Prebiotic utilisation provides Lactiplantibacillus plantarum a competitive advantage in vitro, but is not reflected by an increased intestinal fitness.

Synbiotics combine the concepts of probiotics and prebiotics to synergistically enhance the health-associated effects of both components. Previously, we have shown that the intestinal persistence of inulin-utilizing L. plantarum Lp900 is significantly increased in rats fed an inulin-supplemented, high-calcium diet. Here we employed a competitive population dynamics approach to demonstrate that inulin and GOS can selectively enrich L. plantarum strains that utilize these substrates for growth during in vitro cultivation, but that such enrichment did not occur during intestinal transit in rats fed a GOS or inulin-supplemented diet. The intestinal persistence of all L. plantarum strains increased irrespective of their prebiotic utilization phenotype, which was dependent on the calcium level of the diet. Analysis of fecal microbiota and intestinal persistence decline rates indicated that prebiotic utilization capacity did not selectively stimulate intestinal persistence in prebiotic supplemented diets. Moreover, microbiota and organic acid profile analyses indicate that the prebiotic utilizing probiotic strains are vastly outcompeted by the endogenous prebiotic-utilizing microbiota, and that the collective enhanced persistence of all L. plantarum strains is most likely explained by their well-established tolerance to organic acids.

Dynamic effects of probiotic formula ecologic®825 on human small intestinal ileostoma microbiota: a network theory approach.

The gut microbiota plays a pivotal role in health and disease. The use of probiotics as microbiota-targeted therapies is a promising strategy to improve host health. However, the molecular mechanisms involved in such therapies are often not well understood, particularly when targeting the small intestinal microbiota. In this study, we investigated the effects of a probiotic formula (Ecologic®825) on the adult human small intestinal ileostoma microbiota. The results showed that supplementation with the probiotic formula led to a reduction in the growth of pathobionts, such as Enterococcaceae and Enterobacteriaceae, and a decrease in ethanol production. These changes were associated with significant alterations in nutrient utilization and resistance to perturbations. These probiotic mediated alterations which coincided with an initial increase in lactate production and decrease in pH were followed by a sharp increase in the levels of butyrate and propionate. Moreover, the probiotic formula increased the production of multiple N-acyl amino acids in the stoma samples. The study demonstrates the utility of network theory in identifying novel microbiota-targeted therapies and improving existing ones. Overall, the findings provide insights into the dynamic molecular mechanisms underlying probiotic therapies, which can aid in the development of more effective treatments for a range of conditions.

Potential binding modes of the gut bacterial metabolite, 5-hydroxyindole, to the intestinal L-type calcium channels and its impact on the microbiota in rats.

Intestinal microbiota and microbiota-derived metabolites play a key role in regulating the host physiology. Recently, we have identified a gut-bacterial metabolite, namely 5-hydroxyindole, as a potent stimulant of intestinal motility via its modulation of L-type voltage-gated calcium channels located on the intestinal smooth muscle cells. Dysregulation of L-type voltage-gated calcium channels is associated with various gastrointestinal motility disorders, including constipation, making L-type voltage-gated calcium channels an important target for drug development. Nonetheless, the majority of currently available drugs are associated with alteration of the gut microbiota. Using 16S rRNA sequencing this study shows that, when administered orally, 5-hydroxyindole has only marginal effects on the rat cecal microbiota. Molecular dynamics simulations propose potential-binding pockets of 5-hydroxyindole in the α1 subunit of the L-type voltage-gated calcium channels and when its stimulatory effect on the rat colonic contractility was compared to 16 different analogues, ex-vivo, 5-hydroxyindole stood as the most potent enhancer of the intestinal contractility. Overall, the present findings imply a potential role of microbiota-derived metabolites as candidate therapeutics for targeted treatment of slow intestinal motility-related disorders including constipation.

Gut microbiota transplantation drives the adoptive transfer of colonic genotype-phenotype characteristics between mice lacking catestatin and their wild type counterparts.

