Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Pro-biotics for a healthy gut
    Belzer, C. - \ 2019
    probiotics - intestinal microorganisms - health foods - nutrition and health
    Have you ever noticed the little bottles in your local grocery store that contain living bacteria? When a microorganims is still alive in food and after consumption can have health benefits, it is called “a probiotic”. In this video we will explain how probiotics can promote gut health.
    This lesson is part of the WageningenX MOOC called 'Nutrition and Health: Human Microbiome'
    Causality in microbiome research | WURcast
    Belzer, C. - \ 2019
    Wageningen :
    intestinal microorganisms - microbiology
    How bacteria can keep your gut healthy: beneficial microbes
    Belzer, C. - \ 2019
    Wageningen :
    microorganisms - intestinal microorganisms - metabolism - pathogens
    Interactions and functionalities of the gut revealed by computational approaches
    Benis, Nirupama - \ 2017
    Wageningen University. Promotor(en): M.A. Smits; V.A.P. Martins dos Santos, co-promotor(en): D. Schokker; M. Suarez-Diez. - Wageningen : Wageningen University - ISBN 9789463434546 - 247
    pigs - mice - digestive tract - digestive system - intestinal microorganisms - intestinal mucosa - computational science - immune system - feeds - animal nutrition - nutrition physiology - animal health - varkens - muizen - spijsverteringskanaal - spijsverteringsstelsel - darmmicro-organismen - darmslijmvlies - computational science - immuunsysteem - voer - diervoeding - voedingsfysiologie - diergezondheid

    The gastrointestinal tract is subject of much research for its role in an organism’s health owing to its role as gatekeeper. The tissue acts as a barrier to keep out harmful substances like pathogens and toxins while absorbing nutrients that arise from the digestion of dietary components in in the lumen. There is a large population of microbiota that plays an important role in the functioning of the gut. All these sub-systems of the gastrointestinal tract contribute to the normal functioning of the gut. Due to its various functionalities, the gut is able to respond to different types of stimuli and bring the system back to homeostasis after perturbations.

    The work done in this thesis uses several bioinformatic tools to improve our understanding of the functioning of the gut. This was achieved with data from model animals, mice and pigs which were subjected to changing environments before their gastrointestinal response was measured. Different types of stimuli were studied (eg, antibiotic exposure, changing diets and infection with pathogens) in order to understand the response of the gut to varying environments. This data was analysed using different data integration techniques that provide a holistic view of the gut response.

    Vertical data integration techniques look for associations between different types of ~omics data to highlight possible interactions between the measured variables. Lateral integration techniques allow the study of one type of ~omics data over several time points or several experimental conditions. Using these techniques, we show proof of interactions between different sub-systems of the gut and the functional plasticity of the gut. Of the several hypotheses generated in this thesis we have validated several using existing literature and one using an in-vitro system. Further validation of these hypotheses will increase understanding of the responses of the gut and the interactions involved.

    Mining the human intestinal microbiota for biomarkers associated with metabolic disorders
    Hermes, Gerben - \ 2016
    Wageningen University. Promotor(en): Hauke Smidt; Erwin Zoetendal. - Wageningen : Wageningen University - ISBN 9789462579514 - 205
    gastrointestinal microbiota - metabolic disorders - biomarkers - obesity - intestinal microorganisms - antibiotics - dna sequencing - rna - ribosomal rna - microbiota van het spijsverteringskanaal - stofwisselingsstoornissen - biomarkers - obesitas - darmmicro-organismen - antibiotica - dna-sequencing - rna - ribosomaal rna

    After birth, our gastrointestinal (GI) tract is colonized by a highly complex assemblage of microbes, collectively termed the GI microbiota, that develop intimate interactions with our body. Recent evidence indicates that the GI microbiota and its products may contribute to the development of obesity and related diseases. This, coupled with the current worldwide epidemic of obesity, has moved microbiome research into the spotlight of attention. Although the main cause of obesity and its associated metabolic complications is excess caloric intake compared with expenditure, differences in GI tract microbial ecology between individuals might be an important biomarker, mediator or even new therapeutic target. Nevertheless, it is currently still unclear which bacterial groups play a role in the development of the metabolic syndrome in humans. This might partly be explained by: 1. Biological factors such as the heterogeneity in genotype, lifestyle, diet; and the often complex aetiology of human disease of which the metabolic syndrome is no exception. 2. Technological factors, such as the use of miscellaneous incompatible methods to assess the gut microbiota, often enumerating specific groups rather than using broad 16S rRNA gene surveys or metagenomics. 3. Studies vary greatly in the populations considered, their designs, and the degree of control for potential confounding factors such as lifestyle and diet. Nevertheless, recent research on this matter has shown a conceptual shift by focusing on more homogenous subpopulations, based on stricter control over variables such age range or through the use of both anthropometric (weight, total body fat) as well as biochemical variables (insulin resistance, hyperlipidaemia) to define groups.

    Perturbations in microbial diversity and community structure in adults with overweight and obesity may be partly due to long-term dietary habits or physiological changes in these subjects. As such, exploring the association between the gut microbiota and variation in BMI and weight in early life, prior to or close to the onset of overweight, might provide additional insights into these processes. Therefore, we studied the fecal microbiota of 295 six-seven year old children from the KOALA Birth Cohort, living in the south of the Netherlands. This age range is relatively uncharted microbiota territory. We found that its composition seems to conform to tot same ecosystem rules as that of adults. The bimodal distribution pattern of several bacterial groups as well as their co-correlating groups that were reported previously, including Uncultured Clostridiales II (UCII), Prevotella spp. and Dialister were confirmed. Furthermore, one of the previously described bimodal groups (Uncultured Clostridiales I) was shown before to exhibit very clear shifting state probabilities associated with ageing, where the high abundance state was mainly observed above 40 years of age. This was corroborated as no support for bimodality of this group was observed in the children included in the study described here. A large part of the variation in microbiota composition was explained by the abundance of aforementioned groups in contrast to the anthropometric outcomes, suggesting that in this group of healthy children within a relatively normal weight range, weight and associated parameters were not major drivers of overall genus-level microbial composition or vice versa. Hereafter, multiple linear and logistic regression models with rigorous adjustment for confounders were applied to investigate individual microbiota features association with weight related anthropometric outcomes. Previously reported parameters such as diversity, richness and Bacteroidetes to Firmicutes ratio, were not significantly associated with any of the outcomes. Nevertheless, the abundance of several specific bacterial taxa; Akkermansia, Sutterella wadsworthia et rel. and Bryantella formatexigens et rel. and the dichotomous abundance state of the bi-modally distributed UCII was consistently associated with weight-related outcomes.

    Other biochemical features of the metabolic syndrome have been associated with the gut microbiome. Mainly rodent studies have indicated that antibiotic treatment may improve glucose homeostasis and metabolic impairments. Therefore, the effects of gut microbiota manipulation by antibiotics (7d administration of amoxicillin, vancomycin or a placebo) on tissue-specific insulin sensitivity, energy metabolism, gut permeability and inflammation in 57 obese, pre-diabetic men from the same geographical region, were investigated. Vancomycin decreased bacterial diversity and significantly reduced well known butyrate- producing Firmicutes from Clostridium clusters IV and XIVa and bacterial groups involved in bile acid metabolism. These changes occurred concomitantly with altered plasma and fecal concentrations of these metabolites. In adipose tissue, gene expression of oxidative pathways was upregulated by antibiotics, whereas immune-related pathways were downregulated by vancomycin. However, antibiotic treatment had no significant effects on tissue-specific insulin sensitivity, energy/substrate metabolism, postprandial hormones and metabolites, systemic inflammation, gut permeability and adipocyte size. Importantly, despite a still considerably altered microbial composition at eight weeks follow-up, energy harvesting, adipocyte size and whole-body insulin sensitivity (HOMA-IR) remained unaltered. Overall these data indicate that interference with adult microbiota by antibiotic treatment for 7 days had no clinically relevant impact on metabolic health in obese humans. These data are in contrast with several rodent studies as well as a human intervention. The present study, which was well-powered and placebo-controlled, indicates that the previously reported vancomycin-induced effects on human peripheral insulin sensitivity are probably of minor physiological significance.

