Intermittent Lactobacilli-containing Vaginal Probiotic or Metronidazole Use to Prevent Bacterial Vaginosis Recurrence: A Pilot Study Incorporating Microscopy and Sequencing

Bacterial vaginosis (BV) is associated with HIV acquisition and adverse pregnancy outcomes. Recurrence after metronidazole treatment is high. HIV-negative, non-pregnant Rwandan BV patients were randomized to four groups (n=17/group) after seven-day oral metronidazole treatment: behavioral counseling only (control), or counseling plus intermittent use of oral metronidazole, Ecologic Femi+ vaginal capsule (containing multiple Lactobacillus and one Bifidobacterium species), or Gynophilus LP vaginal tablet (L. rhamnosus 35) for two months. Vaginal microbiota assessments at all visits included Gram stain Nugent scoring and 16S rRNA gene qPCR and HiSeq sequencing. All interventions were safe. BV (Nugent 7-10) incidence was 10.18 per person-year at risk in the control group, and lower in the metronidazole (1.41/person-year; p=0.004), Ecologic Femi+ (3.58/person-year; p=0.043), and Gynophilus LP groups (5.36/person-year; p=0.220). In mixed effects models adjusted for hormonal contraception/pregnancy, sexual risk-taking, and age, metronidazole and Ecologic Femi+ users, each compared to controls, had higher Lactobacillus and lower BV-anaerobes concentrations and/or relative abundances, and were less likely to have a dysbiotic vaginal microbiota type by sequencing. Inter-individual variability was high and effects disappeared soon after intervention cessation. Lactobacilli-based vaginal probiotics warrant further evaluation because, in contrast to antibiotics, they are not expected to negatively affect microbiota or cause antimicrobial resistance

Influence du cycle industriel sur les caractéristiques probiotiques de Lactobacillus rhamnosus (Lcr35®)

Au cours de ces dernières années, le développement de la métagénomique a permis d'étendre notre compréhension du microbiote et de son rôle dans la santé de l'Homme. L'utilisation de probiotiques apparaît comme une opportunité thérapeutique pour lutter contre d'éventuelles dysbioses, mais elle nécessite au préalable une recherche accrue dans la compréhension de leurs mécanismes d'action. La souche probiotique Lactobacillus rhamnosus Lcr35®, produite depuis 1951 par les Laboratoires LYOCENTRE et commercialisée par la société PROBIONOV, constitue le principe actif de produits pharmaceutiques. Notre travail a consisté à comparer certaines propriétés "probiotiques" de cette souche bactérienne à quatre de ses préparations commerciales. Trois de ces formulations sont indiquées pour des applications intestinales : BACILOR (API Lcr Restituo® sachet ou de l'API Lcr Restituo® gélule) et FLOREA (API Lcr Lenio®). Le quatrième produit étudié, GYNOPHILUS (API Lcr Regenerans®), est indiqué pour lutter contre les dysbioses vaginales. Nous avons montré que les différents traitements subis par le micro-organisme au cours des cycles industriels généraient des modifications de ses propriétés. Ainsi, les formes APIs, notamment Lcr Lenio®, sont plus résistantes à un stress gastrique que la souche native. De même, les capacités d'adhésion de la souche vis-à-vis de cellules épithéliales intestinales Caco-2 sont augmentées suite au passage dans un cycle industriel. La caractérisation de l'enveloppe bactérienne et de ses variations de composition en fonction du cycle industriel a permis de montrer que la souche native présentait un profil d'hydrophobicité différent de celui obtenu avec les souches issues des APIs. Ces variations n'ont pourtant pas permis de mettre en évidence de variation dans les capacités d'agrégation de la souche. Inversement, l'analyse du profil d'expression de gènes codant potentiellement des protéines sécrétées ou transmembranaires ou impliquées dans des processus d'adhésion montrent des variations entre la souche native et les APIs testés. Le traitement de la souche par un cycle entraine également des modifications de ses capacités métaboliques. L'API Lcr Regenerans® voit ainsi son aptitude à dégrader le glycogène et son potentiel bactéricide et fongicide fortement augmentés. Ces résultats suggèrent que le processus de fabrication influe sur les propriétés chimiques et la composition des protéines de la paroi cellulaire bactérienne. L'étude des modifications apportées par les processus industriels devra être approfondie afin de mieux adapter les souches probiotiques d'intérêt à la conception de produits manufacturés parfaitement adaptés aux visées thérapeutiques.In recent years, the development of metagenomics has expanded our understanding of the role played by our microbiota on our health. The use of probiotics appears to be a therapeutic opportunity but requires more research to understand their mechanisms of action. The probiotic strain Lactobacillus rhamnosus Lcr35 ®, produced since 1951 by Lyocentre Laboratories and marketed by PROBIONOV, is the active ingredient of pharmaceutical products. Our work was to compare some "probiotic" properties of bacterial strain Lactobacillus rhamnosus Lcr35 ® versus four of its commercial preparations. Three of these formulations are suitable for intestinal applications: BACILOR (API Lcr Restituo ® packet or API Lcr Restituo ® capsule) and FLOREA (API Lcr Lenio ®). The fourth product studied GYNOPHILUS (API Lcr Regenerans ®) which is indicated for the fight against vaginal dysbiosis. We have shown that the different treatments of the microorganism in industrial processes generated changes to its properties. We found that APIs forms increased the resistance of the strain to gastric stress; Lcr Lenio ® offers the most resistance. Similarly, adherence capacities of the strain were increased in Caco-2 cells following industrial processes. Characterization of the bacterial envelope and its composition were changed depending on the industrial cycle and showed that the native strain expressed a hydrophobicity profile different from the APIs. However, these changes have not show variation in the aggregation capabilities of the strain. Conversely, analysis of the expression pattern of genes encoded potentially for secreted or trans-membrane proteins show variations between the native strain and the APIs tested. Treatment of the strain by an industrial manufacturing also allowed for changes in its metabolic capabilities. The API Lcr Regenerans ® sees these abilties as glycogen degradation and its bactericidal and fungicidal potential greatly increased. These results suggest that the manufacturing process affects the chemical properties and protein composition of the cell wall. The study of changes in industrial processes must be thorough in order to better adapt the probiotic strains in the design of manufactured products perfectly suited for therapeutic purposes

