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Abstract Riboflavin (vitamin B2) is an essential water-soluble vitamin that serves as a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FMN and FAD are coenzymes involved in key enzymatic reactions in energy metabolism, biosynthesis, detoxification and electron scavenging pathways. Riboflavin deficiency is prevalent worldwide and impacts women’s health due to riboflavin demands linked to urogenital and reproductive health, hormonal fluctuations during the menstrual cycle, pregnancy, and breastfeeding. Innovative functional foods and nutraceuticals are increasingly developed to meet women’s riboflavin needs to supplement dietary sources. An emerging and particularly promising strategy is the administration of riboflavin-producing lactic acid bacteria, combining the health benefits of riboflavin with those of probiotics and in situ riboflavin production. Specific taxa of lactobacilli are of particular interest for women, because of the crucial role of Lactobacillus species in the vagina and the documented health effects of other Lactobacillaceae taxa in the gut and on the skin. In this narrative review, we synthesize the underlying molecular mechanisms and clinical benefits of riboflavin intake for women’s health, and evaluate the synergistic potential of riboflavin-producing lactobacilli and other microbiota.

Abstract Atopic dermatitis is the most common inflammatory skin condition with a severe negative impact on patients’ quality of life. The etiology of AD is complex and depends on age, genetics, the immune system, environmental factors, and the skin microbiome, with a key role for pathogenic Staphylococcus aureus in the development of severe AD. However, the composition of the skin microbiome in mild AD is understudied. Here, using metagenomic shallow shotgun sequencing, we showed that mild AD lesions did not show a significant difference in the diversity of the skin microbiome compared to samples from non-AD patients and that the relative abundance of S. aureus did not differ in these mild AD lesions. However, when we assessed other taxa, Mycobacterium ostraviense, Pedobacter panaciterrae_A and four Streptomyces species were identified with higher abundances in mild AD lesions and species of 15 genera were decreased in abundance. The highest fold decreases were observed for Paracoccus marcusii, Microbacterium lacticum, Micrococcus luteus, and Moraxella sp002478835. These microbiome compositional insights are a first step towards novel microbiome-based diagnostics and therapeutics for early intervention at the stage of mild AD and provide a path forward for the functional study of species involved in this often-overlooked patient population.

Abstract Background Prematurity remains one of the main causes of neonatal morbidity and mortality. Approximately two thirds of preterm births are spontaneous, i.e. secondary to preterm labour, preterm prelabour rupture of membranes (PPROM) or cervical insufficiency. Etiologically, the vaginal microbiome plays an important role in spontaneous preterm birth (sPTB). Vaginal dysbiosis and bacterial vaginosis are well-known risk factors for ascending lower genital tract infections and sPTB, while a Lactobacillus crispatus-dominated vaginal microbiome is associated with term deliveries. Synbiotics may help to achieve and/or maintain a normal, Lactobacillus-dominated vaginal microbiome. Methods We will perform a multi-centre, double-blind, randomised, placebo-controlled trial. Women aged 18 years or older with a singleton pregnancy are eligible for inclusion at 80/7–106/7 weeks gestational age if they have one or more of the following risk factors for sPTB: previous sPTB at 240/7–356/7 weeks, prior PPROM before 360/7 weeks, or spontaneous pregnancy loss at 140/7–236/7 weeks of gestation. Exclusion criteria are multiple gestation, cervix conisation, inflammatory bowel disease, uterine anomaly, and the use of pro-/pre-/synbiotics. Patients will be randomised to oral synbiotics or placebo, starting before 11 weeks of gestation until delivery. The oral synbiotic consists of eight Lactobacillus species (including L. crispatus) and prebiotics. The primary outcome is the gestational age at delivery. Vaginal microbiome analysis once per trimester (at approximately 9, 20, and 30 weeks) and delivery will be performed using metataxonomic sequencing (16S rRNA gene) and microbial culture. Secondary outcomes include PPROM, the use of antibiotics, antenatal admission information, and neonatal outcomes. Discussion This study will evaluate the effect of oral synbiotics on the vaginal microbiome during pregnancy in a high-risk population and correlate the microbial changes with the gestational age at delivery and relevant pregnancy outcomes. Trial registration ClinicalTrials.gov, NCT05966649. Registered on April 5, 2024.

