2-Fucosyllactose Metabolism by Bifidobacteria Promotes Lactobacilli Growth in Co-Culture.

Breastfeeding is recognized as the gold standard in infant nutrition, not only because of breastmilk's intrinsic nutritional benefits but also due to the high content of different bioactive components such as 2-fucosyllactose (2'FL) in the mother's milk. It promotes the growth of its two major consumers, Bifidobacterium longum ssp. infantis and Bifidobacterium bifidum, but the effect on other intestinal microorganisms of infant microbiota remains incompletely understood. pH-uncontrolled fecal cultures from infants donors identified as "fast 2'FL -degrader" microbiota phenotype were used for the isolation of 2'FL-associated microorganisms. The use of specific selective agents allowed the successful isolation of B. bifidum IPLA20048 and of Lactobacillus gasseri IPLA20136. The characterization of 2'FL consumption and its moieties has revealed more pronounced growth, pH drop, and lactic acid production after 2'FL consumption when both microorganisms were grown together. The results point to an association between B. bifidum IPLA20048 and L. gasseri IPLA20136 in which L. gasseri is able to use the galactose from the lactose moiety after the hydrolysis of 2'FL by B. bifidum. The additional screening of two groups of bifidobacteria (n = 38), fast and slow degraders of 2'FL, in co-culture with lactobacilli confirmed a potential cross-feeding mechanism based on degradation products released from bifidobacterial 2'FL break-down. Our work suggests that this phenomenon may be widespread among lactobacilli and bifidobacteria in the infant gut. More investigation is needed to decipher how the ability to degrade 2'FL and other human milk oligosaccharides could influence the microbiota establishment in neonates and the evolution of the microbiota in adult life.

Commensal Fecal Microbiota Profiles Associated with Initial Stages of Intestinal Mucosa Damage: A Pilot Study.

Progressive intestinal mucosal damage occurs over years prior to colorectal cancer (CRC) development. The endoscopic screening of polyps and histopathological examination are used clinically to determine the risk and progression of mucosal lesions. We analyzed fecal microbiota compositions using 16S rRNA gene-based metataxonomic analyses and the levels of short-chain fatty acids (SCFAs) using gas chromatography in volunteers undergoing colonoscopy and histopathological analyses to determine the microbiota shifts occurring at the early stages of intestinal mucosa alterations. The results were compared between diagnosis groups (nonpathological controls and polyps), between samples from individuals with hyperplastic polyps or conventional adenomas, and between grades of dysplasia in conventional adenomas. Some microbial taxa from the Bacillota and Euryarchaeota phyla were the most affected when comparing the diagnosis and histopathological groups. Deeper microbiota alterations were found in the conventional adenomas than in the hyperplastic polyps. The Ruminococcus torques group was enriched in both the hyperplastic polyps and conventional adenomas, whereas the family Eggerthellaceae was enriched only in the hyperplastic polyps. The abundance of Prevotellaceae, Oscillospiraceae, Methanobacteriaceae, Streptococcaceae, Christensenellaceae, Erysipelotrichaceae, and Clostridiaceae shifted in conventional adenomas depending on the grade of dysplasia, without affecting the major SCFAs. Our results suggest a reorganization of microbial consortia involved in gut fermentative processes.

Dietary xenobiotics, (poly)phenols and fibers: Exploring associations with gut microbiota in socially vulnerable individuals.