The gut microbiota is in continuous interaction with the intestinal mucosa via metabolic, neuro-immunological, and neuroendocrine pathways. Disruption in levels of antimicrobial peptides produced by the enteroendocrine cells, such as catestatin, has been associated with changes in the gut microbiota and imbalance in intestinal homeostasis. However, whether the changes in the gut microbiota have a causational role in intestinal dyshomeostasis has remained elusive. To this end, we performed reciprocal fecal microbial transplantation in wild-type mice and mice with a knockout in the catestatin coding region of the chromogranin-A gene (CST-KO mice). Combined microbiota phylogenetic profiling, RNA sequencing, and transmission electron microscopy were employed. Fecal microbiota transplantation from mice deficient in catestatin (CST-KO) to microbiota-depleted wild-type mice induced transcriptional and physiological features characteristic of a distorted colon in the recipient animals, including impairment in tight junctions, as well as an increased collagen area fraction indicating colonic fibrosis. In contrast, fecal microbiota transplantation from wild-type mice to microbiota-depleted CST-KO mice reduced collagen fibrotic area, restored disrupted tight junction morphology, and altered fatty acid metabolism in recipient CST-KO mice. This study provides a comprehensive overview of the murine metabolic- and immune-related cellular pathways and processes that are co-mediated by the fecal microbiota transplantation and supports a prominent role for the gut microbiota in the colonic distortion associated with the lack of catestatin in mice. Overall, the data show that the gut microbiota may play a causal role in the development of features of intestinal inflammation and metabolic disorders, known to be associated with altered levels of catestatin and may, thus, provide a tractable target in the treatment and prevention of these disorders.

Catestatin selects for colonization of antimicrobial-resistant gut bacterial communities.

The gut microbiota is in continuous interaction with the innermost layer of the gut, namely the epithelium. One of the various functions of the gut epithelium, is to keep the microbes at bay to avoid overstimulation of the underlying mucosa immune cells. To do so, the gut epithelia secrete a variety of antimicrobial peptides, such as chromogranin A (CgA) peptide catestatin (CST: hCgA352-372). As a defense mechanism, gut microbes have evolved antimicrobial resistance mechanisms to counteract the killing effect of the secreted peptides. To this end, we treated wild-type mice and CST knockout (CST-KO) mice (where only the 63 nucleotides encoding CST have been deleted) with CST for 15 consecutive days. CST treatment was associated with a shift in the diversity and composition of the microbiota in the CST-KO mice. This effect was less prominent in WT mice. Levels of the microbiota-produced short-chain fatty acids, in particular, butyrate and acetate were significantly increased in CST-treated CST-KO mice but not the WT group. Both CST-treated CST-KO and WT mice showed a significant increase in microbiota-harboring phosphoethanolamine transferase-encoding genes, which facilitate their antimicrobial resistance. Finally, we show that CST was degraded by Escherichia coli via an omptin-protease and that the abundance of this gene was significantly higher in metagenomic datasets collected from patients with Crohn's disease but not with ulcerative colitis. Overall, this study illustrates how the endogenous antimicrobial peptide, CST, shapes the microbiota composition in the gut and primes further research to uncover the role of bacterial resistance to CST in disease states such as inflammatory bowel disease.

Dietary calcium phosphate strongly impacts gut microbiome changes elicited by inulin and galacto-oligosaccharides consumption.

