Bacterial proteome adaptation during fermentation in dairy environments.

The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.

Detection and classification of the integrative conjugative elements of Lactococcus lactis.

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

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.

The hierarchy of sugar catabolization in Lactococcus cremoris.

IMPORTANCE: The availability of nutrients to microorganisms varies considerably between different environments, and changes can occur rapidly. As a general rule, a fast growth rate-typically growth on glucose-is associated with the repression of other carbohydrate utilization genes, but it is not clear to what extent catabolite repression is exerted by other sugars. We investigated the hierarchy of sugar utilization after substrate transitions in Lactococcus cremoris. For this, we determined the proteome and carbohydrate utilization capacity after growth on different sugars. The results show that the preparedness of cells for the utilization of "slower" sugars is not strictly determined by the growth rate. The data point to individual proteins relevant for various sugar transitions and suggest that the evolutionary history of the organism might be responsible for deviations from a strictly growth rate-related sugar catabolization hierarchy.

L. rhamnosus CNCM I-3690 survival, adaptation, and small bowel microbiome impact in human.

Fermented foods and beverages are a significant source of dietary bacteria that enter the gastrointestinal (GI) tract. However, little is known about how these microbes survive and adapt to the small intestinal environment. Colony-forming units (CFU) enumeration and viability qPCR of Lacticaseibacillus rhamnosus CNCM I-3690 in the ileal effluent of 10 ileostomy subjects during 12-h post consumption of a dairy product fermented with this strain demonstrated the high level of survival of this strain during human small intestine passage. Metatranscriptome analyses revealed the in situ transcriptome of L. rhamnosus in the small intestine, which was contrasted with transcriptome data obtained from in vitro cultivation. These comparative analyses revealed substantial metabolic adaptations of L. rhamnosus during small intestine transit, including adjustments of carbohydrate metabolism, surface-protein expression, and translation machinery. The prominent presence of L. rhamnosus in the effluent samples did not elicit an appreciable effect on the composition of the endogenous small intestine microbiome, but significantly altered the ecosystem's overall activity profile, particularly of pathways associated with carbohydrate metabolism. Strikingly, two of the previously recognized gut-brain metabolic modules expressed in situ by L. rhamnosus (inositol degradation and glutamate synthesis II) are among the most dominantly enriched activities in the ecosystem's activity profile. This study establishes the survival capacity of L. rhamnosus in the human small intestine and highlights its functional adjustment in situ, which we postulate to play a role in the probiotic effects associated with this strain.

Early feeding leads to molecular maturation of the gut mucosal immune system in suckling piglets.

Introduction: Diet-microbiota-host interactions are increasingly studied to comprehend their implications in host metabolism and overall health. Keeping in mind the importance of early life programming in shaping intestinal mucosal development, the pre-weaning period can be utilised to understand these interactions in suckling piglets. The objective of this study was to investigate the consequences of early life feeding on the time-resolved mucosal transcriptional program as well as mucosal morphology. Methods: A customised fibrous feed was provided to piglets (early-fed or EF group; 7 litters) from five days of age until weaning (29 days of age) in addition to sow's milk, whereas control piglets (CON; 6 litters) suckled mother's milk only. Rectal swabs, intestinal content, and mucosal tissues (jejunum, colon) were obtained pre- and post-weaning for microbiota analysis (16S amplicon sequencing) and host transcriptome analysis (RNA sequencing). Results: Early feeding accelerated both microbiota colonisation as well as host transcriptome, towards a more "mature state", with a more pronounced response in colon compared to jejunum. Early feeding elicited the largest impact on the colon transcriptome just before weaning (compared to post-weaning time-points), exemplified by the modulation of genes involved in cholesterol and energy metabolism and immune response. The transcriptional impact of early feeding persisted during the first days post-weaning and was highlighted by a stronger mucosal response to the weaning stress, via pronounced activation of barrier repair reactions, which is a combination of immune activation, epithelial migration and "wound-repair" like processes, compared to the CON piglets. Discussion: Our study demonstrates the potential of early life nutrition in neonatal piglets as a means to support their intestinal development during the suckling period, and to improve adaptation during the weaning transition.

