Comprehensive pan-genome analysis of Lactiplantibacillus plantarum complete genomes.

AIMS: The aim of this work was to refine the taxonomy and the functional characterization of publicly available Lactiplantibacillus plantarum complete genomes through a pan-genome analysis. Particular attention was paid in depicting the probiotic potential of each strain. METHODS AND RESULTS: Complete genome sequence of 127 L. plantarum strains, without detected anomalies, was downloaded from NCBI. Roary analysis of L. plantarum pan-genome identified 1436 core, 414 soft core, 1858 shell and 13,203 cloud genes, highlighting the 'open' nature of L. plantarum pan-genome. Identification and characterization of plasmid content, mobile genetic elements, adaptative immune system and probiotic marker genes (PMGs) revealed unique features across all the L. plantarum strains included in the present study. Considering our updated list of PMGs, we determined that approximatively 70% of the PMGs belongs to the core/soft-core genome. CONCLUSIONS: The comparative genomic analysis conducted in this study provide new insights into the genomic content and variability of L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a comprehensive pan-genome analysis of L. plantarum, including the largest number (N = 127) of complete L. plantarum genomes retrieved from publicly available repositories. Our effort aimed to determine a solid reference panel for the future characterization of newly sequenced L. plantarum strains useful as probiotic supplements.

Effects of probiotic Lactiplantibacillus plantarum IMC 510 supplementation on metabolic factors in otherwise healthy overweight and obese individuals.

AIMS: Probiotic supplementation approach offers the possibility to shape the gut microbiota (GM), enabling the development of innovative formulations able to improve intestinal well-being and consequently the related body weight modulation and energy metabolism. In the present clinical study, a new potential probiotic supplement based on Lactiplantibacillus plantarum IMC 510 was studied for weight management. METHODS AND RESULTS: Quantitative characterization by qPCR of representative bacterial groups of GM was used to determine the microbiota modulation at different supplementation periods. Furthermore, measurement of the endpoints linked to weight control (body mass index, body weight, waist circumference) was assessed. Specific questionnaires to evaluate the impact on psychological and physiological point of view were performed. Results showed that after 90 days, Lact. plantarum IMC 510 supplementation brought an improvement in endpoints linked to weight control and healthy status, although no significant changes in the microbiota composition were reported for analysed bacterial groups, except for Lactobacillus spp. and Bifidobacterium spp. CONCLUSIONS: We concluded that Lact. plantarum IMC 510 supplementation could be an interesting tool for weight management. More studies are needed to understand the impact on GM, for example, evaluating the production of short-chain fatty acids, since their important role in dietary metabolism. Further research is necessary to better elucidate the relationship between GM and overweight and the mechanism of action by which Lact. plantarum IMC 510 modifies body weight. SIGNIFICANCE AND IMPACT OF THE STUDY: However, these promising outcomes represent a clear advantage of probiotic supplementation and identify a new potential probiotic as a novel and safe therapeutic approach in the obesity prevention and management.

Impact of a probiotic diet on well-being of healthy senior: THE PROBIOSENIOR PROJECT.

AIMS: The aim of this work was to assess the effects of a probiotic diet on well-being of healthy seniors living in boarding and private homes in Marche Region, Italy. In particular, we focused on the modulation of high-sensitivity C-reactive protein (HsCRP), intestinal microbiota and short-chain fatty acids (SCFAs). METHODS AND RESULTS: Ninety-seven healthy seniors took part in a double-blind, placebo-controlled feeding study (59 fed probiotics, 38 fed placebo) for 6 months. Each volunteer ingested daily one food product or a dietary supplement enriched with Synbio® blend (Synbiotec Srl, Camerino, Italy) or the placebo (control group). Blood and faecal samples were collected before and at the end of the intervention period to perform biochemical and microbiological analyses. The serum HsCRP difference value after 6 months of treatment was significantly higher in the probiotic group than placebo (p < 0.05). After the intervention, a significant increase in faecal lactobacilli and a bifidobacteria increase in more participants were observed in the probiotic group. The 16S NGS analysis on the probiotic group showed a decreasing trend of Proteobacteria at the end of the treatment and conversely, an increasing trend of Actinobacteria and Verrucomicrobia phyla, to which the increase of Akkermansiaceae and Bifidobacteriaceae contributes at the family level. Finally, total short-chain fatty acids (SCFAs) and butyric acid were significantly higher in the probiotic group at the end of the treatment respect to the beginning. CONCLUSIONS: Overall, this study emphasizes the beneficial anti-inflammageing effect of a prolonged diet based on functional foods enriched with Synbio® through the modulation of the intestinal microbiota and the consequent increase in the SCFA production. SIGNIFICANCE AND IMPACT OF THE STUDY: Synbio® integration in elderly daily diet may be a preventive strategy to support healthy ageing.

