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 β-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.

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.

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.

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.

The ability to detect probiotic consumption during a human clinical trial is crucial to verify and validate placebo and verum groups in post hoc analysis. While bacterial plating is still a common method for detecting and counting bacteria, when dealing with complex matrices like fecal samples, and given that most probiotics share genera or even species with commensal bacteria, plate counting is not a precise or accurate enough method. Species-specific quantitative real-time polymerase chain reaction (qRT-PCR) has been the most cited method in the literature and when properly validated and optimized remains the high watermark for detecting probiotics from fecal samples. Recent advancements in PCR technology have given rise to a parallel platform, droplet digital PCR (ddPCR). In this work we aimed to detect the components of a multi-strain probiotic product from a human clinical trial and compare both methods. This work dually demonstrates a process for determining multi-strain detection criteria as well as directly comparing the methods through the lens of sensitivity and specificity or the ability to properly discern true positives and true negatives. We described the optimization and validation of three assays for use in our detection panel and observed that, between qRT-PCR and ddPCR. The two methods were found to be quite congruent with ddPCR demonstrating a 10–100 fold lower limit of detection. Moreover, we discovered that most of the sensitivity and specificity had come from a single assay alone (Bifidobacterium animalis subsp. lactis Bl-04). This is despite all three assays performing well in optimization and validation. This suggests that more work needs to be done in the validation stage when developing novel probiotic detection assays. Taken together we can recommend ddPCR as a method for detecting probiotics from human clinical trials, but that qRT-PCR still performs well and comparably to ddPCR, when properly optimized and validated. However, when novel assays or those with unknown performance in a given biological matrix are needed, employing a strategy that combines multiple assays in a layered discrimination approach can help mitigate the potential underperformance of any single assay.