    The aforementioned group that was relatively homogeneous with regards to phenotype was combined with another cohort with similar phenotypical characteristics (obese, male and pre-diabetic) from another region of the Netherlands, to investigate whether tissue specific insulin sensitivity, as measured by the golden standard hyperinsulinemic-euglycemic clamp technique, is related to a specific microbial pattern. Remarkably, despite the fact that both cohorts were constructed based on comparable recruitment strategies, the average microbiota composition in both cohorts showed pronounced differences. Firstly, we found no consistent and significant association between liver, adipose tissue or skeletal muscle insulin sensitivity and the microbiota in both cohorts. Nevertheless, Random Forests classifiers using microbiota composition as predictors revealed taxa associated with fasting glucose concentrations and HbAc1 but only in one cohort. The top microbial features distinguishing classes were different Proteobacteria and groups involved in butyrogenesis, such as Faecalibacterium prausnitzii, Roseburia intestinalis, and Eubacterium rectale and related species, for fasting glucose levels. For HbAc1 these taxa were Oscillospira guillermondii, Sporobacter termitidis, Lactobacillus gasseri and Peptococcus niger and related species. The striking cohort-specific observations suggest that the relation between microbiota composition and type 2 diabetes mellitus as well as other characteristics of the metabolic syndrome is very dependent on the selected cohort of patients and their respective baseline microbiota composition. Similar observations have been made by other researchers as well. It could be that differences in microbiota composition are not associated with the insulin resistance phenotype when the overweight and/or obese state of the patient is already established, as is the case for our metabolic syndrome patients. In the latter case we cannot exclude that the composition of the fecal microbiota may play a role in the worsening of insulin sensitivity in an early stage in the development from a lean towards an overweight/obese phenotype. Furthermore, the observation of a subgroup- specific microbiota only observed in one of the cohorts might indicate an alternative state of microbiota composition driven by yet unknown forces. Nevertheless, this study clearly demonstrated that cohort-specific microbiota differences hamper finding a consensus biological interpretation between cross-sectional studies. This, combined with the complexity of individual disease pathogenesis, as well as the individual-specific differences in microbiota composition, may explain the inconsistency in observations between different studies concerning the identification of signature microbes for obesity, irritable bowel syndrome and other diseases.

    Besides the biological drivers for cohort specific inconsistencies in identified microbial biomarkers, there are also technological factors. Although high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes, evidence is increasing that methodology rather than the biological variation is responsible for observed sample composition and distribution. Large meta-analyses would aid in elucidating whether the basis for these observed inconsistencies is biological, technical or maybe a combination of both. To facilitate these meta-analyses of microbiota studies we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different Mock Communities (MC). NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies. The analysis of α- and β-diversity of these MC confirmed conclusions guided by biology rather than the methodological aspects. This pipeline was applied to biological samples to monitor the developing communities an in vitro gut model (TIM-2) fed either with a normal diet, or modified versions from which the carbohydrate (MPLC) or protein fraction was diluted (LPMC) for 72h. In combination with global metatranscriptomics and metabolomics this revealed that each diet produced distinct microbial communities and temporal patterns and ratios of metabolites. The microbiota in reactors fed diets containing normal carbohydrate levels were enriched in members of the genera Prevotella, Subdoligranulum, Blautia and Bifidobacterium, all associated with carbohydrate fermentation. In turn, the microbiota in the reactors fed the MPLC diet, containing ten-fold less carbohydrates, was enriched in the genus Bacteroides, which is associated with diets rich in protein and animal fat. This setup allows researchers to study the (trophic) interactions and task division within a community and how they are impacted by diet-related factors under controlled conditions, which may assist in defining causal links between specific diet-derived parameters microbial groups and their activities.

    In conclusion, currently it seems that GI microbiota based biomarkers associated with metabolic impairments and anthropometric variables associated with the metabolic syndrome are cohort specific or possibly individual, which could partly be due to the use of incompatible analytical approaches. Nevertheless, there is growing evidence that human health is a collective property of the human body and its associated microbiome and thus requires to study the interface of two very complex systems, i.e. on one side the extraordinary coding capacity, high inter-individuality and complex dynamics of the microbiome and on the other side the multifactorial individual nature of human disease. In light of these observations the manifestation of individual dynamics of the microbiota with the host when homeostasis is lost seems plausible and likely.

    Interplay between gut microbiota and antibiotics
    Jesus Bello Gonzalez, Teresita de - \ 2016
    Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): M.W.J. van Passel. - Wageningen : Wageningen University - ISBN 9789463430043 - 293
    antibiotics - intestinal microorganisms - aminoglycoside antibiotics - enterococcus - interactions - zoo animals - man - patients - dna sequencing - polymerase chain reaction - antibiotica - darmmicro-organismen - aminoglycoside antibiotica - enterococcus - interacties - dierentuindieren - mens - patiënten - dna-sequencing - polymerase-kettingreactie

    The human body is colonized by a vast number of microorganisms collectively defined as the microbiota. In the gut, the microbiota has important roles in health and disease, and can serve as a host of antibiotic resistance genes. Disturbances in the ecological balance, e.g. by antibiotics, can affect the diversity and dynamics of the microbiota. The extent of the disturbance induced by antibiotics is influenced by, among other factors, the class of antibiotic, the dose, and administration route. One of the most common consequences of excessive antibiotic use is the emergence of antibiotic resistant bacteria and the dissemination of the corresponding resistance genes to other microbial inhabitants of the gut community, in addition to affecting the colonization resistance and promoting the overgrowth of pathogens. These effects are particularly relevant for Intensive Care Unit (ICU) patients, which are frequently exposed to a high risk of hospital-acquired infections associated with antibiotic resistant bacteria.

    Due to the important roles that members of the gut microbiota play in the host, including their role as potential hubs for the dissemination of antibiotic resistance, recent research has focused on determining the composition and function of gut microorganisms and the antibiotic resistance genes associated with them.

    The objectives of the research described in this thesis were to study the diversity and dynamics of the gut microbiota and resistome in ICU patients receiving antibiotic prophylactic therapy, and to assess the colonization dynamics with antibiotic resistant bacteria focusing on the commensal microbiota as a reservoir of antibiotic resistance genes by using culture dependent and independent techniques. Furthermore, the genetic background involved in the subsistence phenotype was investigated to disentangle the links between resistance and subsistence.

    Bacteria harbor antibiotic resistance genes that participate in a range of processes such as resisting the toxic effects of antibiotics, but could also aid in the utilization of antibiotics as sole carbon source, referred to as antibiotic subsistence phenotype. In chapter 2, the potential of gut bacteria from healthy human volunteers and zoo animals to subsist on antibiotics was investigated.

    Various gut isolates of Escherichia coli and Cellulosimicrobium spp. displayed the subsistence phenotype, mainly with aminoglycosides. Although no antibiotic degradation could be detected, the number of colony forming units increased during growth in medium with only the antibiotic as a carbon source. By using different approaches to study the aminoglycoside subsistence phenotype, we observed that laboratory strains carrying the aminoglycoside 3’phosphotransferase II gene also displayed the subsistence phenotype on aminoglycosides and that glycosyl-hydrolases seem to be involved in the subsistence phenotype. As the zoo animals for which the subsistence phenotype was investigated also included a number of non-human primates, the applicability of Human Intestinal Tract Chip (HITChip) to study the gut microbiota composition of these animals was assessed, including a comparison with healthy human volunteers (Chapter 3). It was concluded that the HITChip can be successfully applied to the gut microbiota of closely related hominids, and the microbiota dynamics can therefore be quickly assessed by the HITChip.

    In Chapter 4, a combination of 16S rRNA phylogenetic profiling using the HITChip and metagenomics sequencing was implemented on samples from a single ICU hospitalized patient that received antibiotic prophylactic therapy (Selective Digestive Decontamination - SDD). The different approaches showed a highly dynamic microbiota composition over time and the prevalence of aminoglycoside resistance genes harbored by a member of the commensal anaerobic microbiota, highlighting the role of the commensal microbiota as a reservoir of antibiotic resistance genes. As an extension of this study (Chapter 5), 11 ICU patients receiving SDD were followed using 10 healthy individuals as a control group to compare the diversity and dynamics of the gut microbiota and resistome by HITChip and nanolitre-scale quantitative PCRs, respectively. The microbial diversity of the healthy individuals was higher compared to ICU patients, and it was less dynamic compared to ICU patients under antibiotic treatment. Likewise, the levels of antibiotic resistance genes increased in ICU patients compared to healthy individuals, indicating that during ICU hospitalization and the SDD, gut microbiota diversity and dynamics are profoundly affected, including the selection of antibiotic resistance in anaerobic commensal bacteria.

    This was further expanded in an extensive study focusing on colonization dynamics with antibiotic resistant bacteria as described in Chapter 6. This was performed in the same group of ICU-hospitalized patients receiving SDD therapy and showed that by using a range of culture media and selective conditions a variety of taxonomic groups could be isolated, including aerobic and anaerobic antibiotic resistant bacteria. The overall composition of the faecal microbiota detected by HITChip indicated mainly a decrease of Enterobacteriaceae and an increase of the enterococcal population. Since critically ill patients are susceptible to hospital-acquired infections and the control of the emergence of antibiotic resistance is crucial to improve therapeutic outcomes, an extended analysis of the Enterococcus colonization dynamics in this group of patients by cultivation and phenotypic and genotypic characterization of the isolates provided new information about carriage of antibiotic resistance and virulence factor encoding genes (Chapter 7). It also highlighted the opportunity for the exchange of resistance and virulence genes, which could increase the risk of acquiring nosocomial infections.