Effects of a wholegrain-rich diet on markers of colonic fermentation and bowel function and their associations with the gut microbiome: a randomised controlled cross-over trial.

Background: Diets rich in whole grains are associated with health benefits. Yet, it remains unclear whether the benefits are mediated by changes in gut function and fermentation. Objective: We explored the effects of whole-grain vs. refined-grain diets on markers of colonic fermentation and bowel function, as well as their associations with the gut microbiome. Methods: Fifty overweight individuals with increased metabolic risk and a high habitual intake of whole grains (~69 g/day) completed a randomised cross-over trial with two 8-week dietary intervention periods comprising a whole-grain diet (≥75 g/day) and a refined-grain diet (<10 g/day), separated by a washout period of ≥6 weeks. A range of markers of colonic fermentation and bowel function were assessed before and after each intervention. Results: The whole-grain diet increased the levels of faecal butyrate (p = 0.015) and caproate (p = 0.013) compared to the refined-grain diet. No changes in other faecal SCFA, BCFA or urinary levels of microbial-derived proteolytic markers between the two interventions were observed. Similarly, faecal pH remained unchanged. Faecal pH did however increase (p = 0.030) after the refined-grain diet compared to the baseline. Stool frequency was lower at the end of the refined-grain period compared to the end of the whole-grain diet (p = 0.001). No difference in faecal water content was observed between the intervention periods, however, faecal water content increased following the whole-grain period compared to the baseline (p = 0.007). Dry stool energy density was unaffected by the dietary interventions. Nevertheless, it explained 4.7% of the gut microbiome variation at the end of the refined-grain diet, while faecal pH and colonic transit time explained 4.3 and 5%, respectively. Several butyrate-producers (e.g., Faecalibacterium, Roseburia, Butyriciococcus) were inversely associated with colonic transit time and/or faecal pH, while the mucin-degraders Akkermansia and Ruminococcaceae showed the opposite association. Conclusion: Compared with the refined-grain diet, the whole-grain diet increased faecal butyrate and caproate concentrations as well as stool frequency, emphasising that differences between whole and refined grains affect both colonic fermentation and bowel habits.

Endogenous small intestinal microbiome determinants of transient colonisation efficiency by bacteria from fermented dairy products: a randomised controlled trial.

BACKGROUND: The effects of fermented food consumption on the small intestine microbiome and its role on host homeostasis are largely uncharacterised as our knowledge on intestinal microbiota relies mainly on faecal samples analysis. We investigated changes in small intestinal microbial composition and functionality, short chain fatty acid (SCFA) profiles, and on gastro-intestinal (GI) permeability in ileostomy subjects upon the consumption of fermented milk products. RESULTS: We report the results from a randomised, cross-over, explorative study where 16 ileostomy subjects underwent 3, 2-week intervention periods. In each period, they consumed either milk fermented by Lacticaseibacillus rhamnosus CNCM I-3690, or milk fermented by Streptococcus thermophilus CNCM I-1630 and Lactobacillus delbrueckii subsp. bulgaricus CNCM I-1519, or a chemically acidified milk (placebo) daily. We performed metataxonomic, metatranscriptomic analysis, and SCFA profiling of ileostomy effluents as well as a sugar permeability test to investigate the microbiome impact of these interventions and their potential effect on mucosal barrier function. Consumption of the intervention products impacted the overall small intestinal microbiome composition and functionality, mainly due to the introduction of the product-derived bacteria that reach in several samples 50% of the total microbial community. The interventions did not affect the SCFA levels in ileostoma effluent, or gastro-intestinal permeability and the effects on the endogenous microbial community were negligible. The impact on microbiome composition was highly personalised, and we identified the poorly characterised bacterial family, Peptostreptococcaceae, to be positively associated with a low abundance of the ingested bacteria. Activity profiling of the microbiota revealed that carbon- versus amino acid-derived energy metabolism of the endogenous microbiome could be responsible for the individual-specific intervention effects on the small intestine microbiome composition and function, reflected also on urine microbial metabolites generated through proteolytic fermentation. CONCLUSIONS: The ingested bacteria are the main drivers of the intervention effect on the small intestinal microbiota composition. Their transient abundance level is highly personalised and influenced by the energy metabolism of the ecosystem that is reflected by its microbial composition ( http://www. CLINICALTRIALS: gov , ID NCT NCT02920294). Video Abstract.

