Search

IntroductionThe aim of this work was to characterize a new strain of Ligilactobacillus salivarius (CNCM I-4866) (CNCM I-4866) to address its potential as probiotic with a special focus on intestinal inflammation. Potential anti-inflammatory abilities of this strain were evaluated through in vivo and in vitro experiments.MethodsFirstly, the strain was tested in a murine acute inflammation colitis model induced by DNBS. In vitro characterization was then performed with diverse tests: modulation capability of intestinal permeability; study of the impact on immunity profile through cytokines dosage; capacity to inhibit pathogens and adhere to intestinal cells lines. Production of metabolites, antibiotic resistance and survival to gastro-intestinal tract conditions were also tested.ResultsIn vitro assay has shown a reduction of colonic damage and markers of inflammation after treatment with CNCM I-4866. Transcriptomic analysis performed on colons showed the capacity of the strain to down-regulate pro-inflammatory cytokines. L. salivarius CNCM I-4866 exerted anti-inflammatory profile by reducing IL-8 production by TNF-α stimulated cell and modulated cytokines profile on peripheral blood mononuclear cells (PBMC). It protected intestinal integrity by increasing trans-epithelial electrical resistance (TEER) on Caco-2 TNF-α inflamed cells. Additionally, L. salivarius CNCM I-4866 displayed inhibition capacity on several intestinal pathogens and adhered to eukaryotic cells. Regarding safety and technical concerns, CNCM I-4866 was highly resistant to 0.3% of bile salts and produced mainly L-lactate. Finally, strain genomic characterization allowed us to confirm safety aspect of our strain, with no antibiotic gene resistance found.DiscussionTaken together, these results indicate that L. salivarius CNCM I-4866 could be a good probiotic candidate for intestinal inflammation, especially with its steady anti-inflammatory profile.

Faecalibacterium prausnitzii is a commensal bacterium, ubiquitous in the gastrointestinal tracts of animals and humans. This species is a functionally important member of the microbiota and studies suggest it has an impact on the physiology and health of the host. F. prausnitzii is the only identified species in the genus Faecalibacterium, but a recent study clustered strains of this species in two different phylogroups. Here, we propose the existence of distinct species in this genus through the use of comparative genomics. Briefly, we performed analyses of 16S rRNA gene phylogeny, phylogenomics, whole genome Multi-Locus Sequence Typing (wgMLST), Average Nucleotide Identity (ANI), gene synteny, and pangenome to better elucidate the phylogenetic relationships among strains of Faecalibacterium. For this, we used 12 newly sequenced, assembled, and curated genomes of F. prausnitzii, which were isolated from feces of healthy volunteers from France and Australia, and combined these with published data from 5 strains downloaded from public databases. The phylogenetic analysis of the 16S rRNA sequences, together with the wgMLST profiles and a phylogenomic tree based on comparisons of genome similarity, all supported the clustering of Faecalibacterium strains in different genospecies. Additionally, the global analysis of gene synteny among all strains showed a highly fragmented profile, whereas the intra-cluster analyses revealed larger and more conserved collinear blocks. Finally, ANI analysis substantiated the presence of three distinct clusters—A, B, and C—composed of five, four, and four strains, respectively. The pangenome analysis of each cluster corroborated the classification of these clusters into three distinct species, each containing less variability than that found within the global pangenome of all strains. Here, we propose that comparison of pangenome subsets and their associated α values may be used as an alternative approach, together with ANI, in the in silico classification of new species. Altogether, our results provide evidence not only for the reconsideration of the phylogenetic and genomic relatedness among strains currently assigned to F. prausnitzii, but also the need for lineage (strain-based) differentiation of this taxon to better define how specific members might be associated with positive or negative host interactions.

