Interactions with M cells and macrophages as key steps in the pathogenesis of enterohemorrhagic Escherichia coli infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases

Can dynamic in vitro digestion systems mimic the physiological reality?

During the last decade, there has been a growing interest in understanding the fate of food during digestion in the gastrointestinal tract in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore simple static in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments but these models are quite basic and hardly recreate the complexity of the digestive tract. In contrast, dynamic models that allow pH regulation, flow of the food and injection in real time of digestive enzymes in the different compartments of the gastrointestinal tract are more promising to accurately mimic the digestive process. Most of the systems developed so far have been compared for their performances to in vivo data obtained on animals and/or humans. The objective of this article is to review the validation towards in vivo data of some of the dynamic digestion systems currently available in order to determine what aspects of food digestion they are able to mimic. Eight dynamic digestion systems are presented as well as their validation towards in vivo data. Advantages and limits of each simulator is discussed. This is the result of a cooperative international effort made by some of the scientists involved in Infogest, an international network on food digestion

Characterization of the behavior of carotenoids from pitanga (Eugenia uniflora) and buriti (Mauritia flexuosa) during microemulsion production and in a dynamic gastrointestinal system

Uncommon tropical fruits are emerging as raw-material for new food products with health benefits. This work aimed at formulating and processing microemulsions from pitanga (Eugenia uniflora) and buriti (Mauritia flexuosa) fruits, since they are very rich in carotenoids (particularly lycopene and -carotene), in order to encapsulate and increase carotenoids bioaccessibility. Pitanga and buriti microemulsions were produced by applying a direct processing (high-speed homogenization at 15,000 rpm and ultrasound with 20 kHz probe at 40% amplitude) of the whole pulp together with surfactant (Tween 80 or Whey Protein Isolate at 2%) and corn oil (5%). All treatments (HSHUS for 04, 40, 44, 48 minmin) applied were able to increase the amount of carotenoid released. However, the processing also decreased the total amount of carotenoids in the whole pulp of studied fruits. The impact of processing during microemulsion production was not severe. The overall data suggest that the presence of surfactant and oil during processing may protect the carotenoids in fruits and microemulsions. Final recovery of total carotenoids, after passing the samples through a dynamic gastrointestinal system that simulates the human digestion, was higher for microemulsions than for whole pulps. High losses of total carotenoids in buriti and -carotene and lycopene in pitanga occurred during jejunum and ileum phases. The present work confirms that it is possible to increase -carotene and lycopene bioaccessibility from fruits by directly processing microemulsions (p<0.01).This work was supported by the São Paulo Research Foundation—FAPESP through research funding [Grant #2015/15507-9] and Ph.D. scholarship for Paulo Berni [Grant #2014/15119-6] and a Research Internships Abroad (BEPE) support [Grant #2016/13355-0]. The author Ana C. Pinheiro is recipient of a fellowship from the Portuguese Foundation for Science and Technology (FCT) [Grant SFRH/BPD/101181/2014]info:eu-repo/semantics/publishedVersio

Importance of oral phase in in vitro starch digestibility related to wholegrain versus refined pastas and mastication impairment

International audienceStarch represents the main source of carbohydrates in human diet and its digestibility is suspected to be involved in the control of glycemic response. The low glycemic index caused by pastas is mainly attributed to the starch-protein network constituting their compact structure. A significant part of the physico-chemical digestive process probably occurs during mastication with exposure to amylase. However, the respective accountability of oral and intestinal phases in digestion is not clearly established, and this knowledge would especially benefit to health management of people suffering of impaired mastication. Food boluses were produced for in vitro gastrointestinal digestion either by in vitro normal (NM) or deficient mastication (DM) of wholegrain and refined pastas. Boluses were first characterized for physical properties. Many large particles were obtained in DM boluses whatever the pastas. Starch and hydrolysis products were then determined in boluses and gastrointestinal digestas. The beginning of starch hydrolysis was confirmed in the mouth with a production of maltose in the NM boluses, around 1.6 g/100g of cooked pastas, significantly decreased to 1.2 g/100g in the DM boluses, whatever the pastas. Even if the negative effect of DM on gastrointestinal starch hydrolysis into glucose was observed for both pastas, the greatest impact occurred in refined ones with 55.9 ± 0.82 g glucose/100g pastas after NM versus 53.00 ± 0.95 g/100g after DM. This study highlighted the importance to consider the oral phase in digestion studies, regarding the impact of food structure and oral disruption, and especially in case of DM

