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.

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.

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.

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.

Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria.

Spontaneous vegetable fermentations, with their rich flavors and postulated health benefits, are regaining popularity. However, their microbiology is still poorly understood, therefore raising concerns about food safety. In addition, such spontaneous fermentations form interesting cases of man-made microbial ecosystems. Here, samples from 38 carrot juice fermentations were collected through a citizen science initiative, in addition to three laboratory fermentations. Culturing showed that Enterobacteriaceae were outcompeted by lactic acid bacteria (LAB) between 3 and 13 days of fermentation. Metabolite-target analysis showed that lactic acid and mannitol were highly produced, as well as the biogenic amine cadaverine. High-throughput 16S rRNA gene sequencing revealed that mainly species of Leuconostoc and Lactobacillus (as identified by 8 and 20 amplicon sequence variants [ASVs], respectively) mediated the fermentations in subsequent order. The analyses at the DNA level still detected a high number of Enterobacteriaceae, but their relative abundance was low when RNA-based sequencing was performed to detect presumptive metabolically active bacterial cells. In addition, this method greatly reduced host read contamination. Phylogenetic placement indicated a high LAB diversity, with ASVs from nine different phylogenetic groups of the Lactobacillus genus complex. However, fermentation experiments with isolates showed that only strains belonging to the most prevalent phylogenetic groups preserved the fermentation dynamics. The carrot juice fermentation thus forms a robust man-made microbial ecosystem suitable for studies on LAB diversity and niche specificity.IMPORTANCE The usage of fermented food products by professional chefs is steadily growing worldwide. Meanwhile, this interest has also increased at the household level. However, many of these artisanal food products remain understudied. Here, an extensive microbial analysis was performed of spontaneous fermented carrot juices which are used as nonalcoholic alternatives for wine in a Belgian Michelin star restaurant. Samples were collected through an active citizen science approach with 38 participants, in addition to three laboratory fermentations. Identification of the main microbial players revealed that mainly species of Leuconostoc and Lactobacillus mediated the fermentations in subsequent order. In addition, a high diversity of lactic acid bacteria was found; however, fermentation experiments with isolates showed that only strains belonging to the most prevalent lactic acid bacteria preserved the fermentation dynamics. Finally, this study showed that the usage of RNA-based 16S rRNA amplicon sequencing greatly reduces host read contamination.

Comparing the Healthy Nose and Nasopharynx Microbiota Reveals Continuity As Well As Niche-Specificity.

To improve our understanding of upper respiratory tract (URT) diseases and the underlying microbial pathogenesis, a better characterization of the healthy URT microbiome is crucial. In this first large-scale study, we obtained more insight in the URT microbiome of healthy adults. Hereto, we collected paired nasal and nasopharyngeal swabs from 100 healthy participants in a citizen-science project. High-throughput 16S rRNA gene V4 amplicon sequencing was performed and samples were processed using the Divisive Amplicon Denoising Algorithm 2 (DADA2) algorithm. This allowed us to identify the bacterial richness and diversity of the samples in terms of amplicon sequence variants (ASVs), with special attention to intragenus variation. We found both niches to have a low overall species richness and uneven distribution. Moreover, based on hierarchical clustering, nasopharyngeal samples could be grouped into some bacterial community types at genus level, of which four were supported to some extent by prediction strength evaluation: one intermixed type with a higher bacterial diversity where Staphylococcus, Corynebacterium, and Dolosigranulum appeared main bacterial members in different relative abundances, and three types dominated by either Moraxella, Streptococcus, or Fusobacterium. Some of these bacterial community types such as Streptococcus and Fusobacterium were nasopharynx-specific and never occurred in the nose. No clear association between the nasopharyngeal bacterial profiles at genus level and the variables age, gender, blood type, season of sampling, or common respiratory allergies was found in this study population, except for smoking showing a positive association with Corynebacterium and Staphylococcus. Based on the fine-scale resolution of the ASVs, both known commensal and potential pathogenic bacteria were found within several genera - particularly in Streptococcus and Moraxella - in our healthy study population. Of interest, the nasopharynx hosted more potential pathogenic species than the nose. To our knowledge, this is the first large-scale study using the DADA2 algorithm to investigate the microbiota in the "healthy" adult nose and nasopharynx. These results contribute to a better understanding of the composition and diversity of the healthy microbiome in the URT and the differences between these important URT niches. Trial Registration: Ethical Committee of Antwerp University Hospital, B300201524257, registered 23 March 2015, ClinicalTrials.gov Identifier: NCT02 933983.

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.