    Next, chapter 8 described the implementation of high-throughput cultivation-based screening using the Microdish platform combined with high-throughput sequencing (MiSeq) using faecal samples from ICU patients receiving SDD. This allowed for the recovery of previously uncultivable bacteria, including a pure culture of a close relative of Sellimonas intestinalis BR72T that was isolated from media containing tobramycin, cefotaxime and polymyxin E. This strain could therefore represent a potential antibiotic resistance reservoir.

    In conclusion, this thesis provides broad insight into the diversity and dynamics of the gut microbiota and resistome in ICU hospitalized patients receiving SDD therapy as well as the dynamics of colonization with antibiotic resistant bacteria. Especially our extensive study of the colonization dynamics of Enterococcus spp. during ICU stay reinforced the notion that SDD therapy does not cover this group of bacteria and highlights the importance of a critical control of the emergence of antibiotic resistance in enterococci and their spread and dissemination as known potential pathogens.

    Furthermore, the extensive use of antibiotics could select for an increase in the rate of antibiotic resistance against aminoglycosides and beta-lactams, indicating that a control in the use of broad spectrum antibiotics needs to be considered. In addition, this thesis provides evidence regarding the possible genetic background involved in the subsistence phenotype, however, future studies on metabolic pathways could provide novel insight into the underlying mechanisms.

    Antibiotic resistance reservoirs : the cases of sponge and human gut microbiota
    Versluis, Dennis - \ 2016
    Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Mark van Passel; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462579057 - 197
    antibiotic resistance - reservoirs - intestinal microorganisms - luffa - forest soils - sediment - escherichia coli - penicillium - faecal examination - antibioticaresistentie - reservoirs - darmmicro-organismen - luffa - bosgronden - sediment - escherichia coli - penicillium - fecesonderzoek

    One of the major threats to human health in the 21st century is the emergence of pathogenic bacteria that are resistant to multiple antibiotics, thereby limiting treatment options. An important route through which pathogens become resistant is via acquisition of resistance genes from environmental and human-associated bacteria. Yet, it is poorly understood to what extent and by what mechanisms these so-called reservoirs contribute to emerging resistance. Therefore, the work described in this thesis focussed on generating novel insights into different niches as sources of resistance, with a particular focus on the human gut microbiota as well as on microbial communities associated with marine sponges, especially because the latter have been described as one of the richest sources of bioactive secondary metabolites, including a broad range of antimicrobials. Cultivation-based methods were complemented with culture-independent approaches in order to study bacterial taxa that are not readily cultivated.

    Using metatranscriptomics it was found that clinically relevant antibiotic resistance genes are expressed in a broad range of environmental niches including human, mouse and pig gut microbiota, sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment. The diversity of resistance gene transcripts differed greatly per niche indicating that the environment contains a rich reservoir of functional resistance that could be accessible by pathogens. Even though resistance gene expression might be linked to the presence of natural antibiotics, we did not detect expression of the corresponding secondary metabolite biosynthesis clusters.

    Thirty-one antibiotic-resistant bacteria, amongst which three belonging to potentially novel Flavobacteriaceae spp., were isolated from the Mediterranean sponges Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis. Isolates were identified in a high throughput manner by double-barcoded 16S rRNA gene amplicon sequencing. Furthermore, analysis of sponge tissue-derived bacterial biomass growing on agar media showed that many novel bacterial taxa can still be isolated by conventional cultivation methods. Genomic DNA from the 31 antibiotic resistant bacteria was interrogated with respect to the presence of active resistance genes by functional metagenomics. In addition, we also screened metagenomic libraries prepared from DNA directly isolated from sponge tissue in order to circumvent the need for cultivation. In total, 37 unique resistance genes were identified, and the predicted gene products of 15 of these shared <90% amino acid identity with known gene products. One resistance gene (blaPSV-1), which was classified into a new β-lactamase family, was found to be exclusive to the marine specific genus Pseudovibrio. These findings raised questions as to the functional roles of these genes in sponges, but more importantly, the functionality of these genes in E. coli shows that they can potentially be harnessed by phylogenetically distinct bacteria in other environments, including human pathogens. As such, it is a wake-up call as to the significance of marine resistance reservoirs.

    Pseudovibrio, a genus of α-Proteobacteria, was studied in more detail by comparative genomics as it comprises bacteria that potentially play a role as sponge symbionts and marine hubs of antibiotics resistance. Based on gene content, members of the genus Pseudovibrio were found to cluster by sponge sampling location indicating geographic speciation. Furthermore, Pseudovibrio spp. isolated from sponges near the Spanish coast clustered by sponge, suggesting host-specific colonization or adaptation. Strong support for Pseudovibrio spp. forming symbiotic relations with sponges came from the presence of a plethora of (predicted) conserved symbiosis-related functions in their genomes.

    A final study aimed to isolate novel antibiotic resistant reservoir species from the human gut microbiota using a targeted approach. Faecal samples from hospitalized patients that received Selective Digestive Decontamination (SDD), a prophylactic treatment with a cocktail of different antibiotics (tobramycin, polymyxin E, amphotericin B and cefotaxime), were inoculated anaerobically on agar media, after which bacterial biomass was analysed by 16S rRNA gene amplicon sequencing. Six novel taxa were identified that, based on their growth on media supplemented with the SDD antibiotics, could serve as clinically relevant reservoirs of antibiotic resistance. For one of these six taxa a member was obtained in pure culture by targeted isolation. The abundance of antibiotic resistant uncultivated taxa in the human gut microbiota warrants further research as to their potential roles in resistance dissemination.

    In conclusion, this thesis provides deeper insights into different environmental niches as reservoirs of antibiotic resistance. The results can serve to prime and inspire future research.

    Microbial interactions in the fish gut
    Giatsis, Christos - \ 2016
    Wageningen University. Promotor(en): Johan Verreth, co-promotor(en): Marc Verdegem; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462578777 - 196
    fishes - tilapia - larvae - microbial interactions - intestinal microorganisms - intestines - dynamics - fish feeding - probiotics - fish culture - aquaculture - vissen - tilapia - larven - microbiële interacties - darmmicro-organismen - darmen - dynamica - visvoeding - probiotica - visteelt - aquacultuur

    Aquaculture has realized considerable growth over the past years while the world demand on seafood has been increasing. As aquaculture intensifies, the production sector needs to tackle major bottlenecks such as suboptimal growth and high and unpredictable mortality, especially in larval cultures. Fish-microbe interactions are closely related to overall fish health. To obtain a healthy and resilient microbial community (MC), it is important to understand the underlying mechanisms of microbial colonization in the fish gut.

    The goal of this thesis was to investigate the role of water and feed microbial communities on shaping gut communities during early development of Nile tilapia.

    To determine the contribution of stochasticity to overall variation, we first characterized the spatio-temporal variation in MC composition between individuals reared within the same or in replicate recirculating or active suspension systems (RAS vs. AS). Highly similar MCs developed in the gut when larvae shared the same water and diet. Rearing larvae in replicate production systems resulted in significantly different gut communities indicating that compositional replication of the MCs of an ecosystem is not fully predictable. We found that mainly water MCs, and to a lesser degree feed MCs, were associated with changes in MCs. Thus, we could conclude that steering gut MCs can be possible through water MC management tailored on the specifications of the rearing system in use.

    Next, the possibility of early life steering of gut communities via microbial manipulations of feed MCs was explored. We hypothesized that gut microbial composition is strongly shaped by selective pressures in the gut and by the MCs present in the water. Thus similar MCs should develop between treatments regardless of the dietary treatments. Fish larvae were fed either a control feed or the control feed containing MCs derived from aerobic, methanogenic or denitrifying sludge reactors. We found that gut microbiota shared a much higher number of operational taxonomic units (OTUs) with microbiota in sludge-based feeds than with water, resulting in distinct gut MCs between treatments. Our findings suggest that Nile tilapia gut MC has a certain plasticity, which makes it amenable to interventions through proper feed microbial management.

    Subsequently, we tested the imprinting effect of early exposure to the probiotic Bacillus subtilis on shaping gut MC composition even after the administration of the probiotic discontinues. For this, we constrained the initial contact with microbes from the environment by producing axenic tilapia larvae, which were then exposed to normal husbandry conditions. Early life probiotic exposure affected gut MC composition during B. subtilis administration but also within the first two weeks after its administration stopped, thus indicating that early exposure to the probiotic strain via the water had a sustained impact on gut MC composition.