Supplementation with Lactiplantibacillus plantarum IMC 510 Modifies Microbiota Composition and Prevents Body Weight Gain Induced by Cafeteria Diet in Rats.

Changes in functionality and composition of gut microbiota (GM) have been associated and may contribute to the development and maintenance of obesity and related diseases. The aim of our study was to investigate for the first time the impact of Lactiplantibacillus (L.) plantarum IMC 510 in a rat model of diet-induced obesity, specifically in the cafeteria (CAF) diet. This diet provides a strong motivation to voluntary overeat, due to the palatability and variety of selected energy-dense foods. The oral administration for 84 days of this probiotic strain, added to the CAF diet, decreased food intake and body weight gain. Accordingly, it ameliorated body mass index, liver and white adipose tissue weight, hepatic lipid accumulation, adipocyte size, serum parameters, including glycemia and low-density lipoprotein levels, in CAF fed rats, potentially through leptin control. In this scenario, L. plantarum IMC 510 showed also beneficial effects on GM, limiting the microbial imbalance established by long exposure to CAF diet and preserving the proportion of different bacterial taxa. Further research is necessary to better elucidate the relationship between GM and overweight and then the mechanism of action by which L. plantarum IMC 510 modifies weight. However, these promising results prompt a clear advantage of probiotic supplementation and identify a new potential probiotic as a novel and safe therapeutic approach in obesity prevention and management.

Impact of Probiotic SYNBIO(®) Administered by Vaginal Suppositories in Promoting Vaginal Health of Apparently Healthy Women.

The purpose of this study was to investigate whether vaginal administration of probiotic Lactobacillus results in their colonization and persistence in the vagina and whether it promotes normalization and maintenance of pH and Nugent score. A single-arm, open-label controlled towards the baseline (pre-post) study including 35 apparently healthy women was conducted. Each woman was examined three times during the study. Women were instructed to receive daily for 7 days, the probiotic suppositories SYNBIO(®) gin (Lactobacillus rhamnosus IMC 501(®) and Lactobacillus paracasei IMC 502(®)). Vaginal swabs were collected during visit 1, 2, and 3 to determine the total lactobacilli count, the presence of the two administered bacteria, the measure of the pH, and the estimation of Nugent score. Evaluation of treatment tolerability was based on analysis of the type and occurrence of adverse events. The probiotic vaginal suppository was well tolerated and no side effects were reported. Intermediate Nugent score was registered in 40 % of women at visit 1 and these intermediate scores reverted to normal at day 7 (end of treatment) in 20 % of subjects. Administration of SYNBIO(®) gin contributed to a significant increase in the lactobacilli level at visit 2. Molecular typing revealed the presence of the two strains originating from SYNBIO(®) gin in 100 % of women at visit 2 and 34 % at visit 3. No significant changes were registered for pH between visits. The SYNBIO(®) gin product is safe for daily use in healthy women and it could be useful to restore and maintain a normal vaginal microbiota.

In Vitro Assessment of Postbiotic and Probiotic Commercial Dietary Supplements Recommended for Counteracting Intestinal Dysbiosis in Dogs.

Many environmental aspects influence the preservation of a beneficial microbiome in dogs, and gut dysbiosis occurs when imbalances in the intestinal ecosystem cause functional changes in the microbial populations. The authors evaluated the effects of two specific commercial dietary supplements: a combination of a postbiotic and prebiotics (Microbiotal cane®) and a probiotic product (NBF 1®) recommended for counteracting intestinal dysbiosis in dogs, on the gut canine microbiota composition and its metabolic activities (production of short-chain fatty acids). The investigation was performed using an in vitro fermentation system inoculated with dog fecal samples. Microbiotal cane® promoted a more immediate increase in Lactobacillus spp. after the first 6 h of fermentation, whereas NBF 1® promoted the increase at the end of the process only. The two supplements supported an increase in the Bifidobacterium spp. counts only after 24 h. The in vitro abilities of Microbiotal cane® and NBF 1® to increase selectively beneficial bacterial groups producing acetic, propionic, and butyric acids suggest a possible positive effect on the canine gut microbiota, even if further in vivo studies are needed to confirm the beneficial effects on the intestinal health.

