Role of Klebsiella pneumoniae Type VI secretion system (T6SS) in long-term gastrointestinal colonization.

Type VI secretion systems (T6SS), recently described in hypervirulent K. pneumoniae (hvKp) strains, are involved in bacterial warfare but their role in classical clinical strains (cKp) has been little investigated. In silico analysis indicated the presence of T6SS clusters (from zero to four), irrespective of the strains origin or virulence, with a high prevalence in the K. pneumoniae species (98%). In the strain CH1157, two T6SS-apparented pathogenicity islands were detected, T6SS-1 and -2, harboring a phospholipase-encoding gene (tle1) and a potential new effector-encoding gene named tke (Type VI Klebsiella effector). Tle1 expression in Escherichia coli periplasm affected cell membrane permeability. T6SS-1 isogenic mutants colonized the highest gastrointestinal tract of mice less efficiently than their parental strain, at long term. Comparative analysis of faecal 16S sequences indicated that T6SS-1 impaired the microbiota richness and its resilience capacity. Oscillospiraceae family members could be specific competitors for the long-term gut establishment of K. pneumoniae.

Gut-Kidney Axis Investigations in Animal Models of Chronic Kidney Disease.

Chronic kidney disease (CKD) is an incurable disease in which renal function gradually declines, resulting in no noticeable symptoms during the early stages and a life-threatening disorder in the latest stage. The changes that accompany renal failure are likely to influence the gut microbiota, or the ecosystem of micro-organisms resident in the intestine. Altered gut microbiota can display metabolic changes and become harmful to the host. To study the gut-kidney axis in vivo, animal models should ideally reproduce the disorders affecting both the host and the gut microbiota. Murine models of CKD, but not dog, manifest slowed gut transit, similarly to patient. Animal models of CKD also reproduce altered intestinal barrier function, as well as the resulting leaky gut syndrome and bacterial translocation. CKD animal models replicate metabolic but not compositional changes in the gut microbiota. Researchers investigating the gut-kidney axis should pay attention to the selection of the animal model (disease induction method, species) and the setting of the experimental design (control group, sterilization method, individually ventilated cages) that have been shown to influence gut microbiota.

Approaches to discern if microbiome associations reflect causation in metabolic and immune disorders.

Our understanding of microorganisms residing within our gut and their roles in the host metabolism and immunity advanced greatly over the past 20 years. Currently, microbiome studies are shifting from association and correlation studies to studies demonstrating causality of identified microbiome signatures and identification of molecular mechanisms underlying these interactions. This transformation is crucial for the efficient translation into clinical application and development of targeted strategies to beneficially modulate the intestinal microbiota. As mechanistic studies are still quite challenging to perform in humans, the causal role of microbiota is frequently evaluated in animal models that need to be appropriately selected. Here, we provide a comprehensive overview on approaches that can be applied in addressing causality of host-microbe interactions in five major animal model organisms (Caenorhabditis elegans, Drosophila melanogaster, zebrafish, rodents, and pigs). We particularly focused on discussing methods available for studying the causality ranging from the usage of gut microbiota transfer, diverse models of metabolic and immune perturbations involving nutritional and chemical factors, gene modifications and surgically induced models, metabolite profiling up to culture-based approached. Furthermore, we addressed the impact of the gut morphology, physiology as well as diet on the microbiota composition in various models and resulting species specificities. Finally, we conclude this review with the discussion on models that can be applied to study the causal role of the gut microbiota in the context of metabolic syndrome and host immunity. We hope this review will facilitate important considerations for appropriate animal model selection.

Lacticaseibacillus casei CNCM I-5663 supplementation maintained muscle mass in a model of frail rodents.

