Randomised, Placebo-Controlled Investigation of the Impact of Probiotic Consumption on Gut Microbiota Diversity and the Faecal Metabolome in Seniors.

Aging has been associated with a changed composition and function of the gut microbiota (GM). Here, we investigate the effects of the multi-strain probiotic HOWARU® Restore on GM composition and function in seniors. Ninety-eight healthy adult volunteers aged ≥75 years were enrolled in a randomised, double-blinded intervention (NCT02207140), where they received HOWARU Restore (1010 CFU) or the placebo daily for 24 weeks, with 45 volunteers from each group completing the intervention. Questionnaires monitoring the effects on gastro-intestinal discomfort and bowel movements were collected. Faecal samples for GM characterisation (qPCR, 16S rRNA gene amplicon sequencing) and metabolomics (GC-FID, 1H NMR) were collected at the baseline and after 24 weeks. In the probiotic group, self-reported gastro-intestinal discomfort in the form of flatulence was significantly decreased during the intervention. At the baseline, 151 'core species' (present in ≥95% of samples) were identified. Most core species belonged to the Lachnospiraceae and Ruminococcaceae families. Neither alpha diversity nor beta diversity or faecal metabolites was affected by probiotic intake. On the contrary, we observed high intra-individual GM stability, with 'individual' accounting for 72-75% of variation. In conclusion, 24 weeks of HOWARU Restore intake reduced gastro-intestinal discomfort in the form of flatulence in healthy seniors without significantly influencing GM composition or activity.

The Effect of Bifidobacterium animalis subsp. lactis Bl-04 on Influenza A Virus Infection in Mice.

Influenza A virus infection is a major global disease requiring annual vaccination. Clinical studies indicate that certain probiotics may support immune function against influenza and other respiratory viruses, but direct molecular evidence is scarce. Here, mice were treated with a placebo or Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04) orally via food (cereal) and also by gavage and exposed to Influenza A virus H1N1 (H1N1). The symptoms of the infection were observed, and tissues and digesta were collected for viral load RT-qPCR, transcriptomics, and microbiomics. The treatment decreased the viral load by 48% at day 3 post-infection in lungs and symptoms of infection at day 4 compared to placebo. Tissue transcriptomics showed differences between the Bl-04 and placebo groups in the genes in the Influenza A pathway in the intestine, blood, and lungs prior to and post-infection, but the results were inconclusive. Moreover, 16S rRNA gene profiling and qPCR showed the presence of Bl-04 in the intestine, but without major shifts in the microbiome. In conclusion, Bl-04 treatment may influence the host response against H1N1 in a murine challenge model; however, further studies are required to elucidate the mechanism of action.

Efficacy and safety of Lacticaseibacillus paracasei Lpc-37® in students facing examination stress: A randomized, triple-blind, placebo-controlled clinical trial (the ChillEx study).

Lacticaseibacillus paracasei Lpc-37 (Lpc-37) has previously shown to reduce perceived stress in healthy adults. The ChillEx study investigated whether Lpc-37 reduces stress in a model of chronic examination stress in healthy students. One hundred ninety university students (18-40 y) were randomized to take 1.56 × 1010 colony-forming units of Lpc-37 or placebo (1:1) each day for 10 weeks, in a triple-blind, parallel, multicenter clinical trial consisting of six visits: two screening visits, a baseline visit, and visits at 4, 8, and 10 weeks after baseline. The primary objective was to demonstrate that Lpc-37 reduces stress, as measured by the change in state anxiety from baseline to just before the first examination, after 8 weeks using the State Trait Anxiety Inventory (STAI-state). Secondary objectives aimed to demonstrate that Lpc-37 modulates psychological stress-induced symptoms and biomarkers related to mood and sleep. An exploratory analysis of fecal microbiota composition was also conducted. There was no difference between Lpc-37 and placebo groups in the change of STAI-state score (estimate 1.03; 95% confidence interval [CI]: -1.62, 3.67; p = 0.446). None of the secondary outcomes resulted in significant results when corrected for multiplicity, but exploratory results were notable. Results showed an improvement in sleep-disturbance scores (odds ratio 0.30; 95% CI: 0.11, 0.82; p = 0.020) and reduction in duration of sleep (odds ratio 3.52; 95% CI: 1.46, 8.54; p = 0.005) on the Pittsburgh Sleep Quality Index questionnaire after 8 weeks in the Lpc-37 group compared to placebo. A reduction in Bond Lader VAS-alertness was also demonstrated in the Lpc-37 group compared to placebo (estimate -3.97; 95% CI: -7.78, -0.15; p = 0.042) just prior to the examination. Analysis of fecal microbiota found no differences between study groups for alpha and beta diversity or microbiota abundance. Adverse events were similar between groups. Vital signs, safety-related laboratory measures, and gastrointestinal parameters were stable during the trial. In conclusion, probiotic Lpc-37 was safe but had no effect on stress, mood, or anxiety in healthy university students in this model of chronic academic stress. ClinicalTrials.gov: NCT04125810.