Abstract Background In this study we shed light on ongoing trends in contraceptive use in Flanders (Belgium). Building on the fundamental cause theory and social diffusion of innovation theory, we examine socio-economic gradients in contraceptive use and the relationship to health behaviours. Methods Using the unique and recently collected (2020) ISALA data, we used multinomial logistic regression to model the uptake of contraceptives and its association to educational level and health behaviour (N:4316 women). Results Higher educated women, and women with a healthy lifestyle especially, tend to use non-hormonal contraceptives or perceived lower-dosage hormonal contraceptives that are still trustworthy from a medical point of view. Moreover, we identified a potentially vulnerable group in terms of health as our results indicate that women who do not engage in preventive health behaviours are more likely to use no, or no modern, contraceptive method. Discussion The fact that higher educated women and women with a healthy lifestyle are less likely to use hormonal contraceptive methods is in line with patient empowerment, as women no longer necessarily follow recommendations by healthcare professionals, and there is a growing demand for naturalness in Western societies. Conclusion The results of this study can therefore be used to inform policy makers and reproductive healthcare professionals, since up-to-date understanding of women’s contraceptive choices is clearly needed in order to develop effective strategies to prevent sexually transmitted infections and unplanned pregnancies, and in which women can take control over their sexuality and fertility in a comfortable and pleasurable way.

Abstract Strategies against the opportunistic fungal pathogen Candida albicans based on probiotic microorganisms represent a promising alternative to traditional antifungals. Here, we investigated the effects of Lactobacillaceae isolates from fermented foods or the human vagina, alone or in combination with the probiotic yeast Saccharomyces cerevisiae CNCM I-3856, against C. albicans in vitro. Nine out of nineteen tested strains of Lactobacillaceae inhibited growth of C. albicans with inhibition zones of 1–3 mm in spot assays. Five out of nineteen lactobacilli tested as such or in combination with S. cerevisiae CNCM I-3856 also significantly inhibited C. albicans hyphae formation, including Limosilactobacillus fermentum LS4 and L. fermentum LS5 resulting in respectively 62% and 78% hyphae inhibition compared to the control. Thirteen of the tested nineteen lactobacilli aggregated with the yeast form of C. albicans, with Lactiplantibacillus carotarum AMBF275 showing the strongest aggregation. The aggregation was enhanced when lactobacilli were combined with S. cerevisiae CNCM I-3856. No significant antagonistic effects were observed between the tested lactobacilli and S. cerevisiae CNCM I-3856. The multifactorial activity of Lactobacillaceae strains alone or combined with the probiotic S. cerevisiae CNCM I-3856 against C. albicans without antagonistic effects between the beneficial strains, paves the way for developing consortium probiotics for in vivo applications.

Environment and lifestyle can affect the epigenome passed down from generation to generation. A mother’s nutrition can impact the methylation levels of her offspring’s epigenome, but it’s unclear which genes may be affected by malnutrition during gestation or early development. In this study, we examined the levels of methylated GC in the promoter region of HLA-C in mothers and infants from the Kichwa community in Ecuador. To do this, we analyzed saliva samples using bisulfite DNA sequencing. While we did not observe any significant differences in the mean methylation percentages in exon 1 of HLA-C between mothers and their infants after the first two years of lactation and life, respectively, we did find that infants tended to increase their methylation level during the first two years of life, while mothers tended to decrease it after the first two years of breastfeeding. When we compared methylation levels between mothers and infants using an ANOVA/posthoc Tukey test, we found that the average methylation for the entire population was less than 3% at T1 and T2. Although there was a tendency for infants to have higher methylation levels during their first two years of life and for mothers to have lower methylation levels after the first two years of breastfeeding, the mean values were not significantly different. However, we found a significant difference when we contrasted the data using a Kruskal-Wallis test at 0.05 for T1 AND T2 (p-value: 0.0148). Specifically, mothers had an average of X̅ = 2.06% and sons had X̅ = 1.57% at T2 (p-value: 0.7227), while the average for mothers was X̅ = 1.83% and for sons X̅ =1.77%. Finally, we identified three CpG motif nucleotide positions (32–33, 43–44, and 96–97) along the 122 bp analysis of HLA-C exon one, which was found to retain methylation patterns over time and is inherited from mother to offspring. Finally, our small pilot study did not reveal significant correlations between maternal and offspring nutritional status and DNA methylation levels of HLA-C exon one.

