Multifactorial inhibition of Candida albicans by combinations of lactobacilli and probiotic Saccharomyces cerevisiae CNCM I-3856.

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.

A citizen-science-enabled catalogue of the vaginal microbiome and associated factors.

Understanding the composition and function of the vaginal microbiome is crucial for reproductive and overall health. Here we established the Isala citizen-science project to analyse the vaginal microbiomes of 3,345 women in Belgium (18-98 years) through self-sampling, 16S amplicon sequencing and extensive questionnaires. The overall vaginal microbiome composition was strongly tied to age, childbirth and menstrual cycle phase. Lactobacillus species dominated 78% of the vaginal samples. Specific bacterial taxa also showed to co-occur in modules based on network correlation analysis. Notably, the module containing Lactobacillus crispatus, Lactobacillus jensenii and Limosilactobacillus taxa was positively linked to oestrogen levels and contraceptive use and negatively linked to childbirth and breastfeeding. Other modules, named after abundant taxa (Gardnerella, Prevotella and Bacteroides), correlated with multiple partners, menopause, menstrual hygiene and contraceptive use. With this resource-rich vaginal microbiome map and associated health, life-course, lifestyle and dietary factors, we provide unique data and insights for follow-up clinical and mechanistic research.

Salivary microbiome of healthy women of reproductive age.

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.

Comparing Vaginal and Endometrial Microbiota Using Culturomics: Proof of Concept.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal-cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

Selective targeting of skin pathobionts and inflammation with topically applied lactobacilli.

Tailored skin microbiome modulation approaches with probiotics are highly challenging. Here, we show that lactobacilli are underestimated members of the skin microbiota. We select specific strains of nomadic lactobacilli for their functional applicability on the skin and capacity to inhibit growth and inflammation by skin pathobionts. The strains are formulated as microcapsules for topical formulations and tested in patients with mild-to-moderate acne. The selected lactobacilli are able to reduce inflammatory lesions in a pilot and placebo-controlled study. Daily application for 8 weeks is associated with an in vivo temporary modulation of the microbiome, including a reduction in relative abundance of staphylococci and Cutibacterium acnes, and an increase in lactobacilli. The reduction in inflammatory lesions is still apparent 4 weeks after the topical application of the lactobacilli ended, indicating a possible additional immunomodulatory effect. This study shows that carefully selected and formulated lactobacilli are a viable therapeutic option for common acne lesions.

Global maps of soil temperature.

Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km2 resolution for 0-5 and 5-15 cm soil depth. These maps were created by calculating the difference (i.e. offset) between in situ soil temperature measurements, based on time series from over 1200 1-km2 pixels (summarized from 8519 unique temperature sensors) across all the world's major terrestrial biomes, and coarse-grained air temperature estimates from ERA5-Land (an atmospheric reanalysis by the European Centre for Medium-Range Weather Forecasts). We show that mean annual soil temperature differs markedly from the corresponding gridded air temperature, by up to 10°C (mean = 3.0 ± 2.1°C), with substantial variation across biomes and seasons. Over the year, soils in cold and/or dry biomes are substantially warmer (+3.6 ± 2.3°C) than gridded air temperature, whereas soils in warm and humid environments are on average slightly cooler (-0.7 ± 2.3°C). The observed substantial and biome-specific offsets emphasize that the projected impacts of climate and climate change on near-surface biodiversity and ecosystem functioning are inaccurately assessed when air rather than soil temperature is used, especially in cold environments. The global soil-related bioclimatic variables provided here are an important step forward for any application in ecology and related disciplines. Nevertheless, we highlight the need to fill remaining geographic gaps by collecting more in situ measurements of microclimate conditions to further enhance the spatiotemporal resolution of global soil temperature products for ecological applications.

The nasal mutualist Dolosigranulum pigrum AMBR11 supports homeostasis via multiple mechanisms.