Caenorhabditis elegans, a Host to Investigate the Probiotic Properties of Beneficial Microorganisms

International audienceCaenorhabditis elegans, a non-parasitic nematode emerges as a relevant and powerful candidate as an in vivo model for microorganisms-microorganisms and microorganisms-host interactions studies. Experiments have demonstrated the probiotic potential of bacteria since they can provide to the worm a longer lifespan, an increased resistance to pathogens and to oxidative or heat stresses. Probiotics are used to prevent or treat microbiota dysbiosis and associated pathologies but the molecular mechanisms underlying their capacities are still unknown. Beyond safety and healthy aspects of probiotics, C. elegans represents a powerful way to design large-scale studies to explore transkingdom interactions and to solve questioning about the molecular aspect of these interactions. Future challenges and opportunities would be to validate C. elegans as an in vivo tool for high-throughput screening of microorganisms for their potential probiotic use on human health and to enlarge the panels of microorganisms studied as well as the human diseases investigated

Etudes in vivo des propriétés probiotiques et anti-Candida albicans de Lactobacillus rhamnosus Lcr35®

National audienceLes probiotiques sont définis par la FAO et l’OMS comme des microorganismes vivants conférant des effets bénéfiques à l’hôte comme par exemple Lactobacillus rhamnosus Lcr35®. Ce dernier est notamment commercialisé pour ses propriétés anti-Candida albicans. De plus, ses propriétés sont potentialisées par des composés inclus dans les formulations. Cependant, comme pour la plupart des probiotiques, les mécanismes d’action de Lcr35® ne sont pas encore décrits. Les identifier devient aujourd’hui une priorité. Dans ce but, des études in vivo utilisant un organisme modèle, Caenorhabditis elegans, ont été réalisées : (i) étude de la survie du nématode en présence du probiotique potentialisé ou non, (ii) étude des effets préventifs et curatifs de Lcr35® contre une infection à Candida chez C. elegans et (iii) approches transcriptomiques pour identifier les voies de signalisation impliquées dans les effets anti-Candida de Lcr35®.La pathogénicité de C. albicans est généralement liée à la filamentation induisant la destruction des cellules de l’hôte. De Barros et al. (2018) ont montré que C. elegans, prétraité avec L. paracasei, était moins sensible à une infection à C. albicans. Cependant, une réduction significative de la filamentation n’a pas été observée. De ce fait, des expérimentations sur la réduction de la filamentation de C. albicans par Lcr35® seront réalisées