    Finally, overall conclusions and practical implications of our results for aquaculture production were presented. A meta-analysis was also performed to examine (1) the phylogenetic similarity among gut MCs of the same and different fish species reared in different habitats, fed different diets and at different developmental stages and (2) the factors primarily shaping gut MCs. We showed that the selective pressure responsible in shaping gut MC composition highly depends on the host as gut communities clustered primarily together by host and to a lesser extent reflected differences in habitat and diet. The phylogenetic analysis of gut communities revealed a clear clustering by study thus indicating that manipulation of gut communities is conceivable. Study-to-study variation could be attributed to the methodology used for MC analysis highlighting also the importance of methodological uniformity when comparisons between studies are made.

    Overall, this thesis provided fundamental knowledge on MC composition and development in aquaculture rearing systems. Although the insights generated by this thesis are still premature to fully explain, predict or steer MC composition, and though additional studies are needed, we believe that, in the long run, this approach will facilitate the development of safe and effective methods for manipulating gut microbial composition to promote fish health in aquaculture rearing systems.

    Phenotypic and genetic diversity of the species Lactobacillus rhamnosus
    Ceapa, C.D. - \ 2016
    Wageningen University. Promotor(en): Michiel Kleerebezem; Jan Knol; J. Lambert. - Wageningen : Wageningen University - ISBN 9789462576285 - 195
    fermentation products - probiotics - intestinal microorganisms - lactic acid bacteria - strains - medicinal properties - genomics - nutrition and health - fermentatieproducten - probiotica - darmmicro-organismen - melkzuurbacteriën - stammen (biologisch) - medicinale eigenschappen - genomica - voeding en gezondheid

    The thesis explores the diversity of Lactobacillus rhamnosus, a species from which strains are studied for their anti-inflammatory, anti-allergic, and diarrhea preventing effects. The work combines observations on the behavior of the bacteria in a simplified laboratory setting (use of carbohydrates, immune modulation effects, anti-pathogenic effects) with genomic information obtained by sequencing, with the aim to pinpoint genes that could be relevant for bacterial survival and metabolic capacities. Phenotypic and genotypic profiling analyses congruently revealed that carbohydrate metabolism and transport is essential for this species’ adaptation to the environment. Genotype–phenotype correlation analysis enabled us to predict and then experimentally verify genes responsible for the utilization of L-Sorbose, L-Fucose α-D-Methyl Glycoside.

    Effect of fructooligosaccharides on gut health in neonatal piglets : VDI-3 Piglet experimen
    Schokker, D. ; Jansen, R. ; Jansman, A.J.M. ; Vastenbouw, S. ; Bree, F.M. de; Bossers, A. ; Rebel, J.M.J. ; Smits, M.A. - \ 2015
    Wageningen : Wageningen UR Livestock Research (Livestock Research report 913) - 39
    piglets - intestines - animal health - oligosaccharides - intestinal microorganisms - immunology - biggen - darmen - diergezondheid - oligosacchariden - darmmicro-organismen - immunologie
    Gut microbial colonization and immune competence development are affected by early-life environmental and dietary interventions. The interplay between microbiota in the intestinal tract and the gut mucosal surfaces of the host is critical for the development of an accurate immune competence. In the present study we intervened during early life of suckling piglets by a daily oral administration of fructooligosaccharides (FOS solution) from day 2 – 14 and investigated the effects on intestinal microbiota composition (by 16S rDNA sequencing) and biological processes of the intestinal mucosal tissue (by genome-wide intestinal gene expression analysis) during the suckling phase.
    Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila
    Ottman, N.A. - \ 2015
    Wageningen University. Promotor(en): Willem de Vos; Hauke Smidt, co-promotor(en): Clara Belzer. - Wageningen : Wageningen University - ISBN 9789462574564 - 208
    akkermansia muciniphila - akkermansia - microbiota van het spijsverteringskanaal - darmmicro-organismen - probiotica - immunostimulerende eigenschappen - immunostimulerende middelen - moedermelk - metabolische studies - akkermansia muciniphila - akkermansia - gastrointestinal microbiota - intestinal microorganisms - probiotics - immunostimulatory properties - immunostimulants - human milk - metabolic studies

    Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila

    Noora A. Ottman

    The human gastrointestinal tract is colonized by a complex community of micro-organisms, the gut microbiota. The majority of these are bacteria, which perform various functions involved in host energy metabolism and immune system stimulation. The field of gut microbiology is continuously expanding as novel species are isolated and high-throughput techniques are developed. The research focus is shifting from DNA-based techniques, looking at microbial community composition, to techniques relying on analysis of RNA and proteins, which reveal more about the activity and functionality of the microbiota.

    The mucosa-associated microbiota forms a distinct population in the gut, and is influenced by the close proximity of the epithelial layer and nutrients present in the mucus layer. One of the key players in this community is the mucus degrader Akkermansia muciniphila. This Gram-negative, anaerobic bacterium can use mucin, the main component of mucus, as the sole carbon and nitrogen source for growth. A. muciniphila belongs to the phylum Verrucomicrobia and is present in the majority of humans, starting from early life. Interestingly, the levels of A. muciniphila are negatively correlated with several disorders, including inflammatory bowel diseases and diabetes. A. muciniphila lives in a symbiosis with its host, harvesting energy from mucin; whether the relationship is mutualistic, and thereby also beneficial to the host, remains to be discovered. In this thesis, the ability of A. muciniphila to utilize the host-derived glycans mucin and human milk oligosaccharides was studied in detail. In addition, the host-bacterial interactions were examined by immunological assays, focusing especially on the effect of A. muciniphila outer membrane proteins on host immune response.

    The genome of A. muciniphila encodes numerous enzymes involved in mucin degradation. Transcriptome analysis comparing the gene expression of A. muciniphila grown on mucin or the non-mucin sugar glucose confirmed the activity of these genes and revealed most of them to be upregulated in the presence of mucin. This was also confirmed by a proteome analysis, reinforcing the adaptation of A. muciniphila to the mucosal environment. A genome-based metabolic model was constructed to test amino acid auxotrophy, vitamin biosynthesis, and sugar-degrading capacities of A. muciniphila. The model predicted A. muciniphila to be able to synthesize all the essential amino acids, with the exception of threonine, which was added to the mucin-free medium designed to test A. muciniphila growth on single sugars. A. muciniphila was able to individually metabolize all the main monomeric sugars present in mucin, albeit with limited efficiency in comparison to mucin. As mucin shares structural similarities with human milk oligosaccharides (HMOs), which stimulate the bacterial community colonizing the gut in early life, growth of A. muciniphila on human milk and its components was tested. A. muciniphila showed metabolic activity on human milk and one of the HMOs, 2’-fucosyllactose. Comparison of A. muciniphila activity during growth on human milk or mucin revealed that the expression of genes involved in mucin degradation was similar for both experimental conditions, suggesting that A. muciniphila might be capable of also using the corresponding gene products for utilization of human milk glycans. The capacity to survive in the early life environment by degrading and consuming human milk components would be beneficial for A. muciniphila during initial colonization before reaching the mucosal layer in the intestine.

    Several mouse studies have indicated that A. muciniphila is able to modulate the host immune system, possibly to the benefit of the host, but not much is known about its immunological mechanism of action. The cell envelope structures of bacteria can have a big influence on their immunostimulatory capacities, and therefore the outer membrane (OM) proteome of A. muciniphila was characterized. The membrane structure of A. muciniphila is also of interest because it belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum, which contains bacteria with features that differentiate them from classical Gram-negative bacteria, including a complex endomembrane system. Mass spectrometry data, coupled with bioinformatics analysis, revealed the presence of highly abundant OM proteins involved in secretion, transport and biogenesis of the Gram-negative membranes, as well as proteins predicted to take part in formation of the fimbriae-like structures observed in A. muciniphila by electron microscopy. Live A. muciniphila and the identified OM proteins induced production of a wide range of cytokines and activated the intestinal Toll-like receptors 2 and 4. Moreover, a 30 kDa protein that was predicted to form a part of the fimbriae, increased transepithelial resistance, indicating it may be involved in improving gut barrier function.

    Based on the evidence from in vitro and in vivo studies, A. muciniphila is a promising candidate for a next-generation probiotic. However, further confirmation of causal relationships between disease development and presence of this species in the gut is required. The findings of this thesis provide valuable insight into the bacterial lifestyle and host interactions of the gut symbiont A. muciniphila.