Objectives: Although xenobiotics derived from food processing may cause modifications in the composition of the gut microbiota (GM) evidence is scarce. The aim of this study is to evaluate the impact of potential dietary carcinogens as heterocyclic amines (HAs), polycyclic aromatic hydrocarbons (PAHs), nitrates, nitrites, nitroso compounds and acrylamide, in combination to fibers (poly)phenols on the GM composition in a group of materially deprived subjects. Study design: Transversal observational study in a sample of 19 subjects recipients of Red Cross food aid. Dietary information was recorded by means of 3 non-consecutive 24 h recalls. Questions focused on the type of cooking and the extent of cooking and roasting were included. Information on potential carcinogens was mainly obtained from the European Prospective Investigation into Cancer and Nutrition (EPIC) and Computerized Heterocyclic Amines Resource for Research in Epidemiology of Disease (CHARRED) Carcinogen Databases. Microbial composition was determined by 16S ribosomal RNA gene sequencing in fecal samples. Results: Higher levels of Lachnospiraceae and Eggerthellaceae families were found in individuals consuming less than 50 ng/day of 2-amino-3,8 dimethylimidazo (4,5,f) quinoxaline (MeIQx) (considered as lower risk dose for colorectal adenoma) while those consuming more than 40 ng/day of 2-amino-1-methyl-6-phenylimidazo (4,5,b) pyridine (PhIP) (higher risk for colorectal adenoma) showed lower relative abundance of Muribaculaceae and greater presence of Streptococcaceae and Eubacterium coprostanoligenes group. Conclusion: The associations identified between diet and processing by-products on GM in this study could be used as potential targets for the designing of dietary interventions tailored to this collective.

In Vitro Probiotic Modulation of the Intestinal Microbiota and 2'Fucosyllactose Consumption in Fecal Cultures from Infants at Two Months of Age.

2'-fucosyllactose (2'FL) is one of the most abundant oligosaccharides in human milk, with benefits on neonatal health. Previous results point to the inability of the fecal microbiota from some infants to ferment 2'FL. We evaluated a probiotic formulation, including the strains Lactobacillus helveticus Rosell®-52 (R0052), Bifidobacterium longum subsp. infantis Rosell®-33 (R0033), and Bifidobacterium bifidum Rosell®-71 (R0071), individually or in an 80:10:10 combination on the microbiota and 2'FL degradation. Independent batch fermentations were performed with feces from six full-term infant donors of two months of age (three breastfed and three formula-fed) with added probiotic formulation or the constituent strains in the presence of 2'FL. Microbiota composition was analyzed by 16S rRNA gene sequencing. Gas accumulation, pH decrease and 2'FL consumption, and levels of different metabolites were determined by chromatography. B. bifidum R0071 was the sole microorganism promoting a partial increase of 2'FL degradation during fermentation in fecal cultures of 2'FL slow-degrading donors. However, major changes in microbiota composition and metabolic activity occurred with L. helveticus R0052 or the probiotic formulation in cultures of slow degraders. Further studies are needed to decipher the role of the host intestinal microbiota in the efficacy of these strains.

Role of Bifidobacteria on Infant Health.

Bifidobacteria are among the predominant microorganisms during infancy, being a dominant microbial group in the healthy breastfed infant and playing a crucial role in newborns and infant development. Not only the levels of the Bifidobacterium genus but also the profile and quantity of the different bifidobacterial species have been demonstrated to be of relevance to infant health. Although no definitive proof is available on the causal association, reduced levels of bifidobacteria are perhaps the most frequently observed alteration of the intestinal microbiota in infant diseases. Moreover, Bifidobacterium strains have been extensively studied by their probiotic attributes. This review compiles the available information about bifidobacterial composition and function since the beginning of life, describing different perinatal factors affecting them, and their implications on different health alterations in infancy. In addition, this review gathers exhaustive information about pre-clinical and clinical studies with Bifidobacterium strains as probiotics in neonates.

Influence of 2'-Fucosyllactose on the Microbiota Composition and Metabolic Activity of Fecal Cultures from Breastfed and Formula-Fed Infants at Two Months of Age.