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut.

BACKGROUND: Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by integrating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived components such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. RESULTS: We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the transcription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidoreductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehensive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi114) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose-dependent increase of neutrophils and macrophages in the gut area. CONCLUSION: The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life.

The Pleiotropic Effects of Carbohydrate-Mediated Growth Rate Modifications in Bifidobacterium longum NCC 2705.

Bifidobacteria are saccharolytic bacteria that are able to metabolize a relatively large range of carbohydrates through their unique central carbon metabolism known as the "bifid-shunt". Carbohydrates have been shown to modulate the growth rate of bifidobacteria, but unlike for other genera (e.g., E. coli or L. lactis), the impact it may have on the overall physiology of the bacteria has not been studied in detail to date. Using glucose and galactose as model substrates in Bifidobacterium longum NCC 2705, we established that the strain displayed fast and slow growth rates on those carbohydrates, respectively. We show that these differential growth conditions are accompanied by global transcriptional changes and adjustments of central carbon fluxes. In addition, when grown on galactose, NCC 2705 cells were significantly smaller, exhibited an expanded capacity to import and metabolized different sugars and displayed an increased acid-stress resistance, a phenotypic signature associated with generalized fitness. We predict that part of the observed adaptation is regulated by the previously described bifidobacterial global transcriptional regulator AraQ, which we propose to reflect a catabolite-repression-like response in B. longum. With this manuscript, we demonstrate that not only growth rate but also various physiological characteristics of B. longum NCC 2705 are responsive to the carbon source used for growth, which is relevant in the context of its lifestyle in the human infant gut where galactose-containing oligosaccharides are prominent.

Stool energy density is positively correlated to intestinal transit time and related to microbial enterotypes.

BACKGROUND: It has been hypothesised that the gut microbiota causally affects obesity via its capacity to extract energy from the diet. Yet, evidence elucidating the role of particular human microbial community structures and determinants of microbiota-dependent energy harvest is lacking. RESULTS: Here, we investigated whether energy extraction from the diet in 85 overweight adults, estimated by dry stool energy density, was associated with intestinal transit time and variations in microbial community diversity and overall structure stratified as enterotypes. We hypothesised that a slower intestinal transit would allow for more energy extraction. However, opposite of what we expected, the stool energy density was positively associated with intestinal transit time. Stratifications into enterotypes showed that individuals with a Bacteroides enterotype (B-type) had significantly lower stool energy density, shorter intestinal transit times, and lower alpha-diversity compared to individuals with a Ruminococcaceae enterotype (R-type). The Prevotella (P-type) individuals appeared in between the B- and R-type. The differences in stool energy density between enterotypes were not explained by differences in habitual diet, intake of dietary fibre or faecal bacterial cell counts. However, the R-type individuals showed higher urinary and faecal levels of microbial-derived proteolytic metabolites compared to the B-type, suggesting increased colonic proteolysis in the R-type individuals. This could imply a less effective colonic energy extraction in the R-type individuals compared to the B-type individuals. Notably, the R-type had significantly lower body weight compared to the B-type. CONCLUSIONS: Our findings suggest that gut microbial energy harvest is diversified among individuals by intestinal transit time and associated gut microbiome ecosystem variations. A better understanding of these associations could support the development of personalised nutrition and improved weight-loss strategies. Video Abstract.

Perspective: Leveraging the Gut Microbiota to Predict Personalized Responses to Dietary, Prebiotic, and Probiotic Interventions.

