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Gnotobiotic rats harboring human intestinal microbiota as a model for studying cholesterol-to-coprostanol conversion
Abstract
The efficiency of microbial reduction of cholesterol to coprostanol in human gut is highly variable among population and mechanisms remain unexplored. In the present study, we investigated whether microbial communities and their cholesterol metabolism characteristics can be transferred to germ-free rats. Two groups of six, initially germ-free rats were associated with two different human microbiota, exhibiting high and low cholesterol-reducing activities. Four months after inoculation, enumeration of coprostanoligenic bacteria, fecal coprostanol levels and composition of the fecal microbial communities were studied in gnotobiotic rats and compared with those of the human donors. Combination of culture (most probable number enumeration of active bacteria) and biochemical approaches (extraction followed by gas chromatography of sterols) showed that gnotobiotic rats harbored a coprostanoligenic bacterial population level and exhibited coprostanoligenic activities similar to those of the corresponding human donor. On the other hand, molecular approaches (whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes, and temporal temperature gradient gel electrophoresis of bacterial 16S rRNA gene amplicons) demonstrated that gnotobiotic rats reproduced a stable microbial community, close to the human donor microbiota at the group or genus levels but different at the dominant species level. These results suggest that the gnotobiotic rat model can be used to explore the still unknown human intestinal microbiota involved in luminal cholesterol metabolism, including regulation of expression of its activity and impact on health.
... Therefore we aimed at investigating whether other bacterial groups modulated by the ingredient are correlated with host metabolism by using a humanized mice model. This model provides a reliable microbiota stability over time and can be used to test the effects of different diets, including the addition of fermentable oligosaccharides, on the composition of the microbiota [19,20]. The effect of scFOS will be particularly studied in a high fat diet known to induce obesity and impair glucose metabolism [21]. ...
... A fresh stool sample was provided in an anaerobic box (Anaerocult; Merck, Darmstadt, Germany) by a healthy lean adult (40 years, 19, BMI ,25) on an unrestricted Western diet, without laxative or antibiotic use for the previous 3 months, who was involved in this study and who gave his/her verbal consent. A 10 22 dilution of this stool was administered to 48 germ-free (GF) male C57BL/6J 4-week old mice (initial body weight 20.662.9 g) as previously described [19]. As the major components of the human microbiota can be transferred into the ex-germfree animals, these animals are considered the better model to investigate interactions between human intestinal microbiota, host factors and dietary manipulations [19,20]. ...
... A 10 22 dilution of this stool was administered to 48 germ-free (GF) male C57BL/6J 4-week old mice (initial body weight 20.662.9 g) as previously described [19]. As the major components of the human microbiota can be transferred into the ex-germfree animals, these animals are considered the better model to investigate interactions between human intestinal microbiota, host factors and dietary manipulations [19,20]. Mice were kept in sterile Plexiglas isolators, 3 per cage, at ANAXEM, the GF animal facilities of Micalis Institute (INRA, Jouy-en-Josas, France) for the 10-week duration of the study and had ad libitum access to irradiated feeds and sterile water. ...
... Therefore we aimed at investigating whether other bacterial groups modulated by the ingredient are correlated with host metabolism by using a humanized mice model. This model provides a reliable microbiota stability over time and can be used to test the effects of different diets, including the addition of fermentable oligosaccharides, on the composition of the microbiota [19,20]. The effect of scFOS will be particularly studied in a high fat diet known to induce obesity and impair glucose metabolism [21]. ...
... A fresh stool sample was provided in an anaerobic box (Anaerocult; Merck, Darmstadt, Germany) by a healthy lean adult (40 years, 19, BMI ,25) on an unrestricted Western diet, without laxative or antibiotic use for the previous 3 months, who was involved in this study and who gave his/her verbal consent. A 10 22 dilution of this stool was administered to 48 germ-free (GF) male C57BL/6J 4-week old mice (initial body weight 20.662.9 g) as previously described [19]. As the major components of the human microbiota can be transferred into the ex-germfree animals, these animals are considered the better model to investigate interactions between human intestinal microbiota, host factors and dietary manipulations [19,20]. ...
... A 10 22 dilution of this stool was administered to 48 germ-free (GF) male C57BL/6J 4-week old mice (initial body weight 20.662.9 g) as previously described [19]. As the major components of the human microbiota can be transferred into the ex-germfree animals, these animals are considered the better model to investigate interactions between human intestinal microbiota, host factors and dietary manipulations [19,20]. Mice were kept in sterile Plexiglas isolators, 3 per cage, at ANAXEM, the GF animal facilities of Micalis Institute (INRA, Jouy-en-Josas, France) for the 10-week duration of the study and had ad libitum access to irradiated feeds and sterile water. ...
... Therefore we aimed at investigating whether other bacterial groups modulated by the ingredient are correlated with host metabolism by using a humanized mice model. This model provides a reliable microbiota stability over time and can be used to test the effects of different diets, including the addition of fermentable oligosaccharides, on the composition of the microbiota [19,20]. The effect of scFOS will be particularly studied in a high fat diet known to induce obesity and impair glucose metabolism [21]. ...
... A fresh stool sample was provided in an anaerobic box (Anaerocult; Merck, Darmstadt, Germany) by a healthy lean adult (40 years, 19, BMI ,25) on an unrestricted Western diet, without laxative or antibiotic use for the previous 3 months, who was involved in this study and who gave his/her verbal consent. A 10 22 dilution of this stool was administered to 48 germ-free (GF) male C57BL/6J 4-week old mice (initial body weight 20.662.9 g) as previously described [19]. As the major components of the human microbiota can be transferred into the ex-germfree animals, these animals are considered the better model to investigate interactions between human intestinal microbiota, host factors and dietary manipulations [19,20]. ...
... A 10 22 dilution of this stool was administered to 48 germ-free (GF) male C57BL/6J 4-week old mice (initial body weight 20.662.9 g) as previously described [19]. As the major components of the human microbiota can be transferred into the ex-germfree animals, these animals are considered the better model to investigate interactions between human intestinal microbiota, host factors and dietary manipulations [19,20]. Mice were kept in sterile Plexiglas isolators, 3 per cage, at ANAXEM, the GF animal facilities of Micalis Institute (INRA, Jouy-en-Josas, France) for the 10-week duration of the study and had ad libitum access to irradiated feeds and sterile water. ...
Prebiotic fibres like short-chain fructo-oligosaccharides (scFOS) are known to selectively modulate the composition of the intestinal microbiota and especially to stimulate Bifidobacteria. In parallel, the involvement of intestinal microbiota in host metabolic regulation has been recently highlighted. The objective of the study was to evaluate the effect of scFOS on the composition of the faecal microbiota and on metabolic parameters in an animal model of diet-induced obesity harbouring a human-type microbiota. Forty eight axenic C57BL/6J mice were inoculated with a sample of faecal human microbiota and randomly assigned to one of 3 diets for 7 weeks: a control diet, a high fat diet (HF, 60% of energy derived from fat)) or an isocaloric HF diet containing 10% of scFOS (HF-scFOS). Mice fed with the two HF gained at least 21% more weight than mice from the control group. Addition of scFOS partially abolished the deposition of fat mass but significantly increased the weight of the caecum. The analysis of the taxonomic composition of the faecal microbiota by FISH technique revealed that the addition of scFOS induced a significant increase of faecal Bifidobacteria and the Clostridium coccoides group whereas it decreased the Clostridium leptum group. In addition to modifying the composition of the faecal microbiota, scFOS most prominently affected the faecal metabolome (e.g. bile acids derivatives, hydroxyl monoenoic fatty acids) as well as urine, plasma hydrophilic and plasma lipid metabolomes. The increase in C. coccoides and the decrease in C. leptum, were highly correlated to these metabolic changes, including insulinaemia, as well as to the weight of the caecum (empty and full) but not the increase in Bifidobacteria. In conclusion scFOS induce profound metabolic changes by modulating the composition and the activity of the intestinal microbiota, that may partly explain their effect on the reduction of insulinaemia.
... Human to rat: when GF rats are colonized with human microbiota the transplanted community resembles its community of origin in terms of the group or genus levels but differences at the dominant species level occur. Thus, a selective pressure of the host is imposed on the gut habitat [82]. However, certain metabolic characteristics (high equolproducing and low equol-producing status or cholesterol-to-coprostanol conversion) of human intestinal floras can be transferred to GF rats [82,83]. ...
... Thus, a selective pressure of the host is imposed on the gut habitat [82]. However, certain metabolic characteristics (high equolproducing and low equol-producing status or cholesterol-to-coprostanol conversion) of human intestinal floras can be transferred to GF rats [82,83]. In CONV rats, the colonic mucus layer is twice as thick as in GF rats [84], and the mucin chemical composition is altered [85,86]. ...
... However, in order to causally link dysbiosis with human diseases, these animal models need to be gnotobiotically transplanted further with 'humanized' microbiota. The latter poses additional potential pitfalls, as many microbial species have evolved to fill host-specific niches [55,82,97] and the topology of the mouse GIT is, for example, distinct from that of humans, rendering the mimicking of human-specific niches challenging if not impossible. Consequently, xenograft microbiota may not representatively colonize the GIT of a humanized animal model. ...
Large-scale 'meta-omic' projects are greatly advancing our knowledge of the human microbiome and its specific role in governing health and disease states. A myriad of ongoing studies aim at identifying links between microbial community disequilibria (dysbiosis) and human diseases. However, due to the inherent complexity and heterogeneity of the human microbiome, cross-sectional, case–control and longitudinal studies may not have enough statistical power to allow causation to be deduced from patterns of association between variables in high-resolution omic datasets. Therefore, to move beyond reliance on the empirical method, experiments are critical. For these, robust experimental models are required that allow the systematic manipulation of variables to test the multitude of hypotheses, which arise from high-throughput molecular studies. Particularly promising in this respect are microfluidics-based in vitro co-culture systems, which allow high-throughput first-pass experiments aimed at proving cause-and-effect relationships prior to testing of hypotheses in animal models. This review focuses on widely used in vivo, in vitro, ex vivo and in silico approaches to study host-microbial community interactions. Such systems, either used in isolation or in a combinatory experimental approach, will allow systematic investigations of the impact of microbes on the health and disease of the human host. All the currently available models present pros and cons, which are described and discussed. Moreover, suggestions are made on how to develop future experimental models that not only allow the study of host-microbiota interactions but are also amenable to high-throughput experimentation.
... In this study, we compared the fermentation patterns of water-extractable LC-AX with the well-established prebiotic IN. By introducing a human faecal microbiota in axenic rats, the animals can be reproducibly colonized with a microbiota that closely resembles the human inoculum (Gérard et al., 2004), allowing the analysis of dietary saccharide fermentation by human-derived microbes in an in vivo environment. Using this model, we demonstrated that the two structurally different carbohydrates specifically altered the intestinal microbiota in terms of metabolic activity and local community composition, coinciding with changes in host physiology. ...
... The experiment was conducted in the germfree animal facilities (ANAXEM platform) of INRA UMR 1319 MICALIS (Jouy-en-Josas, France) (Gérard et al., 2004). A 10 -2 dilution of a fresh stool provided by a healthy human volunteer (male, 24 years) who had no history of antibiotic treatment 6 months prior to the study was anaerobically prepared in (in g l -1 ) NaCl (5.0) -glucose (2.0) -cysteine-HCl (0.3) and immediately administered to 24 male germfree Fisher 344 albino rats (eight weeks of age) through a single oral gavage (1 ml per rat). ...
There is increasing interest in gastro-intestinal microbiology to improve the balance between putative beneficial and harmful commensal microbiota. Mucosal microbes make closer contact with the host than luminal ones and could therefore more strongly affect host health. A short term in vitro adhesion assay was used to examine initial mucin colonisation by bacteria derived from the mixed microbial community of the ascending colon of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Further, long term prebiotic effects on the luminal and mucosa-associated microbial community (MAMC) were assessed in vitro and in vivo with respectively the SHIME and gnotobiotic rats. During the short term experiment, adhesion capacity differed substantially between bacteria and decreased from lactobacilli over faecal coliforms, bifidobacteria and clostridia to total anaerobes. In addition, intestinal water lowered adhesion compared to phosphate buffered saline. By processing the data to an Adhesion Related Prebiotic Index (AR-PI), it was found that intestinal water stimulated adherence of positive commensals. Arabinoxylans (AX) decreased the bacterial adhesion capacity (matrix independently), whereas inulin had less or no influence. During the long term experiment, group-specific DGGE analysis showed that although inoculated with the same human faecal microbiota, the in vivo and in vitro model both harbour distinct microbial communities. Both carbohydrates were shown to exert long term prebiotic effects on the luminal and mucosal microbial community. Interestingly, AX induced a shift towards a specific Bifidobacterium sp, both in the in vitro and in vivo model. The developed methodologies could be useful for the characterisation of the relevant but barely investigated mucosa-associated microbial community in health and disease (e.g. IBD) as well as for its modulation with functional foods like prebiotics.