IntroductionThe pathogenesis of Attention-Deficit Hyperactivity Disorder (ADHD) is thought to be multifactorial, with a potential role for the bidirectional communication between the gut microbiome and brain development and function. Since the “golden-standard” medication therapy with methylphenidate (MPH) is linked to multiple adverse effects, there is a need for alternative treatment options such as dietary polyphenols. These secondary plant metabolites exert antioxidant and anti-inflammatory effects, but much less is known about their impact on the gut microbiota. Since polyphenols are believed to modulate gut microbial composition, interventions might be advantageous in ADHD therapy. Therefore, intervention studies with polyphenols in ADHD therapy investigating the gut microbial composition are highly relevant.MethodsBesides the primary research questions addressed previously, this study explored a potential prebiotic effect of the polyphenol-rich French Maritime Pine Bark Extract (PBE) compared to MPH and a placebo in pediatric ADHD patients by studying their impact on the gut microbiota via amplicon sequencing of the full length 16S rRNA gene ribosomal subunit (V1-V9).ResultsOne interesting finding was the high relative abundance of Bifidobacteria among all patients in our study cohort. Moreover, our study has identified that treatment (placebo, MPH and PBE) explains 3.94% of the variation in distribution of microbial taxa (adjusted p-value of 0.011).DiscussionOur small sample size (placebo: n = 10; PBE: n = 13 and MPH: n = 14) did not allow to observe clear prebiotic effects in the patients treated with PBE. Notwithstanding this limitation, subtle changes were noticeable and some limited compositional changes could be observed.Clinical Trial Registrationdoi: 10.1186/S13063-017-1879-6

ABSTRACTIn patients with presumptive tuberculosis (TB) in whom the diagnosis of TB was excluded, understanding the bacterial etiology of lower respiratory tract infections (LRTIs) is important for optimal patient management. A secondary analysis was performed on a cohort of 250 hospitalized patients with symptoms of TB. Bacterial DNA was extracted from sputum samples for Illumina 16S rRNA sequencing to identify bacterial species based on amplicon sequence variant level. The bacterial pathogen most likely to be responsible for the patients’ LRTI could only be identified in a minority (6.0%, 13/215) of cases based on 16S rRNA amplicon sequencing: Mycoplasma pneumoniae (n = 7), Bordetella pertussis (n = 2), Acinetobacter baumanii (n = 2), and Pseudomonas aeruginosa (n = 2). Other putative pathogens were present in similar proportions of Xpert Ultra-positive and Xpert Ultra-negative sputum samples. The presence of Streptococcus (pseudo)pneumoniae appeared to increase the odds of radiological abnormalities (aOR 2.5, 95% CI 1.12–6.16) and the presence of S. (pseudo)pneumoniae (aOR 5.31, 95% CI 1.29–26.6) and Moraxella catarrhalis/nonliquefaciens (aOR 12.1, 95% CI 2.67–72.8) increased the odds of 6-month mortality, suggesting that these pathogens might have clinical relevance. M. pneumoniae, B. pertussis, and A. baumanii appeared to be the possible causes of TB-like symptoms. S. (pseudo)pneumoniae and M. catarrhalis/nonliquefaciens also appeared of clinical relevance based on 16S rRNA amplicon sequencing. Further research using tools with higher discriminatory power than 16S rRNA sequencing is required to develop optimal diagnostic and treatment strategies for this population.IMPORTANCEThe objective of this study was to identify possible bacterial lower respiratory tract infection (LRTI) pathogens in hospitalized patients who were initially suspected to have TB but later tested negative using the Xpert Ultra test. Although 16S rRNA was able to identify some less common or difficult-to-culture pathogens such as Mycoplasma pneumoniae and Bordetella pertussis, one of the main findings of the study is that, in contrast to what we had hypothesized, 16S rRNA is not a method that can be used to assist in the management of patients with presumptive TB having a negative Xpert Ultra test. Even though this could be considered a negative finding, we believe it is an important finding to report as it highlights the need for further research using different approaches.