SYNBIO® Probiotic and Antioxidant Dietary Supplementation: Clinical Trial Evaluation of Potential Effects on Airline Flight Crew Members' Well-Being.

The irregular lifestyle of airline crew members, wide/adverse job-related exposures, and the impact of temporary hypoxia on gut microbiota well-being have increased concern about the daily recommended dose of certain nutrients among flight crew. The aim of this study was to determine if daily consumption of a SYNBIO® probiotics-elderberry extract supplement (ACTIVE) may contribute to the well-being of flight attendants. Forty healthy crew members enrolled in a double-blind, randomized, placebo-controlled study consumed one ACTIVE capsule/day or placebo for 30 days. Bowel well-being, health-related quality of life, and gastrointestinal tolerance were assessed by validated questionnaires. Saliva and fecal samples were analyzed to determine secretory immunoglobulin-A (sIgA) levels and to characterize gut microbiota composition, respectively. ACTIVE subjects presented a physiological improvement and a statistically significant higher Psychological General Well-Being Index (PGWBI) global score compared to PLACEBO subjects. The ACTIVE subjects showed significantly increased levels of lactobacilli and bifidobacteria compared to the PLACEBO group, while a significant increase in lactobacilli and a significant reduction in Enterobacteriaceae were registered when compared with the beginning of supplementation, confirming the persistence of probiotics in the gastrointestinal tract and the direct antagonism and competitive exclusion effects. Additionally, sIgA levels were significantly higher in the ACTIVE group compared to the baseline and to the PLACEBO group at the end of supplementation. The ACTIVE supplementation might be beneficial to airline crew members, improving their physiological state, their immune defenses, and the strength and efficiency of their gastrointestinal tract when responding to stressful conditions.

Effects of the probiotic Lactiplantibacillus plantarum IMC 510® on body composition, biochemical parameters, gut microbiota composition and function, and clinical symptoms of overweight/obese subjects.

Background and aim: In recent decades, obesity prevalence has reached epidemic proportions and considering the pivotal role of gut microbiota (GM) in the regulation of energy balance, alternative non-pharmacological approaches involving probiotics' administration have been proposed. The aim of the present study was to evaluate the effect of Lactiplantibacillus plantarum IMC 510® supplementation on anthropometric and biochemical parameters, GM composition and functionality, and gastrointestinal and general symptoms of overweight/obese subjects. Methods: Forty overweight/obese subjects were randomly assigned to daily consume the probiotic Lactiplantibacillus plantarum IMC 510® or placebo for 3 months. Before and after the administration period, anthropometric and biochemical parameters, self-administered questionnaires, and plasma and stool samples were obtained from each participant. The GM characterization was performed with 16S rRNA sequencing, while fecal short (SCFAs) and medium (MCFAs) chain fatty acids were analyzed with a gas chromatography-mass spectrometry protocol. Results: Compared to placebo, probiotic supplementation determined a significant decrease in body weight, BMI, waist circumference, waist-to-height ratio, and blood glucose. Moreover, probiotic administration produced a significant decrease of the genera Hafnia-Obesumbacterium and Romboutsia and an increase of Succiniclasticum spp.; conversely, placebo administration resulted in the decrease of Actinomycetaceae and an increase of both Alloprevotella spp. and of the levels of pro-inflammatory hexanoic and heptanoic acids. Conclusion: Thanks to its effect in increasing some beneficial gut bacteria and lowering effects on waist circumference, fasting glucose levels and gastrointestinal symptoms of obese subjects, Lactiplantibacillus plantarum IMC 510® supplementation could represent a future and encouraging strategy for the prevention or treatment of obesity.

Exploratory Study for Probiotic Enrichment of a Sea Fennel (Crithmum maritimum L.) Preserve in Brine.