The aim of this study was to identify a probiotic-based strategy for maintaining muscle anabolism in the elderly. In previous research, we found that individuals experiencing short bowel syndrome (SBS) after an intestinal resection displayed beneficial metabolic adjustments that were mediated by their gut microbes. Thus, these bacteria could potentially be used to elicit similar positive effects in elderly people, who often have low food intake and thus develop sarcopenia. Gut bacterial strains from an SBS patient were evaluated for their ability to (1) maintain Caenorhabditis elegans survival and muscle structure and (2) promote protein anabolism in a model of frail rodents (18-month-old rats on a food-restricted diet: 75% of ad libitum consumption). We screened a first set of bacteria in C. elegans and selected two Lacticaseibacillus casei strains (62 and 63) for further testing in the rat model. We had four experimental groups: control rats on an ad libitum diet (AL); non-supplemented rats on the food-restricted diet (R); and two sets of food-restricted rats that received a daily supplement of one of the strains (∼109 CFU; R+62 and R+63). We measured lean mass, protein metabolism, insulin resistance, cecal short-chain fatty acids (SCFAs), and SCFA receptor expression in the gut. Food restriction led to decreased muscle mass [-10% vs. AL (p < 0.05)]. Supplementation with strain 63 tempered this effect [-2% vs. AL (p > 0.1)]. The mechanism appeared to be the stimulation of the insulin-sensitive p-S6/S6 and p-eIF2α/eIF2α ratios, which were similar in the R+63 and AL groups (p > 0.1) but lower in the R group (p < 0.05). We hypothesize that greater SCFA receptor sensitivity in the R+63 group promoted gut-muscle cross talk [GPR41: +40% and GPR43: +47% vs. R (p < 0.05)]. Hence, strain 63 could be used in association with other nutritional strategies and exercise regimes to limit sarcopenia in frail elderly people.

In vivo investigation of Lcr35® anti-candidiasis properties in Caenorhabditis elegans reveals the involvement of highly conserved immune pathways.

Lactic acid bacteria, including the microorganisms formerly designated as Lactobacillus, are the major representatives of Live Biotherapeutic Microorganisms (LBM) when used for therapeutic purposes. However, in most cases, the mechanisms of action remain unknown. The antifungal potential of LBM has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding their mechanisms of action is strategic for the development of new therapeutics for humans. Here, Caenorhabditis elegans was used as an in vivo model to analyze pro-longevity, anti-aging and anti-candidiasis effects of the LBM Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) Lcr35®. A high-throughput transcriptomic analysis revealed a specific response of C. elegans depending on whether it is in the presence of the LBM L. rhamnosus Lcr35® (structural response), the yeast Candida albicans (metabolic response) or both (structural and metabolic responses) in a preventive and a curative conditions. Studies on C. elegans mutants demonstrated that the p38 MAPK (sek-1, skn-1) and the insulin-like (daf-2, daf-16) signaling pathways were involved in the extended lifespan provided by L. rhamnosus Lcr35® strain whereas the JNK pathway was not involved (jnk-1). In addition, the anti C. albicans effect of the bacterium requires the daf-16 and sek-1 genes while it is independent of daf-2 and skn-1. Moreover, the anti-aging effect of Lcr35®, linked to the extension of longevity, is not due to protection against oxidative stress (H2O2). Taken together, these results formally show the involvement of the p38 MAP kinase and insulin-like signaling pathways for the longevity extension and anti-Candida albicans properties of Lcr35® with, however, differences in the genes involved. Overall, these findings provide new insight for understanding the mechanisms of action of a probiotic strain with antimicrobial potential.

Corrigendum: Lacticaseibacillus casei CNCM I-5663 supplementation maintained muscle mass in a model of frail rodents.

[This corrects the article DOI: 10.3389/fnut.2022.928798.].

A Mechanistic Study of the Antiaging Effect of Raw-Milk Cheese Extracts.

Many studies have highlighted the relationship between food and health status, with the aim of improving both disease prevention and life expectancy. Among the different food groups, fermented foods a have huge microbial biodiversity, making them an interesting source of metabolites that could exhibit health benefits. Our previous study highlighted the capacity of raw goat milk cheese, and some of the extracts recovered by the means of chemical fractionation, to increase the longevity of the nematode Caenorhabditis elegans. In this article, we pursued the investigation with a view toward understanding the biological mechanisms involved in this phenomenon. Using mutant nematode strains, we evaluated the implication of the insulin-like DAF-2/DAF-16 and the p38 MAPK pathways in the phenomenon of increased longevity and oxidative-stress resistance mechanisms. Our results demonstrated that freeze-dried raw goat milk cheese, and its extracts, induced the activation of the DAF-2/DAF-16 pathway, increasing longevity. Concerning oxidative-stress resistance, all the extracts increased the survival of the worms, but no evidence of the implication of both of the pathways was highlighted, except for the cheese-lipid extract that did seem to require both pathways to improve the survival rate. Simultaneously, the cheese-lipid extract and the dried extract W70, obtained with water, were able to reduce the reactive oxygen species (ROS) production in human leukocytes. This result is in good correlation with the results obtained with the nematode.