The Effect of Delivery Matrix on Bifidobacterium animalis subsp. lactis HN019 Survival through In Vitro Human Digestion.

Bifidobacterium animalis subsp. lactis HN019 is a probiotic with several documented human health benefits. Interest in probiotics has led to the development of new formats that probiotics, including HN019, can be supplemented into. In this study, we looked at common HN019 formats such as frozen culture and freeze-dried powder as well as supplementing it into the following food matrices: yogurts (dairy, soy, and oat based), xanthan gum-based tablets, pulpless orange juice, whey sports drink, and dark chocolate (70% cocoa). In this work, our aim was to investigate whether the food matrix that carried HN019 via simulated human digestion (a dual model system mimicking both upper and lower gastrointestinal digestion) influenced probiotic delivery. To that end, we validated and used a real-time qPCR assay to detect HN019 after simulated digestion. In addition, we also measured the effect on a panel of metabolites. After simulated digestion, we were able to detect HN019 from all the matrices tested, and the observed changes to the metabolite profile were consistent with those expected from the food matrix used. In conclusion, this work suggests that the food matrix supplemented with HN019 did not interfere with delivery to the colon via simulated human digestion.

A Healthy Vaginal Microbiota Remains Stable during Oral Probiotic Supplementation: A Randomised Controlled Trial.

The primary objective of this randomised, placebo-controlled, triple-blind study was to assess whether orally consumed Lactobacillus acidophilus La-14 (La-14) and Lacticaseibacillus rhamnosus HN001 (HN001) colonise a healthy human vagina. Furthermore, potential effects on vaginal microbiota and immune markers were explored. Fifty women devoid of vaginal complaints (Nugent score 0-3 and vaginal pH ≤ 4.5) were randomised into a 2-week intervention with either La-14 and HN001 as the verum product or a comparable placebo. Vaginal swab samples were collected at baseline, after one and two weeks of intervention, and after a one-week follow-up, for assessing colonisation of the supplemented lactobacilli, vaginal microbiota, and six specific immune markers. Colonisation of L. acidophilus and L. rhamnosus was not observed above the assay detection limit (5.29 and 5.11 log 10 genomes/swab for L. acidophilus and L. rhamnosus, respectively). Vaginal microbiotas remained stable and predominated by lactobacilli throughout the intervention, and vaginal pH remained optimal (at least 90% of participants in both groups had pH 4.0 or 4.5 throughout the study). Immune markers elafin and human ß-defensin 3 (HBD-3) were significantly decreased in the verum group (p = 0.022 and p = 0.028, respectively) but did not correlate with any microbiota changes. Adverse events raised no safety concerns, and no undesired changes in the vaginal microbiota or immune markers were detected.

Characterization of vaginal fungal communities in healthy women and women with bacterial vaginosis (BV); a pilot study.

The role of the vaginal fungal community, the mycobiota, in women's health is an emerging area of research. Utilization of novel molecular technology enables more in-depth characterization and identification of fungal diversity, and their potential associations to health. The present study is a substudy of a larger observational clinical trial investigating the vaginal microbiota composition before and after antibiotic treatment for Bacterial Vaginosis (BV) infection in comparison to the microbiota of healthy women (Clinicaltrials.gov identifier: NCT03187). Here, we characterized the vaginal mycobiota by sequencing the internal transcribed spacer (ITS) 2 region from vaginal microbial DNA collected from healthy women and women with BV and in relation to their treatment with oral metronidazole. Interestingly, both ascomycetous and basidiomycetous yeasts and filamentous fungi consisting of more than 30 different species were detectable from 21 out of 94 vaginal swab samples. The mycobiota was dominated by Candida species (>60% of relative abundance) and especially with Candida albicans in both study groups. The abundance of C. albicans was inversely correlated with fungal diversity but did not correlate with Nugent scores. Metronidazole did not seem to have a major effect on the relative abundance of C. albicans. The results revealed the diversity of the fungal community within healthy and BV-infected women, which is worth exploring further.

Cholesterol loading suppresses the atheroinflammatory gene polarization of human macrophages induced by colony stimulating factors.