Although breast milk is considered the gold standard of nutrition for infant feeding, some circumstances may make breastfeeding difficult. Several commercial milk preparations include synthetic human milk oligosaccharides (HMOs) in their composition. However, the effect of HMOs on the establishment of the intestinal microbiota remains incompletely understood. Independent batch fermentations were performed with feces from six full-term infant donors of two months of age (three breastfed and three formula-fed, exclusively) in the presence of 2'fucosyllactose (2'FL), one of the most abundant HMOs in human milk. Microbiota composition was analyzed by 16S rRNA gene sequencing at baseline and at 24 h of incubation. The 2'FL consumption, gas accumulation, and levels of different metabolites were determined by chromatography. Microbiota profiles at baseline were clearly influenced by the mode of feeding and by the intrinsic ability of microbiotas to degrade 2'FL. The 2'FL degradation rate clustered fecal cultures into slow and fast degraders, regardless of feeding type, this being a determinant factor influencing the evolution of the microbiota during incubation, although the low number of donors precludes drawing sound conclusions. More studies are needed to decipher the extent to which the early intervention with HMOs could influence the microbiota as a function of its ability to utilize 2'FL.

Early-Life Development of the Bifidobacterial Community in the Infant Gut.

The establishment of the gut microbiota poses implications for short and long-term health. Bifidobacterium is an important taxon in early life, being one of the most abundant genera in the infant intestinal microbiota and carrying out key functions for maintaining host-homeostasis. Recent metagenomic studies have shown that different factors, such as gestational age, delivery mode, or feeding habits, affect the gut microbiota establishment at high phylogenetic levels. However, their impact on the specific bifidobacterial populations is not yet well understood. Here we studied the impact of these factors on the different Bifidobacterium species and subspecies at both the quantitative and qualitative levels. Fecal samples were taken from 85 neonates at 2, 10, 30, 90 days of life, and the relative proportions of the different bifidobacterial populations were assessed by 16S rRNA-23S rRNA internal transcribed spacer (ITS) region sequencing. Absolute levels of the main species were determined by q-PCR. Our results showed that the bifidobacterial population establishment is affected by gestational age, delivery mode, and infant feeding, as it is evidenced by qualitative and quantitative changes. These data underline the need for understanding the impact of perinatal factors on the gut microbiota also at low taxonomic levels, especially in the case of relevant microbial populations such as Bifidobacterium. The data obtained provide indications for the selection of the species best suited for the development of bifidobacteria-based products for different groups of neonates and will help to develop rational strategies for favoring a healthy early microbiota development when this process is challenged.

Effect of inulin-type fructans and galactooligosaccharides on cultures of Lactobacillus strains isolated in Algeria from camel's milk and human colostrum.

Bacteria from the genus Lactobacillus are responsible for spontaneous food fermentations. Some species, such as Lactobacillus casei and Lactobacillus brevis, have the "Qualified Presumption of Safety" status recognized by the European Food Safety Authority. Several of their strains are used as probiotics in foods and sometimes are included in synbiotic combinations together with prebiotics. New microbial strains isolated from different sources represent an opportunity to use them for the production of traditional food products. The capacity of three selected strains (one isolated from Camel's milk and identified by partial 16 S rRNA gene sequencing as L. brevis, and two isolated from human colostrum and identified as L. paracasei/L. casei and L. brevis, respectively) was assessed in vitro for the ability to survive in gastrointestinal conditions (low pH and high bile salts concentrations). We also tested the capacity of growth and the production of organic acids and volatile compounds by high-performance liquid chromatography and gas chromatography, respectively, when these bacteria were incubated anaerobically in the presence of inulin, fructooligosaccharides, or galactooligosaccharides as the main carbon sources. The strains were able to survive in simulated gastrointestinal conditions and to grow in inulin, fructooligosaccharides, and galactooligosaccharides. However, they displayed different profiles of organic acids and volatile compounds, mainly depending on the microbial species and the prebiotic used. The influence that the combined use of strains and different prebiotics could exert on the organic acids and volatiles formed in food and in the gut should be assessed for each synbiotic combination and food product.

Impact of Extreme Obesity and Diet-Induced Weight Loss on the Fecal Metabolome and Gut Microbiota.