Comparing the nasal microbiome of healthy individuals and chronic rhinosinusitis (CRS) patients revealed Dolosigranulum pigrum as a species clearly associated with nasal health, although isolates obtained from healthy individuals are scarce. In this study, we explored the properties of this understudied lactic acid bacterium by integrating comparative genomics, habitat mining, cultivation, and functional characterization of interaction capacities. Mining 10.000 samples from the Earth Microbiome Project of 17 habitat types revealed that Dolosigranulum is mainly associated with the human nasal cavity. D. pigrum AMBR11 isolated from the nose of a healthy individual exerted antimicrobial activity against Staphylococcus aureus, decreased proinflammatory cytokine production in airway epithelial cells, and Galleria mellonella larvae mortality induced by this important nasal pathobiont. Furthermore, the strain protected the nasal barrier function in a mouse model using interleukin-4 as disruptive cytokine. Hence, D. pigrum AMBR11 is a mutualist with high potential as topical live biotherapeutic product.

The Dwindling Microbiota of Aerobic Vaginitis, an Inflammatory State Enriched in Pathobionts with Limited TLR Stimulation.

While bacterial vaginosis (BV) is a well-known type of vaginal dysbiosis, aerobic vaginitis (AV) is an inflammatory condition that remains understudied and under-recognised. It predisposes women to serious complications including urogenital infections and pregnancy problems. Here, we investigated the bacterial community in AV to explore its possible role in AV pathogenesis. We collected vaginal lavage fluid samples of women (n = 58) classified by wet-mount microscopy as suffering from AV or BV and included an asymptomatic reference group without signs of AV or BV. AV samples showed reduced absolute abundances of bacteria in general and specifically of lactobacilli by qPCR, but 16S rRNA gene sequencing and amplicon sequence variant analysis revealed that Lactobacillus remained the dominant taxon in 25% of the AV samples studied. The other AV samples showed high relative abundances of Streptococcus agalactiae and, unexpectedly, the anaerobes Gardnerella vaginalis and Prevotella bivia in more than half of the AV samples studied. Yet, despite increased relative abundance of these potential pathogens or pathobionts in the AV bacterial communities, the AV samples only slightly stimulated Toll-like receptor 4 and showed reduced activation of Toll-like receptor 2/6, receptors of two pathways central to mucosal immunity. Our findings indicate that the reduced total bacterial abundance with associated enrichment in certain pathobionts in AV might be mainly a consequence of the inflammatory conditions and/or altered hormonal regulation rather than bacteria being a major cause of the inflammation.

Lactobacilli Have a Niche in the Human Nose.

Although an increasing number of beneficial microbiome members are characterized for the human gut and vagina, beneficial microbes are underexplored for the human upper respiratory tract (URT). In this study, we demonstrate that taxa from the beneficial Lactobacillus genus complex are more prevalent in the healthy URT than in patients with chronic rhinosinusitis (CRS). Several URT-specific isolates are cultured, characterized, and further explored for their genetic and functional properties related to adaptation to the URT. Catalase genes are found in the identified lactobacilli, which is a unique feature within this mostly facultative anaerobic genus. Moreover, one of our isolated strains, Lactobacillus casei AMBR2, contains fimbriae that enable strong adherence to URT epithelium, inhibit the growth and virulence of several URT pathogens, and successfully colonize nasal epithelium of healthy volunteers. This study thus demonstrates that specific lactobacilli are adapted to the URT and could have a beneficial keystone function in this habitat.

Impact of a lactobacilli-containing gel on vulvovaginal candidosis and the vaginal microbiome.

Vulvovaginal candidosis (VVC) is a common condition with severe symptoms and high recurrence rates. Probiotic lactobacilli are explored as alternatives to azole treatments. Although the vaginal microbiota is generally not depleted in lactobacilli during VVC, studies indicate that the functionality and antimicrobial activity of the lactobacilli is impaired. We selected three strains from the Lactobacillus genus complex (L. rhamnosus GG, L. pentosus KCA1 and L. plantarum WCFS1) based on in vitro evaluation and formulated them in a gel for vaginal application. This gel was evaluated in 20 patients suffering from acute VVC, who were followed for four weeks including a 10-day treatment period. The microbiome was assessed through 16S rRNA (bacteria) and internal transcribed spacer (ITS; fungi) amplicon sequencing, supplemented with quantitative PCR, culture and microscopy for Candida evaluation. 45% of women did not require rescue medication (3×200 mg fluconazole), implying an improvement of their symptoms. These women showed similar end concentrations of fungi as women treated with fluconazole. Moreover, fluconazole appeared to reduce numbers of endogenous lactobacilli. Our study points towards important aspects for future selection of lactobacilli for probiotic use in VVC and the need to investigate possible negative influences of azoles on the vaginal bacterial community.