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Development of an innovative methodology combining chemical fractionation and in vivo analysis to investigate the biological properties of cheese.

With the ever-increasing human lifespan, age-related affections have become a public health issue. The health sector is looking for new bioactive compounds to respond to this demand. The unexplored microbial biodiversity and its metabolites represent a major source of innovative bioactive molecules with health potential. Fermented foods, such as raw-milk cheese, have already been investigated for their rich microbial environment, especially for their organoleptic qualities. But studies remain limited regarding their effects on health and few metabolites of microbial origin have been identified. An efficient methodology was developed in this study to investigate the biological effect of raw-milk cheese, combining a chemical fractionation, to isolate the most metabolites from the cheese matrix, and an in vivo biological test using Caenorhabditis elegans. C. elegans was brought into contact with cheese extracts, obtained by means of chemical fractionation, and with freeze-dried whole cheese by supplementing the nematode growth medium. A longevity assay was performed to evaluate the effects of the extracts on the worms. Our results demonstrate the feasibility of the method developed to bring the worms into contact of the cheese extracts. The evaluation of the effects of the extracts on the longevity was possible. Some extracts showed a beneficial effect as extract W70 for example, obtained with water, which increases the mean lifespan by 16% and extends the longevity by 73% (p < 0.0001).

Identification of sulfur components enhancing the anti-Candida effect of Lactobacillus rhamnosus Lcr35.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

Investigation into In Vitro and In Vivo Caenorhabditis elegans Models to Select Cheese Yeasts as Probiotic Candidates for their Preventive Effects against Salmonella Typhimurium.

The design of multiscale strategies integrating in vitro and in vivo models is necessary for the selection of new probiotics. In this regard, we developed a screening assay based on the investigation of the potential of yeasts from cheese as probiotics against the pathogen Salmonella Typhimurium UPsm1 (ST). Two yeasts isolated from raw-milk cheese (Saccharomyces cerevisiae 16, Sc16; Debaryomyces hansenii 25, Dh25), as well as S. cerevisiae subspecies boulardii (CNCM I-1079, Sb1079), were tested against ST by applying in vitro and in vivo tests. Adherence measurements to Caco-2 and HT29-MTX intestinal cells indicated that the two tested cheese yeasts presented a better adhesion than the probiotic Sb1079 as the control strain. Further, the Dh25 was the cheese yeast most likely to survive in the gastrointestinal tract. What is more, the modulation of the TransEpithelial Electrical Resistance (TEER) of differentiated Caco-2 cell monolayers showed the ability of Dh25 to delay the deleterious effects of ST. The influence of microorganisms on the in vivo model Caenorhabditis elegans was evaluated by measuring the longevity of the worm. This in vivo approach revealed that this yeast increased the worm's lifespan and protected it against ST infection, confirming that this in vivo model can be useful for screening probiotic cheese yeasts.

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

Caenorhabditis 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.

Curative Treatment of Candidiasis by the Live Biotherapeutic Microorganism Lactobacillus rhamnosus Lcr35® in the Invertebrate Model Caenorhabditis elegans: First Mechanistic Insights.

The resistance of Candida albicans to conventional drug treatments, as well as the recurrence phenomena due to dysbiosis caused by antifungal treatments, have highlighted the need to implement new therapeutic methodologies. The antifungal potential of live biotherapeutic products (LBP) has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding their mechanisms of action is strategic for the development of new therapeutics for humans. In this study, we investigated the curative anti-C. albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cell and the in vivo Caenorhabditis elegans models. We showed that Lcr35® does inhibit neither the growth (p = 0.603) nor the biofilm formation (p = 0.869) of C. albicans in vitro. Lcr35® protects the animal from the fungal infection (+225% of survival, p < 2 × 10-16) even if the yeast is detectable in its intestine. In contrast, the Lcr35® cell-free supernatant does not appear to have any antipathogenic effect. At the mechanistic level, the DAF-16/Forkhead Box O transcription factor is activated by Lcr35® and genes of the p38 MAP Kinase signaling pathway and genes involved in the antifungal response are upregulated in presence of Lcr35® after C. albicans infection. These results suggest that the LBM strain acts by stimulating its host via DAF-16 and the p38 MAPK pathway.