ABSTRACT Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified on the basis of human clinical data. The mechanisms underlying its beneficial effects are still unknown. Gnotobiotic mice harboring F. prausnitzii (A2-165) and Escherichia coli (K-12 JM105) were subjected to 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis. The inflammatory colitis scores and a gas chromatography-time of flight (GC/TOF) mass spectrometry-based metabolomic profile were monitored in blood, ileum, cecum, colon, and feces in gnotobiotic mice. The potential anti-inflammatory metabolites were tested in vitro. We obtained stable E. coli and F. prausnitzii-diassociated mice in which E. coli primed the gastrointestinal tract (GIT), allowing a durable and stable establishment of F. prausnitzii. The disease activity index, histological scores, myeloperoxidase (MPO) activity, and serum cytokine levels were significantly lower in the presence of F. prausnitzii after TNBS challenge. The protective effect of F. prausnitzii against colitis was correlated to its implantation level and was linked to overrepresented metabolites along the GIT and in serum. Among 983 metabolites in GIT samples and serum, 279 were assigned to known chemical reactions. Some of them, belonging to the ammonia (α-ketoglutarate), osmoprotective (raffinose), and phenolic (including anti-inflammatory shikimic and salicylic acids) pathways, were associated with a protective effect of F. prausnitzii, and the functional link was established in vitro for salicylic acid. We show for the first time that F. prausnitzii is a highly active commensal bacterium involved in reduction of colitis through in vivo modulation of metabolites along the GIT and in the peripheral blood. IMPORTANCE Inflammatory bowel diseases (IBD) are characterized by low proportions of F. prausnitzii in the gut microbiome. This commensal bacterium exhibits anti-inflammatory effects through still unknown mechanisms. Stable monoassociated rodents are actually not a reproducible model to decipher F. prausnitzii protective effects. We propose a new gnotobiotic rodent model providing mechanistic clues. In this model, F. prausnitzii exhibits protective effects against an acute colitis and a protective metabolic profile is linked to its presence along the digestive tract. We identified a molecule, salicylic acid, directly involved in the protective effect of F. prausnitzii. Targeting its metabolic pathways could be an attractive therapeutic strategy in IBD.

Application of beneficial microorganisms as probiotics targets a broad range of intended uses, from maintaining health and supporting normal bodily functions to curing and preventing diseases. Currently, three main regulatory fields of probiotic products can be defined depending on their intended use: the more similar probiotic foods and probiotic dietary supplements, and live biotherapeutic products. However, it is not always straightforward to classify a probiotic product into one of these categories. The regulatory nuances of developing, manufacturing, investigating and applying each category of probiotic products are not universal, and not always apparent to those unfamiliar with the various global probiotic regulatory guidelines. Various global markets can be significantly different regarding legislation, possible claims, market value and quality requirements for the development and commercialization of probiotic products. Furthermore, different probiotic product categories are also linked with variable costs at different stages of product development. This review outlines the current landscape comparing probiotic foods, probiotic dietary supplements, and live biotherapeutics as probiotic products from a regulatory lens, focusing on product development, manufacturing and production, and clinical research agenda. The aim is to inform and promote a better understanding among stakeholders by outlining the expectations and performance for each probiotic product category, depending on their intended use and targeted geographical region.

The characteristics of the whole PEDV genome that has circulated in Mexico from the first outbreak to the present are unknown. We chose samples obtained from 2013 to 2017 and sequenced them, which enabled us to identify the genetic variation and phylogeny in the virus during the first four years that it circulated in Mexico. A 99% identity was found among the analyzed pandemic strains; however, the 1% difference affected the structure of the S glycoprotein, which is essential for the binding of the virus to the cellular receptor. The S protein induces the most efficacious antibodies; hence, these changes in structure could be implicated in the clinical antecedents of the outbreaks. Antigenic changes could also help PEDV avoid neutralization, even in the presence of previous immunity. The characterization of the complete genome enabled the identification of three circulating strains that have a deletion in ORF1a, which is present in attenuated Asian vaccine strains. The phylogenetic analysis of the complete genome indicates that the first PEDV outbreaks in Mexico were caused by INDEL strains and pandemic strains related to USA strains; however, the possibility of the entry of European strains exists, which may have caused the 2015 and 2016 outbreaks.

Preterm birth is one of the main health problems encountered in the neonatal period, especially because it is also the first cause of death in the critical 1st month of life and the second in children under 5 years of age. Not only preterm birth entails short term health risks due to low weight and underdeveloped organs, but also increases the risk of suffering from non-transmissible diseases in the long term. To date, it is known that medical conditions and lifestyle factors could increase the risk of preterm birth, but the molecular mechanisms that control this process remain unclear. Luteolysis, increased inflammation or oxidative stress have been described as possible triggers for preterm birth and, in some cases, the cause of dysbiosis in preterm neonates. Several murine models have been developed to shed light into the mechanistic of preterm birth but, for the most part, are inflammation-based labor induction models and the offspring health readouts are mainly limited to survival and weight. Using a set of SWISS-CD1 mice born prematurely we analyzed inflammation and gut permeability parameters compared with term pups at weaning age. Overall, preterm mice presented higher systemic inflammation and gastrointestinal tract permeability. In this perspective article, we discuss the recent discoveries on preterm birth and the necessity of non-inflammatory murine models to really understand these phenotypes and be able to design strategies to prevent the sequels of this traumatic event in neonates.

