Microbial colonization of the newborn (primocolonization) plays a crucial role in the development of digestive, metabolic, and immune systems. Disruptions may impair intestinal barrier function and raise the risk of chronic diseases like inflammatory bowel disease, allergies, and obesity. Recent evidence suggests probiotic supplementation in infants may help counter or prevent these effects. This study isolates and characterizes potential probiotic candidates from the gut microbiota of exclusively breastfed infants. Bifidobacterium and Lacticaseibacillus strains were selectively isolated and initially characterized in vitro. To evaluate their ability to reach the colon intact, we assessed their growth and bile tolerance. Strains safety was evaluated in vitro by determining antibiotic resistance profiles, DNase, and β-hemolytic activities. Additionally, their potential effects on the intestinal barrier and their ability to modulate the immune response were assessed in order to highlight potential probiotic functionality. The most promising strains underwent further characterizations. We performed whole genome sequencing (WGS) analysis to examine transferable antibiotic resistance, virulence factors, toxicity, and biogenic amine formation, thereby confirming the safety of the strains. Bile salt hydrolase (BSH) and glycoside hydrolase (GH) genes were also investigated. Furthermore, we explored the ability of certain strains to ferment human milk oligosaccharides (HMOs) and engage in cross-feeding interactions. The study showed that isolated strains exhibit probiotic traits such as acid and bile resistance, immunomodulatory potential, and strong safety with no detected virulence or resistance genes. Bifidobacterium longum A1 stood out for its metabolic strength, safety, ability to degrade human milk oligosaccharides, and support beneficial cross-feeding.

Twelve bacterial strains isolated from two different traditional Algerian products, cow butter and fermented wheat, were genetically identified using PCR and 16S rRNA sequencing. The growth kinetics of these strains were studied with two different carbon sources, lactose and glucose, each at a concentration of 2 %, alongside evaluations of their probiotic properties and safety. Genotypic identification revealed the presence of three species: Levilactobacillus brevis (4 strains), Lactiplantibacillus plantarum (2 strains), and Lacticaseibacillus paracasei (6 strains). Lp. plantarum and L. paracasei strains showed robust growth on both carbon sources, with similar performance, while L. brevis strains exhibited reduced growth in MRS broth containing lactose compared to MRS broth with glucose. Additionally, all strains demonstrated excellent tolerance to simulated gastric conditions and bile salts, with survival rates above 90 %. However, the L. brevis 64 strain was the most affected by bile salts compared to the other strains, showing a significant decrease in survival rate. Safety evaluations revealed no DNase or beta-hemolytic activity, and the strains were generally sensitive to most antibiotics tested. These strains, particularly Lp. plantarum and L. paracasei, isolated from lesser-studied Algerian fermented foods, exhibit promising in vitro probiotic traits and contribute to diversifying available functional microbial resources.

The genus Faecalibacterium is one of the major butyrate-producing bacteria in the human gut. Due to its production of beneficial metabolites and reduced populations in diseased patients, it is regarded as a potential biomarker of healthy gut microbiota. Strains Collection Nationale de Cultures de Microorganismes (CNCM) I-4540 and CNCM I-4541 were isolated from faeces of a healthy elderly and a healthy adult respectively. In this study, we conducted a taxonomic analysis of these strains. Phylogenetic analyses based on 16S rRNA gene and rpoA gene sequences revealed that the two strains belong to the genus Faecalibacterium. A core gene phylogenetic tree further supported their phylogenetic placement. CNCM I-4540 shared an average nucleotide identity (ANI) value of 96.7% and a digital DNA-DNA hybridization (dDDH) value of 74.3% with Faecalibacterium taiwanense HLW78T, indicating that CNCM I-4540 should be classified as F. taiwanense. In contrast, CNCM I-4541 did not exhibit ANI or dDDH values above the species threshold for any known species within the genus Faecalibacterium. These findings indicate that CNCM I-4541 represents a novel species within the genus Faecalibacterium, for which the name Faecalibacterium langellae sp. nov. is proposed. The type strain is CNCM I-4541T (=CIP 112513T=JCM 39552T).