Applications of plant-based fermented foods and their microbes.

Plant-based fermentations and their microbes provide an underexplored source for novel biotechnological applications. Recent advances in DNA sequencing technologies and analyses of sequencing data highlight that a diverse array of lactic acid bacteria (LAB) frequently dominate these plant fermentations. Because of the long history of safe LAB use in fermented foods, we argue here that various novel probiotic, synbiotic and a range of other industrial applications can be produced based on new insights in the functional and genetic potential of these LAB. To aid in this quest, comparative genomics tools are increasingly available enabling a more rational design of wet-lab experiments to screen for the most relevant properties. This is also true for the exploration of useful enzymatic and (secondary) metabolic production capacities of the LAB that can be isolated from these plant-based fermentations, such as the recent discovery of a cellulase enzyme in specific Lactobacillus plantarum group members.

Comparative genome analysis of Lactobacillus mudanjiangensis, an understudied member of the Lactobacillus plantarum group.

The genus Lactobacillus is known to be extremely diverse and consists of different phylogenetic groups that show a diversity that is roughly equal to the expected diversity of a typical bacterial genus. One of the most prominent phylogenetic groups within this genus is the Lactobacillus plantarum group, which contains the understudied Lactobacillus mudanjiangensis species. Before this study, only one L. mudanjiangensis strain, DSM 28402T, had been described, but without whole-genome analysis. In this study, three strains classified as L. mudanjiangensis were isolated from three different carrot juice fermentations and their whole-genome sequence was determined, together with the genome sequence of the type strain. The genomes of all four strains were compared with publicly available L. plantarum group genome sequences. This analysis showed that L. mudanjiangensis harboured the second largest genome size and gene count of the whole L. plantarum group. In addition, all members of this species showed the presence of a gene coding for a cellulose-degrading enzyme. Finally, three of the four L. mudanjiangensis strains studied showed the presence of pili on scanning electron microscopy (SEM) images, which were linked to conjugative gene regions, coded on a plasmid in at least two of the strains studied.

Impact of spray-drying on the pili of Lactobacillus rhamnosus GG.

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.

Inhibition of Candida albicans morphogenesis by chitinase from Lactobacillus rhamnosus GG.

Lactobacilli have been evaluated as probiotics against Candida infections in several clinical trials, but with variable results. Predicting and understanding the clinical efficacy of Lactobacillus strains is hampered by an overall lack of insights into their modes of action. In this study, we aimed to unravel molecular mechanisms underlying the inhibitory effects of lactobacilli on hyphal morphogenesis, which is a crucial step in C. albicans virulence. Based on a screening of different Lactobacillus strains, we found that the closely related taxa L. rhamnosus, L. casei and L. paracasei showed stronger activity against Candida hyphae formation compared to other Lactobacillus species tested. By exploring the activity of purified compounds and mutants of the model strain L. rhamnosus GG, the major peptidoglycan hydrolase Msp1, conserved in the three closely related taxa, was identified as a key effector molecule. We could show that this activity of Msp1 was due to its ability to break down chitin, the main polymer in the hyphal cell wall of C. albicans. This identification of a Lactobacillus-specific protein with chitinase activity having anti-hyphal activity will assist in better strain selection and improved application in future clinical trials for Lactobacillus-based Candida-management strategies.

The microbiome of the invertebrate model host Galleria mellonella is dominated by Enterococcus.

BACKGROUND: The popularity of Galleria mellonella as invertebrate model is increasing rapidly, because it forms an attractive alternative to study bacterial, fungal and viral infections, toxin biology, and to screen antimicrobial drugs. For a number of vertebrate and invertebrate animal and plant models, it has been established that the commensals present within the microbial communities on various host surfaces will influence the host's immune and growth development state and the colonization capacity of newly introduced micro-organisms. The microbial communities of Galleria mellonella larvae have, however, not yet been well characterized. RESULTS: In this study, we present the bacterial communities that were found by 16S rRNA amplicon sequencing on different body sites of G. mellonella larvae. These communities showed very little diversity and were mostly dominated by one Enterococcus taxon. In addition, we found that the production conditions (as 'bait' for fishing or under more controlled 'research grade' conditions - with or without hormones and antibiotics) appear to have little impact on the microbiota of the larvae. CONCLUSIONS: Establishment of the simplicity of the microbiota of G. mellonella larvae underlines the potential of the larvae as a model host system for microbiome-host interactions.