... These conversion patterns were found equally distributed with respect to sex and were independent of age. Only recently has it been demonstrated that the efficiency of cholesterol conversion in the human gut results mainly from the abundance of cholesterol-reducing bacteria (Gérard et al. 2004;Veiga et al. 2005). In a study including 15 human volunteers, it was established that the level of cholesterol-reducing bacteria must be at least 10 6 cells/g (wet weight) of stool to efficiently convert cholesterol in the human gut, while a population containing more than 10 8 cells/g (wet weight) of stool leads to nearly complete conversion (Veiga et al. 2005). ...
... Concurrently, more studies on animal models, specially using gnotobiotic animals, are needed to determine the effect of this bacterial metabolism on plasma cholesterol. Interestingly, it was recently shown that human microbiota retained its level of cholesterol-reducing bacterial population and cholesterolreducing activity in gnotobiotic rats (Gérard et al. 2004). Rats harboring a human gut microbiota could therefore be used as a model to explore the impact on health of microbial cholesterol metabolism. ...
... Memelilerde farklı yollarla (diyet, safra, intestinal hücreler) ince bağırsak boşluğuna gelen ve burada bulunan kolesterolün mikrobiyal flora tarafından metabolize edildiği uzun yıllardan beri bilinmektedir [40,41]. Metabolizasyon işleminde rol oynayan kolesterol redüktaz enzimi kolesterolün koprostanola kimyasal indirgenmesini katalizleyen enzimdir [42]. ...
... Metabolizasyon işleminde rol oynayan kolesterol redüktaz enzimi kolesterolün koprostanola kimyasal indirgenmesini katalizleyen enzimdir [42]. Kolesterol redüktazın katalize ettiği söz konusu metabolizasyon işleminin ara ürünleri 4-kolesten-3-on ve koprostanon iken [41] başlıca son ürün bağırsaklardan zayıf bir şekilde absorbe edilebilen koprostanoldür (5β-kolestan-3β-ol) [40]. Koprostanol'ün yapısı Şekil 2'de gösterilmiştir. ...
Yüksek serum kolesterol düzeyleri (>240 mg/dL) kalp-damar hastalıkları riskini arttırmaktadır. Yapılan in vitro ve in vivo çalışmalar " yeterli miktarlarda alındığında konakçı sağlığına yararlı olan canlı mikroorganizmalar " şeklinde tanımlanan probiyotik mikroorganizmaların bazı suşlarının kolesterol düzeyinin düşürülmesine yardımcı olabileceğini bildirmektedir. Bu makalede probiyotiklerin in vitro ve in vivo koşullarda kolesterol seviyesinin düşürülmesi üzerine etkisi tartışılmıştır.
ABSTRACT High serum cholesterol levels (>240 mg/dL) increase the risk of cardiovascular diseases. Probiotics are defined as " live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host " , and past in vitro and in vivo studies showed that some strains of probitotic microorganisms are effective in the reduction of cholesterol levels. The present review discusses the effects of probiotic microorganisms on reduction of cholesterol levels in vitro and in vivo.
... Fecal samples were collected at various times from alcoholic patients and from mice fed an alcohol diet. Samples were fixed as previously described [7]. A set of 12 probes (Thermo Fisher Scientific GmbH, Ulm, Germany) targeting bacterial 16S rRNA was used to assess the relative proportions of intestinal bacteria from different phylogenetic groups with respect to the total bacteria assessed by an universal bacterial probe (Table S7). ...
... A set of 12 probes (Thermo Fisher Scientific GmbH, Ulm, Germany) targeting bacterial 16S rRNA was used to assess the relative proportions of intestinal bacteria from different phylogenetic groups with respect to the total bacteria assessed by an universal bacterial probe (Table S7). FISH-FCM was performed as described previously [7]. created when there are too many possible orderings of the data to allow complete enumeration in a convenient manner. ...
... These conversion patterns were found equally distributed with respect to sex and were independent of age. Only recently has it been demonstrated that the efficiency of cholesterol conversion in the human gut results mainly from the abundance of cholesterol-reducing bacteria (Gérard et al., 2004;Veiga et al., 2005). In a study including fifteen human volunteers, it was established that the level of cholesterol-reducing bacteria must be at least 10 6 cells g À1 (wet weight) of stool to efficiently convert cholesterol in the human gut, while a population containing more than 10 8 cells g À1 (wet weight) of stool leads to nearly complete conversion (Veiga et al., 2005). ...
... Concurrently, more studies on animal models, specially using gnotobiotic animals, are needed to determine the effect of this bacterial metabolism on plasma cholesterol. Interestingly, it was recently shown that human microbiota retained its level of cholesterol-reducing bacterial population and cholesterolreducing activity in gnotobiotic rats (Gérard et al., 2004). Rats harboring a human gut microbiota could therefore be used as a model to explore the impact on health of microbial cholesterol metabolism. ...
The human gastro-intestinal tract hosts a complex and diverse microbial community, whose microbiome encodes biochemical pathways that humans have not evolved. As a consequence, the gut microbiota produces metabolites from a large range of molecules that host’s enzymes are not able to convert. This is of first importance as these bacterial metabolites may have beneficial or deleterious effects on human health. In particular, cholesterol and bile acids are exposed to the gut microbiota and undergo bacterial metabolism: cholesterol is mainly converted into coprostanol, a non absorbable sterol which is excreted in the feces. Conversely, over twenty different secondary bile acid metabolites are produced by the gut microbiota from the primary bile acids: cholic and chenodeoxycholic acids. The main bile salt conversions in the human gut include deconjugation, oxidation and epimerization of hydroxyl groups at C3, C7 and C12, 7-dehydroxylation, esterification and desulfatation.
... Specific enzymes needed are found in Bacteroides and Eubacterium belonging to phyla Firmicutes. 16 Bile salt hydrolase does deconjugation of PBA in the large intestine, and the enzyme is present in genera Bacteroides, Clostridium, Lactobacillus, and Bifidobacterium (more diverse in the phylum Firmicutes). It acts as a bile acid detoxification mechanism for bacteria. ...
Chronic diarrhea and abdominal pain after radiotherapy continue to be a problem in cancer survivors. Gut microbiomes are essential for preventing intestinal inflammation, maintaining intestinal integrity, maintaining enterohepatic circulation, regulating bile acid metabolism, and absorption of nutrients, including fat-soluble vitamins. Gut microbiome dysbiosis is expected to cause inflammation, bile acid malabsorption, malnutrition, and associated symptoms. Postradiotherapy, Firmicutes and Bacteroidetes phylum are significantly decreased while Fusobacteria and other unclassified bacteria are increased. Available evidence suggests harmful bacteria Veillonella, Erysipelotrichaceae, and Ruminococcus are sensitive to Metronidazole or Ciprofloxacin. Beneficial bacteria lactobacillus and Bifidobacterium are relatively resistant to metronidazole. We hypothesize and provide an evidence-based review that short-course targeted antibiotics followed by specific probiotics may lead to alleviation of radiation enteritis.
... In vivo studies play a major role and provide a powerful tool for studying the underlying pathogenic mechanisms of GM-mediated AD. Various animal models, such as mice, rats, pigs, and zebrafish, have been used for GM research [79][80][81][82]. Furthermore, inbred and knockout techniques are regularly used for some animals like mice or rats [83,84]. ...
Gut microbiota (GM), the microorganisms in the gastrointestinal tract, contribute to the regulation of brain homeostasis through bidirectional communication between the gut and the brain. GM disturbance has been discovered to be related to various neurological disorders, including Alzheimer’s disease (AD). Recently, the microbiota-gut-brain axis (MGBA) has emerged as an enticing subject not only to understand AD pathology but also to provide novel therapeutic strategies for AD. In this review, the general concept of the MGBA and its impacts on the development and progression of AD are described. Then, diverse experimental approaches for studying the roles of GM in AD pathogenesis are presented. Finally, the MGBA-based therapeutic strategies for AD are discussed. This review provides concise guidance for those who wish to obtain a conceptual and methodological understanding of the GM and AD relationship with an emphasis on its practical application.
... Sequence-specific separation of PCR products was performed using TTGE on a DCode Universal Mutation Detection System (Bio-Rad, Paris, France) on polyacrylamide gel (Gérard et al., 2004). The electrophoresis was performed at a fixed voltage of 63 V for 15 h, starting at 66 • C and with a ramp rate of 0.3 • C/h. ...
The intestinal microbiota has a key role in human health via the interaction with the somatic and immune cells in the digestive tract environment. Food, through matrix effect, nutrient and non-nutrient molecules, is a key regulator of microbiota diversity. As a food contaminant, the pesticide chlorpyrifos (CPF) has an effect on the composition of the intestinal microbiota and induces perturbation of microbiota. Prebiotics (and notably inulin) are known for their ability to promote an equilibrium of the microbiota that favours saccharolytic bacteria. The SHIME® dynamic in vitro model of the human intestine was exposed to CPF and inulin concomitantly for 30 days, in order to assess variations in both the bacterial populations and their metabolites. Various analyses of the microbiota (notably temporal temperature gradient gel electrophoresis) revealed a protective effect of the prebiotic through inhibition of the enterobacterial (E. coli) population. Bifidobacteria were only temporarily inhibited at D15 and recovered at D30. Although other potentially beneficial populations (lactobacilli) were not greatly modified, their activity and that of the saccharolytic bacteria in general were highlighted by an increase in levels of short-chain fatty acids and more specifically butyrate. Given the known role of host-microbiota communication, CPF's impact on the body's homeostasis remains to be determined.
... Moreover, the efficiency of cholesterol conversion in the human gut as measured by the ratio of coprostanol to cholesterol in stools was shown to be related to the abundance of cholesterol-reducing bacteria estimated by the Most Probable Number method [34]. That the "low" or "high" phenotype can be transmitted to germ-free born rats colonized with the microbiota of either a low-or high-converter human donor would designate the microbiota structure and functions as a main driving force [100]. But we do not know which particular structural and functional features of the microbiome accompany these different profiles. ...
Every day, up to 1 g of cholesterol, composed of the unabsorbed dietary cholesterol, the biliary cholesterol secretion, and cholesterol of cells sloughed from the intestinal epithelium, enters the colon. All cholesterol arriving in the large intestine can be metabolized by the colonic bacteria. Cholesterol is mainly converted into coprostanol, a non-absorbable sterol that is excreted in the feces. Interestingly, cholesterol-to-coprostanol conversion in human populations is variable, with a majority of high converters and a minority of low or inefficient converters. Two major pathways have been proposed, one involving the direct stereospecific reduction of the Δ5 double bond direct while the indirect pathway involves the intermediate formation of 4-cholelesten-3-one and coprostanone. Despite the fact that intestinal cholesterol conversion was discovered more than a century ago, only a few cholesterol-to-coprostanol-converting bacterial strains have been isolated and characterized. Moreover, the responsible genes were mainly unknown until recently. Interestingly, cholesterol-to-coprostanol conversion is highly regulated by the diet. Finally, this gut bacterial metabolism has been linked to health and disease, and recent evidence suggests it could contribute to lower blood cholesterol and cardiovascular risks.
... Upon ingestion followed by enterohepatic absorption, biliary excretion and circulation, cholesterol is subjected to gut microbial reduction to produce co-prostanol-a major (50%) steroid found in human feces. Eubacterium coprostanoligenes have been documented to be a cholesterol reducing coprostanol synthesizing gut bacteria (Gérard et al. 2004;Kenny et al. 2020;Koppel et al. 2017). ...