Abstract Background and aims We aimed to identify mucin-microbiome signatures shaping the tumor microenvironment in gastric adenocarcinomas and clinical outcomes. Methods We performed high-throughput profiling of the mucin phenotypes present in 108 gastric adenocarcinomas and 20 functional dyspepsia cases using validated mucin-based RT-qPCRs with subsequent immunohistochemistry validation and correlated the data with clinical outcome parameters. The gastric microbiota was assessed by 16S rRNA gene sequencing, taxonomy, and community composition determined, microbial networks analyzed, and the metagenome inferred in association with mucin phenotypes and expression. Results Gastric adenocarcinomas with an intestinal mucin environment or high-level MUC13 expression are associated with poor survival. On the contrary, gastric MUC5AC or MUC6 abundance was associated with a more favorable outcome. The oral taxa Neisseria, Prevotella, and Veillonella had centralities in tumors with intestinal and mixed phenotypes and were associated with MUC13 overexpression, highlighting their role as potential drivers in MUC13 signaling in GC. Furthermore, dense bacterial networks were observed in intestinal and mixed mucin phenotype tumors whereas the lowest community complexity was shown in null mucin phenotype tumors due to higher Helicobacter abundance resulting in a more decreased diversity. Enrichment of oral or intestinal microbes was mucin phenotype dependent. More specifically, intestinal mucin phenotype tumors favored the establishment of pro-inflammatory oral taxa forming strong co-occurrence networks. Conclusions Our results emphasize key roles for mucins in gastric cancer prognosis and shaping microbial networks in the tumor microenvironment. Specifically, the enriched oral taxa associated with aberrant MUC13 expression can be potential biomarkers in predicting disease outcomes. Video Abstract

ABSTRACT The human salivary microbial community plays a crucial role in local and systemic diseases. Biological and lifestyle factors such as menstrual cycle, oral hygiene, and smoking have been documented to impact this community. However, while hormonal contraceptives are the most prescribed drug in healthy women and intimate partners play key roles in microbial exchange between humans, their impact on the salivary microbiome of women of reproductive age have been understudied. Additionally, the role of other lifestyle factors such as diet, allergies, age, and stress on the saliva microbiome of the general population is not well understood. Here, we studied the salivary microbiome of 255 healthy women of reproductive age using self-sampling kits and 16S rRNA amplicon sequencing combined with questionnaires on lifestyle and host-related parameters. A preserved salivary bacterial community of 12 genera (Actinobacillus, Actinomyces, Alloprevotella, Campylobacter, Fusobacterium, Gemella, Granulicatella, Leptotrichia, Neisseria, Prevotella, Streptococcus, and Veillonella) was identified. Contrary to what we expected, the number of intimate partners or specific contraceptive use did not have a major impact on these bacterial communities. However, recent use of oral antibiotics was associated with a significant decrease in richness at genus level and increase in mean relative abundances of several taxa. Being stressed or nervous was associated with a significantly increased richness of the salivary microbiome at the level of amplicon sequencing variants . Nevertheless, these associations with host-related and lifestyle variables only appeared to be subtle, suggesting that the salivary microbiome is mainly driven by the buccal environment and health status of an individual. IMPORTANCE The salivary microbiome has been proven to play a crucial role in local and systemic diseases. Moreover, the effects of biological and lifestyle factors such as oral hygiene and smoking on this microbial community have already been explored. However, what was not yet well understood was the natural variation of the saliva microbiome in healthy women and how this is associated with specific use of hormonal contraception and with the number of different sexual partners with whom microbiome exchange is expected regularly. In this paper, we characterized the salivary microbiome of 255 healthy women of reproductive age using an in-depth questionnaire and self-sampling kits. Using the large metadata set, we were able to investigate the associations of several host-related and lifestyle variables with the salivary microbiome profiles. Our study shows a high preservation between individuals.

Diverse terms have been used in the literature to refer to the health benefits obtained from the administration of non-viable microorganisms or their cell fragments and metabolites. In an effort to provide continuity to this emerging field, the International Scientific Association of Probiotics and Prebiotics (ISAPP) convened a panel of experts to consider this category of substances and adopted the term postbiotic, which they defined as a “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host.” This definition does not stipulate any specific health benefit, finished product, target population or regulatory status. In this perspective article, we focused on postbiotics developed for pharmaceutical uses, including medicinal products and medical devices. We address how this field is regulated for products based on inanimate microorganisms, marketing considerations and existing examples of postbiotics products developed as cosmetics for the skin, for vaginal health, and as orally consumed products. We focus on the European Union for regulatory aspects, but also give examples from other geographical areas.