Lactobacillus rhamnosus Lcr35 as an effective treatment for preventing Candida albicans infection in the invertebrate model Caenorhabditis elegans: First mechanistic insights.

The increased recurrence of Candida albicans infections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols, such as probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals). 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-C. albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cell model and the in vivo Caenorhabditis elegans model. In Caco-2 cells, we showed that the strain Lcr35® significantly inhibited the growth (~2 log CFU.mL-1) and adhesion (150 to 6,300 times less) of the pathogen. Moreover, in addition to having a pro-longevity activity in the nematode (+42.9%, p = 3.56.10-6), Lcr35® protects the animal from the fungal infection (+267% of survival, p < 2.10-16) even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signalling 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.

Dialogue between Staphylococcus aureus SA15 and Lactococcus garvieae strains experiencing oxidative stress.

BACKGROUND: Staphylococcus aureus is an important foodborne pathogen. Lactococcus garvieae is a lactic acid bacterium found in dairy products; some of its strains are able to inhibit S. aureus growth by producing H2O2. Three strains of L. garvieae from different origins were tested for their ability to inhibit S. aureus SA15 growth. Two conditions were tested, one in which H2O2 was produced (high aeration) and another one in which it was not detected (low aeration). Several S. aureus genes related to stress, H2O2-response and virulence were examined in order to compare their level of expression depending on the inoculated L. garvieae strain. Simultaneous L. garvieae H2O2 metabolism gene expression was followed. RESULTS: The results showed that under high aeration condition, L. garvieae strains producing H2O2 (N201 and CL-1183) inhibited S. aureus SA15 growth and impaired its ability to deal with hydrogen peroxide by repressing H2O2-degrading genes. L. garvieae strains induced overexpression of S. aureus stress-response genes while cell division genes and virulence genes were repressed. A catalase treatment partially or completely restored the SA15 growth. In addition, the H2O2 non-producing L. garvieae strain (Lg2) did not cause any growth inhibition. The SA15 stress-response genes were down-regulated and cell division genes expression was not affected. Under low aeration condition, while none of the strains tested exhibited H2O2-production, the 3 L. garvieae strains inhibited S. aureus SA15 growth, but to a lesser extent than under high aeration condition. CONCLUSION: Taken together, these results suggest a L. garvieae strain-specific anti-staphylococcal mechanism and an H2O2 involvement in at least two of the tested L. garvieae strains.

New Insights into the Anti-pathogenic Potential of Lactococcus garvieae against Staphylococcus aureus Based on RNA Sequencing Profiling.

The bio-preservation potential of Lactococcus garvieae lies in its capacity to inhibit the growth of staphylococci, especially Staphylococcus aureus, in dairy products and in vitro. In vitro, inhibition is modulated by the level of aeration, owing to hydrogen peroxide (H2O2) production by L. garvieae under aeration. The S. aureus response to this inhibition has already been studied. However, the molecular mechanisms of L. garvieae underlying the antagonism against S. aureus have never been explored. This study provides evidence of the presence of another extracellular inhibition effector in vitro. This effector was neither a protein, nor a lipid, nor a polysaccharide, nor related to an L-threonine deficiency. To better understand the H2O2-related inhibition mechanism at the transcriptome level and to identify other mechanisms potentially involved, we used RNA sequencing to determine the transcriptome response of L. garvieae to different aeration levels and to the presence or absence of S. aureus. The L. garvieae transcriptome differed radically between different aeration levels mainly in biological processes related to fundamental functions and nutritional adaptation. The transcriptomic response of L. garvieae to aeration level differed according to the presence or absence of S. aureus. The higher concentration of H2O2 with high aeration was not associated with a higher expression of L. garvieae H2O2-synthesis genes (pox, sodA, and spxA1) but rather with a repression of L. garvieae H2O2-degradation genes (trxB1, ahpC, ahpF, and gpx). We showed that L. garvieae displayed an original, previously undiscovered, H2O2 production regulation mechanism among bacteria. In addition to the key factor H2O2, the involvement of another extracellular effector in the antagonism against S. aureus was shown. Future studies should explore the relation between H2O2-metabolism, H2O2-producing LAB and the pathogen they inhibit. The nature of the other extracellular effector should also be determined.