The research on human gut microbiome, regarded as the black box of the human body, is still at the stage of infancy as the functional properties of the complex gut microbiome have not yet been understood. Ongoing metagenomic studies have deciphered that the predominant microbial communities belong to eubacterial phyla Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Cyanobacteria, Verrucomicrobia and archaebacterial phylum Euryarchaeota. The indigenous commensal microbial flora prevents opportunistic pathogenic infection and play undeniable roles in digestion, metabolite and signaling molecule production and controlling host’s cellular health, immunity and neuropsychiatric behavior. Besides maintaining intestinal health via short-chain fatty acid (SCFA) production, gut microbes also aid in neuro-immuno-endocrine modulatory molecule production, immune cell differentiation and glucose and lipid metabolism. Interdependence of diet and intestinal microbial diversity suggests the effectiveness of pre- and pro-biotics in maintenance of gut and systemic health. Several companies worldwide have started potentially exploiting the microbial contribution to human health and have translated their use in disease management and therapeutic applications. The present review discusses the vast diversity of microorganisms playing intricate roles in human metabolism. The contribution of the intestinal microbiota to regulate systemic activities including gut–brain–immunity crosstalk has been focused. To the best of our knowledge, this review is the first of its kind to collate and discuss the companies worldwide translating the multi-therapeutic potential of human intestinal microbiota, based on the multi-omics studies, i.e. metagenomics and metabolomics, as ready solutions for several metabolic and systemic disorders.
Graphic abstract
... As this product is not absorbed can be excreted out. Hence help to reduce it, while ingested cholesterol is absorbed in the cholesterol from the serum Gérard et al. (2004), Ren et al. (1996) 6. Reduction of heavy metals ...
Nanotechnology is a revolution in the field of science. Its nanosize (between 100–
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In the agricultural field, nanotechnology offers a solution to satisfy the alarming
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using the photovoltaic technique and plasmonic enhancement.
... L. sakei ADM14 has been shown to reduce the risk of obesity-related factors while lowering total cholesterol, LDL, and fasting blood glucose levels ( Figure 2). It has been reported that the remaining cholesterol not absorbed by the small intestine is converted into other metabolites by the gut microbiota, which helps eliminate cholesterol in the body [42]. In this study, the gut microbiota modulated by L. sakei ADM14 appears to lower blood cholesterol levels. ...
The aim of our study was to evaluate the anti-obesity effects of Lactobacillus sakei (L. sakei) ADM14 administration in a high-fat diet-induced obese mouse model and the resulting changes in the intestinal microbiota. Prior to in vivo testing, L. sakei ADM14 was shown to inhibit adipogenesis through in vitro test and genetic analysis. Subsequently, mice were orally administered 0.85% saline supplemented or not with L. sakei ADM14 to high-fat diet group and normal diet group daily. The results showed that administration of L. sakei ADM14 reduced weight gain, epididymal fat expansion, and total blood cholesterol and glucose levels, and significantly decreased expression of lipid-related genes in the epididymal fat pad. Administration of L. sakei ADM14 showed improvement in terms of energy harvesting while restoring the Firmicutes to Bacteroidetes ratio and also increased the relative abundance of specific microbial taxa such as Bacteroides faecichinchillae and Alistipes, which are abundant in non-obese people. L. sakei ADM14 affected the modulation of gut microbiota, altered the strain profile of short-chain fatty acid production in the cecum and enhanced the stimulation of butyrate production. Overall, L. sakei ADM14 showed potential as a therapeutic probiotic supplement for metabolic disorders, confirming the positive changes of in vivo indicators and controlling gut microbiota in a high-fat diet-induced obese mouse model.
... In a singular way, the rate of conversion of microbial cholesterol to coprostanol in the general human population appears to be multimodal, with an average of 65% of high converters (80% to 100% of luminal cholesterol is converted to coprostanol in the colon), 21% of intermediate converters and 14% of nonconverters (Wilkins and Hackman, 1974;Midtvedt et al., 1990;Veiga et al., 2005;Benno et al., 2005). It was also demonstrated that this phenotypic characteristic was maintained in axenic rodents (without germs) colonized with a high-converter or non-converter human microbiota (Gérard et al., 2004). Finally, several clinical and preclinical studies support the hypothesis that the conversion of cholesterol in coprostanol could influence the bioavailability of cholesterol, leading to modulation of plasma cholesterol levels (Sekimoto et al., 1983;Li et al., 1995;Li et al., 1998). ...
Disruption in cholesterol metabolism, particularly hypercholesterolemia, is a significant cause of atherosclerotic cardiovascular disease. Large interindividual variations in plasma cholesterol levels are traditionally related to genetic factors, and the remaining portion of their variance is accredited to environmental factors. In recent years, the essential role played by intestinal microbiota in human health and diseases has emerged. The gut microbiota is currently viewed as a fundamental regulator of host metabolism and of innate and adaptive immunity. Its bacterial composition but also the synthesis of multiple molecules resulting from bacterial metabolism vary according to diet, antibiotics, drugs used, and exposure to pollutants and infectious agents. Microbiota modifications induced by recent changes in the human environment thus seem to be a major factor in the current epidemic of metabolic/inflammatory diseases (diabetes mellitus, liver diseases, inflammatory bowel disease, obesity, and dyslipidemia). Epidemiological and preclinical studies report associations between bacterial communities and cholesterolemia. However, such an association remains poorly investigated and characterized. The objectives of this review are to present the current knowledge on and potential mechanisms underlying the host-microbiota dialogue for a better understanding of the contribution of microbial communities to the regulation of cholesterol homeostasis.
... TICE is an alternative route to the hepatobiliary pathway, where cholesterol from the blood can directly enter enterocytes through LDL receptors (LDL-R) and is effluxed by ABCG5/G8 and the ATPbinding cassette transporter B1 (ABCB1a/b) into the lumen [22]. The cholesterol content of the lumen is then either absorbed into enterocytes via Niemann-Pick C1like 1 (NPC1L1) and incorporated into chylomicrons for entry into the circulatory system [19], or is reduced by gut microbiota to poorly absorbable coprostanol (5B-Cholestan-3B-ol) [24][25][26], which is mostly excreted. ...
Coronary artery disease (CAD) is the most common health problem worldwide and remains the leading cause of morbidity and mortality. Over the past decade, it has become clear that the inhabitants of our gut, the gut microbiota, play a vital role in human metabolism, immunity, and reactions to diseases, including CAD. Although correlations have been shown between CAD and the gut microbiota, demonstration of potential causal relationships is much more complex and challenging. In this review, we will discuss the potential direct and indirect causal roots between gut microbiota and CAD development via microbial metabolites and interaction with the immune system. Uncovering the causal relationship of gut microbiota and CAD development can lead to novel microbiome-based preventative and therapeutic interventions. However, an interdisciplinary approach is required to shed light on gut bacterial-mediated mechanisms (e.g., using advanced nanomedicine technologies and incorporation of demographic factors such as age, sex, and ethnicity) to enable efficacious and high-precision preventative and therapeutic strategies for CAD.
... Up to 50% of the steroids in human feces are coprostanol [43]. Animal experiments show that GF mice colonized with microbes from high-and low-cholesterol-reducing patients produce particular amounts of coprostanol [44]. Moreover, it has been suggested that cholesterol-reducing bacteria may decrease serum cholesterol [43]. ...
Among gut microbiota’s newly explored roles in human biology is the ability to modify the chemical structures of foreign compounds (xenobiotics). A growing body of evidence has now provided sufficient acumen on the role of the gut microbiota on xenobiotic metabolism, which could have intense impact on therapy for various diseases in future. Gut microbial xenobiotic metabolites have altered bioavailability, bioactivity and toxicity and can intervene with the actions of human xenobiotic-metabolizing enzymes to affect the destiny of other ingested molecules. These modifications are diverse and could lead to physiologically important consequences.
In the current manuscript we aim to review the data currently available on how the gut microbiota directly modifies drugs, dietary compounds, chemicals, pollutants, pesticides and herbal supplements.
... De manière singulière, le taux de conversion du cholestérol microbien en coprostanol dans la population générale humaine est multimodal, avec environ 80 % de hauts convertisseurs (80 % à 100 % du cholestérol luminal est converti en coprostanol dans le colon), 20 % de convertisseurs intermédiaires et 10 % de non-convertisseurs [74,75]. Il est démontré que cette caractéristique phénotypique est transmise chez les rongeurs axéniques colonisés avec des microbiotes humains de haut-convertisseur ou de non-convertisseur [76]. Enfin, des études cliniques et précliniques anciennes suggèrent, une relation inverse entre la capacité de conversion du cholestérol fécal en coprostanol et les concentrations de cholestérol plasmatiques [77][78][79]. ...
Résumé
Les fortes variations interindividuelles de la cholestérolémie sont traditionnellement liées à des facteurs génétiques et la portion résiduelle de leur variance est attribuée à des facteurs environnementaux, dont l’alimentation. Des études précliniques suggèrent que les bactéries commensales jouent un rôle dans les pathologies inflammatoires et métaboliques. Des altérations de la composition du microbiote intestinal associées à des changements de cholestérolémie sont observés chez les patients dysmétaboliques et des données épidémiologiques font état d’associations entre des phyla bactériens et la cholestérolémie. Le métabolisme du cholestérol et la microbiologie sont des domaines de recherche inexplorés bien que les endocrinologues et les microbiologistes accordent de l’attention aux relations nutrition-cholestérolémie ou nutrition-microbes. L’objectif de cette revue est de présenter les mécanismes potentiels qui sous-tendent ce dialogue afin de stimuler la recherche sur la contribution du monde procaryote à la régulation de l’homéostasie du cholestérol chez l’hôte.
... The production of coprostanone and coprostanol by the intestinal microbiota can contribute to reduction in serum cholesterol levels via increase in fecal cholesterol excretion (70). The underlying mechanisms for this effect on cholesterol involve cholesterol reductases causing direct reduction of the double-bond 5-6 to give coprostanol, and cholesterol oxidases or 3-hydroxysteroid dehydrogenases/isomerases causing oxidation of the 3-hydroxy group and the isomerization of the double bond to produce 4-cholesten-3-one, followed by two reductions to produce coprostanone and finally coprostanol (39,40). ...
The gut microbiome is the natural intestinal inhabitant that has been recognized recently as a major player in the maintenance of human health and the pathophysiology of many diseases. Those commensals produce metabolites that have various effects on host biologic functions. Therefore, alterations in the normal composition or diversity of microbiome have been implicated in various diseases, including liver cirrhosis and nonalcoholic fatty liver disease. Moreover, accumulating evidence suggests that progression of dysbiosis can be associated with worsening of liver disease. Here, we review the possible roles for gut microbiota in the development, progression and complication of liver disease.
... It is important to note that we did not sequence the original donor communities and therefore we cannot characterize precisely the true extent of engraftment of strains or the resulting compositional similarity to the original fecal sample. We have based our conclusions on this model protocol as it is established in our facility in multiple studies that replicate phenotypic traits of the donor [36][37][38][39], as well as from other groups, where seminal findings have established that up to 85% of human taxa can be established following oral gavage [40]. However this study and other similar studies have used higher initial concentrations, and other studies have used higher quantities again [41], as well as repeated dosing schedules [42]. ...
Background:
Normal mammalian development and homeostasis are dependent upon the gut microbiota. Antibiotics, essential for the treatment and prophylaxis of bacterial infections, can have collateral effects on the gut microbiota composition, which can in turn have far-reaching and potentially deleterious consequences for the host. However, the magnitude and duration of such collateral effects appear to vary between individuals. Furthermore, the degree to which such perturbations affect the host response is currently unclear. We aimed to test the hypothesis that different human microbiomes have different responses to a commonly prescribed antibiotic and that these differences may impact the host response.
Methods:
Germ-free mice (n = 30) humanized with the microbiota of two unrelated donors (A and B) were subjected to a 7-day antibiotic challenge with amoxicillin-clavulanate ("co-amoxiclav"). Microbiome and colonic transcriptome analysis was performed, pre (day 0) and post antibiotics (day 8) and subsequently into recovery (days 11 and 18).
Results:
Unique community profiles were evident depending upon the donor, with donor A recipient mice being dominated by Prevotella and Faecalibacterium and donor B recipient mice dominated by Bacteroides and Parabacteroides. Donor A mice underwent a marked destabilization of their microbiota following antibiotic treatment, while donor B mice maintained a more stable profile. Dramatic and overlapping alterations in the host transcriptome were apparent following antibiotic challenge in both groups. Despite this overlap, donor A mice experienced a more significant alteration in gene expression and uniquely showed correlations between host pathways and key microbial genera.
Conclusions:
Germ-free mice humanized by different donor microbiotas maintain distinct microbiome profiles, which respond in distinct ways to antibiotic challenge and evince host responses that parallel microbiome disequilibrium. These results suggest that inter-individual variation in the gut microbiota may contribute to personalized host responses following microbiota perturbation.
... In fact, we previously reported the identification of the first human bacterium (Bacteroides sp. D8) that displayed a high coprostanol production level in human feces [45,54]. Furthermore, feeding the cholesterol-reducing bacterium Eubacterium coprostanoligenes significantly reduced plasma cholesterol levels in hypercholesterolemic rabbits [27]. ...