In atherosclerotic lesions, blood-derived monocytes differentiate into distinct macrophage subpopulations, and further into cholesterol-filled foam cells under a complex milieu of cytokines, which also contains macrophage-colony stimulating factor (M-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF). Here we generated human macrophages in the presence of either M-CSF or GM-CSF to obtain M-MØ and GM-MØ, respectively. The macrophages were converted into cholesterol-loaded foam cells by incubating them with acetyl-LDL, and their atheroinflammatory gene expression profiles were then assessed. Compared with GM-MØ, the M-MØ expressed higher levels of CD36, SRA1, and ACAT1, and also exhibited a greater ability to take up acetyl-LDL, esterify cholesterol, and become converted to foam cells. M-MØ foam cells expressed higher levels of ABCA1 and ABCG1, and, correspondingly, exhibited higher rates of cholesterol efflux to apoA-I and HDL2. Cholesterol loading of M-MØ strongly suppressed the high baseline expression of CCL2, whereas in GM-MØ the low baseline expression CCL2 remained unchanged during cholesterol loading. The expression of TNFA, IL1B, and CXCL8 were reduced in LPS-activated macrophage foam cells of either subtype. In summary, cholesterol loading converged the CSF-dependent expression of key genes related to intracellular cholesterol balance and inflammation. These findings suggest that transformation of CSF-polarized macrophages into foam cells may reduce their atheroinflammatory potential in atherogenesis.

Recovery of Vaginal Microbiota After Standard Treatment for Bacterial Vaginosis Infection: An Observational Study.

Vaginal microbiota dysbiosis and bacterial vaginosis (BV) affect negatively women's health. Understanding vaginal microbiota fluctuations in BV during and after antibiotic treatment would facilitate accurate decision-making on the treatment regimen, avoid unnecessary antibiotic use, and potentially mitigate recurrence. We investigated vaginal microbiota composition of 30 women with BV before and after 5-day metronidazole treatment and compared the results with 30 healthy women. Vaginal microbiota was assessed by Nugent score and analyzed by 16S rRNA gene sequencing in swabs on baseline Day 1, and on Day 8 and 15, after completion of antibiotic treatment by women with BV. Prior to antibiotic treatment (Day 1), BV-positive women were dominated by Lactobacillus iners (25.8%), Prevotella timonensis/bivia (18.0%), and Gardnerella vaginalis (14.6%), whereas healthy women were dominated by L. iners (37.5%) and Lactobacillus crispatus/acidophilus (19.2%). On Day 8, L. iners abundance increased in BV-treated women being significantly higher compared with healthy women (67.8% vs. 37.5%, p = 0.049). On Day 15, the relative abundance of all microbial taxa was similar between the groups. Vaginal microbiota of women with BV shifted to resemble that of healthy controls after metronidazole. Sequencing analysis provides more in-depth understanding of changes in vaginal microbiota. The role of L. iners in vaginal health and dysbiosis requires further investigations.

Fecal Recovery of Probiotics Administered as a Multi-Strain Formulation during Antibiotic Treatment.

The present study aimed to investigate whether probiotic recovery is affected when consumed together with antibiotics. Fecal samples were collected from an earlier antibiotic associated diarrhea, randomized, placebo-controlled study with a product consisting of a combination of Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, and Bifidobacterium lactis Bi-07, B. lactis Bl-04 at equal numbers and at a total dose of 1010 CFU. Fecal samples were collected during the screening visit (T0), i.e., at the time of antibiotic prescription, and then on the last day of the antibiotic treatment (T1) as well as seven days after the subject had stopped taking the antibiotic treatment (T2) and at two weeks after completing antibiotic treatment and one week after probiotic/placebo consumption stopped (T3). Samples were analyzed for the presence of the four administered strains. The study was registered at clinicaltrials.gov as NCT01596829. Detection levels of all four strains were significantly increased from T0 to T1 and returned to baseline level from T2 to T3. There were also significantly more subjects with detectable levels of L. paracasei Lpc-37, B. lactis Bi-07, and B. lactis Bl-04 at T1 and T2 compared to T0 and T3, and compared to placebo. Each of the four strains could be detected in the feces of patients apparently unaffected by the simultaneous consumption of antibiotics.

Nasal microbiota clusters associate with inflammatory response, viral load, and symptom severity in experimental rhinovirus challenge.