    Impact on gut development of an early life oral antibiotic intervention in broilers
    Schokker, D. ; Jansman, A.J.M. ; Bruin, N. de; Vastenhouw, S.A. ; Bree, F.M. de; Rebel, J.M.J. ; Smits, M.A. - \ 2015
    Wageningen : Wageningen UR Livestock Research (Livestock Research report 859) - 30
    vleeskuikens - antibiotica - adequate immuniteit - immuunsysteem - darmmicro-organismen - microbiota van het spijsverteringskanaal - dierziektepreventie - diergezondheid - pluimveehouderij - broilers - antibiotics - immune competence - immune system - intestinal microorganisms - gastrointestinal microbiota - animal disease prevention - animal health - poultry farming
    The current report describes an experiment that was conducted to investigate the effect of a shortterm antibiotic intervention in early life on microbial colonization and gut development in broilers. Such an intervention is expected to have negative effects on immune competence. This experiment was performed within the frame work of the Feed4Foodure program line “Nutrition, Intestinal Health, and Immunity” in combination with partners from Breed4Food.
    The genus Romboutsia : genomic and functional characterization of novel bacteria dedicated to life in the intestinal tract
    Gerritsen, J. - \ 2015
    Wageningen University. Promotor(en): Hauke Smidt; Willem de Vos, co-promotor(en): G.T. Rijkers. - Wageningen : Wageningen University - ISBN 9789462572423 - 280
    darmmicro-organismen - voeding en gezondheid - microbiota van het spijsverteringskanaal - darmziekten - moleculaire technieken - probiotica - intestinal microorganisms - nutrition and health - gastrointestinal microbiota - intestinal diseases - molecular techniques - probiotics
    The genus Romboutsia: genomic and functional characterization of novel bacteria dedicated to life in the intestinal tract

    PhD thesis Jacoline Gerritsen, 2015

    Abstract

    Humans, like other mammals, are not single-species organisms, but they constitute in fact very complex ecosystems. The extensive network of host-microbe and microbe-microbe interactions is tremendously important for our health, and we are just starting to unravel the mechanisms by which microbes contribute to host health and disease.

    Especially the intestinal tract of both humans and mammals contains an enormous diversity of microbial species of which many still remain to be cultured and characterized. There are numerous diseases for which aberrations in composition and diversity of the intestinal microbiota have been reported. Probiotic microorganism defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” have the potential to modulate the intestinal microbiota and thereby contribute to health and well-being. To this end, the relative abundance of a specific bacterial phylotype, named CRIB, was found to be associated with probiotic-induced changes in gut microbiota and decreased severity of pancreatitis and associated sepsis in an experimental rat model for acute pancreatitis studies. Later, a representative of this phylotype (strain CRIB) was isolated, and characterized using a polyphasic taxonomic approach. The taxonomy of several closely related members of the family Peptostreptococcaceae was revised in order to provide a valid systematic name to the isolate, for which Romboutsia ilealis was chosen. It was found that the majority of Romboutsia-associated 16S rRNA gene sequences have an intestinal origin, however, the specific roles that Romboutsia species play in the intestinal tract are largely unknown. To gain more insight in metabolic and functional capabilities of members of the genus Romboutsia, efforts towards the isolation of additional representatives were undertaken. This ultimately led to the isolation of a human small intestine-derived representative (strain FRIFI) of another novel Romboutsia species which was given the name R. hominis. Characterization of both novel species of intestinal origin, i.e. R. ilealis and R. hominis, belonging to the genus Romboutsia at the genomic and functional level provided first insights into the genetic diversity within the genus Romboutsia and their adaptation to a life in the (upper) intestinal tract. To this end, Romboutsia species are flexible anaerobes that are adapted to a nutrient-rich environment in which carbohydrates and exogenous sources of amino acids and vitamins are abundantly available.

    Microbiomic approaches such as those employed in this study can be used to pinpoint specific commensal microbes that might have a beneficial effect on the health of the host. In addition, the combination of genomic and functional analyses with single organisms and complex communities can be used to identify microbial functionalities that are related to health and disease, which in turn can be used to select potential probiotic strains based on specific functional properties. Ultimately, these approaches will lead to the characterization of (new) beneficial commensal microbes that exert health-promoting effects, with the ultimate possibility for them to be exploited as next-generation probiotics.

    Development of gut microbiota in pigs and the effect of diet, antibiotics and other environmental factors
    Zhang, J. - \ 2014
    Wageningen University. Promotor(en): Hauke Smidt; Willem de Vos. - Wageningen : Wageningen University - ISBN 9789462570924 - 245
    varkens - darmmicro-organismen - microbiota van het spijsverteringskanaal - antibiotica - dieet - microbiologie - pigs - intestinal microorganisms - gastrointestinal microbiota - antibiotics - diet - microbiology

    The intestinal tract of humans and animals is colonized by trillions of microorganisms that constitute a community or ecosystem known as the gut microbiota. The gut microbiota undergoes remarkable alterations during early age, reaches a relative stable state in adulthood, and is driven by internal and external factors such as genotype of the host, diet and antibiotics. The objective of this research was to determine the effects of antibiotic treatment, microbial exposure and diet on the development of intestinal microbiota, focusing on the pig as an important production animal as well as a model for human. To achieve this objective, a series of intervention experiments were performed both in piglets and adult pigs.

    To determine the impact of antibiotic treatment on the development of intestinal microbiota of piglets, two experiments were performed. The first experiment aimed to determine the effect of perinatal maternal antibiotic treatment on the intestinal microbiota of piglets. In this experiment, the sows received amoxicillin orally around parturition, and their offspring was serially sacrificed up to 42 days of age for analysis of ileal and colonic microbiota. It was observed that amoxicillin treatment drastically impacted the sows’ faecal microbiota, and furthermore influenced specific microbial groups in the ileum and colon of the piglets before and after weaning. These findings indicated that maternal amoxicillin treatment may indirectly affect the gut microbiota of offspring through disturbing the maternal microbiota and the transfer of maternal microbiota to the offspring. In a second experiment, we determined the effect of early antibiotic treatment on intestinal microbial colonization and immune development of piglets. Additionally, the effect of stress factors associated with routine farm practice was investigated. Antibiotic treatment affected the composition and diversity of jejunal microbiota, and reduced the expression of a large number of genes involved in immune-related processes. The cumulative effect of management procedures on top of the use of an antibiotic was limited. This study reinforced the notion that the early phase of life is critical for intestinal immune development, also under regular production circumstances.

    Apart from antibiotic treatment, the effect of early microbial association on the development of intestinal microbiota and immune system of piglets was also studied in this thesis. One group of caesarean derived piglets was inoculated with a mixture of three microbial species (Lactobacillus amylovorus, Clostridium glycolicum, and Parabacteroides sp. ASF519) at day 1 and 2 after birth (the simple microbial association group), whereas a second group of piglets was inoculated with the above mixture at day 1 and 2 after birth as well as diluted adult sow faeces at day 3 and 4 after birth as the complex microbial association (CA) group. CA caused an increase of faecal microbial diversity and accelerated the faecal microbiota to develop into a stable and diverse microbiota. CA significantly affected luminal microbial composition and gene expression in jejunal and ileal mucosa, albeit in different ways. In the pig ileum, CA led to an increased relative abundance of microbial groups known to have beneficial effects, whereas it reduced the relative contribution of potential pathobionts. CA also induced the enrichment of immune-related gene sets in the ileal mucosa.

    Another research goal of this thesis was to determine the influence of diet on the microbiota in the large intestine of adult pigs. To this end, the effect of resistance starch (RS) was evaluated in two studies. In the first study, pigs were either assigned to an RS diet or a digestible starch (DS) diet for two weeks. Samples from along the intestine were collected for measuring luminal microbiota composition, short chain fatty acid (SCFA) concentrations and the expression of host genes involved in SCFA uptake, SCFA signalling, and satiety regulation in mucosal tissue. In both the caecum and colon, differences in microbiota composition and SCFA concentrations were observed between DS- and RS-fed pigs. Caecal tissue expression of genes encoding monocarboxylate transporter 1 and glucagon was induced by RS. Based on these results, an additional experiment was performed. In this study, ten pigs, fitted with a cannula in the proximal colon for repeated collection of tissue biopsies and luminal content, were fed a DS diet, or a diet high in RS (34%) for two consecutive periods of 14 days in a crossover design. RS increased the relative abundance of several butyrate-producing microbial groups and reduced that of potentially pathogenic members of the genus Leptospira and the phylum of Proteobacteria. Concentrations of acetate, propionate and butyrate in carotid plasma were significantly higher after RS consumption. Upon RS feeding, oxidative metabolic pathways, such as TCA cycle and beta-oxidation, were induced, whereas many immune response pathways, including adaptive and innate immune system, as well as cell division were suppressed. The nuclear receptor PPARG was identified as a potential key upstream regulator.