Staphylococcus aureus transcriptomic response to inhibition by H2O2-producing Lactococcus garvieae.

Growth of the foodborne pathogen Staphylococcus aureus can be inhibited in milk and in cheese by the hydrogen peroxide-producing Lactococcus garvieae N201 dairy strain. Transcriptomic responses of two S. aureus strains, the S. aureus SA15 dairy strain and the MW2 human pathogenic strain, to this growth inhibition were investigated in Brain-Heart Infusion broth under a high or a low aeration level. We demonstrated that S. aureus MW2 had a higher resistance to L. garvieae inhibition under the high aeration level: this correlated to a higher survival under hydrogen peroxide exposure. Conversely, the two strains were similarly inhibited under the low aeration level. Expression of S. aureus genes involved in response to H2O2 or other stresses as well as in cell division was generally repressed by L. garvieae. However, differential expressions between the two S. aureus strains were observed, especially under the high aeration level. Additionally, expression of virulence-related genes (enterotoxins, regulatory genes) was modulated by L. garvieae depending on the aeration level and on the S. aureus strain. This study led to new insights into potential molecular mechanisms of S. aureus inhibition by Lactic Acid Bacteria via H2O2 production.

Influence of manufacturing processes on cell surface properties of probiotic strain Lactobacillus rhamnosus Lcr35®.

The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products' properties would therefore represent an essential step in evaluating the effects of probiotic strains.

Study of the Lactobacillus rhamnosus Lcr35® properties after compression and proposition of a model to predict tablet stability.

The beneficial effects of probiotic bacteria on human health are now widely acknowledged, and this has prompted growing interest in research and development in the pharmaceutical field. However, to be viable when they reach their target, the bacteria must be able to survive during the manufacturing process and the biological pathway. Tablet form best meets the requirements for protecting acid labile drugs, but the tableting process could be an additional stress for the bacteria. This study evaluated the initial effect of compression pressure on the Lcr35® strain in a vaginal (Lcr regenerans®) and an intestinal (Lcr restituo®) formulation. A stability study was also performed on the tablets and revealed a beneficial effect of this form. The obtained destruction rates (k) demonstrated that the bacterial stability was greater in tablets than in powders (kpowders>ktablets). A new mathematical model was developed combining compression and temperature parameters to predict the bacterial viability at any pressure and time. Moreover, the genetic profile of Lcr35® (Rep-PCR, microarrays), its resistance to acidity and its ability to inhibit Candidaalbicans growth, after compression, were determined to evaluate the target product profile (TPP) in a Quality by Design (QbD) approach. The Rep-PCR analysis validated the strain identity and the microarrays demonstrated the genetic stability of Lcr35® strain after compaction. Additionally, ability to inhibit the C. albicans growth was maintained and the resistance to gastric conditions of Lcr35® was even improved by tableting. As a dosage form, tablets containing probiotic can guarantee that an adequate amount of bacteria reaches the therapeutic target (intestinal or vaginal) and that the product remains stable until the time of consumption.

Annotation of microsporidian genomes using transcriptional signals.

High-quality annotation of microsporidian genomes is essential for understanding the biological processes that govern the development of these parasites. Here we present an improved structural annotation method using transcriptional DNA signals. We apply this method to re-annotate four previously annotated genomes, which allow us to detect annotation errors and identify a significant number of unpredicted genes. We then annotate the newly sequenced genome of Anncaliia algerae. A comparative genomic analysis of A. algerae permits the identification of not only microsporidian core genes, but also potentially highly expressed genes encoding membrane-associated proteins, which represent good candidates involved in the spore architecture, the invasion process and the microsporidian-host relationships. Furthermore, we find that the ten-fold variation in microsporidian genome sizes is not due to gene number, size or complexity, but instead stems from the presence of transposable elements. Such elements, along with kinase regulatory pathways and specific transporters, appear to be key factors in microsporidian adaptive processes.