No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Gastrointestinal Tract: Microbial Metabolism of Steroids
Abstract
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.
... Gut microbiota have been reported to contribute to the effects of exogenous factors on testicular function though steroid hormone synthesis (Gérard, 2010) and the testis-pituitary axis (Al-Asmakh et al., 2014). In both the ANL and CNL groups, chlorpyrifos significantly decreased the concentrations of LH and FSH, which are gonadotropins secreted by the pituitary (p < .05, ...
... This decrease was partly due to the decrease in LH, a necessary regulator of testosterone synthesis in the testis (Zirkin and Papadopoulos, 2018). Gut microbiota may increase the reabsorption of substrates needed for steroid synthesis from the intestine by affecting cholesterol and bile acid metabolism in the gut (Gérard, 2010). Therefore, the depletion of Turicibacter in the ANL group may lead to the decrease in testosterone although the data was not significant, which was reported to be in company with the decrease of cholesterol, the substrate of testosterone (Dalziel et al., 2017;Kim et al., 2018). ...
Chlorpyrifos is a pesticide frequently detected in food and has been reported to disturb endocrine and gut health, which was regulated by gut microbiota and enteroendocrine cells. In this study, newly weaned (3 week) and adult (8 week) male rats fed a normal- or high- fat diet were chronically exposed to 0.3 mg chlorpyrifos/kg bodyweight/day. The effects of chlorpyrifos exposure on serum hormone levels, proinflammatory cytokines and gut microbiota were evaluated. Chronic exposure to chlorpyrifos significantly decreased the concentrations of luteinizing hormone, follicule stimulating hormone and testosterone, which was found only in the normal-fat diet. The counteracted effect of high-fat diet was also found in gut hormones and proinflammatory cytokines. Significantly higher concentrations of glucagon-like peptide-1, pancreatic polypeptide, peptide tyrosine tyrosine (PYY), ghrelin, gastric inhibitory poly-peptide, IL-6, monocyte chemoattractant protein-1, and TNF-α were found in rats exposed to chlorpyrifos beginning at newly weaned, whereas only the PYY, ghrelin and IL-6 concentrations increased significantly in rats exposed in adulthood. Furthermore, a decrease in epinephrine induced by chlorpyrifos exposure was found in rats exposed to chlorpyrifos beginning at newly weaned, regardless of their diet. Chlorpyrifos-induced disturbances in the microbiome community structure were more apparent in rats fed a high-fat diet and exposed beginning at newly weaned. The affected bacteria included short-chain fatty acid-producing bacteria (Romboutsia, Turicibacter, Clostridium sensu stricto 1, norank_f_Coriobacteriaceae, Faecalibaculum, Parasutterella and norank_f__Erysipelotrichaceae), testosterone-related genus (Turicibacter, Brevibacterium), pathogenic bacteria (Streptococcus), and inflammation-related bacteria (unclassified_f__Ruminococcaceae, Ruminococcaceae_UCG-009, Parasutterella, Oscillibacter), which regulated the endocrine system via the hypothalamic-pituitary-adrenal axis, as well as the immune response and gut barrier. Early exposure accelerated the endocrine-disturbing effect and immune responses of chlorpyrifos, although these effects can be eased or recovered by a high-fat diet. This study helped clarify the relationship between disrupted endocrine function and gut microbiota dysbiosis induced by food contaminants such as pesticides.
... Le côlon reçoit jusqu'à 1 g par jour de cholestérol, dont 70 % proviennent de la bile, 20 % de la fraction de l'alimentation non absorbée au niveau de l'intestin grêle, et les 10 % restants de la desquamation des muqueuses intestinales 18 . Dès les années 1930, il a été montré que le microbiote intestinal était capable de convertir ce cholestérol en coprostanol, non absorbé par l'intestin et éliminé dans les fèces. ...
... Ils sont absorbés au niveau de l'iléon terminal, puis transportés par la veine porte au foie, où ils sont à nouveau excrétés dans la bile (cycle entéro-hépatique). Environ 5 % des sels biliaires (0,2 à 0,3 g par jour) échappent à ce cycle et parviennent au côlon où ils sont métabolisés par le microbiote en acides biliaires dits secondaires, par opposition aux acides biliaires primaires synthétisés par le foie 18,26 . Plus de vingt acides biliaires secondaires ont ainsi été mis en évidence dans les fèces humaines, démontrant la grande variété de conversions possibles des acides biliaires par le microbiote intestinal. ...
... The synthesis of testosterone by Leydig cells requires the action of several enzymes which fall into two categories: the cytochrome P450 enzymes and the hydroxysteroid dehydrogenases (HSD) [16]. Earlier studies have documented that the gut microbiota is involved in the deconjugation and metabolism of steroid hormones [17]. In the present study we demonstrate that the gut microbiota affect the testis-pituitary axis, regulates lumen formation of the seminiferous tubules and the cellcell adhesion and also the permeability of the BTB. ...