Interplay between Lactobacillus rhamnosus GG and Candida and the involvement of exopolysaccharides.

A number of clinical studies have shown protective effects of lactobacilli against Candida species in the gastrointestinal tract, the urogenital tract and the oral cavity, while others did not show clear effects. Evidence on the mode of action of lactobacilli against Candida is also still lacking. In this study, the anti-Candida activity of the model probiotic strain Lactobacillus rhamnosus GG was explored in different assays to determine molecular interactions. We found that L. rhamnosus GG was able to interfere with Candida growth, morphogenesis and adhesion. These three aspects of Candida's physiology are all crucial to its opportunistic pathogenesis. In follow-up assays, we compared the activity of L. rhamnosus GG wild-type with its exopolysaccharide (EPS)-deficient mutant and purified EPS to evaluate the involvement of this outer carbohydrate layer. Our data demonstrate that purified EPS can both interfere with hyphal formation and adhesion to epithelial cells, which indicates that EPS is part of a combined molecular mechanism underlying the antihyphal and anti-adhesion mechanisms of L. rhamnosus GG.

Large-Scale Phylogenomics of the Lactobacillus casei Group Highlights Taxonomic Inconsistencies and Reveals Novel Clade-Associated Features.

Although the genotypic and phenotypic properties of the Lactobacillus casei group have been studied extensively, the taxonomic structure has been the subject of debate for a long time. Here, we performed a large-scale comparative analysis by using 183 publicly available genomes supplemented with a Lactobacillus strain isolated from the human upper respiratory tract. On the basis of this analysis, we identified inconsistencies in the taxonomy and reclassified all of the genomes according to their most closely related type strains. This led to the identification of a catalase-encoding gene in all 10 L. casei sensu stricto strains, making it the first described catalase-positive species in the Lactobacillus genus. Moreover, we found that 6 of 10 L. casei genomes contained a SecA2/SecY2 gene cluster with two putative glycosylated surface adhesin proteins. Altogether, our results highlight current inconsistencies in the taxonomy of the L. casei group and reveal new clade-associated functional features. IMPORTANCE The closely related species of the Lactobacillus casei group are extensively studied because of their applications in food fermentations and as probiotics. Our results show that many strains in this group are incorrectly classified and that reclassifying them to their most closely related species type strain improves the functional predictive power of their taxonomy. In addition, our findings may spark increased interest in the L. casei species. We find that after reclassification, only 10 genomes remain classified as L. casei. These strains show some interesting properties. First, they all appear to be catalase positive. This suggests that they have increased oxidative stress resistance. Second, we isolated an L. casei strain from the human upper respiratory tract and discovered that it and multiple other L. casei strains harbor one or even two large, glycosylated putative surface adhesins. This might inspire further exploration of this species as a potential probiotic organism.

Characterization of Novel Bacteriophages for Biocontrol of Bacterial Blight in Leek Caused by Pseudomonas syringae pv. porri.

Pseudomonas syringae pv. porri, the causative agent of bacterial blight in leek (Allium porrum), is increasingly frequent causing problems in leek cultivation. Because of the current lack of control measures, novel bacteriophages were isolated to control this pathogen using phage therapy. Five novel phages were isolated from infected fields in Flanders (vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, vB_PsyM_KIL4, and vB_PsyM_KIL5), and were complemented with one selected host range mutant phage (vB_PsyM_KIL3b). Genome analysis of the phages revealed genome sizes between 90 and 94 kb and an average GC-content of 44.8%. Phylogenomic networking classified them into a novel clade, named the "KIL-like viruses," related to the Felixounalikevirus genus, together with phage phiPsa374 from P. syringae pv. actinidiae. In vitro characterization demonstrated the stability and lytic potential of these phages. Host range analysis confirmed heterogeneity within P. syringae pv. porri, leading to the development of a phage cocktail with a range that covers the entire set of 41 strains tested. Specific bio-assays demonstrated the in planta efficacy of phages vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, and vB_PsyM_KIL3b. In addition, two parallel field trial experiments on three locations using a phage cocktail of the six phages showed variable results. In one trial, symptom development was attenuated. These data suggest some potential for phage therapy in controlling bacterial blight of leek, pending optimization of formulation and application methods.