The gut microbiota plays a key role in cholesterol metabolism, mainly through the reduction of cholesterol to coprostanol. The latter sterol exhibits distinct physicochemical properties linked to its limited absorption in the gut. Few bacteria were reported to reduce cholesterol into coprostanol. Three microbial pathways of coprostanol production were described based on the analysis of reaction intermediates. However, these metabolic pathways and their associated genes remain poorly studied. In this review, we shed light on the microbial metabolic pathways related to coprostanol synthesis. Moreover, we highlight current strategies and future directions to better characterize these microbial enzymes and pathways.
... The immunoregulatory activities of short-chain fatty acids (SCFA) produced by gut bacteria via the fermentation of carbohydrates and proteins has been already studied and reviewed extensively 141,142 and will be described only briefly in the next section. The gut microbiota can also metabolize dietary lipids, cholesterol, and host-derived cholesterol metabolites such as steroids and bile acids.[143][144][145] Hence, gut bacteria have the potential to alter the bioavailability and bioactivities of these compounds and thereby alter corresponding signaling networks. ...
The community of microorganisms in the mammalian gastrointestinal tract, referred to as the gut microbiota, influences host physiology and immunity. The last decade of microbiome research has provided significant advancements for the field and highlighted the importance of gut microbes to states of both health and disease. Novel molecular techniques have unraveled the tremendous diversity of intestinal symbionts that potentially influence the host, many proof-of-concept studies have demonstrated causative roles of gut microbial communities in various pathologies, and microbiome-based approaches are beginning to be implemented in the clinic for diagnostic purposes or for personalized treatments. However, several challenges for the field remain: purely descriptive reports outnumbering mechanistic studies and slow translation of experimental results obtained in animal models into the clinics. Moreover, there is a dearth of knowledge regarding how gut microbes, including novel species that have yet to be identified, impact host immune responses. The sheer complexity of the gut microbial ecosystem makes it difficult, in part, to fully understand the microbiota-host networks that regulate immunity. In the present manuscript, we review key findings on the interactions between gut microbiota members and the immune system. Because culturing microbes allows performing functional studies, we have emphasized the impact of specific taxa or communities thereof. We also highlight underlying molecular mechanisms and discuss opportunities to implement minimal microbiome-based strategies.
... This transformation therefore effectively removes cholesterol from circulation. Coprostanol comprises up to 50% of the steroids in human feces (61), and germ-free (GF) mice colonized with microbes from high and low cholesterol-reducing patients produce distinct amounts of coprostanol (62). Animal experiments also suggest that cholesterol-reducing bacteria may decrease serum cholesterol (61). ...
One person's meat is another's poison
The human gut is packed with actively metabolizing microorganisms. These have a transformative effect on what we ingest—whether food, drugs, or pollutants. Koppel et al. review the distinguishing features of microbial xenobiotic metabolism, its interaction with somatic metabolism, and interindividual variation. Depending on the functional composition of microorganisms in the gut, the subsequent products may have nutritionally beneficial effects, modify pharmaceuticals, or be toxic. All of these consequences of our companion microbes can have important impacts on human health and well-being.
Science , this issue p. eaag2770
... Total bacterial DNA and Bifidobacterium-specific DNA were amplified using HotStar Taq DNA polymerase (Qiagen, Courtaboeuf, France) and specific primer sets and temperature-time programs (Table 3 and Table S1). PCR products were submitted to TTGE for sequence-specific separation using the DCode Universal Mutation Detection System (Bio-Rad, Paris, France), a 1 mm-thick, 16 × 16 polyacrylamide gel (as described by [42]) and 7 L of 1.25× Tris-acetate-EDTA electrophoresis buffer. The electrophoresis was performed at a fixed voltage of 63 V for 15 h, with an initial temperature of 66 • C and a ramp rate of 0.3 • C/h. ...
Abstract: The presence of pesticide residues in food is a public health problem. Exposure to these substancesindailylifecouldhaveseriouseffectsontheintestine—thefirstorgantocomeintocontact withfoodcontaminants. Thepresentstudyinvestigatedtheimpactofalowdose(1mg/dayinoil)of the pesticide chlorpyrifos (CPF) on the community structure, diversity and metabolic response of the human gut microbiota using the SHIME® model (six reactors, representing the different parts of the gastrointestinal tract). The last three reactors (representing the colon) were inoculated with a mixture of feces from human adults. Three time points were studied: immediately before the first dose of CPF, and then after 15 and 30 days of CPF-oil administration. By using conventional bacterial culture and molecular biology methods, we showed that CPF in oil can affect the gut microbiota. It had the greatest effects on counts of culturable bacteria (with an increase in Enterobacteria, Bacteroides spp. and clostridia counts, and a decrease in bifidobacterial counts) and fermentative activity, which were colon-segment-dependent. Ourresultssuggestthat: (i)CPFinoiltreatmentaffectsthegutmicrobiota (although there was some discordance between the culture-dependent and culture-independent analyses); (ii) the changes are “SHIME®-compartment” specific; and (iii) the changes are associated with minor alterations in the production of short-chain fatty acids and lactate
... Concurrently, more studies on animal models, especially using gnotobiotic animals, are needed to determine the effect of this bacterial metabolism on plasma cholesterol. Interestingly, it was shown that human microbiota retained its level of cholesterol-reducing bacterial population and cholesterol-reducing activity in gnotobiotic rats [26]. Rodents harboring a human gut microbiota could therefore be used as a model to explore the impact on cholesterolemia and atherosclerosis development of gut microbial cholesterol metabolism and to decipher if a dysbiosis may change host cholesterol metabolism. ...
... However, antibiotic treatment can influence the rate of conversion [33]. Healthy individuals can be classified as high or low coprostanol formers [34,35], and these high or low metabolic phenotypes could be replicated in animals by transplantation of human fecal material into gnotobiotic rats [36]. These observations suggest that coprostanol-associated phenotype is determined by the composition of gut microbiota. ...
Clostridium difficile infection (CDI) is characterized by dysbiosis of the intestinal microbiota and a profound derangement in the fecal metabolome. However, the contribution of specific gut microbes to fecal metabolites in C. difficile-associated gut microbiome remains poorly understood. Using gas-chromatography mass spectrometry (GC-MS) and 16S rRNA deep sequencing, we analyzed the metabolome and microbiome of fecal samples obtained longitudinally from subjects with Clostridium difficile infection (n = 7) and healthy controls (n = 6). From 155 fecal metabolites, we identified two sterol metabolites at >95% match to cholesterol and coprostanol that significantly discriminated C. difficile-associated gut microbiome from healthy microbiota. By correlating the levels of cholesterol and coprostanol in fecal extracts with 2,395 bacterial operational taxonomic units (OTUs) determined by 16S rRNA sequencing, we identified 63 OTUs associated with high levels of coprostanol and 2 OTUs correlated with low coprostanol levels. Using indicator species analysis (ISA), 31 of the 63 coprostanol-associated bacteria correlated with health, and two Veillonella species were associated with low coprostanol levels that correlated strongly with CDI. These 65 bacterial taxa could be clustered into 12 sub-communities, with each community containing a consortium of organisms that co-occurred with one another. Our studies identified 63 human gut microbes associated with cholesterol-reducing activities. Given the importance of gut bacteria in reducing and eliminating cholesterol from the GI tract, these results support the recent finding that gut microbiome may play an important role in host lipid metabolism.
... Récemment, il a été montré que cette répartition était directement liée au nombre de bactéries réductrices de cholestérol présentes dans le tube digestif. Une population minimale de 10 6 /g de contenu digestif est ainsi nécessaire pour une conversion partielle du cholestérol, tandis qu'une conversion totale nécessite une population de bactéries réductrices de cholestérol supérieure ou égale à 10 8 /g 22,23 . Par ailleurs, si ce métabolisme pouvait limiter l'absorption du cholestérol et donc le risque de maladies cardiovasculaires, il a également été suggéré que le taux fécal de coprostanol pourrait être corrélé à la cancérogenèse colique. ...
... Finally, we asked whether the bile cholesterol could be excreted into the feces. Studies on cholesterol generally make it clear that cholesterol is primarily transformed into coprostanol by the intestinal microbiota [29][30][31]. Coprostanol comprised the largest amount of the feces and was produced from cholesterol principally by direct The data are shown as the mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 vs the ND controls; ## P < 0.01 vs the BBR-treated HFD hamsters (100 mg/kg/day). saturation of the 5, 6-double bond of cholesterol [32]. ...
Berberine (BBR), as a new medicine for hyperlipidemia, can reduce the blood lipids in patients. Mechanistic studies have shown that BBR activates the extracellular-signal regulated kinase pathway by stabilizing low-density-lipoprotein receptor mRNA. However, aside from inhibiting the intestinal absorption of cholesterol, the effects of BBR on other metabolic pathways of cholesterol have not been reported. This study aimed to investigate the action of BBR on the excretion of cholesterol in high-fat diet-induced hyperlipidemic hamsters.
Golden hamsters were fed a high-fat diet (HFD) for 6 weeks to induce hyperlipidemia, followed by oral treatment with 50 and 100 mg/kg/day of BBR or 10 and 30 mg/kg/day of lovastatin for 10 days, respectively. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), transaminases, and total bile acid in the serum, liver, bile and feces were measured using an enzyme-linked immunosorbent assay. The cholesterol (as well as coprostanol) levels in the liver, bile and feces were determined by gas chromatography-mass spectrometry.
The HFD hamsters showed significantly hyperlipidemic characteristics compared with the normal hamsters. Treatment with BBR for 10 days reduced the serum TC, TG and LDL-C levels in HFD hamsters by 44-70, 34-51 and 47-71 %, respectively, and this effect was both dose- and time-dependent. Initially, a large amount of cholesterol accumulated in the hyperlipidemic hamster livers. After BBR treatment, reductions in the liver cholesterol were observed by day 3 and became significant by day 7 at both doses (P < 0.001). Meanwhile, bile cholesterol was elevated by day 3 and significantly increased at day 10 (P < 0.001). BBR promoted cholesterol excretion from the liver into the bile in hyperlipidemic hamsters but not in normal hamsters, and these results provide a link between the cholesterol-lowering effect of BBR with cholesterol excretion into the bile.
We conclude that BBR significantly promoted the excretion of cholesterol from the liver to the bile in hyperlipidemic hamsters, which led to large decreases in the serum TC, TG and LDL-C levels. Additionally, compared with lovastatin, the BBR treatment produced no obvious side effects on the liver function.
... DNA was extracted from each bacterial culture as in (Tomas et al., 2013). Primers GCclamp-U968 (GCclamp-5 0 -GAACGCGAAGAACCTTAC-3 0 ) and L1401 (5 0 -GCGTGTGTACAAGACCC-3 0 ) were used to amplify the V6-V8 regions of bacterial 16S rRNA genes (Gerard et al., 2004). PCR products were purified using the Promega Wizard SV PCR clean-up system (Promega, Lyon, France) and sequenced by Eurofins MGW Operon (Ebersberg, Germany). ...
We investigated the effects of early colonizing bacteria on the colonic epithelium. We isolated dominant bacteria, Escherichia coli, Enterococcus faecalis, Lactobacillus intestinalis, Clostridium innocuum and a novel Fusobacterium spp., from the intestinal contents of conventional suckling rats and transferred them in different combinations into germfree (GF) adult rats. Animals were investigated after various times up to 21 days. Proliferative cell markers (Ki67, proliferating cell nuclear antigen, phospho-histone H3, cyclin A) were higher in rats monocolonized with E. coli than in GF at all time points, but not in rats monocolonized with E. faecalis. The mucin content of goblet cells declined shortly after E. coli administration whereas the mucus layer doubled in thickness. Fluorescence in situ hybridization analyses revealed that E. coli resides in this mucus layer. The epithelial mucin content progressively returned to baseline, following an increase in KLF4 and in the cell cycle arrest-related proteins p21(CIP1) and p27(KIP1). Markers of colonic differentiated cells involved in electrolyte (carbonic anhydrase II and slc26A3) and water (aquaglyceroporin3 (aqp3)) transport, and secretory responses to carbachol were modulated after E. coli inoculation suggesting that ion transport dynamics were also affected. The colonic responses to simplified microbiotas differed substantially according to whether or not E. coli was combined with the other four bacteria. Thus, proliferation markers increased substantially when E. coli was in the mix, but very much less when it was absent. This work demonstrates that a pioneer strain of E. coli elicits sequential epithelial remodeling affecting the structure, mucus layer and ionic movements and suggests this can result in a microbiota-compliant state.The ISME Journal advance online publication, 11 July 2014; doi:10.1038/ismej.2014.111.