Considering the increasing consumer demand for vegan and vegetarian health foods, different vegetables have been already exploited to produce non-dairy probiotic foods. In addition to being rich in bioactive compounds, sea fennel (Crithmum maritimum L.), also known as rock samphire, represents a valuable candidate in the production of probiotic-enriched foods, and, to the authors' knowledge, it has not yet been explored as carrier for probiotics. Hence, the present study was aimed at evaluating the survival of a commercially available probiotic formulation, SYNBIO®, and Lactiplantibacillus plantarum IMC 509 in an artificially acidified, pasteurized sea fennel preserve in brine during a refrigerated storage of 44 days. Despite slight reductions in the microbial loads, at the end of the storage, both the probiotic formulations showed loads higher than 7.0 Log CFU g-1 of sea fennel or mL-1 of brine, above the recommended administration dose to exert beneficial health effects. Thus, acidified sea fennel sprouts in brine represent a potential vehicle for probiotics delivery to humans.

Oral Intake of the Commercial Probiotic Blend Synbio® for the Management of Vaginal Dysbiosis.

A healthy vaginal microbiota is Lactobacillus-dominated. Several factors can interfere with the state of balance leading to dysbiosis, such as vaginal infections caused by bacteria and Candida species. The present single-arm, uncontrolled open-label study aimed to evaluate the ability of the SYNBIO® probiotic combination, taken as an oral formulation, to contribute to vaginal health. Thirty pre-menopausal participants were included in the study. Participants were instructed for daily oral intake of SYNBIO® probiotic capsules for 15 days. Vaginal swabs were collected at baseline (T0), 15 days after the start of the treatment (T1), and 7 days after the end of the treatment (T2). Amsel criteria, Nugent score, and vaginal pH were evaluated at each sampling time. In addition, the participants' quality of life was assessed by the WHOQOL-BREF questionnaire. The administration of SYNBIO® once daily for 15 days resulted in a substantial improvement in the vaginal flora in terms of an increase in lactobacilli and a decrease in enterococci, staphylococci, Gardnerella spp., and Candida spp. According to the results, statistically significant changes in leucorrhoea, itching, and vulvo-vaginal erythema/edema as well as a decrease in all the Amsel criteria were recorded. The oral consumption of SYNBIO® demonstrated enhanced benefits for vaginal health.

Modulation of cortisol levels, endocannabinoid receptor 1A, proopiomelanocortin and thyroid hormone receptor alpha mRNA expressions by probiotics during sole (Solea solea) larval development.

In the present study, we investigated whether the use of Enterococcus faecium IMC 511 as a probiotic can modulate neuroendocrine system responses during the larval rearing of Solea solea; to this end, the gene expression patterns of proopiomelanocortin (POMC), endocannabinoid receptor 1A (CB1A), and thyroid receptor alpha (TRα) were quantified, and whole-body cortisol levels were measured. Probiotic treatment up-regulated transcription of all selected genes and cortisol concentrations on day 10 post hatch (ph), while on day 30 ph experimental groups showed significantly lower levels of both POMC and CB1A compared to those of the control group. These changes were no longer evident on day 60 ph, when POMC, CB1A, TRα gene expression and cortisol titers were found to be similar in all experimental groups. Our results suggest that metabolic responses to probiotic treatment can be modulated through the activation of genes selected for functional interaction between the hypothalamic-pituitary-thyroid (HPT) axis and the melanocortin and the endocannabinoid systems. Furthermore, the observed (30 ph) down-regulation of both POMC and CB1A gene expression coupled with up-regulation of TRα mRΝΑ levels suggest the activation of a compensatory mechanism that promotes growth and development and perhaps modulates food intake.

Effect of a probiotic intake on oxidant and antioxidant parameters in plasma of athletes during intense exercise training.