Abstract This study investigates an evolutionary hypothesis of gossip postulating that in humans it serves a similar function as social grooming in other primates. It examines whether gossip decreases physiological markers of stress and increases markers of positive emotionality and sociability. Dyads of friends (N = 66) recruited at the university, participated in an experiment where they experienced a stressor followed by social interaction (gossip or control task). Individual levels of salivary cortisol and $$\beta$$ β -endorphins were assessed at before and after social interactions. Sympathetic activity and parasympathetic activity were monitored throughout the experiment. Individual differences in Tendency and Attitude towards Gossip were investigated as potential covariates. Gossip condition was characterized with increased sympathetic and parasympathetic activity, but did not differ in cortisol or $$\beta$$ β -endorphins levels. However, high Tendency to Gossip was associated with decreases in cortisol. Gossip was shown to be more emotionally salient than non-social talk, but the evidence with regard to lowering stress was not sufficient to support an analogy to social grooming.

Abstract Several oral diseases are characterized by a shift within the oral microbiome towards a pathogenic, dysbiotic composition. Broad-spectrum antimicrobials are often part of patient care. However, because of the rising antibiotic resistance, alternatives are increasingly desirable. Alternatively, supplying beneficial species through probiotics is increasingly showing favorable results. Unfortunately, these probiotics are rarely evaluated comparatively. In this study, the in vitro effects of three known and three novel Lactobacillus strains, together with four novel Streptococcus salivarius strains were comparatively evaluated for antagonistic effects on proximal agar growth, antimicrobial properties of probiotic supernatant and the probiotic’s effects on in vitro periodontal biofilms. Strain-specific effects were observed as differences in efficacy between genera and differences within genera. While some of the Lactobacillus candidates were able to reduce the periodontal pathobiont A. actinomycetemcomitans, the S. salivarius strains were not. However, the S. salivarius strains were more effective against periodontal pathobionts P. intermedia, P. gingivalis, and F. nucleatum. Vexingly, most of the Lactobacillus strains also negatively affected the prevalence of commensal species within the biofilms, while this was lower for S. salivarius strains. Both within lactobacilli and streptococci, some strains showed significantly more inhibition of the pathobionts, indicating the importance of proper strain selection. Additionally, some species showed reductions in non-target species, which can result in unexpected and unexplored effects on the whole microbiome.

Abstract Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut‐targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up‐scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three‐step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier‐enhancing and anti‐pathogenic properties and the two well‐studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV‐229E coronavirus in co‐culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway‐inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad‐acting live biotherapeutic strategy against respiratory viral diseases.

In 2020, a taxonomic reorganization of the lactic acid bacteria reclassified over 300 species in 7 genera and 2 families into one family, the Lactobacillaceae, with 31 genera including 23 new genera to include organisms formerly classified as Lactobacillus species. This communication aims to provide a debrief on the taxonomic reorganization of lactobacilli to identify shortcomings in the proposed taxonomic framework, and to outline perspectives and opportunities provided by the current taxonomy of the Lactobacillaceae. The current taxonomy of lactobacilli not only necessitates becoming familiar with 23 new genus names but also provides substantial new opportunities in scientific discovery and regulatory approval of these organisms. First, description of new species in the Lactobacillaceae is facilitated and a solid framework for description of novel genera is provided. Second, the current taxonomy greatly enhances the resolution of genus-level sequencing approaches (e.g., 16S rRNA–based metagenomics) when identifying the composition and function of microbial communities. Third, the current taxonomy greatly facilitates the formulation of hypotheses linking phylogeny to metabolism and ecology of lactobacilli.

ABSTRACT Primary care urgently needs treatments for coronavirus disease 2019 (COVID-19) patients because current options are limited, while these patients who do not require hospitalization encompass more than 90% of the people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a throat spray containing three Lactobacillaceae strains with broad antiviral properties in a randomized, double-blind, placebo-controlled trial. Before the availability of vaccines, 78 eligible COVID-19 patients were randomized to verum (n = 41) and placebo (n = 37) within 96 h of a positive PCR-based SARS-CoV-2 diagnosis, and a per-protocol analysis was performed. Symptoms and severity were reported daily via an online diary. Combined nose-throat swabs and dried blood spots were collected at regular time points in the study for microbiome, viral load, and antibody analyses. The daily reported symptoms were highly variable, with no added benefit for symptom resolution in the verum group. However, based on 16S V4 amplicon sequencing, the acute symptom score (fever, diarrhea, chills, and muscle pain) was significantly negatively associated with the relative abundance of amplicon sequence variants (ASVs) that included the applied lactobacilli (P