... Surprisingly, colonization with CBUT enhanced expression of these genes. One explanation for the normalization of testosterone levels is that bacterial colonization can affect cholesterol and bile acid metabolism in the gut, resulting in an increased reabsorption of substrate for steroid synthesis from the intestine [17]. Furthermore, testosterone levels are also controlled by gonadotropins secreted from the pituitary glands and bacterial metabolites such as butyrate have been shown to increase the levels of LH [43] and FSH [44]. ...
Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.
... Le rôle principal des acides biliaires est d'aider à l'absorption des lipides alimentaires mais il est également reconnu qu'ils agissent en tant que molécules signal et peuvent ainsi influencer les métabolismes lipidique et glucidique. Environ 5 % des sels biliaires (0,2 à 0,5 g par jour) échappent à ce cycle et parviennent au côlon où ils sont convertis par le microbiote en acides biliaires dits secondaires [25,26]. Plus de 20 acides biliaires secondaires ont ainsi été mis en évidence dans les fèces humaines, démontrant la grande variété de conversions possibles des acides biliaires par le microbiote intestinal. ...
... Chez l'homme, les deux principaux acides biliaires secondaires sont les acides désoxycholique et lithocholique, produits de la 7 ␣-déshydroxylation bactérienne des acides biliaires primaires, acides cholique et chénodésoxycholique. Les bactéries intestinales d'origine humaine possédant cette activité ont toutes été identifiées comme appartenant au genre Clostridium [25]. Il est à noter que ces activités de conversion des acides biliaires portées par les bactéries intestinales et exprimées pour part dans l'intestin grêle, peuvent modifier l'absorption et l'excrétion des lipides alimentaires, et de ce fait, influer sur le niveau de lipides sériques. ...
The human intestine harbours a complex bacterial community called the gut microbiota. This microbiota is specific of each individual despite the existence of several bacterial species shared by the majority of adults. The influence of the gut microbiota on host's physiology and its lipid metabolism has been largely studied using germfree (devoid of a gut microbiota) animals. Indeed, these animals are resistant to diet induced obesity along with improved insulin sensitivity and reduced dyslipidemia or steatosis development. In humans, several grams of lipids reach the colon each day. These lipids have an impact on the gut microbiota composition and activities. Concurrently, the gut microbiota is able to convert lipids, including fatty acids or cholesterol, leading to the production of metabolites with potential health effects.
... strain GDN1 can decompose androgens in the intestinal tract of mice, hinder the hepatic intestinal circulation of androgens, and lower serum androgen levels [102]. However, as a steroid metabolite similar to bile acids, the existence of hepatointestinal circulation in androgens requires intensive study [103]. ...
Reproductive health is an important issue for humanity. In the context of the increasing incidence rate of male infertility, it is essential to find the factors that affect male reproductive health. Gastrointestinal health is closely related to reproductive health. Gastrointestinal hormones (GIH) and gut microbiota (GM), as important material foundations for gastrointestinal function, can promote or inhibit testicular reproductive function, including spermatogenesis, sperm maturation, androgen synthesis, and even broader male diseases such as sexual function, prostate cancer, etc. On the contrary, the functional health of the testes is also of great significance for the stability of gastrointestinal function. This review mainly discusses the important regulatory effects of GIH and GM on male reproductive function.
... Then, primary BAs are conjugated to glycine or taurine, and over 95% primary BAs are reabsorbed and recirculated back to the liver [115]. The nonreabsorbed BAs can be deconjugated by catalyzed enzyme bile salt hydrolases (BSHs), which are expressed by several commensal gut bacteria, including the Gram-positive Bifidobacterium, Clostridium, Enterococcus, Lactobacillus and the Gram-negative Bacteroides [116,117]. Besides deconjugation, gut microbes such as Clostridium and Eubacterium are a source of 7-dehydoxylase to generate secondary BAs including lithocholic acid (LCA) from CDCA and deoxycholic acid (DCA) from CA [118]. ...
Cardiovascular diseases (CVD) are a group of disorders of the heart and blood vessels and remain the leading cause of morbidity and mortality worldwide. Over the past decades, accumulating studies indicated that the gut microbiota, an indispensable “invisible organ”, plays a vital role in human metabolism and disease states including CVD. Among many endogenous and exogenous factors that can impact gut microbial communities, the dietary nutrients emerge as an essential component of host-microbiota relationships that can be involved in CVD susceptibility. In this review, we summarize the major concepts of dietary modulation of the gut microbiota and the chief principles of the involvement of this microbiota in CVD development. We also discuss the mechanisms of diet-microbiota crosstalk that regulate CVD progression, including endotoxemia, inflammation, gut barrier dysfunction and lipid metabolism dysfunction. In addition, we describe how metabolites produced by the microbiota, including trimethylamine-N-oxide (TMAO), secondary bile acids (BAs), short chain fatty acids (SCFAs) as well as aromatic amino acids (AAAs) derived metabolites play a role in CVD pathogenesis. Finally, we present the potential dietary interventions which interacted with gut microbiota as novel preventive and therapeutic strategies for CVD management.