... Concurrently, more studies on animal models, especially using gnotobiotic animals, are needed to determine the effect of this bacterial metabolism on plasma cholesterol. Interestingly, it was shown that human microbiota retained its level of cholesterol-reducing bacterial population and cholesterol-reducing activity in gnotobiotic rats [26]. Rodents harboring a human gut microbiota could therefore be used as a model to explore the impact on cholesterolemia and atherosclerosis development of gut microbial cholesterol metabolism and to decipher if a dysbiosis may change host cholesterol metabolism. ...
The human gastro-intestinal tract hosts a complex and diverse microbial
community, whose collective genetic coding capacity vastly exceeds that of the human
genome. As a consequence, the gut microbiota produces metabolites from a large range of
molecules that host’s enzymes are not able to convert. Among these molecules, two main
classes of steroids, cholesterol and bile acids, denote two different examples of bacterial
metabolism in the gut. Therefore, cholesterol is mainly converted into coprostanol, a non
absorbable sterol which is excreted in the feces. Moreover, this conversion occurs in a part
of the human population only. Conversely, the primary bile acids (cholic and
chenodeoxycholic acids) are converted to over twenty different secondary bile acid
metabolites by the gut microbiota. The main bile salt conversions, which appear in the gut
of the whole human population, include deconjugation, oxidation and epimerization of
hydroxyl groups at C3, C7 and C12, 7-dehydroxylation, esterification and desulfatation.
If the metabolisms of cholesterol and bile acids by the gut microbiota are known for
decades, their consequences on human health and disease are poorly understood and only
start to be considered.
... In general, cholesterol not absorbed in the small intestine can be metabolized by the resident intestinal microbiota. It has been suggested that cholesterol-to-coprostanol conversion by the intestinal microbiota may facilitate the removal of body cholesterol (35,36). However, the major factors that modulate the cholesterollowering microbial activity have not yet been elucidated. ...
Objective:
To determine the effects of naturally derived probiotic strains individually or combination on a short-term diet-induced obesity model.
Design and methods:
C57BL/6J mice (n = 50) were randomly divided into five groups, then fed a high-fat high-cholesterol diet (HFCD), HFCD and Lactobacillus plantarum KY1032 (PL, 10(10) cfu/day), HFCD and Lactobacillus curvatus HY7601 (CU, 10(10) cfu/day), HFCD and in combination with PL+CU (10(10) cfu/day), or a normal diet (ND) for 9 weeks.
Results:
PL and CU showed distinct and shared metabolic activity against a panel of 50 carbohydrates. Fat accumulation in adipose tissue and liver was significantly reduced by probiotic strains CU or PL+CU. Probiotic strains CU or PL+CU reduced cholesterol in plasma and liver, while PL+CL had a synergistic effect on hepatic triglycerides. Probiotic strains PL+CU combination was more effective for inhibiting gene expressions of various fatty acid synthesis enzymes in the liver, concomitant with decreases in fatty acid oxidation-related enzyme activities and their gene expressions.
Conclusions:
Multi-strain probiotics may prove more beneficial than single-strain probiotics to combat fat accumulation and metabolic alterations in diet-induced obesity.
Research conducted in the past couple of decades has showcased the importance of the gut microbiota in human health and well-being. While many studies have reported on the differences in community membership between a disease state and a healthy state, few have investigated the mechanisms through which an aberrant microbiota contributes to a disease phenotype. One of the primary reasons for this are the many technical and ethical barriers to conducting the necessary studies directly in human individuals. Human microbiota-associated (HMA) porcine models have the potential to become important research tools which can enable the testing of hypotheses regarding host-microbiota interactions in human health and disease without directly involving humans. However, relatively few microbiome studies have utilized porcine models in this capacity. Through multiple studies, we evaluated HMA porcine models in terms of their suitability for use in gut microbiota studies. Results demonstrated that (1) compared to an HMA C3H/HeN mouse model, a higher percentage of donor taxa from donors of different age groups were able to persistently colonize HMA piglets, (2) while a majority of donor taxa in infant donors were able to colonize HMA piglets, rare/low-abundance taxa found in the infant donors enriched once engrafted into the piglets, and (3) the potential for using HMA piglets for studying host-microbiota interactions related to obesity. We believe that further improvements to address some of the shortcoming and challenges associated with HMA piglets will facilitate more wide-spread use of this animal model in the field of gut microbiome research. Advisor: Samodha C. Fernando
Le microbiote intestinal est associé à plusieurs troubles métaboliques de l'hôte, comme la cholestérolémie. Dans ce contexte, le microbiote intestinal pourrait également avoir un impact bénéfique par le biais de différentes voies microbiennes, notamment : le piégeage du cholestérol, la production de coprostanol et les activités hydrolases des sels biliaires. Cependant, de nombreux éléments restent à élucider pour comprendre les mécanismes de l'influence du microbiote intestinal sur le métabolisme du cholestérol de l'hôte. Ce travail de thèse a permis d’enrichir nos connaissances conceptuelles à travers des travaux de recherche basés sur ces trois mécanismes microbiens impliqués dans le métabolisme du cholestérol. Par la suite, nous avons choisi d’étendre cette approche conceptuelle à la modélisation mathématique, pour comprendre la dynamique et les interactions de systèmes complexes. Nous avons développé pour la première fois, un modèle humain du métabolisme du cholestérol incluant le microbiote intestinal pour étudier l'impact relatif des voies de l'hôte et celle microbiote. Ce modèle montre que ces voies bactériennes apparaissent comme un moteur important de la régulation du cholestérol. Pour renforcer ces observations, nous avons identifié trois nouvelles bactéries intestinales présentant chacune de ces 3 voies métaboliques microbiennes et tester l'impact de ces trois voies métaboliques microbiennes dans un modèle animal, la souris. Cette application montre que les hydrolases des sels biliaires (BSH) constituent le levier le plus important pour réguler la cholestérolémie. Pour consolider ces données expérimentales, nous avons confirmé le potentiel hypocholestérolémique attractif des bactéries dotées de BSH en utilisant un modèle plus proche de l’homme, le lapin. L'administration de la bactérie présentant une BSH, a permis une diminution de l’hypercholestérolémie et de ses troubles associés (lésions hépatiques et développement de l'athérosclérose). L’ensemble de ces travaux mettent en évidence l'utilisation de bactéries intestinales ayant une activité BSH comme stratégie probiotique pour faire face à l'hypercholestérolémie.
The microbiomes, including bacteria, fungi, and viruses, exist within and on all the organisms, which is the current field of research. Particularly of interest are microbiome of human and its direct impact on human health. The health and fitness of animals, including humans, are influenced by the existence and composition of microbial communities of the host. To date, maximum microbiome research has been focused on the mouse as a model organism for studying the mechanisms of different processes occurring in the microbial communities. Mouse microbiome models have also been the primary choice for performing preclinical tests for studying relationships between the microbiomes and host physiological, metabolic, immune, and neurologic phenotypes. These were also used for developing methodologies to correct functional abnormalities in these communities that lead to disease. The mouse, however, is not a perfect model for studying different aspects of the microbiome and for studying the host stimuli and environmental responses. Hence, researchers have been conducting microbiome studies using other animals as well, for example, zebrafish, pigs, and Drosophila. This chapter summarizes the microbiome studies conducted using different models and an insight into its advantages.
Gastric cancer is the fifth most commonly diagnosed cancer in the world. With over one million diagnosed cases per year, it is the third leading cause of cancer-related deaths worldwide. Since the establishment of cell culture in the early 1900s along with the generation of the first cell lines to study gastric cancer and disease, the breadth of knowledge and resources has grown substantially. A key element of the evolution of modeling for gastric carcinogenesis, in particular, has been the advent of genetic engineering and organoid culture. Genetic engineering has allowed the study of specific genes and “humanizing” model systems for preclinical use, while organoid culture provides a physiologically similar in vitro cell culture of patient-derived tissue. This chapter describes the generation and use of commonly used models in vitro and in vivo to study gastric disease and recent developments in methods and technology.
Bile is a biological fluid synthesized in the liver, mainly constituted by bile acids and cholesterol, which functions as a biological detergent that emulsifies and solubilizes lipids, thereby playing an essential role in fat digestion. Besides, bile acids are important signaling molecules that regulate key functions at intestinal and systemic levels in the human body, affecting glucose and lipid metabolism, and immune homeostasis. Apart from this, due to their amphipathic nature, bile acids are toxic for bacterial cells and, thus, exert a strong selective pressure on the microbial populations inhabiting the human gut, decisively shaping the microbial profiles of our gut microbiota, which has been recognized as a metabolic organ playing a pivotal role in host health. Remarkably, bacteria in our gut also display a range of enzymatic activities capable of acting on bile acids and, to a lesser extent, cholesterol. These activities can have a direct impact on host physiology as they influence the composition of the intestinal and circulating bile acid pool in the host, affecting bile homeostasis. Given that bile acids are important signaling molecules in the human body, changes in the microbiota-residing bile biotransformation ability can significantly impact host physiology and health status. Elucidating ways to fine-tune microbiota-bile acids-host interplay are promising strategies to act on bile and cholesterol-related disorders. This manuscript summarizes the current knowledge on bile and cholesterol metabolism by intestinal bacteria, as well as its influence on host physiology, identifying knowledge gaps and opportunities to guide further advances in the field.
Gut microbiome studies have been gaining popularity over the years, especially with the development of new technologies (e.g. metataxonomics, metagenomics, metatranscriptomics, and metabonomics) that makes it easier for researchers to characterize the composition and functionality of these complex microbial communities. The goal of these studies is to identify a microorganism, group of microbes, or microbial metabolite which correlates with a disease state (e.g. inflammatory bowel disease, colorectal cancer, and obesity). Many of these are cross-sectional studies, where fecal samples from a group of diseased individuals are compared with those from a group of healthy individuals at a single time point. However, there are a wide range of variables that can affect the gut microbiota of humans which make mechanistic studies challenging. Longitudinal studies are required for research to more reliably correlate interventions or disease status to microbiota composition and functionality. However, longitudinal studies in humans and animals are difficult, expensive, and time-consuming. This review will discuss in vitro gut fermentation models and how they can be used to perform longitudinal studies that complement in vivo microbiome studies. Gut fermentation models support the growth of stable, reproducible, and diverse microbial communities in a tightly controlled environment set to mimic the conditions microbes encounter in the gastrointestinal tract. Gut fermentation models will make it easier for researchers to perform mechanistic studies and aid in the development of novel treatments that are both targeted and maintained over time.
4. International Dietary Fibre Conference. Vienne. 2009/07/01-03
The gastrointestinal (GI) tract ensures food digestion and nutrient and water absorption, but it has also a crucial barrier function. The GI tract harbors a complex microbiota with underestimated metabolic potential and influences. Dysregulation in microbiota-GI cross talk is increasingly suspected to be involved in inflammatory, functional, and proliferative GI diseases. This chapter analyzes the evidence for early influences on GI epithelium epigenetic modifications in connection with the “developmental origin of health and disease” theory; the possible role of the gut microbiota; the links between early epigenetic modifications and chronic diseases such as inflammatory bowel diseases, irritable bowel syndrome, and colorectal cancer in later life; and, finally, dietary modulation of the GI epigenome. Available data indicate epigenetic modifications to operate at a GI epithelial level, but our understanding of these changes and their consequences is still fragmentary. Massive research efforts are needed to decipher the roles and mechanisms of dietary, microbial, and environmental factors in epigenetic changes that could affect GI epithelial function in the long term.
The exploitation of dietary oligosaccharides as prebiotic compounds depends largely upon their survival rates after digestion, their fermentation by gut microbiota, and selective stimulation in the growth and/or activities of one or a limited number of intestinal bacteria associated with health and well-being. This chapter summarizes the scientific evidence, based on animal models and well-controlled human intervention studies carried out with healthy volunteers, of dietary oligosaccharides in order to fulfil these three main criteria. The use of different in vivo approaches as well as the relevance of certain factors, including the full characterization of oligosaccharides tested and definition of the target population group, is reported.
The intestine of the human contains a dynamic population of microbes that have a symbiotic relationship with the host. In addition, there is an effect of the intestinal microbiota on metabolism and digestion. Non-alcoholic fatty liver disease (NAFLD) is a common cause worldwide of hepatic pathology and is thought to be the hepatic manifestation of the metabolic syndrome. In this review we examine the effect of the human microbiome on the components and pathogenesis of the metabolic syndrome. We are now on the threshold of therapeutic interventions on the human microbiome in order to effect human disease including NAFLD.