Humans often show variable responses to dietary, prebiotic, and probiotic interventions. Emerging evidence indicates that the gut microbiota is a key determinant for this population heterogeneity. Here, we provide an overview of some of the major computational and experimental tools being applied to critical questions of microbiota-mediated personalized nutrition and health. First, we discuss the latest advances in in silico modeling of the microbiota-nutrition-health axis, including the application of statistical, mechanistic, and hybrid artificial intelligence models. Second, we address high-throughput in vitro techniques for assessing interindividual heterogeneity, from ex vivo batch culturing of stool and continuous culturing in anaerobic bioreactors, to more sophisticated organ-on-a-chip models that integrate both host and microbial compartments. Third, we explore in vivo approaches for better understanding of personalized, microbiota-mediated responses to diet, prebiotics, and probiotics, from nonhuman animal models and human observational studies, to human feeding trials and crossover interventions. We highlight examples of existing, consumer-facing precision nutrition platforms that are currently leveraging the gut microbiota. Furthermore, we discuss how the integration of a broader set of the tools and techniques described in this piece can generate the data necessary to support a greater diversity of precision nutrition strategies. Finally, we present a vision of a precision nutrition and healthcare future, which leverages the gut microbiota to design effective, individual-specific interventions.

Manganese Modulates Metabolic Activity and Redox Homeostasis in Translationally Blocked Lactococcus cremoris, Impacting Metabolic Persistence, Cell Culturability, and Flavor Formation.

Manganese (Mn) is an essential trace element that is supplemented in microbial media with varying benefits across species and growth conditions. We found that growth of Lactococcus cremoris was unaffected by manganese omission from the growth medium. The main proteome adaptation to manganese omission involved increased manganese transporter production (up to 2,000-fold), while the remaining 10 significant proteome changes were between 1.4- and 4-fold. Further investigation in translationally blocked (TB), nongrowing cells showed that Mn supplementation (20 µM) led to approximately 1.5 X faster acidification compared with Mn-free conditions. However, this faster acidification stagnated within 24 h, likely due to draining of intracellular NADH that coincides with substantial loss of culturability. Conversely, without manganese, nongrowing cells persisted to acidify for weeks, albeit at a reduced rate, but maintaining redox balance and culturability. Strikingly, despite being unculturable, α-keto acid-derived aldehydes continued to accumulate in cells incubated in the presence of manganese, whereas without manganese cells predominantly formed the corresponding alcohols. This is most likely reflecting NADH availability for the alcohol dehydrogenase-catalyzed conversion. Overall, manganese influences the lactococcal acidification rate, and flavor formation capacity in a redox dependent manner. These are important industrial traits especially during cheese ripening, where cells are in a non-growing, often unculturable state. IMPORTANCE In nature as well as in various biotechnology applications, microorganisms are often in a nongrowing state and their metabolic persistence determines cell survival and functionality. Industrial examples are dairy fermentations where bacteria remain active during the ripening phases that can take up to months and even years. Here we investigated environmental factors that can influence lactococcal metabolic persistence throughout such prolonged periods. We found that in the absence of manganese, acidification of nongrowing cells remained active for weeks while in the presence of manganese it stopped within 1 day. The latter coincided with the accumulation of amino acid derived volatile metabolites. Based on metabolic conversions, proteome analysis, and a reporter assay, we demonstrated that the manganese elicited effects were NADH dependent. Overall the results show the effect of environmental modulation on prolonged cell-based catalysis, which is highly relevant to non-growing cells in nature and biotechnological applications.

The growth-survival trade-off is hard-wired in the Lactococcus lactis gene regulation network.

Most microbes reside in oligotrophic environments for extended periods of time, requiring survival strategies that maintain proliferative capacity. We demonstrate that the non-spore-forming Lactococcus lactis KF147 progressively activates the expression of stress-associated functions in response to the declining growth rate elicited by prolonged retentostat cultivation, which coincides with up to 104 -fold increased stress tolerance. Our findings provide a quantified view of the transcription and stress-tolerance adaptations underlying the growth-survival trade-off in L. lactis, and exemplify the hard-wiring of this trade-off in the lactococcal gene regulation network.