Riboflavin-producing lactic acid bacteria represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin (vitamin B2) overproduction of 18.36 μg/ml, biomass production up to 6 × 1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy, and oat) and cow milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut beverage and butter milk fermentations, and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or in a coconut beverage survived in gastric juice and in a simulated gastrointestinal dialysis model with colon phase (GIDM-colon system) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM-colon system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents a promising candidate to provide riboflavin fortification of plant-based and dairy foods, and has a high application potential in the human gastrointestinal tract.

Lactic acid bacteria (LAB) form a group of bacteria to which most probiotics belong and are commonly found in fermented dairy products. Fermented foods and beverages are foods made through desired microbial growth and enzymatic conversions of food components. In this study, 43 LAB were isolated from Ethiopian traditional cottage cheese, cheese, and yogurt and evaluated for their functional and safety properties as candidate probiotics. Twenty-seven isolates, representative of each fermented food type, were selected and identified to the species level. Limosilactobacillus fermentum was found to be the predominant species in all samples studied (70.4%), while 11.1% of isolates were identified as Lactiplantibacillus plantarum. All 27 isolates tested showed resistance to 0.5% bile salt, while 26 strains were resistant to pH 3. The LAB isolates were also evaluated for antagonistic properties against key pathogens, with strain-specific features observed for their antimicrobial activity. Five strains from cottage cheese (Lactiplantibacillus plantarum 54B, 54C, and 55A, Lactiplantibacillus pentosus 55B, and Pediococcus pentosaceus 95E) showed inhibitory activity against indicator pathogens that are key causes of gastrointestinal infections in Ethiopia, i.e., Escherichia coli, Salmonella enterica subsp. enterica var. Typhimurium, Staphylococcus aureus, Shigella flexneri, and Listeria monocytogenes. Strain-specific immunomodulatory activity monitored as nuclear factor kappa B (NF-κB) and interferon regulatory factor (IRF) activation was documented for Lactiplantibacillus plantarum 54B, 55A and P. pentosaceus 95E. Antibiotic susceptibility testing confirmed that all LAB isolates were safe concerning their antibiotic resistance profiles. Five isolates (especially Lactiplantibacillus plantarum 54B, 54C, and 55A, Lactiplantibacillus pentosus 55B, and P. pentosaceus 95E) showed promising results in all assays and are novel probiotic candidates of interest for clinical trial follow-up.

We have previously shown that lysates of Lacticaseibacillus rhamnosus GG confer protection to human keratinocytes against Staphylococcus aureus. L. rhamnosus GG inhibits the growth of S. aureus as well as competitively excludes and displaces the pathogen from keratinocytes. In this study, we have specifically investigated the anti-adhesive action. We have tested the hypothesis that this activity is due to quenching of S. aureus binding sites on keratinocytes by molecules within the Lacticaseibacillus lysate. Trypsinisation or heat treatment removed the protective effect of the lysate suggesting the involvement of proteins as effector molecules. Column separation of the lysate and analysis of discrete fractions in adhesion assays identified a fraction of moderate hydrophobicity that possessed all anti-adhesive functions. Immunoblotting demonstrated that this fraction contained the pilus protein, SpaC. Recombinant SpaC inhibited staphylococcal adhesion to keratinocytes in a dose-dependent manner and improved keratinocyte viability following challenge with viable S. aureus. However, SpaC did not confer the full anti-adhesive effects of the LGG lysate and excluded but did not displace S. aureus from keratinocytes. Further purification produced four protein-containing peaks (F1–F4). Of these, F4, which had the greatest column retention time, was the most efficacious in anti-staphylococcal adhesion and keratinocyte viability assays. Identification of proteins by mass spectrometry showed F4 to contain several known “moonlighting proteins”—i.e., with additional activities to the canonical function, including enolase, Triosephosphate isomerase (TPI), Glyceraldehyde 3 phosphate dehydrogenase (G3P) and Elongation factor TU (EF-Tu). Of these, only enolase and TPI inhibited S. aureus adhesion and protected keratinocytes viability in a dose-dependent manner. These data suggest that inhibition of staphylococcal binding by the L. rhamnosus GG lysate is mediated by SpaC and specific moonlight proteins.