Abstract The aim of this review is to summarize the effect in host energy metabolism of the production of B group vitamins and short chain fatty acids (SCFA) by commensal, food-grade and probiotic bacteria, which are also actors of the mammalian nutrition. The mechanisms of how these microbial end products, produced by these bacterial strains, act on energy metabolism will be discussed. We will show that these vitamins and SCFA producing bacteria could be used as tools to recover energy intakes by either optimizing ATP production from foods or by the fermentation of certain fibers in the gastrointestinal tract (GIT). Original data are also presented in this work where SCFA (acetate, butyrate and propionate) and B group vitamins (riboflavin, folate and thiamine) production was determined for selected probiotic bacteria.

Murine colitis models are crucial tools for understanding intestinal homeostasis and inflammation. However, most current models utilize a highly inbred strain of mice, and often only one sex is employed to limit bias. This targeted approach, which in itself is biased, means that murine genetic diversity and sex-related differences are ignored, making it even more difficult to extend findings to humans, who are highly heterogeneous. Furthermore, most models do not examine the chronic form of colitis, an important fact taking into account the chronic nature of the inflammatory bowel diseases (IBD). Here, we attempted to create a more realistic murine colitis model by addressing these three issues. Using chemically induced chronic colon inflammation in an outbred strain of mice (RjOrl:SWISS [CD-1]), we (i) mimicked the relapsing nature of the disease, (ii) better represented normal genetic variability, and (iii) employed both female and male mice. Colitis was induced by intrarectal administration of dinitrobenzene sulfonic acid (DNBS). After a recovery period and 3 days before the mice were euthanized, colitis was reactivated by a second administration of DNBS. Protocol length was 24 days. Colitis severity was assessed using body mass, macroscopic scores, and histological scores. Myeloperoxidase (MPO) activity, cytokine levels, and lymphocyte populations were also characterized. Our results show that the intrarectal administration of DNBS effectively causes colitis in both female and male CD-1 mice in a dose-dependent manner, as reflected by loss of body mass, macroscopic scores and histological scores. Furthermore, colon cytokine levels and mesenteric lymph node characteristics indicate that this model involves immune system activation. Although some variables were sex-specific, most of the results support including both females and males in the model. Our ultimate goal is to make this model available to researchers for testing candidate anti-inflammatory agents, such as classical or next-generation probiotics; we also aim for the results to be more easily transferrable to human trials.

Faecalibacterium prausnitzii represents approximately 5% of the total fecal microbiota in healthy adults being one of the most abundant bacterium in the human intestinal microbiota of healthy adults. Furthermore, this bacterium has been proposed to be a sensor and a major actor of the human intestinal health because of its importance in the gut ecosystem. In this context, F. prausnitzii population levels have been found to be reduced in patients suffering from several syndromes and diseases such as inflammatory bowel diseases. These diseases are characterized by a breakage of the intestinal homeostasis called dysbiosis and the use of F. prausnitzii as a next generation probiotic (also called live biotherapeutics) has been proposed as a natural tool to restore such dysbiosis within the gut. Nevertheless, despite the potential importance of this bacterium in human health, little is known about its main effectors underlying its beneficial effects. In this perspective note, we aim to present the actual state in the research about F. prausnitzii effectors and the future milestones in this field.

Faecalibacterium prausnitzii is a major member of the Firmicutes phylum and one of the most abundant bacteria in the healthy human microbiota. F. prausnitzii depletion has been reported in several intestinal disorders, and more consistently in Crohn's disease (CD) patients. Despite its importance in human health, only few microbiological studies have been performed to isolate novel F. prausnitzii strains in order to better understand the biodiversity and physiological diversity of this beneficial commensal species. In this study, we described a protocol to isolate novel F. prausnitzii strains from feces of healthy volunteers as well as a deep molecular and metabolic characterization of these isolated strains. These F. prausnitzii strains were classified in two phylogroups and three clusters according to 16S rRNA sequences and results support that they would belong to two different genomospecies or genomovars as no genome sequencing has been performed in this work. Differences in enzymes production, antibiotic resistance and immunomodulatory properties were found to be strain-dependent. So far, all F. prausnitzii isolates share some characteristic such as (i) the lack of epithelial cells adhesion, plasmids, anti-microbial, and hemolytic activity and (ii) the presence of DNAse activity. Furthermore, Short Chain Fatty Acids (SCFA) production was assessed for the novel isolates as these products influence intestinal homeostasis. Indeed, the butyrate production has been correlated to the capacity to induce IL-10, an anti-inflammatory cytokine, in peripheral blood mononuclear cells (PBMC) but not to the ability to block IL-8 secretion in TNF-α-stimulated HT-29 cells, reinforcing the hypothesis of a complex anti-inflammatory pathway driven by F. prausnitzii. Altogether, our results suggest that some F. prausnitzii strains could represent good candidates as next-generation probiotic.