Highlighting of the molecular mechanisms involved in probiotic properties of Lactobacillus rhamnosus Lcr35

National audienceProbiotics are defined by the World Health Organization as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (1). Although their beneficial effects on health are proven, knowledge on their mechanisms of action is becoming a priority today. This is the case with L. rhamnosus Lcr35, whose probiotic and anti- Candida albicans properties have been demonstrated (2, 3). Lcr35 is genetically and physiologically well-characterized and it has been proven that ingredients included in the formulations is able to potentialize Lcr35 probiotic effect. In order to investigate the molecular mechanisms involved in the Lcr35 strain probiotic properties, in vivo studies have been carried out in C. elegans : the analysis of the nematode survival in the presence of the probiotic strain potentialized or not for the anti- C. albicans properties, and a preliminary analysis of the modulation of the immune response by Lcr35.The survival analyzes showed that the strain Lcr35 allowed a significant increased lifespan of the nematode in comparison with E. coli OP50 whereas C. albicans impaired survival. The activation of insulin and p38 MAPK pathways has been shown using RT-qPCR. Then we will go further into the investigations to understand the molecular mechanisms related to these properties by studying in particular the preventive and curative effects of Lcr35 with respect to a C. albicans infection. In addition, a complete analysis of the transcriptome of C. elegans will be carried out by RNA sequencing in order to precisely identify the signaling pathways involved in the interaction between the host and the probiotic or the pathogen microorganisms. Moreover, because of C. elegans genetic homology with humans and in particular its intestinal physiology close to humans, our work lead us to evaluate the potential of C. elegans to be a relevant in vivo model to characterize the molecular mechanisms involved in the probiotic potential of a microorganisms and then to screen microorganisms collections for their potential probiotic capacities.1. FAO, WHO. 2001. Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Food Nutr Pap 85:71.2. Nivoliez A et al. 2012. Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35. J Biotechnol 160:236–241.3. Nivoliez A et al. 2015. Influence of manufacturing processes on cell surface properties of probiotic strain Lactobacillus rhamnosus Lcr35. Appl Microbiol Biotechnol 99:399–41

Caractérisation in vivo des mécanismes probiotiques de Lactobacillus rhamnosus Lcr35®

National audienceLa levure Candida albicans est un pathogène opportuniste retrouvée dans la flore commensale de 80% de la population. Elle est l’agent d’un grand nombre d’infections digestives et gynécologiques appelées candidoses. La prise d’antifongique, bien qu’efficace, peut entrainer une dysbiose favorable à une récidive ultérieure. Lactobacillus rhamnosus Lcr35® est un probiotique dont les propriétés antimicrobiennes, notamment anti-C. albicans, ont été démontrées à la fois in vitro et lors d’essais cliniques. Cependant, les mécanismes moléculaires expliquant ces propriétés n’ont pas encore été décrits. L’objectif de ma thèse est d’identifier ces mécanismes en utilisant un modèle in vivo physiologiquement proche de l’Homme, le ver Caenorhabditis elegans.Ce nématode modèle sera présenté suivi des différentes stratégies expérimentales mises en œuvre : (i) l’étude du temps de vie en présence du probiotique ou du pathogène, (ii) l’étude des effets préventifs et curatifs du probiotique vis-à-vis d’une infection fongique puis (iii) des études omiques (transcriptomique et métabolomique).Les premiers résultats ont mis en évidence que Lcr35® induit un effet positif sur la durée de vie de C. elegans à l’inverse de C. albicans qui induit un effet négatif. De plus, un effet protecteur du probiotique vis-à-vis d’une infection à C. albicans est observé. En effet, en présence de Lcr35®, le nématode résiste à l’infection et voit son temps de vie augmenter. En perspective, une étude transcriptomique de la réponse globale du ver à l’administration du probiotique et/ou du pathogène est envisagée dans le but d’identifier les mécanismes moléculaires responsables des effets observés

Mechanistic approach to stability studies as a tool for the optimization and development of new products based on L. rhamnosus Lcr35® in compliance with current regulations.