SCOPE: A limited number of human studies have characterized fecal microbiota and metabolome in extreme obesity and after diet-induced weight loss. METHODS AND RESULTS: Fecal samples from normal-weight and extremely obese adults and from obese participants before and after moderate diet-induced weight loss are evaluated for their interaction with the intestinal adenocarcinoma cell line HT29 using an impedance-based in vitro model, which reveals variations in the interaction between the gut microbiota and host linked to obesity status. Microbiota composition, short chain fatty acids, and other intestinal metabolites are further analyzed to assess the interplay among diet, gut microbiota, and host in extreme obesity. Microbiota profiles are distinct between normal-weight and obese participants and are accompanied by fecal signatures in the metabolism of biliary compounds and catecholamines. Moderate diet-induced weight loss promotes shifts in the gut microbiota, and the primary fecal metabolomics features are associated with diet and the gut-liver and gut-brain axes. CONCLUSIONS: Analyses of the fecal microbiota and metabolome enable assessment of the impact of diet on gut microbiota composition and activity, supporting the potential use of certain fecal metabolites or members of the gut microbiota as biomarkers for the efficacy of weight loss in extreme obesity.

An Overview on Fecal Branched Short-Chain Fatty Acids Along Human Life and as Related With Body Mass Index: Associated Dietary and Anthropometric Factors.

Short-chain fatty acids (SCFA) are the main bacterial products of the catabolism of carbohydrates and proteins in the gut, and their role is essential in host-microbiota interactions. Acetic, propionic, and butyric acids are the major SCFA produced in the gut, and they have been extensively studied. In contrast, branched short-chain fatty acids (BCFA), mainly isovaleric and isobutyric acids, are produced in less amounts and their fecal levels in different human groups, intestinal microbial producing populations, and influence on health are insufficiently known. They have been proposed as markers of protein fermentation, which leads to the concomitant production of other fermentation products that can be harmful for the colon epithelium. In this context, the aim of this study was to shed light into the production of BCFA by the human intestinal microbiota, as related to age, body mass index (BMI), and diet. Fecal levels of the different SCFA were analyzed by gas chromatography in 232 healthy individuals with ages between 3 months and 95 years, and BMI in adults ranging from 19 to 54. Dietary assessments in adults were obtained through a food frequency questionnaire (FFQ). Molar proportions of BCFA in feces were strongly and positively related with aging. However, not a significant relationship was obtained between BCFA and BMI. A negative correlation was found between the consumption of dietary insoluble fiber and fecal levels of BCFA. More studies are needed for improving our understanding on the relationship of BCFA production profile with the intestinal microbiota composition and human health.

In Vitro Evaluation of Different Prebiotics on the Modulation of Gut Microbiota Composition and Function in Morbid Obese and Normal-Weight Subjects.

The gut microbiota remains relatively stable during adulthood; however, certain intrinsic and environmental factors can lead to microbiota dysbiosis. Its restoration towards a healthy condition using best-suited prebiotics requires previous development of in vitro models for evaluating their functionality. Herein, we carried out fecal cultures with microbiota from healthy normal-weight and morbid obese adults. Cultures were supplemented with different inulin-type fructans (1-kestose, Actilight, P95, Synergy1 and Inulin) and a galactooligosaccharide. Their impact on the gut microbiota was assessed by monitoring gas production and evaluating changes in the microbiota composition (qPCR and 16S rRNA gene profiling) and metabolic activity (gas chromatography). Additionally, the effect on the bifidobacterial species was assessed (ITS-sequencing). Moreover, the functionality of the microbiota before and after prebiotic-modulation was determined in an in vitro model of interaction with an intestinal cell line. In general, 1-kestose was the compound showing the largest effects. The modulation with prebiotics led to significant increases in the Bacteroides group and Faecalibacterium in obese subjects, whereas in normal-weight individuals, substantial rises in Bifidobacterium and Faecalibacterium were appreciated. Notably, the results obtained showed differences in the responses among the tested compounds but also among the studied human populations, indicating the need for developing population-specific products.

Microbiome: Effects of Ageing and Diet.

