.

Lipidomic Profiling of PFOA-Exposed Mouse Liver by Multi-Modal Mass Spectrometry Analysis.

Perfluorooctanoic acid (PFOA) is a synthetic perfluorinated chemical classified as a persistent organic pollutant. PFOA has been linked to many toxic effects, including liver injury. Many studies report that PFOA exposure alters serum and hepatic lipid metabolism. However, lipidomic pathways altered by PFOA exposure are largely unknown and only a few lipid classes, mostly triacylglycerol (TG), are usually considered in lipid analysis. Here, we performed a global lipidomic analysis on the liver of PFOA-exposed (high-dose and short-duration) and control mice by combining three mass spectrometry (MS) techniques: liquid chromatography with tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Among all hepatic lipids identified by LC-MS/MS analysis, more than 350 were statistically impacted (increased or decreased levels) after PFOA exposure, as confirmed by multi-variate data analysis. The levels of many lipid species from different lipid classes, most notably phosphatidylethanolamine (PE), phosphatidylcholine (PC), and TG, were significantly altered. Subsequent lipidomic analysis highlights the pathways significantly impacted by PFOA exposure, with the glycerophospholipid metabolism being the most impacted, and the changes in the lipidome network, which connects all the lipid species together. MALDI-MSI displays the heterogeneous distribution of the affected lipids and PFOA, revealing different areas of lipid expression linked to PFOA localization. TOF-SIMS localizes PFOA at the cellular level, supporting MALDI-MSI results. This multi-modal MS analysis unveils the lipidomic impact of PFOA in the mouse liver after high-dose and short-term exposure and opens new opportunities in toxicology.

O-Mucin-degrading carbohydrate-active enzymes and their possible implication in inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are modern diseases, with incidence rising around the world. They are associated with perturbation of the intestinal microbiota, and with alteration and crossing of the mucus barrier by the commensal bacteria that feed on it. In the process of mucus catabolism and invasion by gut bacteria, carbohydrate-active enzymes (CAZymes) play a critical role since mucus is mainly made up by O- and N-glycans. Moreover, the occurrence of IBD seems to be associated with low-fiber diets. Conversely, supplementation with oligosaccharides, such as human milk oligosaccharides (HMOs), which are structurally similar to intestinal mucins and could thus compete with them towards bacterial mucus-degrading CAZymes, has been suggested to prevent inflammation. In this mini-review, we will establish the current state of knowledge regarding the identification and characterization of mucus-degrading enzymes from both cultured and uncultured species of gut commensals and enteropathogens, with a particular focus on the present technological opportunities available to further the discovery of mucus-degrading CAZymes within the entire gut microbiome, by coupling microfluidics with metagenomics and culturomics. Finally, we will discuss the challenges to overcome to better assess how CAZymes targeting specific functional oligosaccharides could be involved in the modulation of the mucus-driven cross-talk between gut bacteria and their host in the context of IBD.

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.

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.

Lactococcus lactis NCDO2118 exerts visceral antinociceptive properties in rat via GABA production in the gastro-intestinal tract.

Gut disorders associated to irritable bowel syndrome (IBS) are combined with anxiety and depression. Evidence suggests that microbially produced neuroactive molecules, like γ-aminobutyric acid (GABA), can modulate the gut-brain axis. Two natural strains of Lactococcus lactis and one mutant were characterized in vitro for their GABA production and tested in vivo in rat by oral gavage for their antinociceptive properties. L. lactis NCDO2118 significantly reduced visceral hypersensitivity induced by stress due to its glutamate decarboxylase (GAD) activity. L. lactis NCDO2727 with similar genes for GABA metabolism but no detectable GAD activity had no in vivo effect, as well as the NCDO2118 ΔgadB mutant. The antinociceptive effect observed for the NCDO2118 strain was mediated by the production of GABA in the gastro-intestinal tract and blocked by GABAB receptor antagonist. Only minor changes in the faecal microbiota composition were observed after the L. lactis NCDO2118 treatment. These findings reveal the crucial role of the microbial GAD activity of L. lactis NCDO2118 to deliver GABA into the gastro-intestinal tract for exerting antinociceptive properties in vivo and open avenues for this GRAS (Generally Recognized As safe) bacterium in the management of visceral pain and anxious profile of IBS patients.

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.

In vitro digestion of food grade TiO2 (E171) and TiO2 nanoparticles: physicochemical characterization and impact on the activity of digestive enzymes.