Assessing the Impact of Diet on the Mucosa-Adhered Microbiome in Piglets Using Comparative Analysis of Rectal Swabs and Colon Content.

Previously, we demonstrated that rectal swabs provide a legitimate alternative to faecal sampling for the assessment of the intestinal microbiota in young piglets. However, we also reported that mucosa-adhered microbial populations were more represented in rectal swabs compared to faecal samples, albeit to a degree that varied per swab-sample. Here, we explored the possibility to exploit this variable enrichment of adhered populations in the rectal swabs to assess the impact of diet on mucosa-adhered microbiota in pre-weaning piglets. Paired samples of rectal swabs and colon luminal contents were collected from piglets just before weaning during two independent but similarly designed animal experiments [n = 28 piglets (experiment 1); n = 16 piglets (experiment 2)], with an early feeding treatment (EF) group that had access to customised fibrous feed in addition to sow's milk and a control (CON) group exclusively reared on sow's milk. The intestinal microbiome composition in rectal swabs and colon samples collected at 29 days of age were subjected to metataxonomic analysis. The results identified the genera Escherichia-Shigella, Anaerococcus, Peptostreptococcus, Enterococcus, Trueperella, Actinomyces, and Peptoniphilus as discriminative taxa enriched in rectal swabs compared to colon. Apart from Escherichia-Shigella (10-11% average relative abundance), most of these mucosa-adhered microbial genera display relatively low abundance. Rectal swab microbiota was found to be more variable, which is likely due to variable enrichment of mucosa-adhered microbes. Although almost exclusively driven by one of the experiments, the post-weaning diarrhoea-associated taxa Escherichia-Shigella, was enriched in CON compared to the EF group, suggesting that early life feeding may suppress post-weaning-diarrhoea-related problems in piglets. Our findings demonstrate that rectal swabs allow the investigation of the mucosa-adhered microbial populations as a function of dietary treatment in piglets. This offers opportunities to further study dietary approaches that suppress the abundance of the post-weaning diarrhoea associated adherent microbes like Escherichia-Shigella. Furthermore, we demonstrate that the paired swab-colon microbiota information (obtained from a subset of animals) can predict the mucosa-adhered populations or "mucosity factor" in rectal swab samples, facilitating the analysis of the adhered microbiota in large animal cohort studies using readily obtainable rectal swabs.

Development of a reproducible small intestinal microbiota model and its integration into the SHIME®-system, a dynamic in vitro gut model.

The human gastrointestinal tract consists of different regions, each characterized by a distinct physiology, anatomy, and microbial community. While the colonic microbiota has received a lot of attention in recent research projects, little is known about the small intestinal microbiota and its interactions with ingested compounds, primarily due to the inaccessibility of this region in vivo. This study therefore aimed to develop and validate a dynamic, long-term simulation of the ileal microbiota using the SHIME®-technology. Essential parameters were identified and optimized from a screening experiment testing different inoculation strategies, nutritional media, and environmental parameters over an 18-day period. Subjecting a synthetic bacterial consortium to the selected conditions resulted in a stable microbiota that was representative in terms of abundance [8.81 ± 0.12 log (cells/ml)], composition and function. Indeed, the observed community mainly consisted of the genera Streptococcus, Veillonella, Enterococcus, Lactobacillus, and Clostridium (qPCR and 16S rRNA gene targeted Illumina sequencing), while nutrient administration boosted lactate production followed by cross-feeding interactions towards acetate and propionate. Furthermore, similarly as in vivo, bile salts were only partially deconjugated and only marginally converted into secondary bile salts. After confirming reproducibility of the small intestinal microbiota model, it was integrated into the established M-SHIME® where it further increased the compositional relevance of the colonic community. This long-term in vitro model provides a representative simulation of the ileal bacterial community, facilitating research of the ileum microbiota dynamics and activity when, for example, supplemented with microbial or diet components. Furthermore, integration of this present in vitro simulation increases the biological relevance of the current M-SHIME® technology.