The microbial community inhabiting our intestine, known as 'microbiota', and the ensemble of their genomes (microbiome) regulate important functions of the host, being essential for health maintenance. The recent development of next-generation sequencing (NGS) methods has greatly facilitated the study of the microbiota and has contributed to evidence of the strong influence exerted by age and diet. However, the precise way in which the diet and its components modify the functionality of the intestinal microbiome is far from being completely known. Changes in the intestinal microbiota occur during ageing, frequently accompanied by physiological changes of the digestive tract, modification of dietary patterns and impairment of the immune system. Establishing nutritional strategies aiming to counterbalance the specific alterations taking place in the microbiota during ageing would contribute to improved health status in the elderly. This review will analyse changes appearing in the intestinal microbiota from adulthood to old age and their association with dietary patterns and lifestyle factors.

Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.

The gut microbiome is emerging as a key regulator of several metabolic, immune and neuroendocrine pathways1,2. Gut microbiome deregulation has been implicated in major conditions such as obesity, type 2 diabetes, cardiovascular disease, non-alcoholic fatty acid liver disease and cancer3-6, but its precise role in aging remains to be elucidated. Here, we find that two different mouse models of progeria are characterized by intestinal dysbiosis with alterations that include an increase in the abundance of Proteobacteria and Cyanobacteria, and a decrease in the abundance of Verrucomicrobia. Consistent with these findings, we found that human progeria patients also display intestinal dysbiosis and that long-lived humans (that is, centenarians) exhibit a substantial increase in Verrucomicrobia and a reduction in Proteobacteria. Fecal microbiota transplantation from wild-type mice enhanced healthspan and lifespan in both progeroid mouse models, and transplantation with the verrucomicrobia Akkermansia muciniphila was sufficient to exert beneficial effects. Moreover, metabolomic analysis of ileal content points to the restoration of secondary bile acids as a possible mechanism for the beneficial effects of reestablishing a healthy microbiome. Our results demonstrate that correction of the accelerated aging-associated intestinal dysbiosis is beneficial, suggesting the existence of a link between aging and the gut microbiota that provides a rationale for microbiome-based interventions against age-related diseases.

Intestinal Immunomodulation and Shifts on the Gut Microbiota of BALB/c Mice Promoted by Two Bifidobacterium and Lactobacillus Strains Isolated from Human Samples.

Bifidobacterium animalis subsp. lactis IPLA 20020 and Lactobacillus gasseri IPLA 20212, two strains isolated from human samples, were evaluated for safety and influence over the intestinal microbiota and cytokine production by the intestinal tissue of adult BALB/c mice. Mice were divided into four groups receiving during 8 days PBS or a suspension of each strain, prepared fresh or lyophilized (bifidobacteria), at an amount of 4x108 viable cells/day. This dose could be comparable to the probiotic intake of a human adult who consumed about 100-200 mL of functional fermented milk per day, considering the usual level of probiotics in commercial products. No microbial translocation to liver or alterations in food intake, weight, and behavior were observed in treated mice. Intestinal content of secretory immunoglobulin A (s-IgA) was not affected, discarding any adverse effect on the mucosa-associated immunity. The profile of intestinal proinflammatory/regulatory cytokines after intervention evidenced that the microbial strain administered and its cellular state (fresh or lyophilized) as well as the host tissue analyzed (small or large intestine) influenced the immune response and suggests a moderate shift towards a T helper 1 profile (Th1) in the large intestine after the administration of both strains. Changes on relative levels of some intestinal microbial groups were evidenced after intervention. It is noteworthy that butyrate was positively associated with a balanced pro-Th1 immune response. Therefore, B. animalis subsp. lactis IPLA20020 and L. gasseri IPLA 20212 could be considered potential probiotic candidates to be included in functional foods for balancing the intestinal immune response.

Xenobiotics Formed during Food Processing: Their Relation with the Intestinal Microbiota and Colorectal Cancer.