Titanium dioxide is a food additive that has raised some concerns for humans due to the presence of nanoparticles. We were interested in knowing the fate of TiO2 particles in the gastro-intestinal tract and their potential effect on digestive enzymes. For this purpose, we analysed the behaviour of two different food grade TiO2 samples (E171) and one nano-sized TiO2 sample (P25) through a standardized static in vitro digestion protocol simulating the oral, gastric and intestinal phases with appropriate juices including enzymes. Both E171 and P25 TiO2 particles remained intact in the digestive fluids but formed large agglomerates, and especially in the intestinal fluid where up to 500 µm sized particles have been identified. The formation of these agglomerates is mediated by the adsorption of mainly α-amylase and divalent cations. Pepsin was also identified to adsorb onto TiO2 particles but only in the case of silica-covered E171. In the salivary conditions, TiO2 exerted an inhibitory action on the enzymatic activity of α-amylase. The activity was reduced by a factor dependent on enzyme concentrations (up to 34% at 1 mg mL-1) but this inhibitory effect was reduced to hardly 10% in the intestinal fluid. In the gastric phase, pepsin was not affected by any form of TiO2. Our results hint that food grade TiO2 has a limited impact on the global digestion of carbohydrates and proteins. However, the reduced activity specifically observed in the oral phase deserves deeper investigation to prevent any adverse health effects related to the slowdown of carbohydrate metabolism.

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.

Repeated exposure of Caco-2 versus Caco-2/HT29-MTX intestinal cell models to (nano)silver in vitro: Comparison of two commercially available colloidal silver products.

Colloidal silver products are sold for a wide range of disinfectant and health applications. This has increased the potential for human exposure to silver nanoparticles (AgNPs) and ions (Ag+), for which oral ingestion is considered to be a major route of exposure. Our objective was to evaluate and compare the toxicity of two commercially available colloidal silver products on two human intestinal epithelial models under realistic exposure conditions. Mesosilver™ and AgC were characterized and a concentration range between 0.1 and 12 µg/mL chosen. Caco-2 cells vs. co-culture of Caco-2 and mucus-secreting HT29-MTX cells (90/10) were used. Repeated exposure was carried out to determine cell viability over 18 days of cell differentiation in 24-well plates. Selected concentrations (0.1, 1, and 3 µg/mL) were tested on cells cultured in E-plates and Transwells with the same repeated exposure regimen, to determine cell impedance, and cell viability and trans-epithelial electrical resistance (TEER), respectively. Silver uptake, intracellular localisation, and translocation were determined by CytoViva™, HIM-SIMS, and ICP-MS. Genotoxicity was determined on acutely-exposed proliferating Caco-2 cells by γH2AX immunofluorescence staining. Repeated exposure of a given concentration of AgC, which is composed solely of ionic silver, generally exerted more toxic effects on Caco-2 cells than Mesosilver™, which contains a mix of AgNPs and ionic silver. Due to its patchy structure, the presence of mucus in the Caco-2/HT29-MTX co-culture only slightly mitigated the deleterious effects on cell viability. Increased genotoxicity was observed for AgC on proliferating Caco-2 cells. Silver uptake, intracellular localisation, and translocation were similar. In conclusion, Mesosilver™ and AgC colloidal silver products show different levels of gut toxicity due to the forms of distinct silver (AgNPs and/or Ag+) contained within. This study highlights the applicability of high-resolution (chemical) imaging to detect and localize silver and provides insights into its uptake mechanisms, intracellular fate and cellular effects.

The complex puzzle of dietary silver nanoparticles, mucus and microbiota in the gut.

Hundreds of consumer and commercial products containing silver nanoparticles (AgNPs) are currently used in food, personal-care products, pharmaceutical, and many other applications. Human exposure to AgNPs includes oral intake, inhalation, and dermal contact. The aim of this review was to focus on oral intake, intentional and incidental of AgNPs where well-known antimicrobial characteristics that might affect the microbiome and mucus in the gastrointestinal tract (GIT). This critical review summarizes what is known regarding the impacts of AgNPs on gut homeostasis. It is fundamental to understand the forms of AgNPs and their physicochemical characterization before and during digestion. For example, lab-synthesized AgNPs differ from "real" ingestable AgNPs used as food additives and dietary supplements. Similarly, the gut environment alters the chemical and physical state of Ag that is ingested as AgNPs. Emerging research on in vitro and in vivo rodent and human indicated complex multi-directional relationships among AgNPs, the intestinal microbiota, and the epithelial mucus. It may be necessary to go beyond today's descriptive approach to a modeling-based ecosystem approach that might quantitatively integrate spatio-temporal interactions among microbial groups, host factors (e.g., mucus), and environmental factors, including lifestyle-based stressors. It is suggested that future research (1) utilize more representative AgNPs, focus on microbe/mucus interactions, (2) assess the effects of environmental stressors for longer and longitudinal conditions, and (3) be integrated using quantitative modeling.