Faecalibacterium has recently garnered attention for its potential health implications. To better understand its role, we developed and assessed real-time PCR assays for detecting and quantifying various Faecalibacterium species in human stool samples from both healthy individuals and Crohn's disease patients, either in flare or remission. The assays targeted the Microbial Anti-inflammatory Molecule (MAM) genes, which encode MAM proteins. These assays demonstrated 100% species-specificity using strains from six Faecalibacterium species: Faecalibacterium prausnitzii, Faecalibacterium taiwanense, Faecalibacterium duncaniae, Faecalibacterium longum, Faecalibacterium hattori, and Faecalibacterium CNCM4541. They also showed high sensitivity with detection limits of 10^5 bacteria per gram of sample. In healthy individuals, the different Faecalibacterium species varied in abundance. F. taiwanense, F. duncaniae, and F. longum were the most prevalent, around 10^10 bacteria/g of stool. In contrast, F. hattori and CNCM4541 were less abundant, with 10^7 bacteria/g. Despite its low abundance, F. hattori was present in all healthy subjects, while CNCM4541 was detected in only 50% of them. Notably, F. taiwanense, F. duncaniae, and F. longum were found in all healthy individuals. In Crohn's disease patients, both in flare and remission, a decrease in Faecalibacterium species was observed, with no recovery in remission. The most abundant species in Crohn's disease patients were F. prausnitzii and F. duncaniae, around 10^7 bacteria/g, while F. longum, F. hattori, and F. taiwanense were present at lower levels (10^6 bacteria/g), and CNCM4541 was no longer detected. Interestingly, F. prausnitzii showed a smaller decrease in abundance compared with other species. Moreover, F. prausnitzii was significantly more prevalent in patients in remission than in those in flare, suggesting that it may be more resistant to inflammation. These findings highlight the importance of accurately characterizing and quantifying Faecalibacterium species to better understand their role in health and disease.

Goat milk butter, locally known as “Dhan”, from the Sfisfa region of Algeria, holds significant cultural and economic value. This study investigates the probiotic properties of lactic acid bacteria (LAB) present in Dhan, focusing particularly on Lactobacillus strains. Molecular identification using 16S rRNA revealed a dominance of Levilactobacillus brevis and Lactiplantibacillus plantarum, forming a substantial part of the bacterial profile. Three LAB isolates (DC01-A, DC04, and DC06) were selected from fresh samples, and rigorous analyses were performed to evaluate their probiotic properties. Safety assessments confirmed the absence of gelatinase, DNase, and haemolytic activities in all isolates. The isolates demonstrated high tolerance to bile salts and acidic conditions, along with the ability to survive simulated gastrointestinal digestion. Notably, strain DC06 exhibited exceptional survival at low pH (1.5) and high bile salt concentrations (0.15–0.3%). All isolates showed substantial growth in MRS medium with 2% phenol, although growth was significantly decreased at 5% phenol. Furthermore, our strains exhibited high adhesion rates to various solvents, demonstrating their potential for strong interaction with cell membranes. Specifically, adhesion to chloroform was observed at 98.26% for DC01-A, 99.30% for DC04, and 99.20% for DC06. With xylene, the adhesion rates were 75.94% for DC01-A, 61.13% for DC04, and 76.52% for DC06. The LAB strains demonstrated impressive growth in ethanol concentrations up to 12%, but their tolerance did not exceed this concentration. They also exhibited robust growth across temperatures from 10 °C to 37 °C, with strains DC04 and DC06 able to proliferate at 45 °C, though none survived at 50 °C. Additionally, the isolates showed significant resistance to oxidative stress induced by hydrogen peroxide (H2O2) and displayed medium to high autolytic activity, with rates of 50.86%, 37.53%, and 33.42% for DC01-A, DC04, and DC06, respectively. The cell-free supernatant derived from strain DC04 exhibited significant antimicrobial activity against the tested pathogens, while strain DC06 demonstrated moderate antioxidant activity with the highest DPPH scavenging rate at 68.56%, compared to the probiotic reference strain LGG at 61.28%. These collective findings not only suggest the probiotic viability of LAB strains found in Dhan but also highlight the importance of traditional food practises in contributing to health and nutrition. Consequently, this study supports the potential of traditional Dhan butter as a functional food and encourages further exploration of its health benefits.