The colonic epithelium is exposed to a mixture of compounds through diet, among which some are procarcinogens, whereas others have a protective effect. Therefore, the net impact of these compounds on human health depends on the overall balance between all factors involved. Strong scientific evidence has demonstrated the relationship between nitrosamines (NA), heterocyclic amines (HCAs), and polycyclic aromatic hydrocarbons (PAHs), which are the major genotoxins derived from cooking and food processing, and cancer. The mechanisms of the relationship between dietary toxic xenobiotics and cancer risk are not yet well understood, but it has been suggested that differences in dietary habits affect the colonic environment by increasing or decreasing the exposure to mutagens directly and indirectly through changes in the composition and activity of the gut microbiota. Several changes in the proportions of specific microbial groups have been proposed as risk factors for the development of neoplastic lesions and the enrichment of enterotoxigenic microbial strains in stool. In addition, changes in the gut microbiota composition and activity promoted by diet may modify the faecal genotoxicity/cytotoxicity, which can be associated with a higher or lower risk of developing cancer. Therefore, the interaction between dietary components and intestinal bacteria may be a modifiable factor for the development of colorectal cancer in humans and deserves more attention in the near future.

Early microbiota, antibiotics and health.

The colonization of the neonatal digestive tract provides a microbial stimulus required for an adequate maturation towards the physiological homeostasis of the host. This colonization, which is affected by several factors, begins with facultative anaerobes and continues with anaerobic genera. Accumulating evidence underlines the key role of the early neonatal period for this microbiota-induced maturation, being a key determinant factor for later health. Therefore, understanding the factors that determine the establishment of the microbiota in the infant is of critical importance. Exposure to antibiotics, either prenatally or postnatally, is common in early life mainly due to the use of intrapartum prophylaxis or to the administration of antibiotics in C-section deliveries. However, we are still far from understanding the impact of early antibiotics and their long-term effects. Increased risk of non-communicable diseases, such as allergies or obesity, has been observed in individuals exposed to antibiotics during early infancy. Moreover, the impact of antibiotics on the establishment of the infant gut resistome, and on the role of the microbiota as a reservoir of resistance genes, should be evaluated in the context of the problems associated with the increasing number of antibiotic resistant pathogenic strains. In this article, we review and discuss the above-mentioned issues with the aim of encouraging debate on the actions needed for understanding the impact of early life antibiotics upon human microbiota and health and for developing strategies aimed at minimizing this impact.

CFH polymorphisms in a Northern Spanish population with neovascular and dry forms of age-related macular degeneration.

PURPOSE: To elucidate the potential role of single-nucleotide polymorphisms (SNPs) in complement factor H (CFH) gene in Northern Spanish patients with age-related macular degeneration (AMD). METHODS: A case-control study of 130 unrelated native Northern Spanish diagnosed with AMD (46 dry, 35 neovascular and 49 mixed) and 96 healthy controls matched by age and ethnicity were enrolled. DNA was isolated from peripheral blood and genotyped for AMD-associated SNPs (rs3753394, rs529825, rs800292, rs3766404, rs203674, rs10671170, rs3753396 and rs1065489) using TaqMan probes and restriction fragment length polymorphism (RFLP). The association study was performed using the HaploView 4.0 software. RESULTS: The allelic frequency analysis revealed that rs529825, rs800292, rs203674 and rs10671170 were significantly associated with an increased risk for AMD. The haplotypes CGG (rs3753394, rs529825 and rs800292) and GCAG (rs203674, rs1061170, rs3753396 and rs1065489) were significantly associated with AMD while the haplotypes CAA (rs3753394, rs529825 and rs800292) and TTAG (rs203674, rs1061170, rs3753396 and rs1065489) were found to be protective. Small differ-ences in allelic frequencies were found between dry and neovascular cases; however, these differences were not significant and did not distinguish one form the other. CONCLUSIONS: This study found significant association of SNPs rs529825, rs800292, rs203674 and rs1061170 in the CFH gene with susceptibility to AMD. We identified haplotypes that confer protection or increased risk of AMD but not specific genetic variants in CFH capable to distinguish the different clinical forms of AMD in this cohort. Collectively, our results confirmed that CFH represents a strong genetic risk factor for this disease in the Northern Spanish population.