The aim of this study was to evaluate the effect of Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502 on oxidative stress in athletes during a four-week period of intense physical activity. Two groups of twelve subjects each were selected for this analysis. The first group consumed a daily dose of a mixture of the two probiotic strains (1:1 L. rhamnosus IMC 501 and L. paracasei IMC 502; ~10(9) cells/day) for 4 weeks. The second group (control) did not consume any supplements during the 4 weeks. Blood samples collected immediately before and after the supplementation were analyzed, and plasma levels of reactive oxygen metabolites and biological antioxidant potential were determined. Faeces were also collected and analyzed before and at the end of the probiotic supplementation. Antioxidative activity and oxidative stress resistance of the two strains were determined in vitro. Results demonstrated that intense physical activity induced oxidative stress and that probiotic supplementation increased plasma antioxidant levels, thus neutralizing reactive oxygen species. The two strains, L. rhamnosus IMC 501(®) and L. paracasei IMC 502(®), exert strong antioxidant activity. Athletes and all those exposed to oxidative stress may benefit from the ability of these probiotics to increase antioxidant levels and neutralize the effects of reactive oxygen species.

Probiotic properties of Lactobacillus rhamnosus and Lactobacillus paracasei isolated from human faeces.

BACKGROUND: The possibility of using microbes to maintain health, and to prevent or treat disease is a topic as old as microbiology. The research of novel probiotic strains is important in order to satisfy the increasing request of the market and to obtain functional products in which the probiotic cultures are more active and with better probiotic characteristics than those already present on the market. AIM OF THE STUDY: In this study, the probiotic potential of Lactobacillus strains isolated from Italian elderly human faeces was investigated. METHODS: The Lactobacillus strains were identified and examined for resistance to gastric acidity and bile toxicity, adhesion to HT-29 cells, antimicrobial activities, antibiotic susceptibility and plasmid profile. Survival of the strains through human intestine was examined in a 3 months human feeding trial. RESULTS: Two strains, Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502, tolerated well low pH and bile acids. In antimicrobial activity assays, both strains showed inhibitory properties towards selected potential harmful microorganisms, particularly against Candida albicans. The two selected strains expressed high in vitro adherence to HT-29 cells increasing this characteristic when they are used in combination and they were resistant to vamcomycin, colistin sulphate, gentamicin, oxolinic acid and kanamycin. Moreover, the two strains could be recovered from stools of volunteers after the feeding trials. CONCLUSIONS: Lactobacillus rhamnosus IMC 501 and L. paracasei IMC 502 present favourable strain-specific properties for their utilisation as probiotics in functional foods and the high adhesion ability of the L. rhamnosus IMC 501 and L. paracasei IMC 502 used in combination, confirmed by both in vitro and in vivo study, indicate that the two bacterial strains could be used as health-promoting bacteria.

Changes on fecal microbiota in rats exposed to permethrin during postnatal development.

Alteration of the gut microbiota through diet and environmental contaminants may disturb the mammalian digestive system, leading to various diseases. Because most exposure to environmentally pyrethroid pesticides such as permethrin (PERM) occurs through the diet, the commensal gut microbiota is likely to be exposed to PERM. The study aimed at evaluating the effect of low-dose exposure to PERM in early life on the composition of fecal microbiota in rats. Over a 4-month follow-up period, fecal microbiota and short-chain fatty acids were measured in order to identify possible differences between PERM-treated rats and controls. Further in vitro antimicrobial experiments were conducted to establish the antibacterial activity of PERM against different strains to obtain Minimal Inhibitory Concentrations. The main finding focused on the reduced abundance of Bacteroides-Prevotella-Porphyromonas species, increased Enterobacteriaceae and Lactobacillus in PERM-treated rats compared to controls. Changes of acetic and propionic acid levels were registered in PERM-treated group. From in vitro studies, PERM showed higher antibacterial activity against beneficial bacteria such as Bifidobacterium and Lactobacillus paracasei, while to inhibit potential pathogens as Staphylococcus aureus and Escherichia coli PERM concentration needed to be increased. In summary, exposure to PERM could affect the fecal microbiota and could be a crucial factor contributing to the development of diseases.

Effect of buckwheat flour and oat bran on growth and cell viability of the probiotic strains Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and their combination SYNBIO®, in synbiotic fermented milk.