The obesity, diabetes, and cardiovascular diseases like metabolic abnormalities are became major health and public health issues worldwide. Obesity and insulin resistance is associated with significant changes in composition and metabolic function of gut microbiota but the pathophysiological processes having this bidirectional association have not been fully elucidated. In the present investigation, we modulated gut microbiota by giving site-specific delivery of gram positive, gram negative, and broad spectrum antibiotics coated with pH-sensitive polymer. Following high fructose rich diet for the Type II diabetes induction, the gram positive removal showed more profound effect on total fat pad, total cholesterol, and triglyceride and had higher tendency to develop insulin resistance or diabetes in these animals. From the findings, it could be concluded that changes in gut microbiota by partial removal of gram negative bacteria may control metabolic endotoxemia, inflammation, and associated disorders by a mechanism that could increase intestinal permeability. It would thus been useful to develop strategies for changing gut microbiota to control, intestinal permeability, metabolic endotoxemia, and associated disorders.
The human intestinal microbial ecosystem plays an important role in maintaining health. A multitude of diseases including diarrhoea, gastrointestinal inflammatory disorders, such as necrotising enterocolitis (NEC) of neonates, and obesity are linked to microbial composition and metabolic activity. Therefore, research on possible dietary strategies influencing microbial composition and activity, both preventive and curative, is being accomplished. Interest has focused on pre- and probiotics that stimulate the intestinal production of beneficial bacterial metabolites such as butyrate, and beneficially affect microbial composition. The suitability of an animal model to study dietary linked diseases is of much concern. The physiological similarity between humans and pigs in terms of digestive and associated metabolic processes places the pig in a superior position over other non-primate models. Furthermore, the pig is a human-sized omnivorous animal with comparable nutritional requirements, and shows similarities to the human intestinal microbial ecosystem. Also, the pig has been used as a model to assess microbiota-health interactions, since pigs exhibit similar syndromes to humans, such as NEC and partly weanling diarrhoea. In contrast, when using rodent models to study diet-microbiota-health interactions, differences between rodents and humans have to be considered. For example, studies with mice and human subjects assessing possible relationships between the composition and metabolic activity of the gut microbiota and the development of obesity have shown inconsistencies in results between studies. The present review displays the similarities and differences in intestinal microbial ecology between humans and pigs, scrutinising the pig as a potential animal model, with regard to possible health effects.
Dietary phytoestrogens, such as isoflavones, are used as food additives to prevent menopauserelated
disorders. In addition to other factors, their bioavailability strongly depends on the activity of intestinal
bacteria but the underlying interactions remain poorly understood. A randomized, double-blind, placebo-controlled
study was undertaken with 39 postmenopausal women to characterize changes in the dominant microbial
communities of the intestinal tract after 2 mo of isoflavone supplementation with and without pro- or prebiotic. The
diversity and composition of the dominant microbiota were analyzed by temporal temperature-gradient gel
electrophoresis (TTGE) and fluorescent in situ hybridization. Isoflavones alone stimulated dominant microorganisms
of the Clostridium coccoides-Eubacterium rectale cluster, Lactobacillus-Enterococcus group, Faecalibacterium
prausnitzii subgroup, and Bifidobacterium genus. The stimulation of the Clostridium coccoides-Eubacterium
rectale cluster depended on the women’s equol excretion and was transient, with the exception of a prolonged
bifidogenic effect. Lasting changes in the diversity of the dominant species were also observed. The probiotic strain
supplied could be detected by TTGE during its passage through the intestinal tract, and ingestion of fructooligosaccharides
triggered a marked and specific bifidogenic effect. In conclusion, this is the first human study that
shows changes in the diversity and composition of dominant bacterial communities in response to dietary
supplementation with hormone-related compounds combined with functional foods.
A group-specific 16S rRNA-targeted oligonucleotide probe S-G-Lab-0158- a-A20 (Lab158) was designed and validated to quantify species of the phylogenetic group lactobacilli-enterococci. The Lab158 probe detects nearly all species of the genera Lactobacillus, Enterococcus, Pediococcus, Weissella, Vagococcus, Leuconostoc and Oenococcus. The specificity of the probe was tested on various species of the target group and on a range of common intestinal bacteria. For these experiments, procedures to permeabilize these groups of Gram-positive bacteria were optimized and fluorescent in situ hybridization (FISH) conditions for maximum specificity were determined. In addition, we showed that it is possible to distinguish the predominant gut- enterococci i.e. E, faecalis among the Lab158 probe-positive cells with the E. faecalis-specic probe Efs (1). Lactobacilli-enterococci in faecal samples of four Volunteers were enumerated by FISH using the Lab158 probe. With this technique 0.4-0.8 x 108 cells per gram wet weight of faeces were counted The Lab158 probe was also used to identify colonies after culturing faecal bacteria on MRS and Rogosa agar. Only 2% of the colonies hybridized to the lactobacilli-enterococci specific probe. Most of the remaining colonies hybridized to a bifidobacteria specific probe. This shows that FISH with probe Lab158 is a useful method to enumerate lactobacilli-enterococci in faeces and can assist in the identification of lactic acid bacteria grown on plates.
Cultivation methods classically used to describe the faecal flora composition are often too selective for certain groups of bacteria. This study was conducted to determine the human faecal flora composition by fluorescent in situ hybridisation, a direct and culture-independent method. Four group-specific probes and a domain probe Eub 338 targeting the 16S rRNA were used to analyse fixed faecal bacterial suspensions from nine healthy adult volunteers. Epifluorescence microscopy and image analysis were performed to evaluate the relative proportion of cells from each group. After optimisation of hybridisation conditions, the reproducibility of the protocol was evaluated to validate the FISH procedure. The domain probe Eub 338 labelled an average of 80 ± 11% total faecal bacteria. The panel of four probes revealed more than 75% of the flora. The Clostridium coccoides-eubacterium rectale group was the most represented, accounting for 36 ± 7% of the bacteria. The three other probes used, Bacto 1080, Bif 164 and Fprau 645 labelled 17 ± 5%, 15 ± 9% and 23 ± 5% of the total flora, respectively. The two dominant groups belonging to Clostridium and relatives constituted nearly 60% of the total flora. FISH coupled with image analysis is a direct and powerful molecular tool to assess the composition of the human faecal flora.
Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.
The gastrointestinal (GI) tract is one of the most complex ecosystems consisting of microbial and host cells. It is suggested that the host genotype, the physiology of the host and environmental factors affect the composition and function of the bacterial community in the intestine. However, the relative impact of these factors is unknown. In this study, we used a culture-independent approach to analyze the bacterial composition in the GI tract. Denaturing gradient gel electrophoresis (DGGE) profiles of fecal bacterial 16S rDNA amplicons from adult humans with varying degrees of genetic relatedness were compared by determining the similarity indices of the profiles compared. The similarity between fecal DGGE profiles of monozygotic twins were significantly higher than those for unrelated individuals (ts = 2.73, p1-tail = 0.0063, df=21). In addition, a positive relationship (F1, 30 = 8.63, p = 0.0063) between the similarity indices and the genetic relatedness of the hosts was observed. In contrast, fecal DGGE profiles of marital partners, which are living in the same environment and which have comparable feeding habits, showed low similarity which was not significantly different from that of unrelated individuals (ts = 1.03, p1-tail = 0.1561, df=27). Our data indicate that factors related to the host genotype have an important effect on determining the bacterial composition in the GI tract.
To determine the structure of human faecal microbiota, faecal samples from 23 healthy individuals were analysed with a similar set of probes targeting six phylogenetic groups using rRNA dot-blot hybridisation and whole cell fluorescent in situ hybridisation (FISH) combined with flow cytometry. When microbiota compositions derived by each method were compared, the results were not statistically different for Clostridium coccoides, Fusobacterium prausnitzii, Bifidobacterium spp. and Enterobacteria. Conversely, the proportions were significantly different for Bacteroides and Atopobium (P<0.05). The metabolic state of these bacteria within the colon could explain the discrepancy observed between the rRNA level and the actual cell proportion. However, both approaches supplied consistent and complementary information on the structure of the faecal microbiota. FISH combined with flow cytometry appears best suited to future high throughput analysis.
The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.
South Asian immigrants to England and Wales have low mortality from colon cancer and high mortality from coronary heart disease compared with the general population. In a survey of a predominantly Gujarati population in northwest London, both vegetarians and nonvegetarians had similar total dietary fat intake to the native British population but higher dietary fiber intake. Total fecal bile acid and neutral animal sterol concentrations were lower in South Asians than in a native British comparison group. Sixty-two percent of South Asians excreted detectable quantities of free primary bile acids, which were not present in stools from native British subjects. The ratio of fecal coprostanol to total neutral animal sterols was also lower in South Asians. Low risk of colon cancer in this population may be related to reduced microbial activity in the bowel and low levels of tumor-promoting secondary bile acids.
In a metabolic ward 12 healthy male subjects consumed mixed Western (M), lacto-ovovegetarian (L), and vegan (V) diets in a randomized order for 20 d each. The concentrations of deoxycholic acid, isolithocholic acid, and total bile acids in 4-d composites of feces on the L and V diets were significantly lower than on the M diet. The chenodeoxycholic-to-isolithocholic plus lithocholic acid ratio was significantly higher on the V diet. The concentrations of coprostanol and of coprostanol plus cholesterol were highest on M diet and lowest on V diet. The number of fecal lactobacilli and enterococci on the V diet was significantly lower than on the M or the L diets. This study showed a decrease in the concentration of fecal (secondary) bile acids by the L and the V diets and an alteration of the fecal flora composition by the V diet.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
The plasma-cholesterol-lowering effects of some dietary legumes are now well established from animal and human studies, but the mechanism is not completely understood. The present study investigated the effect of baked beans (Phaseolus vulgaris) on steroid metabolism of hypercholesterolaemic pigs. Three groups of four pigs were studied: baseline (BL), normal pigs (NP) and those previously prepared with an ileo-rectal anastomosis to nullify the function of the large intestine (IR). All three groups were given a semi-purified control diet, with about 40% energy as fat (polyunsaturated:saturated fatty acid (P:S) ratio 0.3), supplemented with 10 g cholesterol/kg, for 14 d. Then IR and NP pigs were fed for 28 d on a diet supplemented with 10 g cholesterol/kg and 300 g baked beans/kg (dry-matter basis), so that the 40% contribution to energy from fat was maintained (P:S ratio 0.3). Group BL was fed on the control diet throughout. The intact pigs (NP) fed on baked beans showed considerable differences compared with the other groups, as follows: (a) reduced plasma cholesterol (NS); (b) higher concentration of cholesterol in bile (NS); (c) higher concentration of bile acids, especially secondary bile acids, in bile (P < 0.05); (d) reduced elimination of bile acids in faeces, especially secondary bile acids (P < 0.05); (e) higher excretion of coprostanol and lower elimination of cholesterol in faeces (P < 0.05). From these findings it is proposed that a baked-bean-enriched diet potentiates bacterial fermentation and steroid degradation in the large intestine and enhances conservation of bile acids and cholesterol within the enterohepatic circulation. The high concentration of bile acids and cholesterol in bile may thus promote feedback inhibition of hepatic cholesterol synthesis, and hence, reduce plasma cholesterol.
We designed a panel of four 16S rRNA-targeted oligonucleotide probes specific for bacteria of the phylum cytophaga-flavobacter-bacteroides (CFB). Probes CF319a and CF319b are targeted to members of the flavobacteria-cytophaga group and the genus Porphyromonas, whereas probe BAC303 has a target region characteristic for the genera Prevotella and Bacteroides within the bacteroides group. The probe FFE8b was developed for species-specific hybridizations with Flavobacterium ferrugineum. All probes were designed by computer-assisted sequence analysis and compared to all currently accessible 16S and 23S rRNA sequences. The oligonucleotides were further evaluated by whole-cell and non-radioactive dot-blot hybridization against reference strains of the CFB phylum and other major lineages of Bacteria. The newly developed probes were used together with other higher-order probes to analyse the structure and community composition in complex environments. In activated sludge samples, members of the flavobacteria-cytophaga group were revealed by in situ hybridization as important constituents of sludge flocs and characteristic colonizers of filamentous bacteria. By application of fluorescent probe BAC303, members of the genera Bacteroides and Prevotella could be visualized without prior cultivation as an important part of the human faecal microflora.
The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.
Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 x 10(10) cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 x 10(10) cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 x 10(7) to 7 x 10(8) per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.
The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.
Faecal bulk, pH, water content, the concentrations of neutral sterols and bile acids and dietary intakes were measured in twenty-two Indian vegetarian, twenty-two white omnivorous and eighteen white vegetarian premenopausal women. Faecal bulk and water content were greater and pH lower in the Indian vegetarians. Total faecal animal sterol and coprostanol concentrations expressed on a dry-weight basis were lower in the vegetarians compared with the omnivores. The faecal sterol concentrations were correlated with dietary cholesterol intake. Primary bile acids were detected in six Indian vegetarians, two white vegetarians and two white omnivores; secondary bile acids were detected in all the white omnivores and vegetarian subjects but not in two of the Indian vegetarians. Total faecal free bile acid and conjugated bile acid concentrations were lower in the white vegetarians compared with the omnivores. Faecal lithocholic acid concentrations were lower in both Indian and white vegetarians. The lithocholic: deoxycholic acid ratio and coprostanol: total animal sterols ratio were significantly lower in the Indian vegetarians compared with the omnivores. Both ratios were positively correlated with faecal pH. Stepwise multiple regression analyses were undertaken in order to identify which nutrients influenced faecal pH, lithocholic and deoxycholic acid concentrations. The intakes of starch and dietary fibre were negatively associated with faecal concentrations of lithocholic and deoxycholic acid. Starch intake alone was negatively associated with faecal pH. The results of this study confirm that diets high in dietary fibre decrease faecal bile acid concentrations and suggest that the complex carbohydrates present in Indian vegetarian diets influence faecal pH and inhibit the degradation of faecal steroids.
High fat diets have been implicated in incidence of colon cancer both in epidemiological and animal studies. Present investigation deals with the incidence, location and numbers of large and small bowel tumours induced by 1,2-dimethyl hydrazine (DMH) in rats fed high fat diets and neomycin. Neomycin was used to modify the faecal sterol metabolism and the relationship of the high fat diet and faecal neutral and acid sterols to the large bowel tumorigenesis was evaluated. DMH administered rats were fed with (a) 20% safflower oil; (b) 20% safflower oil and neomycin; (c) 20% safflower oil, cholesterol and cholic acid; and (d) 20% safflower oil, cholesterol, cholic acid and neomycin. Neomycin was found to be associated with both increase and decrease of tumour numbers. The faecal sterols lithocholic and deoxycholic acids were found to have no participation, while cholesterol and cholic acid were found to decrease with increase in tumour numbers. However, faecal coprostanol has been found to have a significant positive correlation with tumorigenesis in all dietary groups. Therefore coprostanol might possibly be associated with colon carcinogenesis in DMH-fed rats and cholesterol metabolism in gut appears to be related to the development of tumours.
To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a
new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted
oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the
total 16S rRNA detected by the bacterial domain probe. The Bacteroidesgroup-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%.Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium andLactobacillus groups made up less than 2%.
Cholesterol precipitation from supersaturated bile is the earliest and determinant step in the formation of cholesterol gallstones, which is thought to be diet-dependent. Bile composition, appearance and growth of cholesterol crystals were studied in fresh gall-bladder biles from pigs adapted to four different protein-containing diets over 3 weeks: 160 g dietary protein/kg as casein (C16; n 6), or as soyabean-protein concentrate (S16; n 6), or a mixture of both protein sources (casein-soyabean protein, 70:30, w/w) (CS16; n 6), or 320 g of the mixed protein/kg (CS32; n 6). Moreover, all four diets contained 3 g cholesterol/kg and 50 g beta-cyclodextrin/kg as modifiers of bile composition towards cholesterol pro-crystallization. Cholesterol precipitation was most active after the high-protein diet, CS32, and the casein diet, C16, and lowest after the soyabean-protein diet, S16. It was intermediate after the mixed diet, CS16, but still much lower than in the former two groups. These diet-induced variations were suggested to be mediated through modifications in the biliary profile of bile acids, whereas all other biliary constituents studied were essentially unchanged. The fasting level of plasma cholesterol was lowest in both 160 g protein/kg diets containing soyabean protein (S16 and CS16), highest for the high-protein diet CS32, and intermediate for the C16 diet. These results should encourage clinical studies on the effect of soyabean protein, or other vegetable proteins, for primary or recurrence prevention of cholelithiasis at its earliest stage.
Two 16S rRNA-targeted probes were developed: one for theCoriobacterium group and the other for theAtopobium cluster (which comprises most of theCoriobacteriaceae species, including theCoriobacterium group). The new probes were based on sequences of three new Coriobacteriaceae strains isolated from human feces and clinical material and sequences from databases. Application of the probes to fecal
samples showed that formula-fed infants had higher numbers of Coriobacterium group cells in their feces than breast-fed infants. In addition, based on the presented results, it is hypothesized that
with the increasing age of a person, the diversity of Atopobium cluster species present in the feces increases.
For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide
probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora.
Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ
hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples
of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers
below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe
detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730
and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were
evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90%
of the total bacterial cells.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 x 10(10) cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 x 10(10) cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 x 10(7) to 7 x 10(8) per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
This chapter presents factors that are considered in developing stringently anaerobic techniques and in describing the procedure and rationale of roll-tube method. A roll-tube method was developed in which agar medium was distributed as a thin layer over the internal surface of test tubes charged with an anaerobic atmosphere for the isolation of obligately anaerobic bacteria of the rumen. In the roll-tube method, exposure of bacteria and culture medium to air is avoided by displacing the air in the culture vessel with an oxygen-free gas, such as carbon dioxide, hydrogen, nitrogen, or mixtures of these gases. Carbon dioxide is the gas of choice because it is heavier than air, relatively cheap, and valuable in buffering. Vessels are stoppered under conditions preventing access of air. The cultures require no special incubators and can be removed and examined with no anaerobic precautions if kept stoppered. If opened, anaerobiosis can be continuously maintained during necessary manipulations, and the culture again closed without exposure to oxygen.
Strains of Lactobacillus isolated from animals of several species were examined for their capacity to colonize the lumens and form layers on the keratinized nonsecreting epithelium in the stomachs of monoassociated ex-germfree mice. All strains tested could be cultured at comparable population levels from the stomachs of the mono-associated mice. With one exception, however, only strains previously isolated from rodents were able to form thick continuous layers on the gastric epithelial surface. The exception was a strain isolated from calf feces. This strain formed a layer on the epithelial surface, comparable to layers seen in animals associated with strains from rodents.
Strains of Lactobacillus isolated from animals of several species were examined for their capacity to colonize the lumens and form layers on the keratinized nonsecreting epithelium in the stomachs of monoassociated ex-germfree mice. All strains tested could be cultured at comparable population levels from the stomachs of the mono-associated mice. With one exception, however, only strains previously isolated from rodents were able to form thick continuous layers on the gastric epithelial surface. The exception was a strain isolated from calf feces. This strain formed a layer on the epithelial surface, comparable to layers seen in animals associated with strains from rodents.
A micro-computer programis described which will calculate the estimate of most probable number and an approximate confidence interval for any combination of dilution levels, numbers of replicates and sample volumes. The use of the program frees the experimenter from the constraints in experimental design often imposed by tables.
The composition of the faecal microbiota and concentrations of faecal short chain fatty acids (SCFAs) and putrefactive products were studied in pig-flora-associated (PFA) mice inoculated with faecal suspensions from pigs before and after weaning. Major faecal bacteria of pigs colonised the intestine of PFA mice, and the composition of microbiota of the PFA mouse faeces were similar to that of the pig faeces inoculum. Unexpectedly, pig faecal lactobacilli also colonised the intestines of PFA mouse groups at levels of 106-109/g faeces. However, faecal concentrations of putrefactive products in PFA mice did not simulate those in the inocula, although the concentrations of the major components of SCFAs of PFA mice were similar to those of the inocula. These findings indicate that PFA mice are a good model for studying the ecosystem of pig faecal microbiota and the control of SCFAs in the pig intestine, but not for studying putrefactive products generated in the pig intestine.
The composition of faecal microbiota, enzyme activities of faecal bacteria and concentrations of faecal putrefactive products and short chain fatty acids (SCFAs) were studied in 'human-flora-associated' (HFA) mice inoculated with six different human faeces. The composition of major faecal bacteria of HFA mice was similar to that of inoculated human faeces, although bifidobacteria were eliminated from some HFA mouse groups. β-Glucuronidase activity of intestinal bacteria in HFA mice was similar to that in humans. However nitroreductase activity was closer to that in conventional mice than in humans. Although the concentrations of faecal putrefactive products and SCFAs in HFA mice were much lower than those in humans, the composition of faecal SCFAs of HFA mice was similar to that of humans. Individual variations of putrefactive products and SCFAs observed among inoculated human faeces were not reflected in HFA mice, and variations among six HFA mice groups were not obvious. Bacterial composition and metabolism in HFA mice were maintained in their offspring by breeding. These findings indicate that HFA mice provide a stable and valuable tool for studying the ecosystem and metabolism of the human faecal microbiota, but they have some limitations as a model.
The adequacy of a human faecal microbiota associated mouse as a model for studying the activities of human intestinal microorganisms was examined. During a 6 month period, several predominant aerobic and anaerobic components of the human faecal bacteria persisted at stable numbers in the intestinal tracts of the mice. However, Bacillus species and both aerobic and anaerobic Lactobacillus species disappeared within 7 d after association. An inverse relationship existed between the presence of short-chain fatty acids and non-fatty organic acids in the caeca of the associated mice. The relative concentrations of the two acid groups changed over a 21 d period, suggesting an alteration in the pattern of metabolism by the bacteria during the course of the study. The total amount of organic acid produced by the microorganisms in the caeca of the associated mice was approximately 25 per cent of the published value for humans, suggesting that the human microbiota retained only a portion of its metabolic activities in the mouse host. When challenged orogastrically with Salmonella typhimurium, associated mice were as resistant to colonisation as conventional mice, but germfree mice were very susceptible.
The results raise questions about the adequacy of the human faecal microorganisms associated mouse as a model to study the ecology of the human intestinal tract. The disappearances of Bacillus and both aerobic and anaerobic Lactobacillus species may influence the outcomes of the studies. Moreover, the greatly reduced metabolic activity of the human faecal microbiota in the mouse may diminish the significance of the results of metabolic studies when extrapolated to the human situation. However, the model might have utility in studies on colonisation resistance against various enteric pathogens.
The intestinal microbial conversion of cholesterol to coprostanol has been measured in groups of healthy subjects before, during and after they received the antibiotics ampicillin, bacitracin, clindamycin, co-trimoxazole, doxycycline, erythromycin, metronidazole, nalidixic acid, ofloxacin or vancomycin orally for 6 days. Before they received antibiotics, the subjects demonstrated two distinct patterns of cholesterol conversion. One pattern was characterised by extensive conversion of cholesterol, the other by little or no conversion. Intake of bacitracin, clindamycin, erythromycin, metronidazole and vancomycin significantly reduced the conversion to coprostanol. In the groups receiving ampicillin or doxycycline, marked reductions were found in most of the subjects. No alterations were found in the groups receiving co-trimoxazole, nalidixic acid or ofloxacin. In 6 subjects no conversion of cholesterol to coprostanol was found up to 5 weeks after the end of the antibiotic intake. We conclude that orally given antibiotics may cause alterations in the intestinal conversion of cholesterol, reflecting changes in the anaerobiC., Gram-positive component of the gut flora.
Ecologists need to analyze their field data to interpret relationships within plant and animal communities and with their environments. The purpose of this book is to show ecologists and environmental scientists what numerical and statistical methods are most useful, how to use them and interpret the results from them, and what pitfalls to avoid. Subjects treated include data requirements, regression analysis, calibration (or inverse regression), ordination techniques, cluster analysis, and spatial analysis of ecological data. The authors take pains to use only elementary mathematics and explain the ecological models behind the techniques. Exercises and solution are provided for practice. This is the only book written specifically for ecologists that explains such techniques as logistic regression, canonical correspondence analysis, and kriging (statistical manipulation of data). This is a reissue of a book first published in 1987 by Pudoc (The Netherlands). Contents List of contributors; Preface to first edition; Acknowledgement; List of symbols; Dune meadow data; 1. Introduction R. H. G. Jongman; 2. Data collection J. C. Jager and C. W. N. Looman; 3. Regression C. J. F. ter Braak and C. W. N. Looman; 4. Calibration C. J. F. ter Braak; 5. Ordination C. J. F. ter Braak; 6. Cluster analysis O. F. R. van Tongeren; 7. Spatial aspects of ecological data P. A. Burrough; 8. Numerical methods in practice: case-studies P. A. Burrough, J. A. F. Oudhof, A. Barendregt, R. H. G. Jongman and T. J. van de Nes; References; Index.