Probiotics are of great current interest in the pharmaceutical industry because of their multiple effects on human health. To beneficially affect the host, an adequate dosage of the probiotic bacteria in the product must be guaranteed from the time of manufacturing to expiration date. Stability test guidelines as laid down by the ICH-Q1A stipulate a minimum testing period of 12 months. The challenge for producers is to reduce this time. In this paper, a mechanistic approach using the Arrhenius model is proposed to predict stability. Applied for the first time to laboratory and industrial probiotic powders, the model was able to provide a reliable mathematical representation of the effects of temperature on bacterial death (R(2)>0.9). The destruction rate (k) was determined according to the manufacturing process, strain and storage conditions. The marketed product demonstrated a better stability (k = 0.08 months(-1)) than the laboratory sample (k = 0.80 months(-1)). With industrial batches, k obtained at 6 months of studies was comparable to that obtained at 12 months, evidence of the model's robustness. In addition, predicted values at 12 months were greatly similar (±30%) to those obtained by real-time assessing the model's reliability. This method could be an interesting approach to predict the probiotic stability and could reduce to 6 months the length of stability studies as against 12 (ICH guideline) or 24 months (expiration date)

Rapid Quantitative PCR Assay for the Detection of the Three Vaginal Pathogens Candida, Gardnerella and Atopobium as well as the Commensal Lactobacillus Genera

International audienceThe vaginal microbiota balance is quite fragile and susceptible to the development of vaginosis and candidiasis. The current diagnostic method for bacterial vaginosis relies on the evaluation of different bacterial morphotypes using the Nugent score. This method is only partially in correlation with a DNA sequencing-based diagnostic or Amsel criteria used by clinicians, suggesting the need for new molecular approaches dedicated to the diagnosis of BV. The objective of this study was to develop and validate a quantitative polymerase chain reaction (qPCR) assay for the specific and rapid detection of three vaginal pathogens, i.e. Candida, Gardnerella and Atopobium and the commensal Lactobacillus genera. For this purpose, four oligonucleotide primer pairs were designed and tested to obtain optimal amplification of the four target genera. The qPCR assay was also tested on the non-target genera and on human DNA. The designed primers allowed specific amplification of the target organisms in vitro and in clinical vaginal samples. The qPCR assay designed in this study is effective to specifically detect these genera in clinical samples as a molecular technique complementary to the Nugent score. It can be used in epidemiological studies for understanding the role of these pathogens and to follow their abundance in the microbiota in disease processes such as the development of vulvovaginal candidiasis and bacterial vaginosis

Lactobacillus rhamnosus Lcr35 as an effective treatment for preventing Candida albicans infection in the preclinical model Caenorhabditis elegans

International audienceIntroduction and aimsThe increased recurrence of Candida albicans infections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols such as the use of probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals) and clinical studies. Understanding the mechanisms of action of probiotic microorganisms has become a strategic need for the development of new therapeutics for humans. In this study, we investigated the prophylactic anti-Candida albicans properties of Lactobacillus rhamnosus Lcr35® using the in vivo Caenorhabditis elegans model. Materials and methodsFor this, we followed the survival of the nematode in the case of a fungal infection, prevented or not by the probiotic Lcr35®. This followed by a gene expression analysis. Also, we are interested in the localization of the DAF-16 transcription factor.Results, discussion and conclusionOn the top of having a pro-longevity activity in the nematode, Lcr35® protects the animal from the fungal infection even if the yeast is still detectable in its intestine. At the mechanistic level, we note the repression of genes of the p38 MAPK signaling pathway and genes involved in the antifungal response induced by Lcr35® suggesting that the pathogen no longer appears to be detected by the worm immune system. However, the DAF-16 / FOXO transcription factor, implicated in the longevity and antipathogenic response of C. elegans, is activated by Lcr35®. These results suggest that the probiotic strain acts by stimulating its host via DAF-16, but also by suppressing the virulence of the pathogen. However, for more precision, an exhaustive study of the C. elegans transcriptome will be carried out in order to decipher the mechanisms of action of Lcr35®