The gut-lung axis is critical during viral respiratory infections such as influenza. Gut dysbiosis during infection translates into a massive drop of microbially produced short-chain fatty acids (SCFAs). Among them, butyrate is important during influenza suggesting that microbiome-based therapeutics targeting butyrate might hold promises. The butyrate-producing bacterium Faecalibacterium duncaniae (formerly referred to as F. prausnitzii) is an emerging probiotic with several health-promoting characteristics. To investigate the potential effects of F. duncaniae on influenza outcomes, mice were gavaged with live F. duncaniae (A2-165 or I-4574 strains) five days before infection. Supplementation of F. duncaniae was associated with less severe disease, a lower pulmonary viral load, and lower levels of lung inflammation. F. duncaniae supplementation impacted on gut dysbiosis induced by infection, as assessed by 16S rRNA sequencing. Interestingly, F. duncaniae administration was associated with a recovery in levels of SCFAs (including butyrate) in infected animals. The live form of F. duncaniae was more potent that the pasteurized form in improving influenza outcomes. Lastly, F. duncaniae partially protected against secondary (systemic) bacterial infection. We conclude that F. duncaniae might serve as a novel next generation probiotic against acute viral respiratory diseases.

The expanding knowledge on the systemic influence of the human microbiome suggests that fecal samples are underexploited sources of new beneficial strains for extra-intestinal health. We have recently shown that acetate, a main circulating microbiota-derived molecule, reduces the deleterious effects of pulmonary Streptococcus pneumoniae and enteric Salmonella enterica serovar Typhimurium bacterial post-influenza superinfections. Considering the beneficial and broad effects of acetate, we intended to isolate a commensal strain, producing acetate and potentially exploitable in the context of respiratory infections. We designed successive steps to select intestinal commensals that are extremely oxygen-sensitive, cultivable after a freezing process, without a proinflammatory effect on IL-8 induction, and producing acetate. We have identified the Blautia faecis DSM33383 strain, which decreased the TNFα-induced production of IL-8 by the intestinal epithelial cell line HT-29. The beneficial effect of this bacterial strain was further studied in two preclinical models of post-influenza Streptococcus pneumoniae (S.p) and Salmonella enterica serovar Typhimurium (S.t) superinfection. The intragastrical administration of Blautia faecis DSM33383 led to protection in influenza-infected mice suffering from an S.p. and, to a lesser extent, from an S.t secondary infection. Altogether, this study showed that Blautia faecis DSM33383 could be a promising candidate for preventive management of respiratory infectious diseases.

The microbiota of the mouth disperses into the lungs, and both compartments share similar phyla. Considering the importance of the microbiota in the maturation of the immunity and physiology during the first days of life, we hypothesized that primo-colonizing bacteria of the oral cavity may induce immune responses in bronchial epithelial cells. Herein, we have isolated and characterized 57 strains of the buccal cavity of two human newborns. These strains belong to Streptococcus, Staphylococcus, Enterococcus, Rothia and Pantoea genera, with Streptococcus being the most represented. The strains were co-incubated with a bronchial epithelial cell line (BEAS-2B), and we established their impact on a panel of cytokines/chemokines and global changes in gene expression. The Staphylococcus strains, which appeared soon after birth, induced a high production of IL-8, suggesting they can trigger inflammation, whereas the Streptococcus strains were less associated with inflammation pathways. The genera Streptococcus, Enterococcus and Pantoea induced differential profiles of cytokine/chemokine/growth factor and set of genes associated with maturation of morphology. Altogether, our results demonstrate that the microorganisms, primo-colonizing the oral cavity, impact immunity and morphology of the lung epithelial cells, with specific effects depending on the phylogeny of the strains.

Fecal microbiota transplantation is now recommended for treating recurrent forms of Clostridioides difficile infection. Recent studies have reported protocols using capsules of either frozen or freeze-dried stool allowing oral administration in in- and out-patient settings. However, a central question remains the viability, engraftment, and efficacy of the microbiome over time during storage life. This study shows that both the freeze-drying and freezing procedures for fecal samples allowed preserving viability, short-chain fatty acids concentration, and anti-Clostridioides difficile properties of microbiota without significant alteration after storage for 12 months. Fecal transplantation with freeze-dried microbiota allowed engraftment of microbiota leading to clearance of Clostridioides difficile infection in a preclinical murine model with a survival rate of 70% versus 53-60% in mice treated with frozen inocula, and 20% in the untreated group. Moreover, the freeze-dried powder can be used to fill oral hard capsules using a very low amount (0.5%) of glidant excipient, allowing oral formulation. Altogether, this study showed that freeze-dried inocula can be used for the treatment of Clostridioides difficile infection with long-lasting stability of the fecal microbiota. This formulation facilitates biobanking and allows the use of hard capsules, an essential step to simplify patient access to treatment.