Because of the potential significance of colonic bacteria and secondary bile acids in the pathogenesis of colon cancer, the present study investigated the effect of different types of dietary fiber on fecal bacterial enzymes, namely, beta-glucuronidase, 7 alpha-dehydroxylase, nitroreductase, and azoreductase, and on bile acids and neutral sterols in premenopausal women. The subjects consumed 13-15 g of wheat, oat, or corn bran daily for 8 weeks in addition to their normal diet. Stools collected during the normal and fiber diet periods were analyzed for the above constituents. Dietary wheat bran decreased the concentrations of fecal deoxycholic acid, lithocholic acid, 12-ketolithocholic acid, and neutral sterols and the activities of all bacterial enzymes. Oat bran had no effect on secondary bile acids and 7 alpha-dehydroxylase but decreased beta-glucuronidase, nitroreductase, and azoreductase levels. Dietary corn bran increased 7 alpha-dehydroxylase, lithocholic acid, and cholesterol levels and decreased deoxycholic acid coprostanol, cholestenone, nitroreductase, and azoreductase levels. These results show that the modifying effect of dietary fiber on secondary bile acids and bacterial enzymes that may play a role in carcinogenesis depends on the type of fiber consumed.
Acceleration of cholesterol catabolism (through feces) has been proposed as one of the mechanisms for the hypocholesterolemic effect of dairy products. This study examined the effects of feeding two milk products (skim milk and skim milk yogurt) on fecal neutral sterol excretions in rats. Six groups of nine rats each were fed iso-caloric Chow-based diets containing water, 45% skim milk (SM), or 45% skim milk yogurt (SMY), without or with cholesterol. The results indicate that both SM and SMY increased the excretion of total neutral sterols under hyperlipemic conditions. The SMY diet (with cholesterol) also increased the excretion of coprostanol, a bacterial metabolite.
The study of colonic flora composition and metabolism presents considerable methodological problems. Attempts to circumvent these problems have led to the development of numerous in vitro and in vivo models to simulate the human colon and its microbial population. In terms of in vivo models, conventional laboratory animals have many limitations. Data of greater relevance to man can be obtained by using germ-free rodents associated with human colonic bacteria. The applications of such animals to studies of toxicity of chemicals and gastrointestinal infections are discussed. The advantages and disadvantages of the various in vitro systems for studying gut microflora and its metabolic activity (from simple static cultures to the more sophisticated continuous and semicontinuous flow models) are reviewed. The apparatus involved is described together with practical information on media, running conditions, and sampling. The bacteriological and metabolic criteria for establishing the similarity of the models to the in situ colonic flora are also discussed. The final sections of the review are devoted to the major applications (current and future) of the models, including fermentation studies on dietary fiber, metabolism of nutrients and foreign compounds (including carcinogens) in food, and the investigation of colonization resistance.
Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.
The intestinal microbial conversion of cholesterol to coprostanol has been measured in groups of healthy subjects before, during and after they received the antibiotics ampicillin, bacitracin, clindamycin, co-trimoxazole, doxycycline, erythromycin, metronidazole, nalidixic acid, ofloxacin or vancomycin orally for 6 days. Before they received antibiotics, the subjects demonstrated two distinct patterns of cholesterol conversion. One pattern was characterised by extensive conversion of cholesterol, the other by little or no conversion. Intake of bacitracin, clindamycin, erythromycin, metronidazole and vancomycin significantly reduced the conversion to coprostanol. In the groups receiving ampicillin or doxycycline, marked reductions were found in most of the subjects. No alterations were found in the groups receiving co-trimoxazole, nalidixic acid or ofloxacin. In 6 subjects no conversion of cholesterol to coprostanol was found up to 5 weeks after the end of the antibiotic intake. We conclude that orally given antibiotics may cause alterations in the intestinal conversion of cholesterol, reflecting changes in the anaerobic, Gram-positive component of the gut flora.
Gnotobiotic rodents are increasingly used as a model for studying in vivo the characteristics of human colonic flora. However, the value of this model has been poorly assessed. In this study fecal bacterial flora provided either by a conventional rat (group RFR) or by man (group RFH) was administered orally to two groups of 6 germ-free rats. One month later, quantitative bacteriological analyses of feces revealed that bacterial populations were close to those of donors in both groups. The metabolic activity of the genuine flora was further compared in groups RFR and RFH with that of the implanted flora: a) concentrations of each fecal volatile fatty acid and of fecal bile acids were similar in conventional and RFR rats as well as the percentage of transformation of cholesterol into coprostanol (48 p. 100 and 54 +/- 5 p. 100 respectively; m +/- SD); b) similar concentrations of fecal volatile fatty acids were obtained from the human donor and RFH rats. Alpha, beta and omega muricholic acids absent in human donor's feces were found in RFH feces. Cholesterol transformation was lower in RFH rats (48 +/- 9 p. 100) than in man (85 p. 100); c) a single dose of lactulose (3 g/kg) increased breath hydrogen excretion in man but not in conventional or in RFR rats and RFH. Chronic lactulose ingestion (3 g/kg d.i.b. for 8 days) had no effect in conventional or RFR rats. Hydrogen excretion was decreased in man, whereas it was significantly increased in RFH rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Fecal bile acids and neutral sterols of spontaneous colon cancer-bearing Wistar-Furth strain rats were examined and compared with those of control rats of the same strain by gas chromatography and gas chromatography-mass spectrometry. The amount of total fecal bile acids and the percentage of fecal lithocholic acid were almost similar in both groups, but the percentage of fecal deoxycholic acid was significantly higher in the colon cancer group than in the controls. The amount of total fecal neutral sterols and the percentage of fecal coprostanol were also markedly higher in the colon cancer group than in the controls. The percentage of cholesterol and the amount of beta-sitosterol were, however, almost similar in the two groups. These results indicate that fecal deoxycholic acid and fecal coprostanol are notably higher in rats with colon cancer than in controls.
Existing methods for the identification of mammalian fecal particles in foods have not been completely satisfactory because visual identification of small particles is difficult. In addition, identification of feces by determining the presence of fecal alkaline phosphatase is limited to specimens in which the enzyme has not been inactivated, and it does not work well with feces from herbivores. A new method has been developed which uses coprostanol as a fecal indicator. Coprostanol is a heat-stable sterol found in the feces of mammals and some birds. A hexane extract of the suspect particle is applied to the preadsorbent zone of a silica gel thin layer chromatography plate which has been impregnated with 5% phosphomolybdic acid in ethanol. The plate is developed in diethyl ether-heptane (55 + 45), heated, and examined visually for the presence of coprostanol. Amounts of rat feces as small as 0.15 mg and cow feces as small as 0.5 mg have been identified using this method.
Analysis of total and individual fecal neutral steroids in different groups of intestinal pathology from 105 patients was performed to investigate a relationship between neutral steroid concentration and malignant potentiality of colon cells. The fecal concentration of total neutral steroids was significantly higher in patients with colon cancer (57.3 +/- 2.4 mmol/kg of wet feces) and patients with malignant degenerative polyps at Stage I or II (49.5 +/- 2.9) compared with controls (36.9 +/- 3.5) as well as patients with adenomatous polyps (22.4 +/- 2.0). Moreover, a significant correlation was found between total neutral steroid concentrations and coprostanol concentrations in patients with colon cancer (r = 0.928) and in patients with malignant polyps (r = 0.915). Coprostanol results only from the biohydrogenation of delta 5-3 beta-OH steroids by anerobic bacteria because the analysis of a malignant colon tumor in comparison with a healthy colon wall showed that cholesterol is the only steroid included in membrane cells, and that the incubation of Clostridium or Bacteroides with delta 5-3 beta-OH steroids, such as cholesterol (steroid of animal origin) or beta-sitosterol (steroid of plant origin) converts these steroids into coprostanol at a level of efficiency greater than 45%. The findings suggest that high levels of coprostanol reflecting a high growth of anerobic bacteria through the gut are related to the malignant potentiality of colon cells. The screening of neutral fecal steroids should provide a valuable parameter for predicting the malignancy of colon cells.
It has been suggested by other investigators that the extent of neutral and acid steroid conversion by human intestinal flora may be correlated with large-bowel carcinogenesis. Within a population of 31 normal North Americans, we found that two distinct patterns of neutral steroid conversion could be detected by gas-liquid Chromatographie procedures. One pattern was characterized by extensive conversion of choles terol, the other by little or no conversion of cholesterol. Similar conversion patterns were also observed for the plant steroids, sitosterol and campesterol. These patterns were found to be relatively stable over long periods of time. It is possible that there may be differences in the risk level for colon cancer between these two groups.
Eight strains of lactobacilli isolated from conventional mice, rats, fowls, and pigs were tested for their ability to colonize squamous epithelia in the gastrointestinal tracts of gnotobiotic animals. Two of the strains isolated from rodents could colonize the nonsecretory epithelium of the stomach of gnotobiotic rodents, and one strain isolated from a fowl could colonize the crop epithelium of gnotobiotic chickens. The specificity of colonization of squamous epithelia by lactobacilli was demonstrated by these in vivo studies; strains of lactobacilli isolated from rodents could only colonize rodent epithelium; the strain from a fowl could only colonize crop epithelium.
A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of nonspecific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA-targeted oligonucleotide probes to well-growing microbial cells. Two probes of different specificities--one labeled with fluorescein, the other with tetramethylrhodamine--could be applied simultaneously for dual color analysis.
Plasma cholesterol concentrations were significantly lower in rats fed a cholesterol-free diet containing white wheat flour than those fed the diet with whole wheat or wheat bran. Concentrations of total bile acids and neutral sterols in cecal digesta were significantly higher in rats fed wheat flour than in those fed whole wheat, wheat pollard or wheat bran. Digesta bile acids and neutral sterol pools correlated negatively with plasma cholesterol, indicating that excretion was regulating plasma concentration. Total cecal volatile fatty acid (VFA) concentrations were unaffected by diet but cecal propionate was higher and butyrate lower in rats fed wheat flour than in those fed whole wheat. Cecal digesta butyrate concentrations correlated negatively with the cholesterol metabolite, coprostanol, and with secondary bile acids. Cecal propionate correlated negatively with plasma cholesterol concentration, but butyrate correlated equally positively, suggesting these VFA were indicators rather than regulators of altered cecal steroid metabolism. Effects of white wheat flour on steroid metabolism and cecal VFA resemble those of oat bran and support the observation that wheat flour might be hypocholesterolemic in humans.
Male Wistar rats were fed on a conventional diet containing normal corn starch or 6% enzyme-resistant starch originating from either raw or retrograded high-amylose corn starch. Furthermore, the diets were either cholesterol-free or contained 1% cholesterol and 0.1% cholic acid. The main objective of this study was to investigate whether the addition of enzyme-resistant starch to a rat conventional diet had any effect on cholesterol metabolism. Therefore, plasma and liver cholesterol concentrations, plasma HDL:LDL cholesterol ratios and neutral steroid and bile acid excretion were determined. No significant effect of enzyme-resistant starch feeding on plasma and liver cholesterol concentrations was found. However, consumption of raw or retrograded high-amylose corn starch resulted in a decrease in esterified and total liver cholesterol concentrations of 24 and 22%, respectively. This was accompanied by a reduction in plasma esterified and total cholesterol levels of 4% and a tendency to higher daily faecal coprostanol and total bile acid excretion.
The human gut microflora plays a key role in nutrition and health. It has been extensively studied by conventional culture techniques. However these methods are difficult, time consuming and their results not always consistent. Furthermore microscopic counts indicate that only 20 to 40% of the total flora can be cultivated. Among the predominant species of the human gut, Fusobacterium prausnitzii was reported either as one of the most frequent and numerous species or was seldom retrieved. We designed and validated a specific rRNA-targeted oligonucleotide probe, called S-*-Fprau-0645-a-A-23, to accurately detect and quantify F. prausnitzii and relatives within the human fecal microflora. The target group accounted for 5.3 +/- 3% of total bacterial 16S rRNA using dot blot hybridization (10 human fecal samples) and 16.5 +/- 7% of cells stained with Dapi using in situ hybridization (10 other human fecal samples). A specific morphology seemed to be typical and dominant: two cells forming an asymmetrical double droplet. This work showed that F. prausnitzii and phylogenetically related species represent a dominant group within the human fecal flora.