    In conclusion, this thesis provides direct evidence that maternal antibiotic treatment, early antibiotic admistration and microbial exposure affect the development of intestinal microbiota of the piglets. Moreover, both early antibiotic admistration and microbial exposure affected piglet mucosal tissue gene expression. These findings reinforce the notion that the early phase of life is critical for the development of intestinal microbiota and immune system. Furthermore, it is proposed that manipulation of the microbial association at early age may be a way of supporting functional gut development. In addition to the above discussed early life envents, a diet with RS can also affect the microbiota in the large intestine of adult pigs. This thesis provides an enhanced understanding of the interaction between diet, microbiota and host in a number of complementary pig models and revealed the impact of antibiotics in early life microbial colonization. The gained insight is expected to be instrumental in improving sustainable pig management. Moreover, it may also be useful in understanding similar processes in the human gut.

    Galacto-oligosaccharides to counter the side effects of antibiotic treatments
    Ladirat, S.E. - \ 2014
    Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789461738394 - 160
    oligosacchariden - darmmicro-organismen - bifidobacterium longum - antibiotica - prebiotica - amoxicilline - oligosaccharides - intestinal microorganisms - bifidobacterium longum - antibiotics - prebiotics - amoxicillin

    Antibiotic treatments are known to disturb the composition and metabolic activity of the human gut microbiota and, therefore, may lead to gut disorders. In this thesis, it was investigated whether and by which mechanisms galacto-oligosaccharides (GOS), a prebiotic known to stimulate the growth of bifidobacteria and to positively influence human health, may counter the negative effects of antibiotics on the microbiota.

    First, a high throughput approach combining the in vitro fermentation screening platform with a phylogenetic microarray read-outs was shown to be reliable to simultaneously analyse the effects of several often-used antibiotics on the intestinal microbiota. Then, using the same approach, the recovery of the composition and metabolic activity of the microbiota treated with four selected antibiotics upon GOS addition was shown to be antibiotic and dose dependant. The addition of GOS to an amoxicillin (AMX)-treated microbiota was considered successful as, after a decrease of the levelofBifidobacterium species, the recovery of mainly Bifidobacterium longum, was observed. The growth of bifidobacteria and the production of the beneficial butyrate tended to be higher upon addition of small GOS (dimers-trimers) than upon large GOS in non-treated microbiota (tetramers to hexamers). On the contrary in AMX-treated microbiota, the growth of bifidobacteria and production of butyrate tented to be higher upon addition of large GOS than upon addition of small GOS. The positive results of GOS on AMX-treated microbiota during in vitro experiments were evidenced in a double-blind randomized parallel intervention study involving 12 healthy adults.

    Overall, the addition of GOS, especially the large oligosaccharides, allowed the recovery of B. longum and, subsequently, stimulated the activity of the microbiota through cross-feeding after an AMX treatment.

    Darmbacteriën vormen de mens
    Vos, Willem de - \ 2013
    intestinal microorganisms - gastrointestinal microbiota - microbiology - health - bacteria - babies
    Community and genomic analysis of the human small intestine microbiota
    Bogert, B. van den - \ 2013
    Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Erwin Zoetendal. - S.l. : s.n. - ISBN 9789461736628 - 214
    darmmicro-organismen - dunne darm - dna microarrays - dna-sequencing - microbiële ecologie - immunomodulerende eigenschappen - intestinal microorganisms - small intestine - dna microarrays - dna sequencing - microbial ecology - immunomodulatory properties

    Our intestinal tract is densely populated by different microbes, collectively called microbiota, of which the majority are bacteria. Research focusing on the intestinal microbiota often use fecal samples as a representative of the bacteria that inhabit the end of the large intestine. These studies revealed that the intestinal bacteria contribute to our health, which has stimulated the interest in understanding their dynamics and activities. However, bacterial communities in fecal samples are different compared to microbial communities at other locations in the intestinal tract, such as the small intestine. Despite that the small intestine is the first region where our food and intestinal microbiota meet, we know little about the bacteria in the small intestine and how they influence our overall well-being. This is mainly attributable to difficulties in obtaining samples with the small intestine being located between the stomach and the large intestine. Therefore, the work in this thesis aimed at providing a better understanding of the composition and dynamics of the human small intestinal microbiota and to provide insight in the metabolic potential as well as immunomodulatory properties of some of its typical commensal inhabitants. Small intestinal samples used in the work described in this thesis were collected from ileostomy subjects, individuals that had their large intestine surgically removed and the end of the small intestine connected to an abdominal stoma, providing access to luminal content of the small intestine.

    Considering the importance of molecular techniques in contemporary ecological surveys of microbial communities, first of all, two technologies, barcoded pyrosequencing and phylogenetic microarray analysis were compared in terms of their capacity to determine the bacterial composition in fecal and small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, the use of different primer pairs in the amplification step was assessed in terms of its impact on the outcome of microbial profiling. The analyses revealed that the different primer pairs and the two profiling technologies provide overall similar results for samples of fecal and terminal ileum origin. In contrast, the microbial profiles obtained for small intestinal samples by barcoded pyrosequencing and phylogenetic microarray analysesdiffered considerably. This is most likely attributable to the constraints that are intrinsic to the use of the microarray to enable the detection of predefined microbiota members only, which is due to the probe design that is largely based on large intestinal microbiota communities. However, the pyrosequencing technology also allows for identification of bacteria that were not in advance known to inhabit our intestinal tract.

    The pyrosequencing technology was used as the method of choice to study the total and active small intestinal communities in ileostoma effluent samples from four different subjects through sequencing the 16S ribosomal RNA gene (rDNA) and ribosomal RNA (rRNA) contentcombined with metatranscriptome analysis by Illumina sequencing of cDNA derived from enriched mRNAof the same sample set to investigate the activities of the small intestinal bacteria. The composition of the small intestinal bacterial communities as assessed from rDNA, rRNA, and mRNA patterns appeared to be similar, indicating that the dominant bacteria in the small intestine are also highly active in this ecosystem. Streptococcusspp. were among the bacterial species that were detected in each ileostoma effluent sample, albeit that their abundance varied greatly between samples from the same subject as well as samples from different subjects. Veillonellaspp. frequently co-occurred with Streptococcus spp., indicating that the Streptococcusand Veillonellapopulations play a prominent role in the human small intestine ecosystem and their co-occurrence suggests a metabolic relation between these genera.

    Therefore, cultivation and molecular typing methodologies were employed to zoom-in on these groups, which revealed that the richness of the small intestinal streptococci strongly exceeded the diversity that could be estimated on basis of 16S rRNA analyses, and could be extended to the genomic lineage level (anticipated to resemble strain-level). From ileostoma samples 3 different Streptococcusspecies were recovered belonging to the S. mitisgroup, S. bovisgroup, and S. salivariusgroup, which could be further divided in 7 genomic lineages. Notably, the Streptococcuslineages that were isolated displayed distinct carbohydrate utilization capacities, which may imply that their growth and relative community composition may respond quite strongly to differences in the dietary intake of simple carbohydrates over time. This notion is in good agreement with the observation that the Streptococcuslineage populations fluctuated in time with only one Streptococcuslineage being cultivated from both ileostoma samples collected in a one-year time frame. Conversely, the cultivated Veillonellaisolates from samples during that same time-interval consistently encompassed a single lineage. Furthermore, this Veillonellalineage could be isolated from both the oral cavity as well as the ileostoma effluent. Analogously, three Streptococcuslineages that belong to a single phylotype also appeared to be present in bacterial communities from the oral cavity as well as the small intestine. These observations suggest the representatives of the Veillonellaand Streptococcusgenera that are encountered in the oral and small intestinal microbial ecosystems are closely related and indicate that the oral microbiota may serve as an inoculum for the upper GI tract.

    The metabolic capacity of 6 small intestinal Streptococcus lineages, that were obtained from a single ileostoma effluent sample, was further investigated through the determination of genomic sequences of these lineages. The small-intestinal Streptococcusgenomes were found to encode different carbohydrate transporters and the necessary enzymes to metabolize different sugars, which was in excellent agreement with what carbohydrates could be used by representative strains of the Streptococcuslineages.

    To further our understanding how the different streptococci as representatives of the dominant small intestinal bacterial populations may influence our immune system, human dendritic cells were stimulated with strains of the different Streptococcuslineages to study their immunomodulatory properties. The Streptococcuslineages differed significantly in their capacity to modulate cytokine responses of blood-monocyte derived immature dendritic cells. As Streptococcusand Veillonellafrequently co-occur in the small intestinal ecosystem, pair-wise combinations of strains of these two species were also tested for their combined immunomodulatory properties. This resulted in considerably different cytokine responses as those that could be predicted from the stimulations with either Streptococcusor Veillonella, indicating that it is not trivial to predict gut mucosal associated immune responses and thatthe composition of the intestinal microbiota as a whole may have a distinct influence on an individual’s immune status.