Correlation between brain function and ADHD symptom changes in children with ADHD following a few-foods diet: an open-label intervention trial.

Research into the effect of nutrition on attention-deficit hyperactivity disorder (ADHD) in children has shown that the few-foods diet (FFD) substantially decreases ADHD symptoms in 60% of children. However, the underlying mechanism is unknown. In this open-label nutritional intervention study we investigated whether behavioural changes after following an FFD are associated with changes in brain function during inhibitory control in 79 boys with ADHD, aged 8-10 years. Parents completed the ADHD Rating Scale before (t1) and after the FFD (t2). Functional magnetic resonance imaging (fMRI) scans were acquired during a stop-signal task at t1 and t2, and initial subject-level analyses were done blinded for ARS scores. Fifty (63%) participants were diet responders, showing a decrease of ADHD symptoms of at least 40%. Fifty-three children had fMRI scans of sufficient quality for further analysis. Region-of-interest analyses demonstrated that brain activation in regions implicated in the stop-signal task was not associated with ADHD symptom change. However, whole-brain analyses revealed a correlation between ADHD symptom decrease and increased precuneus activation (pFWE(cluster) = 0.015 for StopSuccess > Go trials and pFWE(cluster) < 0.001 for StopSuccess > StopFail trials). These results provide evidence for a neurocognitive mechanism underlying the efficacy of a few-foods diet in children with ADHD.

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.

Colonizing Microbes, IL-10 and IL-22: Keeping the Peace at the Mucosal Surface.

Our world is filled with microbes. Each multicellular organism has developed ways to interact with this microbial environment. Microbes do not always pose a threat; they can contribute to many processes that benefit the host. Upon colonization both host and microbes adapt resulting in dynamic ecosystems in different host niches. Regulatory processes develop within the host to prevent overt inflammation to beneficial microbes, yet keeping the possibility to respond when pathogens attempt to adhere and invade tissues. This review will focus on microbial colonization and the early (innate) host immune response, with special emphasis on the microbiota-modifying roles of IL-10 and IL-22 in the intestine. IL-10 knock out mice show an altered microbial composition, and spontaneously develop enterocolitis over time. IL-22 knock out mice, although not developing enterocolitis spontaneously, also have an altered microbial composition and increase of epithelial-adherent bacteria, mainly caused by a decrease in mucin and anti-microbial peptide production. Recently interesting links have been found between the IL-10 and IL-22 pathways. While IL-22 can function as a regulatory cytokine at the mucosal surface, it also has inflammatory roles depending on the context. For example, lack of IL-22 in the IL-10-/- mice model prevents spontaneous colitis development. Additionally, the reduced microbial diversity observed in IL-10-/- mice was also reversed in IL-10/IL-22 double mutant mice (Gunasekera et al., 2020). Since in early life, host immunity develops in parallel and in interaction with colonizing microbes, there is a need for future studies that focus on the effect of the timing of colonization in relation to the developmental phase of the host. To illustrate this, examples from zebrafish research will be compared with studies performed in mammals. Since zebrafish develop from eggs and are directly exposed to the outside microbial world, timing of the development of host immunity and subsequent control of microbial composition, is different from mammals that develop in utero and only get exposed after birth. Likewise, colonization studies using adult germfree mice might yield different results from those using neonatal germfree mice. Lastly, special emphasis will be given to the need for host genotype and environmental (co-housing) control of experiments.

Complete Closed Genome Sequence of the Inulin-Utilizing Lactiplantibacillus plantarum Strain Lp900, Obtained Using a Hybrid Nanopore and Illumina Assembly.

Lactiplantibacillus plantarum is a genetically and phenotypically diverse lactic acid bacterium species. We announce the hybrid de novo assembly of Oxford Nanopore Technologies and Illumina DNA sequence reads, producing a closed circular chromosome of 3,206,992 bp and six plasmids of the inulin-utilizing L. plantarum strain Lp900.