BACKGROUND: Fructo-oligosaccharides (FOS), inulin, and galacto-oligosaccharides (GOS) are widely recognized prebiotics that profoundly affect the intestinal microbiota, including stimulation of bifidobacteria and lactobacilli, and are reported to elicit several health benefits. The combination of dietary FOS and inulin with calcium phosphate was reported to stimulate commensal Lactobacillus populations and protect the host against pathogenic Enterobacteriaceae, but little is known about the effects of GOS in diets with a different level of calcium phosphate. METHODS: We investigated the microbiome changes elicited by dietary supplementation with GOS or inulin using diets with high (100 mmol/kg) and low (30 mmol/kg) calcium phosphate levels in adult Wistar rats. Rats were acclimatized to the respective experimental diets for 14 days, after which fecal material was collected, DNA was extracted from fecal material, and the V3­V4 region of the bacterial 16S rRNA gene was amplified with PCR, followed by microbial composition analysis. In tandem, the organic acid profiles of the fecal material were analyzed. RESULTS: Feeding rats non-supplemented (no prebiotic-added) diets revealed that diets rich in calcium phosphate favored members of the Firmicutes and increased fecal lactic, succinic, acetic, propionic, and butyric acid levels. In contrast, relatively low dietary calcium phosphate levels promoted the abundance of mucin degrading genera like Akkermansia and Bacteroides, and resulted in increased fecal propionic acid levels and modest increases in lactic and butyric acid levels. Irrespective of the calcium phosphate levels, supplementation with GOS or inulin strongly stimulated Bifidobacterium, while only high calcium phosphate diets increased the endogenous Faecalibaculum populations. CONCLUSIONS: Despite the prebiotic's substantial difference in chemical structure, sugar composition, oligomer size, and the microbial degradation pathway involved in their utilization, inulin and GOS modulated the gut microbiota very similarly, in a manner that strongly depended on the dietary calcium phosphate level. Therefore, our study implies that the collection of detailed diet information including micronutrient balance is necessary to correctly assess diet-driven microbiota analysis. Video Abstract.

Stability of Methylphenidate under Various pH Conditions in the Presence or Absence of Gut Microbiota.

Methylphenidate is one of the most widely used oral treatments for attention-deficit/hyperactivity disorder (ADHD). The drug is mainly absorbed in the small intestine and has low bioavailability. Accordingly, a high interindividual variability in terms of response to the treatment is known among ADHD patients treated with methylphenidate. Nonetheless, very little is known about the factors that influence the drug's absorption and bioavailability. Gut microbiota has been shown to reduce the bioavailability of a wide variety of orally administered drugs. Here, we tested the ability of small intestinal bacteria to metabolize methylphenidate. In silico analysis identified several small intestinal bacteria to harbor homologues of the human carboxylesterase 1 enzyme responsible for the hydrolysis of methylphenidate in the liver into the inactive form, ritalinic acid. Despite our initial results hinting towards possible bacterial hydrolysis of the drug, up to 60% of methylphenidate is spontaneously hydrolyzed in the absence of bacteria and this hydrolysis is pH-dependent. Overall, our results indicate that the stability of methylphenidate is compromised under certain pH conditions in the presence or absence of gut microbiota.

Dietary Inulin Increases Lactiplantibacillus plantarum Strain Lp900 Persistence in Rats Depending on the Dietary-Calcium Level.

Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit health benefits in the consumer. Lactiplantibacillus plantarum strains display high survival during transit through the mammalian gastrointestinal tract and were shown to have health-promoting properties. Growth on the fructose polysaccharide inulin is relatively uncommon in L. plantarum, and in this study we describe FosE, a plasmid-encoded ß-fructosidase of L. plantarum strain Lp900 which has inulin-hydrolyzing properties. FosE contains an LPxTG-like motif involved in sortase-dependent cell wall anchoring but is also (partially) released in the culture supernatant. In addition, we examined the effect of diet supplementation with inulin on the intestinal persistence of Lp900 in adult male Wistar rats in diets with distinct calcium levels. Inulin supplementation in high-dietary-calcium diets significantly increased the intestinal persistence of L. plantarum Lp900, whereas this effect was not observed upon inulin supplementation of the low-calcium diet. Moreover, intestinal persistence of L. plantarum Lp900 was determined when provided as a probiotic (by itself) or as a synbiotic (i.e., in an inulin suspension) in rats that were fed unsupplemented diets containing the different calcium levels, revealing that the synbiotic administration increased bacterial survival and led to higher abundance of L. plantarum Lp900 in rats, particularly in a low-calcium-diet context. Our findings demonstrate that inulin supplementation can significantly enhance the intestinal delivery of L. plantarum Lp900 but that this effect strongly depends on calcium levels in the diet.IMPORTANCE Synbiotics combine probiotics with prebiotics to synergistically elicit a health benefit in the consumer. Previous studies have shown that prebiotics can selectively stimulate the growth in the intestine of specific bacterial strains. In synbiotic supplementations the prebiotics constituent could increase the intestinal persistence and survival of accompanying probiotic strain(s) and/or modulate the endogenous host microbiota to contribute to the synergistic enhancement of the health-promoting effects of the synbiotic constituents. Our study establishes a profound effect of dietary-calcium-dependent inulin supplementation on the intestinal persistence of inulin-utilizing L. plantarum Lp900 in rats. We also show that in rats on a low-dietary-calcium regime, the survival and intestinal abundance of L. plantarum Lp900 are significantly increased by administering it as an inulin-containing synbiotic. This study demonstrates that prebiotics can enhance the intestinal delivery of specific probiotics and that the prebiotic effect is profoundly influenced by the calcium content of the diet.

Gut bacteria-derived 5-hydroxyindole is a potent stimulant of intestinal motility via its action on L-type calcium channels.

Microbial conversion of dietary or drug substrates into small bioactive molecules represents a regulatory mechanism by which the gut microbiota alters intestinal physiology. Here, we show that a wide variety of gut bacteria can metabolize the dietary supplement and antidepressant 5-hydroxytryptophan (5-HTP) to 5-hydroxyindole (5-HI) via the tryptophanase (TnaA) enzyme. Oral administration of 5-HTP results in detection of 5-HI in fecal samples of healthy volunteers with interindividual variation. The production of 5-HI is inhibited upon pH reduction in in vitro studies. When administered orally in rats, 5-HI significantly accelerates the total gut transit time (TGTT). Deciphering the underlying mechanisms of action reveals that 5-HI accelerates gut contractility via activation of L-type calcium channels located on the colonic smooth muscle cells. Moreover, 5-HI stimulation of a cell line model of intestinal enterochromaffin cells results in significant increase in serotonin production. Together, our findings support a role for bacterial metabolism in altering gut motility and lay the foundation for microbiota-targeted interventions.

Phenotypic and genetic characterization of differential galacto-oligosaccharide utilization in Lactobacillus plantarum.

Several Lactobacillus plantarum strains are marketed as probiotics for their potential health benefits. Prebiotics, e.g., galacto-oligosaccharides (GOS), have the potential to selectively stimulate the growth of L. plantarum probiotic strains based on their phenotypic diversity in carbohydrate utilization, and thereby enhance their health promoting effects in the host in a strain-specific manner. Previously, we have shown that GOS variably promotes the strain-specific growth of L. plantarum. In this study we investigated this variation by molecular analysis of GOS utilization by L. plantarum. HPAEC-PAD analysis revealed two distinct GOS utilization phenotypes in L. plantarum. Linking these phenotypes to the strain-specific genotypes led to the identification of a lac operon encoding a ß-galactosidase (lacA), a permease (lacS), and a divergently oriented regulator (lacR), that are predicted to be involved in the utilization of higher degree of polymerization (DP) constituents present in GOS (specifically DP of 3-4). Mutation of lacA and lacS in L. plantarum NC8 resulted in reduced growth on GOS, and HPAEC analysis confirmed the role of these genes in the import and utilization of higher-DP GOS constituents. Overall, the results enable the design of highly-selective synbiotic combinations of L. plantarum strain-specific probiotics and specific GOS-prebiotic fractions.

Kmasker plants - a tool for assessing complex sequence space in plant species.

Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high-throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k-mer counting methods has proved to be reliable. Easy-to-use applications utilizing k-mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k-mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command-line and web-based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site-directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre-computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de.