    In conclusion, the work described this thesis provides an expansion to the accumulating knowledge on the human intestine microbiota. Whereas most studies focus on the microbiota present in the distal regions of the intestinal tract, this study targeted the microbiota of the poorly proximal regions of the intestine and also addressed its capacity to interact with the local mucosal tissue. The data presented here can be exploited to guide the design of future studies that aim to elucidate the interplay between diet, microbiota and the mucosal tissues in the human small intestinal tract.

    Molecular effects of fermentation in the gut and its relevance for metabolism and satiety
    Haenen, D. - \ 2013
    Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Guido Hooiveld; Bas Kemp. - S.l. : s.n. - ISBN 9789461736673 - 210
    dikke darm - fermentatie - voedingsvezels - verzadigdheid - darmmicro-organismen - vetzuren met een korte keten - metabolisme - large intestine - fermentation - dietary fibres - satiety - intestinal microorganisms - short chain fatty acids - metabolism

    Dietary fibres, the edible parts of plants that are resistant to digestion and absorption in the human small intestine, were shown to be important in the prevention of obesity and the metabolic syndrome. This association can partially be attributed to a fibre-induced increase in satiety. Dietary fibres can be fermented by bacteria, collectively referred to as the microbiota, in the large intestine (i.e. caecum and colon), resulting in the production of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate. Part of the effect of dietary fibres on satiety is thought to be mediated via the production of SCFAs.

    The objective of the research described in this thesis was to reveal the effects of fermentation in the large intestine using comprehensive approaches with focus on metabolism and satiety.

    The effect of 2-wk-consumption of resistant starch (RS), a dietary fibre highly fermentable by the gut microbiota, was studied in 2 pig experiments. In the first experiment, performed in adult female pigs, intestinal samples were collected from different areas of the gastrointestinal tract to measure luminal microbiota composition, luminal SCFA concentrations and the expression of host genes involved in SCFA uptake, SCFA signalling, and satiety regulation in mucosal tissue. In an additional study the effects of RS were investigated in young growing pigs fitted with a cannula in the proximal colon for repeated collection of tissue biopsies for whole-genome expression profiling and luminal content for measurement of SCFA concentrations and microbiota composition. To limit inter-individual variation, the RS diet was provided to the pigs in a 2 x 2 crossover design for 2 wk per diet. Furthermore, the behaviour of the pigs was monitored and the postprandial plasma response of satiety-related hormones and metabolites was measured at the end of each 2 wk period using repeated peripheral blood sampling via catheters.

    In order to determine the potential differences in post-prandial plasma protein profiles, minipigs were assigned to a control (C) diet or a diet containing the bulky fibre lignocellulose (LC), the viscous and fermentable fibre pectin (PEC) or RS for periods of 8 d in a 4 x 4 Latin square design. Portal and carotid blood samples were collected from catheters on d 8 of each treatment, both before and at several time points after an ad libitum morning meal.

    Male C57BL/6J mice were used to study the effect of background diet and SCFAs on colonic gene expression. Mice were fed a semi-synthetic low fat or high fat diet starting 2 wk before the treatment period. During treatment, mice received a rectal infusion of either an acetate, propionate, butyrate, or a control saline solution on 6 consecutive days, after which colon was collected to perform comprehensive gene expression profiling.

    RS enhanced satiety based on behavioural observations, as RS-fed pigs showed less feeder-directed and drinking behaviours than pigs fed a digestible starch (DS) diet. In both caecum and colon, differences in microbiota composition were observed between RS-fed pigs and DS-fed pigs. In the colon these included the induction of the healthy gut-associated butyrate-producing Faecalibacterium prausnitzii, whereas potentially pathogenic members of the Gammaproteobacteria were reduced in relative abundance in RS-fed pigs. Caecal and colonic SCFA concentrations were significantly higher in RS-fed pigs. Geneexpression profiling of the proximal colon revealed a shift upon RS consumption from the regulation of immune response towards lipid and energy metabolism. The nuclear receptor PPARG was identified as a potential key upstream regulator. At plasma level, SCFA concentrations were higher in RS-fed pigs throughout the day. Postprandial glucose, insulin and glucagon-like peptide 1 (GLP-1) responses were lower in RS-fed than in DS-fed pigs, whereas triglyceride levels were higher in RS-fed than in DS-fed pigs.

    In minipigs, plasma protein profiles were found to be most similar with C and LC consumption and with PEC and RS consumption, indicating that the consumption of diets with fermentable fibres results in a different plasma protein profile compared to a diet containing non-fermentable fibres or a diet without fibres.

    In mice we observed that dietary fat content had a major impact on colonic gene expression responses to SCFAs, especially after propionate treatment. Moreover, the diet- and SCFA-dependent gene expression changes pointed towards the modulation of several metabolic processes. Genes involved in oxidative phosphorylation, lipid catabolism, lipoprotein metabolism and cholesterol transport were suppressed by acetate and butyrate treatment, whereas propionate resulted in changes in fatty acid and sterol biosynthesis, and in amino acid and carbohydrate metabolism.Furthermore, SCFA infusion on the high fat diet background appeared to partially reverse the gene expression changes induced by high fat feeding without SCFA infusion.

    In conclusion, this thesis showed that the consumption of RS changed the microbiota composition in the colonic lumen, with a decrease in the abundance of potentially pathogenic bacteriaand an increase in the abundance of SCFA-producing populations. Furthermore, colonic gene expression changes were observed after RS consumption and after colonic administration of SCFAs. With both treatments,among the changes inthe transcriptional profileof the host were adaptations inmetabolic processes, such as energy and lipid metabolism, and immune response.We also showed that fat content in the background diet had a major impact on gene expression responses to SCFAs in colon. Overall, this thesis supports the implementation of fermentable dietary fibres into the human diet to improve colonic health and to reduce energy intake and body weight gain, which ultimately may prevent obesity and type 2 diabetes. Additional research is required to further elucidate the mechanisms via which fermentable dietary fibres can improve human health.

    Interactions of commensal bacteria with the host immune system
    Rossi, O. - \ 2013
    Wageningen University. Promotor(en): Jerry Wells, co-promotor(en): J.N. Samsom. - S.l. : s.n. - ISBN 9789461735218 - 182
    commensalen - darmmicro-organismen - gastheren (dieren, mensen, planten) - faecalibacterium prausnitzii - immuunsysteem - commensals - intestinal microorganisms - hosts - faecalibacterium prausnitzii - immune system

    The intestinal microbiota plays role in intestinal homeostasis via interactions with the epithelium and innate and adaptive immune mechanisms of the gut thereby profoundly shaping mammalian mucosal immunity and tolerance. However, in some diseases, such as inflammatory bowel disease (IBD), the microbiota may assume a pathologic character accentuating the damage to the host. Despite the recent advances in our understanding of the microbiota structure at different stages of life and the perturbations associated with disease, our understanding of its functional impact and the contribution of individual microbial components on health is still in its infancy. The aim of this thesis was to contribute to our knowledge and understanding of several aspects of the interactions between gut commensal bacteria and host, focusing on one of the most abundant colonic microbes, F. prausnitzii, for the detailed mechanistic studies.

    In Chapter 2, we investigated whether polarized intestinal epithelial cells regulate inflammatory responses to microbes by secreting the chemokine IL-8 in a vectorial fashion depending on the location of the Toll-like receptor (TLR) stimulus. In the Caco-2 model of polarized epithelium, apical stimulation with TLR2 and TLR5 ligands resulted in the apical secretion of IL-8. The CXCR1 receptor for IL-8 was expressed only on the apical membrane of Caco-2 cells and differentiated epithelial cells in the human small intestine and colon suggesting that IL-8 has an autocrine function. Transcriptome analyses revealed that Caco-2 cells respond to stimulation with IL-8, supporting the hypothesis of autocrine signalling via CXCR1. We speculated that this mechanism might be an evolutionary adaptation to the coexistence with microbiota whereby bacterial microbe-associated molecular patterns are utilized to trigger repair mechanisms in epithelial cells.

    In Chapter 3, mono-associated and conventional mice were used to gain new insights into the interaction of bacteria with the host mucosa and mucosal-associated lymphoid tissue. The vast majority of the bacteria are kept at a distance by the host physical and chemical barriers except for segmented filamentous bacteria which appear to have unique and specialized physical interactions with the epithelium.

    Chapter 4, 5 and 6 focused on the immunoregulatory mechanisms of the human commensalF. prausnitzii, which has been proposed to have anti-inflammatory properties and is underrepresented in IBD patients with active disease.

