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

Health 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.

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.

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

Recent 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.

Anti-infectious properties of the probiotic Saccharomyces cerevisiae CNCM I-3856 on enterotoxigenic E. coli (ETEC) strain H10407.

Enterotoxigenic Escherichia coli (ETEC) are major food-borne pathogens responsible for traveler's diarrhea. The production of adhesins and the secretion of enterotoxins constitute the major virulence traits of the bacteria. Treatments are mainly symptomatic and can involve antibiotherapy. However, given the rise of antibiotic resistance worldwide, there is an urgent need for the development of new preventive strategies for the control of ETEC infections. Among them, a promising approach is the use of probiotics. The aim of this study was to investigate, using complementary in vitro and in vivo approaches, the inhibitory potential of the yeast Saccharomyces cerevisiae CNCM I-3856 against the human ETEC reference strain H10407. In conventional culture media, S. cerevisiae significantly reduced ETEC growth and toxin production. The yeast also inhibited bacterial adhesion to mucin-agar and intestinal Caco-2/TC7 cells in a dose-dependent manner. Lastly, pre-treatment with S. cerevisiae inhibited interleukin-8 production by ETEC-infected intestinal cells. In streptomycin-treated mice, the probiotic yeast decreased bacterial colonization, mainly in the ileum, the main site of ETEC pathogenesis. For the first time, this study shows that the probiotic yeast S. cerevisiae CNCM I-3856 can exert an anti-infectious activity against a human ETEC strain through a multi-targeted approach, including inhibition of bacterial growth and toxin production, reduction of bacterial adhesion to mucins and intestinal epithelial cells, and suppression of ETEC-induced inflammation. Interestingly, the highest activity was obtained with a prophylactic treatment. Further studies will aim to assess the effect of the yeast on ETEC survival and virulence under human simulated digestive conditions.

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

Colistin is often used in piglets but underdosing and overdosing are frequent. The impact of such administrations on fecal microbiota was studied. Piglets were given either underdoses of colistin by oral gavage for five days or overdoses by in-feed medication for 14days. The composition of fecal microbiota was studied by quantitative PCR, 16S rRNA sequencing, culture of Enterobacteriaceae, and quantification of short-chain fatty acids (SCFAs). The mean colistin concentrations during the treatment for underdosed and overdosed groups were 14.4µg/g and 64.9µg/g of feces respectively. Whatever the piglet and the sampling day, the two main phyla were Firmicutes and Bacteroidetes, The main families were Lactobacillaceae, Clostridiales, Lachnospiraceae and Ruminococcaceae. The main perturbation was the significant but transitory decrease in the Escherichia coli population during treatment, yet all the E. coli isolates were susceptible to colistin. Moreover, colistin did not affect the production of SCFAs. These results show that under- or overdoses of colistin do not result in any major disturbance of piglet fecal microbiota and rarely select for chromosomal resistance in the dominant E. coli population.

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

In 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 ceftiofur injection on gut microbiota and Escherichia coli resistance in pigs.

Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, bla CTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the bla CTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the bla CTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

Enterohemorrhagic Escherichia coli infection has donor-dependent effect on human gut microbiota and may be antagonized by probiotic yeast during interaction with Peyer's patches.

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens responsible for serious infections ranging from mild diarrhea to hemorrhagic colitis and life-threatening complications. Shiga toxins (Stxs) are the main virulence factor of EHEC. The antagonistic effect of a prophylactic treatment with the probiotic strain Saccharomyces cerevisiae against EHEC O157:H7 was investigated using complementary in vitro human colonic model and in vivo murine ileal loop assays. In vitro, the probiotic treatment had no effect on O157:H7 survival but favorably influenced gut microbiota activity through modulation of short-chain fatty acid production, increasing acetate production and decreasing that of butyrate. Both pathogen and probiotic strains had individual-dependent effects on human gut microbiota. For the first time, stx expression was followed in human colonic environment: at 9 and 12 h post EHEC infection, probiotic treatment significantly decreased stx mRNA levels. Besides, in murine ileal loops, the probiotic yeast specifically exerted a trophic effect on intestinal mucosa and inhibited O157:H7 interactions with Peyer's patches and subsequent hemorrhagic lesions. Taken together, the results suggest that S. cerevisiae may be useful in the fight against EHEC infection and that host associated factors such as microbiota could influence clinical evolution of EHEC infection and the effectiveness of probiotics.

Genetically engineered yeasts as a new delivery vehicle of active compounds to the digestive tract: in vivo validation of the concept in the rat.

An innovative "biodrug" concept based on oral administration of living recombinant microorganisms as a vehicle to deliver active compounds directly into the digestive tract has recently been developed. To validate this concept, we studied a recombinant Saccharomyces cerevisiae strain in order to investigate its viability and its ability to produce a protein (glutathione-S-transferase (GST)-V(5)H(6)) in the rat. Following oral administration, the recombinant yeast showed a survival rate of around 40% in the upper parts of the digestive tract, but was more sensitive to the conditions in the large intestinal, where viability dropped to 1%. Western blot analysis was able to detect the model protein throughout the digestive tract, including stomach, duodenum, jejunum (proximal, median and distal), ileum, cecum and colon. The gastrointestinal sac technique was employed to quantify GST-V(5)H(6) in all the digestive compartments. These results suggest that S. cerevisiae may represent a useful host for producing compounds of interest directly in the digestive tract.

Enumeration of some cultivable bacterial groups and characterization of some abiotic variables in the jejunoileal content of Prim'Holstein veal calves.

A study was conducted to characterize the bacterial and biochemical composition of the jejunoileal content of veal calves and the effect of pre-slaughter fasting time. At 22 wk of age, 22 preruminant Prim'Holstein calves fed milk replacer and pellets (mainly composed of corn) were slaughtered at 6, 12, or 24 h after their last meal. Chyme samples were collected from the jejunoileal compartment just after slaughter, and pH and redox potential were immediately measured. Culture-based methods were used to determine the concentrations of total anaerobic microflora, lactate-utilizing bacteria, Bacteroides fragilis group, Lactobacilli, Bifidobacteria, Enterococci, and 2 potential pathogenic species, Escherichia coli and Clostridium perfringens. Concentrations of l-lactate, ammonia, and short-chain fatty acids (SCFA) were determined on frozen samples. The biochemical composition (DM, total protein, lactose, galactose, glucose, minerals, AA profile, and fatty acid profile) of the jejunoileal content was determined only on samples from the 6-h fasted group. Microflora concentrations were greater (P < 0.01) in the 6-h fasted group compared with the 12- and 24-h fasted groups, involving a decreased pH (P < 0.05) and greater lactate and SCFA concentrations, both linked directly to the fermentative state of the microorganisms. The 6-h fasted group showed the least interanimal variability in bacterial group levels, except for Cl. perfringens, which presented increased interanimal variability regardless of fasting time. At 6 h postprandial, the jejunoileal content of veal calves seemed to be in a stable state, allowing the creation of a database on its biochemical composition. This study is a key first step in the development of an in vitro system for modeling the jejunoileal ecosystem of veal calves. This model will provide a useful tool for assessing the effects of feed additives on intestinal microflora.