Fermented foods have a great significance since they provide and preserve large quantities of nutritious foods in a wide diversity of flavors, aromas and texture, which enrich the human diet. Originally fermented milks were developed as a means of preserving nutrients and are the most representatives of the category. The first aim of this study was to screen the effect of buckwheat flour and oat bran as prebiotics on the production of probiotic fiber-enriched fermented milks, by investigating the kinetics of acidification of buckwheat flour- and oat bran-supplemented milk fermented by Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and their 1:1 combination named SYNBIO®. The probiotic strains viability, pH and sensory characteristics of the fermented fiber-enriched milk products, stored at 4 °C for 28 days were also monitored. The results showed that supplementation of whole milk with the tested probiotic strains and the two vegetable substrates results in a significant faster lowering of the pH. Also, the stability of L. rhamnosus IMC 501®, L. paracasei IMC 502® and SYNBIO® during storage at 4 °C for 28 days in buckwheat flour- and oat bran-supplemented samples was remarkably enhanced. The second aim of the study was to develop a new synbiotic product using the best combination of probiotics and prebiotics by promoting better growth and survival and be acceptable to the consumers with high concentration of probiotic strain. This new product was used to conduct a human feeding trial to validate the fermented milk as a carrier for transporting bacterial cells into the human gastrointestinal tract. The probiotic strains were recovered from fecal samples in 40 out of 40 volunteers fed for 4 weeks one portion per day of synbiotic fermented milk carrying about 10(9) viable cells.

Functional foods as carriers for SYNBIO®, a probiotic bacteria combination.

The popularity of functional foods continues to increase as consumers desire flavorful foods that will fulfil their health needs. Among these foods, probiotics may exert positive effects on the composition of gut microbiota and overall health. However, in order to be beneficial, the bacterial cultures have to remain live and active at the time of consumption. The aim of this study was to develop new probiotic food products, such as seasoned cheeses, salami, chocolate and ice-cream with a final probiotic concentration of approximately 109CFU/daily dose of Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® mixed 1:1 (SYNBIO®). The survival and viability of probiotics were determined during the foods shelf-life. The values of viable probiotic bacteria of all dairy and non-dairy foods were between 107 and 109CFU/g of food at the end of the shelf-life and for some of them the values were maintained even after the expiry date. Based on the results of the current study, all the dairy ("Caciotta" cheese, "Pecorino" cheese, "Büscion" Swiss cheese and "Fiordilatte" ice-cream) and non-dairy ("Ciauscolo" salami, Larded salami, Swiss small salami, milk chocolate, dark chocolate, organic jam and chocolate mousse) food products studied would be excellent vehicles to deliver the probiotic health effects because of the high viability of probiotics during the shelf-life of foods and in some cases even after their expiry date.

Pilot-scale production and viability analysis of freeze-dried probiotic bacteria using different protective agents.

The functional food industry requires an improvement of probiotic strain stability during storage, especially when they are stored at room temperature. In this study, the viability of freeze-dried Lactobacillus rhamnosus IMC 501(®) and Lactobacillus paracasei IMC 502(®) using different protective agents (i.e., glycerine, mannitol, sorbitol, inulin, dextrin, Crystalean(®)) was determined and compared with semi skimmed milk (SSM) control. No significant differences were observed between the tested protectants and the control (SSM) during storage at refrigerated conditions. During storage at room temperature, only glycerine was found to stabilize viability better than other tested substances.

Differences in fecal microbiota in different European study populations in relation to age, gender, and country: a cross-sectional study.

A cross-sectional study on intestinal microbiota composition was performed on 230 healthy subjects at four European locations in France, Germany, Italy, and Sweden. The study participants were assigned to two age groups: 20 to 50 years (mean age, 35 years; n = 85) and >60 years (mean age, 75 years; n = 145). A set of 14 group- and species-specific 16S rRNA-targeted oligonucleotide probes was applied to the analysis of fecal samples by fluorescence in situ hybridization coupled with flow cytometry. Marked country-age interactions were observed for the German and Italian study groups. These interactions were inverse for the predominant bacterial groups Eubacterium rectale-Clostridium coccoides and Bacteroides-Prevotella. Differences between European populations were observed for the Bifidobacterium group only. Proportions of bifidobacteria were two- to threefold higher in the Italian study population than in any other study group, and this effect was independent of age. Higher proportions of enterobacteria were found in all elderly volunteers independent of the location. Gender effects were observed for the Bacteroides-Prevotella group, with higher levels in males than in females. In summary, age-related differences in the microbiota makeup were detected but differed between the study populations from the four countries, each showing a characteristic colonization pattern.