Comparison of conventional plating, PMA-qPCR, and flow cytometry for the determination of viable enterotoxigenic Escherichia coli along a gastrointestinal in vitro model

International audienceRecent technological advances for bacterial viability assessment using molecular methods or flow cytometry can provide meaningful interest for the demarcation between live and dead microorganisms. Nonetheless, these methods have been scarcely applied to foodborne pathogens and never for directly assessing their viability within the human digestive environment. The purpose of this study was to compare two methods based on membrane integrity (propidium monoazide (PMA)q-PCR and Live/Dead flow cytometry) and the classical plate-count method to determine the viability of a common foodborne pathogen, enterotoxigenic Escherichia coli (ETEC), during its transit trough simulated human gastrointestinal environment. Viable ETEC counts in the gastric and small intestinal compartments of the gastrointestinal TIM model indicated a consensus between the three tested methods (PMA-qPCR, flow cytometry, and plate counts). In a further step, flow cytometry analysis appeared as the preferred method to elucidate ETEC physiological states in the in vitro digestive environment by discriminating four subpopulations, while PMA-qPCR can only distinguish two. The defined viable/altered ETEC population was found during all in vitro digestions, but mainly in the gastric compartment. Being able to discriminate the particular physiological states of pathogenic microorganisms in the digestive environment is of high interest, because if some cells are not observable on culture media, they might keep their ability to express virulence functions

Digestion of cooked meat proteins is slightly affected by age as assessed using the dynamic gastrointestinal TIM model and mass spectrometry

International audienceIn humans, meat ensures the supply of proteins with high nutritional value and indispensable amino acids. The main goal of the present study was to compare the degradation of meat proteins in adult and elderly digestive conditions. Cooked meat was subjected to in vitro digestion in the dynamic multi-compartmental TIM (TNO gastroIntestinal Model) system. Digestibility and bioaccessibility were determined using nitrogen balance and digestion products were identified using mass spectrometry. The TIM model was adapted according to in vivo data to mimic the specific digestive conditions of elderly people. Meat protein digestibility and bioaccessibility were around 96 and 60% respectively and were not influenced by age (P > 0.05). As much as 800 peptides were identified in the duodenal and jejunal compartments issued from 50 meat proteins with a percentage of coverage varying from 13 to 69%. Six proteins, mainly from the cytosol, were differentially hydrolyzed under the adult and elderly digestive conditions. Pyruvate kinase was the only protein clearly showing a delay in its degradation under elderly digestive conditions. This study provides significant insights into the understanding of meat protein dynamic digestion. Such data will be helpful to design in vivo studies aiming to evaluate dietary strategies that can attenuate muscle mass loss and more generally maintain a better quality of life in the elderly population

Impact of oral galenic formulations of Lactobacillus salivarius on probiotic survival and interactions with microbiota in human in vitro gut models

International audienceHealth benefits of probiotics in humans essentially depend on their ability to survive during gastrointestinal (GI) transit and to modulate gut microbiota. To date, there is few data on the impact of galenic formulations of probiotics on these parameters. Even if clinical studies remain the gold standard to evaluate the efficacy of galenic forms, they stay hampered by technical, ethical and cost reasons. As an alternative approach, we used two complementary in vitro models of the human gut, the TNO gastrointestinal (TIM-1) model and the Artificial Colon (ARCOL), to study the effect of three oral formulations of a Lactobacillus salivarius strain (powder, capsule and sustained-release tablet) on its viability and interactions with gut microbiota. In the TIM-1 stomach, no or low numbers of bacteria were respectively released from the capsule and tablet, confirming their gastro-resistance. The capsule was disintegrated in the jejunum on average 76 min after administration while the core of sustained-release tablet was still intact at the end of digestion. Viability in TIM-1 was significantly influenced by the galenic form with survival percentages of 0.003±0.004%, 2.8±0.6% and 17.0±1.8% (n=3) for powder, capsule and tablet, respectively. In the ARCOL, the survival of the strain tended to be higher in the post-treatment phase with the tablet compared to capsule, but gut microbiota composition and activity were not differently modulated by the two formulations. In conclusion, the sustained-release tablet emerged as the formulation that most effectively preserved viability of the tested strain during GI passage. This study highlights the usefulness of in vitro gut models for the pre-screening of probiotic pharmaceutical forms. Their use could also easily be extended to the evaluation of the effects of food matrices and age on probiotic survival and activity during GI transit

Impact of a more realistic fermentation medium on microbiota composition and metabolic activities in two well-validated in vitro human colon models.

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Impact of two different colistin dosing strategies on healthy piglet fecal microbiota

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