Every year, millions of people around the world benefit from radiation therapy to treat cancers localized in the pelvic area. Damage to healthy tissue in the radiation field can cause undesirable toxic effects leading to gastrointestinal complications called pelvic radiation disease. A change in the composition and/or function of the microbiota could contribute to radiation-induced gastrointestinal toxicity. In this study, we tested the prophylactic effect of a new generation of probiotic like Faecalibacterium prausnitzii (F. prausnitzii) on acute radiation-induced colonic lesions. Experiments were carried out in a preclinical model of pelvic radiation disease. Rats were locally irradiated at 29 Gray in the colon resulting in colonic epithelial barrier rupture. Three days before the irradiation and up to 3 d after the irradiation, the F. prausnitzii A2-165 strain was administered daily (intragastrically) to test its putative protective effects. Results showed that prophylactic F. prausnitzii treatment limits radiation-induced para-cellular hyperpermeability, as well as the infiltration of neutrophils (MPO+ cells) in the colonic mucosa. Moreover, F. prausnitzii treatment reduced the severity of the morphological change of crypts, but also preserved the pool of Sox-9+ stem/progenitor cells, the proliferating epithelial PCNA+ crypt cells and the Dclk1+/IL-25+ differentiated epithelial tuft cells. The benefit of F. prausnitzii was associated with increased production of IL-18 by colonic crypt epithelial cells. Thus, F. prausnitzii treatment protected the epithelial colonic barrier from colorectal irradiation. New-generation probiotics may be promising prophylactic treatments to reduce acute side effects in patients treated with radiation therapy and may improve their quality of life.

Alterations in the gut microbiota have been associated with a wide range of pathologies and conditions. Maintaining a well-balanced microbiota is a key factor in sustaining good health. Our aim was to investigate the impact of a resistant starch-containing dietary supplement (SymbioIntest®) on the composition of the human gut microbiota and on intestinal short chain fatty acid (SCFA) concentration. Human microbiota-associated mice were used. Ex-germ-free mice were inoculated with fecal suspensions from four different donors. Three weeks later, the mice were orally gavaged for 1 month with either a daily dose of 10 mg of SymbioIntest® or the vehicle (water) for the negative control group. The composition of the microbiota and SCFA levels were analyzed by 16S rRNA gene sequencing and gas chromatography, respectively. In three groups of mice, SymbioIntest® supplementation increased the concentration of caecal butyrate. This was in conjunction with a remodeling of the gut microbiota. OTUs belonging to the Bacteroidaceae, Porphyromonadaceae, Lachnospiraceae and Ruminococcaceae families were affected. In two groups of mice the greatest changes in OTUs were seen in the Faecalibacterium genus. The supplementation’s highest impact was observed in mice inoculated with gut microbiota containing a lower number of Ruminococcaceae and Faecalibacterium and a higher number of Prevotellaceae. SymbioIntest® supplementation elicited a beneficial effect on the healthy adult gut microbiota by increasing caecal butyrate production and health-promoting taxa. We highlight the fact that screening the gut microbiota may be used for predicting individualized responses to dietary interventions and thus developing personalized nutritional strategies.

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.

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.

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 Grampositive/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.

The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6 mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.

Lactic acid bacteria as Lactococcus lactis are used as vector for protein but also DNA delivery into intestinal cells in vitro and in vivo. For the plasmid delivery strategy, the plasmid copy number per bacteria (PCN) is thus of great importance. The aim of this paper is to determine the physiological conditions when PCN is the highest in the bacteria. PCN was characterized by qPCR in five different recombinant Lactococcus lactis strains, containing one (mono-) or two different plasmids (biplasmidic), at exponential or stationary phase. We showed that in all cases but one, PCN is higher at exponential than stationary phase. PCN seems to depend on (i) monoplasmidic or biplasmidic strain; (ii) origin of replication of the plasmid; and (iii) the DNA load of the bacteria. Then we studied plasmid transfer in vitro from recombinant L. lactis to eukaryotic COS-7 cells using culture at exponential or stationary phase. We showed that plasmid transfer can be improved in vitro by using bacteria at exponential phase.

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.