Structure and biological activities of a hexosamine-rich cell wall polysaccharide isolated from the probiotic Lactobacillus farciminis.

Lactobacillus farciminis CIP 103136 is a bacterial strain with recognized probiotic properties. However, the mechanisms underlying such properties have only been partially elucidated. In this study, we isolated and purified a cell-wall associated polysaccharide (CWPS), and evaluated its biological role in vitro. The structure of CWPS and responses from stimulation of (i) human macrophage-like THP-1 cells, (ii) human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR2 or TLR4) and (iii) human colonocyte-like T84 intestinal epithelial cells, upon exposure to CWPS were studied. The structure of the purified CWPS from L. farciminis CIP 103136 was analyzed by nuclear magnetic resonance (NMR), MALDI-TOF-TOF MS, and methylation analyses in its native form and following Smith degradation. It was shown to be a novel branched polysaccharide, composed of linear backbone of trisaccharide repeating units of: [→6αGlcpNAc1 → 4ßManpNAc1 → 4ßGlcpNAc1→] highly substituted with single residues of αGlcp, αGalp and αGlcpNAc. Subsequently, the lack of pro- or anti-inflammatory properties of CWPS was established on macrophage-like THP-1 cells. In addition, CWPS failed to modulate cell signaling pathways dependent of TLR2 and TLR4 in transfected HEK-cells. Finally, in T84 cells, CWPS neither influenced intestinal barrier integrity under basal conditions nor prevented TNF-α/IFN-γ cytokine-mediated epithelium impairment.

Characterization of Mucus-Related Properties of Streptococcus thermophilus: From Adhesion to Induction.

Mucus is a major component of the intestinal barrier involved both in the protection of the host and the fitness of commensals of the gut. Streptococcus thermophilus is consumed world-wide in fermented dairy products and is also recognized as a probiotic, as its consumption is associated with improved lactose digestion. We determined the overall effect of S. thermophilus on the mucus by evaluating its ability to adhere, degrade, modify, or induce the production of mucus and/or mucins. Adhesion was analyzed in vitro using two types of mucins (from pig or human biopsies) and mucus-producing intestinal HT29-MTX cells. The induction of mucus was characterized in two different rodent models, in which S. thermophilus is the unique bacterial species in the digestive tract or transited as a sub-dominant bacterium through a complex microbiota. S. thermophilus LMD-9 and LMG18311 strains did not grow in sugars used to form mucins as the sole carbon source and displayed weak binding to mucus/mucins relative to the highly adhesive TIL448 Lactococcus lactis. The presence of S. thermophilus as the unique bacteria in the digestive tract of gnotobiotic rats led to accumulation of lactate and increased the number of Alcian-Blue positive goblet cells and the amount of the mucus-inducer KLF4 transcription factor. Lactate significantly increased KLF4 protein levels in HT29-MTX cells. Introduction of S. thermophilusvia transit as a sub-dominant bacterium (103 CFU/g feces) in a complex endogenous microbiota resulted in a slight increase in lactate levels in the digestive tract, no induction of overall mucus production, and moderate induction of sulfated mucin production. We thus show that although S. thermophilus is a poor mucus-adhesive bacterium, it can promote mucus pathway at least in part by producing lactate in the digestive tract.

Food-grade TiO2 is trapped by intestinal mucus in vitro but does not impair mucin O-glycosylation and short-chain fatty acid synthesis in vivo: implications for gut barrier protection.

BACKGROUND: Titanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. RESULTS: We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside "patchy" regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular "islands" located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. CONCLUSIONS: Food-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under "healthy gut" conditions.

Mucus: An Underestimated Gut Target for Environmental Pollutants and Food Additives.

Synthetic chemicals (environmental pollutants, food additives) are widely used for many industrial purposes and consumer-related applications, which implies, through manufactured products, diet, and environment, a repeated exposure of the general population with growing concern regarding health disorders. The gastrointestinal tract is the first physical and biological barrier against these compounds, and thus their first target. Mounting evidence indicates that the gut microbiota represents a major player in the toxicity of environmental pollutants and food additives; however, little is known on the toxicological relevance of the mucus/pollutant interplay, even though mucus is increasingly recognized as essential in gut homeostasis. Here, we aimed at describing how environmental pollutants (heavy metals, pesticides, and other persistent organic pollutants) and food additives (emulsifiers, nanomaterials) might interact with mucus and mucus-related microbial species; that is, "mucophilic" bacteria such as mucus degraders. This review highlights that intestinal mucus, either directly or through its crosstalk with the gut microbiota, is a key, yet underestimated gut player that must be considered for better risk assessment and management of environmental pollution.