ABSTRACT Every year, deciduous trees shed their leaves, and when new leaves emerge next spring, they establish a characteristic bacterial leaf community. In this exploratory study, we assessed the bacterial phyllosphere (aboveground plant surfaces) of eight London plane trees (Platanus × acerifolia) in Antwerp and Milan by sampling weekly during leaf emergence and expansion. We sampled the surfaces of different tree compartments: leaves, leaf buds, branches, and trunk, for up to 6 weeks. Phyllosphere community composition was most strongly determined by tree compartment. Only the communities on the emerging leaves showed changing dynamics over time. The rate of change in the leaf phyllosphere composition, expressed as the beta dissimilarity between consecutive time points, was very high following leaf emergence, with decreasing speed over time, indicating that these communities stabilize over time. We also identified cooccurring groups of bacteria associated with potential stages of ecological succession on the leaves and accordingly named them general cluster, early cluster, middle cluster, and late cluster. Taxa of the general cluster were not only more abundant than the others on leaves, but they were also widespread on other tree compartments. The late cluster was most pronounced in trees surrounded by trafficked urban land use. This study mainly generates hypotheses on the ecological succession on the emerging leaves of deciduous trees in urban environments and contributes to understanding the development of the tree leaf phyllosphere in spring. IMPORTANCE Improving our understanding of phyllosphere ecology is key in successfully applying bacterial biological agents or modulating the leaf microbiome in order to achieve valuable ecosystem services, such as plant protection, plant growth, air purification, and developing a healthy human immune system. Modulation of the phyllosphere microbiome in the field works only with variable success. To improve the impact of our applications in the field, a better understanding of the ecological principles governing phyllosphere dynamics is required. This exploratory study demonstrates how the combination of different analyses of a chronosequence of bacterial communities can provide new ecological insights. With a limited number of sampled trees, we demonstrated different indications of ecological succession of bacterial communities in the leaves and observed a potential impact of intensely trafficked land use becoming apparent in the leaf bacterial communities approximately 3 weeks after leaf emergence, consisting of a separate stage in community development.

Pelvic irradiation-induced mucositis secondarily leads to dysbiosis, which seriously affects patients’ quality of life after treatment. No safe and effective radioprotector or mitigator has yet been approved for clinical therapy. Here, we investigated the potential protective effects of fresh biomass of Limnospira indica PCC 8005 against ionizing irradiation-induced mucositis and dysbiosis in respect to benchmark probiotic Lacticaseibacillus rhamnosus GG ATCC 53103. For this, mice were supplemented daily before and after 12 Gy X-irradiation of the pelvis. Upon sacrifice, food supplements’ efficacy was assessed for intestinal barrier protection, immunomodulation and changes in the microbiota composition. While both could not confer barrier protection or significant immunomodulatory effects, 16S microbial profiling revealed that L. indica PCC 8005 and L. rhamnosus GG could prevent pelvic irradiation-induced dysbiosis. Altogether, our data show that—besides benchmarked L. rhamnosus GG—L. indica PCC 8005 is an interesting candidate to further explore as a radiomitigator counteracting pelvic irradiation-induced dysbiosis in the presented in vivo irradiation–gut–microbiota platform.

Summary: Few validated protocols are available for large-scale collection, storage, and analysis of microbiome samples from the vagina, skin, and mouth. To prepare for a large-scale study on the female microbiome by remote self-sampling, we investigated the impact of sample collection, storage, and host DNA depletion on microbiome profiling. Vaginal, skin, and saliva samples were analyzed using 16S rRNA gene amplicon and metagenomic shotgun sequencing, and qPCR. Of the two tested storage buffers, the eNAT buffer could keep the microbial composition stable during various conditions. All three tested host DNA-depletion approaches showed a bias against Gram-negative taxa. However, using the HostZERO Microbial DNA and QIAamp DNA Microbiome kits, samples still clustered according to body site and not by depletion approach. Therefore, our study showed the effectiveness of these methods in depleting host DNA. Yet, a suitable approach is recommended for each habitat studied based on microbial composition.