The role of nasal and fecal microbiota in viral respiratory infections has not been established. We collected nasal swabs and washes, and fecal samples in a clinical study assessing the effect of probiotic Bifidobacterium animalis subsp. lactis Bl-04 on experimental rhinovirus infection. The nasal and fecal microbiota were characterized by 16S rRNA gene sequencing. The resulting data were compared with nasal inflammatory marker concentrations, viral load, and clinical symptoms. By using unsupervised clustering, the nasal microbiota divided into six clusters. The clusters predominant of Staphylococcus, Corynebacterium/Alloiococcus, Moraxella, and Pseudomonadaceae/Mixed had characteristic inflammatory marker and viral load profiles in nasal washes. The nasal microbiota clusters of subjects before the infection associated with the severity of clinical cold symptoms during rhinovirus infection. Rhinovirus infection and probiotic intervention did not significantly alter the composition of nasal or fecal microbiota. Our results suggest that nasal microbiota may influence the virus load, host innate immune response, and clinical symptoms during rhinovirus infection, however, further studies are needed.

Probiotic With or Without Fiber Controls Body Fat Mass, Associated With Serum Zonulin, in Overweight and Obese Adults-Randomized Controlled Trial.

BACKGROUND: The gut microbiota is interlinked with obesity, but direct evidence of effects of its modulation on body fat mass is still scarce. We investigated the possible effects of Bifidobacterium animalisssp. lactis 420 (B420) and the dietary fiber Litesse® Ultra polydextrose (LU) on body fat mass and other obesity-related parameters. METHODS: 225 healthy volunteers (healthy, BMI 28-34.9) were randomized into four groups (1:1:1:1), using a computer-generated sequence, for 6months of double-blind, parallel treatment: 1) Placebo, microcrystalline cellulose, 12g/d; 2) LU, 12g/d; 3) B420, 1010CFU/d in microcrystalline cellulose, 12g/d; 4) LU+B420, 12g+1010CFU/d. Body composition was monitored with dual-energy X-ray absorptiometry, and the primary outcome was relative change in body fat mass, comparing treatment groups to Placebo. Other outcomes included anthropometric measurements, food intake and blood and fecal biomarkers. The study was registered in Clinicaltrials.gov (NCT01978691). FINDINGS: There were marked differences in the results of the Intention-To-Treat (ITT; n=209) and Per Protocol (PP; n=134) study populations. The PP analysis included only those participants who completed the intervention with >80% product compliance and no antibiotic use. In addition, three participants were excluded from DXA analyses for PP due to a long delay between the end of intervention and the last DXA measurement. There were no significant differences between groups in body fat mass in the ITT population. However, LU+B420 and B420 seemed to improve weight management in the PP population. For relative change in body fat mass, LU+B420 showed a-4.5% (-1.4kg, P=0.02, N=37) difference to the Placebo group, whereas LU (+0.3%, P=1.00, N=35) and B420 (-3.0%, P=0.28, N=24) alone had no effect (overall ANOVA P=0.095, Placebo N=35). A post-hoc factorial analysis was significant for B420 (-4.0%, P=0.002 vs. Placebo). Changes in fat mass were most pronounced in the abdominal region, and were reflected by similar changes in waist circumference. B420 and LU+B420 also significantly reduced energy intake compared to Placebo. Changes in blood zonulin levels and hsCRP were associated with corresponding changes in trunk fat mass in the LU+B420 group and in the overall population. There were no differences between groups in the incidence of adverse events. DISCUSSION: This clinical trial demonstrates that a probiotic product with or without dietary fiber controls body fat mass. B420 and LU+B420 also reduced waist circumference and food intake, whereas LU alone had no effect on the measured outcomes.

Safety evaluation of AB-LIFE(®) (Lactobacillus plantarum CECT 7527, 7528 and 7529): Antibiotic resistance and 90-day repeated-dose study in rats.

AB-LIFE(®) is a probiotic product consisting of equal parts of three strains of Lactobacillus plantarum (CECT 7527, 7528, and 7529) blended with inert excipients. Whole genome sequencing was performed on each of the three strains. Antibiotic resistance was evaluated by genomic mining for resistance genes, and assessment for transferability. No risk of transfer potential was identified for any antibiotic resistance genes in the three strains. AB-LIFE(®) was evaluated for potential subchronic oral toxicity in rats, with dosages of 300 and 1000 mg/kg BW/day (equivalent to 5.55 × 10(10) and 1.85 × 10(11) CFU/kg BW/day). Survival of the three test strains through the gastrointestinal tract was supported by fecal analysis. No adverse effects were identified with respect to in-life parameters, clinical or anatomic pathology, translocation, or fecal chemical analyses. The no-observed-adverse-effect level (NOAEL) for AB-LIFE(®) in male and female rats was 1000 mg/kg BW/day (1.85 × 10(11) CFU of AB-LIFE(®)/kg BW/day), the highest dose level evaluated. These results, in conjunction with a previous acute toxicity study in rats, support the conclusion that AB-LIFE(®) is safe for human consumption.

Quantitative changes in Gimap3 and Gimap5 expression modify mitochondrial DNA segregation in mice.

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.