Toxicity of Food-Grade TiO2 to Commensal Intestinal and Transient Food-Borne Bacteria: New Insights Using Nano-SIMS and Synchrotron UV Fluorescence Imaging.

Titanium dioxide (TiO2) is commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, a risk of intestinal barrier disruption, including dysbiosis of the gut microbiota, is increasingly suspected because of the presence of a nano-sized fraction in this additive. We hypothesized that food-grade E171 and Aeroxyde P25 (identical to the NM-105 OECD reference nanomaterial in the European Union Joint Research Centre) interact with both commensal intestinal bacteria and transient food-borne bacteria under non-UV-irradiated conditions. Based on differences in their physicochemical properties, we expect a difference in their respective effects. To test these hypotheses, we chose a panel of eight Gram-positive/Gram-negative bacterial strains, isolated from different biotopes and belonging to the species Escherichia coli, Lactobacillus rhamnosus, Lactococcus lactis (subsp. lactis and cremoris), Streptococcus thermophilus, and Lactobacillus sakei. Bacterial cells were exposed to food-grade E171 vs. P25 in vitro and the interactions were explored with innovative (nano)imaging methods. The ability of bacteria to trap TiO2 was demonstrated using synchrotron UV fluorescence imaging with single cell resolution. Subsequent alterations in the growth profiles were shown, notably for the transient food-borne L. lactis and the commensal intestinal E. coli in contact with food-grade TiO2. However, for both species, the reduction in cell cultivability remained moderate, and the morphological and ultrastructural damages, observed with electron microscopy, were restricted to a small number of cells. E. coli exposed to food-grade TiO2 showed some internalization of TiO2 (7% of cells), observed with high-resolution nano-secondary ion mass spectrometry (Nano-SIMS) chemical imaging. Taken together, these data show that E171 may be trapped by commensal and transient food-borne bacteria within the gut. In return, it may induce some physiological alterations in the most sensitive species, with a putative impact on gut microbiota composition and functioning, especially after chronic exposure.

A Sensitive and Rapid Method to Determin the Adhesion Capacity of Probiotics and Pathogenic Microorganisms to Human Gastrointestinal Mucins.

Mucus is the habitat for the microorganisms, bacteria and yeast that form the commensal flora. Mucins, the main macromolecules of mucus, and more specifically, the glycans that cover them, play essential roles in microbial gastrointestinal colonization. Probiotics and pathogens must also colonize mucus to have lasting positive or deleterious effects. The question of which mucin-harboured glycan motifs favour the adhesion of specific microorganisms remains very poorly studied. In the current study, a simple test based on the detection of fluorescent-labeled microorganisms raised against microgram amounts of mucins spotted on nitrocellulose was developed. The adhesion of various probiotic, commensal and pathogenic microorganisms was evaluated on a panel of human purified gastrointestinal mucins and compared with that of commercially available pig gastric mucins (PGM) and of mucins secreted by the colonic cancer cell line HT29-MTX. The latter two proved to be very poor indicators of adhesion capacity on intestinal mucins. Our results show that the nature of the sialylated cores of O-glycans, determined by MALDI MS-MS analysis, potentially enables sialic acid residues to modulate the adhesion of microorganisms either positively or negatively. Other identified factors affecting the adhesion propensity were O-glycan core types and the presence of blood group motifs. This test should help to select probiotics with enhanced adhesion capabilities as well as deciphering the role of specific mucin glycotopes on microbial adhesion.

Mucus and microbiota as emerging players in gut nanotoxicology: The example of dietary silver and titanium dioxide nanoparticles.

Given the growing use of nanotechnology in many common consumer products, including foods, evaluation of the consequences of chronic exposure to nanoparticles in humans has become a major public health issue. The oral route of exposure has been poorly explored, despite the presence of a fraction of nanosized particles in certain food additives/supplements and the incorporation of such particles into packaging in contact with foods. After their ingestion, these nanoparticles pass through the digestive tract, where they may undergo physicochemical transformations, with consequences for the luminal environment, before crossing the epithelial barrier to reach the systemic compartment. In this review, we consider two examples, nanosilver and nanotitanium dioxide. Despite the specific features of these particles and the differences between them, both display a close relationship between physicochemical reactivity and bioavailability/biopersistence in the gastrointestinal tract. Few studies have focused on the interactions of nanoparticles of silver or titanium dioxide with the microbiota and mucus. However, the microbiota and mucus play key roles in intestinal homeostasis and host health and are undoubtedly involved in controlling the distribution of nanoparticles in the systemic compartment.