    In Chapter 4, we compared the effects of F. prausnitziiand other commensal bacterial strains on human monocyte derived dendritic cells (hDCs). F. prausnitziistrain A2-165 induced a very different cytokine profile in hDCs compared with four other F. prausnitziistrains and eight other commensal strains, this was due to the high levels of secreted IL-10 and low levels of IL-12.

    The effects of F. prausnitziiA2-165 and another commensal strain on the induction of mucosal T cell responses to the model antigen ovalbumin (OVA) in vitroand in vivowere studied in Chapter 5. Here we demonstrated that F. prausnitziiA2-165 had immunoregulatory properties in vitrowhich may be related to its strong capacity to induce IL-10 by both DCs and T cells. These effects translated in vivointo enhanced OVA-specific T cell proliferation in the nose-draining cervical lymph nodes (CLNs) after combined nasal application of F. prausnitziiA2-165 and OVA. Strikingly, this was associated with a reduction in the differentiation of IFN-γ secreting T cells in the CLNs. In contrast, a different commensal, Clostridium hathewayi82-B, significantly decreased the percentage of dividing OVA-specific T cells in the CLNs and spleen.

    In Chapter 6, we investigated the immunomodulatory effects of F. prausnitziistrain A2-615, strain HTF-F which forms a biofilm in liquid culture and the extracellular polymeric matrix (EPM) purified from strain HTF-F. The protective capacity of the two strains and the EPM were investigated in the DSS-induced colitis model. Both F. prausnitziistrains attenuated the disease activity index, but the biofilm producing strain HTF-F conferred increased protection compared to strain A2-165 and this was partly due to the immunomodulatory properties of the EPM.

    Chapter 7summarizes and discusses the results of the thesis in the context of wider literature on host-microbe interactions and mucosal immunology. Furthermore, the implications of this work on intestinal health and the possible directions for future research are discussed.

    Red meat and colon cancer : how dietary heme initiates hyperproliferation
    IJssennagger, N. - \ 2012
    Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Roelof van der Meer. - S.l. : s.n. - ISBN 9789461733931 - 160
    vlees - karteldarmkanker - haem - weefselproliferatie - dieet - colorectaal kanker - darmmicro-organismen - darmslijmvlies - meat - colon cancer - haem - tissue proliferation - diet - colorectal cancer - intestinal microorganisms - intestinal mucosa

    Colorectal cancer is a leading cause of cancer deaths in Western countries. The risk to develop colorectal cancer is associated with the intake of red meat. Red meat contains the porphyrin pigment heme. Heme is an irritant for the colonic wall and it is previously shown that the addition of heme to the diet of rats induces hyperproliferation. Hyperproliferation increases the risk of endogenous mutations, which subsequently increases the risk to develop colon cancer. The aim of this thesis was to elucidate the diet-modulated signaling from an injured surface epithelium to the proliferative stem cells in the crypt to initiate compensatory hyperproliferation.

    In chapter 2 we showed that when heme is added to the diet of mice, there is an increased cytotoxicity of the colonic contents. Heme-fed mice showed decreased apoptosis and increased compensatory epithelial hyperproliferation resulting in hyperplasia. Gene expression levels of mouse colon after heme feeding were analyzed by microarrays and showed 3,710 differentially expressed genes (q<0.01) of which many were involved in proliferation and stress response. Stainings for the enzyme Heme oxygenase-1 and expression levels of heme- and stress-related genes showed that heme affected the epithelial surface cells, but that heme did not reach the crypt cells. Heme caused injury of the surface epithelial cells, and as proliferation originates from the stem cells in the crypts this implied that there must be a signaling mechanism from the injured surface to the stem cells in the crypts to start the hyperproliferation. In chapter 2 several surface to crypt signaling molecules were identified. Heme downregulated inhibitors of proliferation, such as Wnt inhibitory factor 1, Indian hedgehog and Bone morphogenetic protein 2. Furthermore, heme downregulated the cytokine Interleukin-15. Heme upregulated the expression of the growth factors Amphiregulin, Epiregulin and of Cyclooxygenase-2 mRNA in the surface. However, their protein/metabolite levels were not increased as heme induced surface-specific inhibition of translation by increasing the levels of the translation inhibitor 4E-BP1. We concluded that heme induced colonic hyperproliferation and hyperplasia by downregulating the surface to crypt signaling of feedback inhibitors of proliferation.

    Besides many proliferation and stress-related genes, many PPARα target genes were upregulated upon heme feeding. As PPARα is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized in chapter 3 that absence of PPARα leads to more colonic surface injury, which subsequently leads to increased compensatory hyperproliferation in colonic crypts upon heme-feeding. This hypothesis was tested using wild-type and PPARα knockout mice receiving a heme diet. Proliferation levels and gene expression profiles were determined. Heme induced luminal cytotoxicity and lipid peroxidation to the same extent in wild-type and PPARα knockout mice. We showed that PPARα does not play a role in the heme-induced hyperproliferation, as heme induced hyperproliferation both in wild-type as well as in PPARα knockout mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in the heme-fed PPARα knockout mice. We concluded that PPARα plays a protective role in colon against oxidative stress, but PPARα does not mediate heme-induced hyperproliferation. This implied that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia.

    Heme was shown to increase both reactive oxygen species as well as cytotoxicity of the colonic contents of mice. So far, the time dependency of the heme-induced oxidative stress and cytotoxic stress on the initiation of hyperproliferation was not studied. Therefore, in chapter 4 the effects of dietary heme on the colonic mucosa after 2, 4, 7 and 14 days of heme feeding were determined. This study showed that the effects of dietary heme on the colonic mucosa can be separated in acute and delayed effects. Acutely, heme increased oxidative stress which caused an increase in lipid peroxidation products. Besides, there was an acute activation of PPARα target genes, most probable induced by the generated oxidized lipids. Nrf2 target genes were activated acutely which played a role in the protection against oxidative stress. Delayed effects which occurred after day 4 of heme feeding, were increased luminal cytotoxicity and the induction of hyperproliferation. This suggested that the cytotoxicity, rather than oxidative stress, induced hyperproliferation. Remarkably, the surface epithelial cells sensed heme after day 4, although heme was present in the colon several hours after ingestion of the heme diet. This suggested that the mucus barrier played a role in the protection of the surface epithelium the first days of heme feeding.

    As the colon is densely populated by bacteria, the microbiota might play a role in modulating the surface to crypt signaling inducing hyperproliferation. To explore the role of the colonic microbiota we simultaneously investigated the effects of dietary heme on colonic microbiota and on the host mucosa of mice (chapter 5). Using 16S rRNA phylogenetic microarrays, it was determined that heme increased Bacteroidetes and decreased Firmicutes in colonic contents. This shift in the microbiota was most likely caused by a selective susceptibility of Gram-positive bacteria to heme cytotoxic fecal water. This susceptibility was not observed for Gram-negative bacteria and allowed the expansion of the Gram-negative community. The increased amount of Gram-negative bacteria, which likely caused an increased mucosal exposure to lipopolysaccharide (LPS), did not elicit a detectable immune reaction in the host mucosa. The absence of an immune reaction might be influenced by the strong upregulation of Secretory leukocyte peptidase inhibitor (Slpi) at gene and protein level, which is known to suppress excessive immune reactions. We showed that there was no functional change in the sensing of the bacteria by the mucosa, as changes in inflammation pathways and Toll- like receptor signaling were not detected. In conclusion, the change in microbiota did not cause the observed hyperproliferation and hyperplasia via inflammation pathways.

    In the study described in chapter 6 we investigated whether microbiota play a causal role in the heme-induced hyperproliferation. In this study mice received a control or a heme diet with or without broad spectrum antibiotics (Abx). Similar to previous experiments, heme induced epithelial hyperproliferation. Interestingly, when heme was administered together with Abx there was no induction of hyperproliferation. Heme induced oxidative stress in the heme group as well as in the heme plus Abx group. Cytotoxicity was also induced in both heme groups. As bacteria were decreased by 100 to 1000 fold in abundance upon Abx treatment it is unlikely that bacteria play a major role in the formation of the cytotoxic factor. Whole genome transcriptomics showed that Abx blocked the heme-induced differential expression of oncogenes, tumor suppressors and cell turnover genes. Moreover, Abx blocked the mucosal sensing of luminal cytotoxicity indicating that Abx increased the mucus barrier. Abx eliminated mucin-degrading bacteria, such as Akkermansia, and sulfate-reducing bacteria (SRBs) that produce sulfide. In-vitro studies showed that sulfide is more potent than N-acetylcysteine and cysteine in splitting disulfide bonds, indicating that SRB generated sulfide can denature mucins and thus open the mucus barrier. This study showed that the microbiota plays an important facilitating role in the heme-induced hyperproliferation and hyperplasia by breaking the mucus barrier and thereby decreasing the protection against luminal irritants such as the toxic heme metabolite.

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