... The bottleneck is, however, our limited knowledge about cholesterol metabolism by the gut microbiota, as regards to only few isolated coprostanoligenic bacterial strains and unidentified responsible genes. If cholesterol-to-coprostanol conversion has been mentioned or described in numerous previous reviews [10][11][12][13][14][15][16][17][18][19][20], the present one is the first one entirely dedicated to this gut bacterial metabolism. It constitutes an opportunity to reassess what we know or suspect about this bacterial metabolism, which, in spite of being probably as old as appearance of mammals on earth, still remains so mysterious. ...
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.
... The alteration of gut microbiota, as well as their metabolites, such as short-chain fatty acids (SCFAs), has been reported to play a role in the regulation of the testis-pituitary axis [63] by disrupting steroid hormone synthesis [64]. It has been verified in cells that butyrate acid may increase the level of estradiol [65]. ...
Organophosphorus pesticides (OPs) can be metabolized to diethyl phosphate (DEP) in the gut environment, which may affect the immune and endocrine systems and the microbiota. Correlations between OPs and diseases have been established by epidemiological studies, mainly based on the contents of their metabolites, including DEP, in the serum or urine. However, the effects of DEP require further study. Therefore, in this study, adult male rats were exposed to 0.08 or 0.13 mg/kg DEP for 20 weeks. Serum levels of hormones, lipids, and inflammatory cytokines as well as gut microbiota were measured. DEP significantly enriched opportunistic pathogens, including Paraprevotella, Parabacteroides, Alloprevotella, and Helicobacter, leading to a decrease in interleukin-6 (IL-6). Exposure to the high dose of DEP enriched the butyrate-producing genera, Alloprevotella and Intestinimonas, leading to an increase in estradiol and a resulting decrease in total triglycerides (TGs) and low-density lipoprotein cholesterol (LDL-C); meanwhile, DEP-induced increases in peptide tyrosine‒tyrosine (PYY) and ghrelin were attributed to the enrichment of short-chain fatty acid-producing Clostridium sensu stricto 1 and Lactobacillus. These findings indicate that measuring the effects of DEP is not a proxy for measuring the effects of its parent compounds.
... Although primate diets are typically very low in fat, fruits and seeds are important lipid sources (Rothman et al., 2012; Reiner et al., 2014). Indeed, taxa related to Coriobacteriaceae, Clostridium, Bifidobacterium and Lactobacillus, significantly enriched in lowland gorillas when ripe fruit was consumed, can also impact cholesterol and bile acid turnover in the colon (Gerard, 2010; Martin et al., 2007). This observation may explain the significant interactions observed between these taxa and abundances of coprostanol, cholestanol, hydrocholestane and ursodeoxycholic acid in the lowland gorilla metabolome. ...
Although the critical role that our gastrointestinal microbes play in host physiology is now well established, we know little about the factors that influenced the evolution of primate gut microbiomes. To further understand current gut microbiome configurations and diet-microbe co-metabolic fingerprints in primates, from an evolutionary perspective, we characterized fecal bacterial communities and metabolomic profiles in 228 fecal samples of lowland and mountain gorillas (G. g. gorilla and G. b. beringei, respectively), our closest evolutionary relatives after chimpanzees. Our results demonstrate that the gut microbiomes and metabolomes of these two species exhibit significantly different patterns. This is supported by increased abundance of metabolites and bacterial taxa associated with fiber metabolism in mountain gorillas, and enrichment of markers associated with simple sugar, lipid and sterol turnover in the lowland species. However, longitudinal sampling shows that both species' microbiomes and metabolomes converge when hosts face similar dietary constraints, associated with low fruit availability in their habitats. By showing differences and convergence of diet-microbe co-metabolic fingerprints in two geographically isolated primate species, under specific dietary stimuli, we suggest that dietary constraints triggered during their adaptive radiation were potential factors behind the species-specific microbiome patterns observed in primates today.
... Animal and in vitro studies have shown that the intestinal microbiota can regulate host lipid metabolism via numerous microbial activities (Fava, 2006;Martin et al., 2007). The best characterized mechanism is through the biotransformation of bile acids, which regulates the digestion and absorption of fats, and profoundly affects the cholesterol and other lipid metabolism in the body (Gérard, 2010;Ridlon, Kang & Hylemon, 2006). However, global monitoring of the serum and organ lipid profiles of germ-free and conventionally raised mice suggests an even more widespread and profound influence of the intestinal microbiota on host lipid metabolism, in particular on triglycerides and phosphatidylcholines (Velagapudi et al., 2010;Orešič, Hänninen, & Vidal-Puig, 2008). ...
Accumulating evidence indicates that the intestinal microbiota regulates our physiology and metabolism. Bacteria marketed as probiotics confer health benefits that may arise from their ability to affect the microbiota. Here high-throughput screening of the intestinal microbiota was carried out and integrated with serum lipidomic profiling data to study the impact of probiotic intervention on the intestinal ecosystem, and to explore the associations between the intestinal bacteria and serum lipids. We performed a comprehensive intestinal microbiota analysis using a phylogenetic microarray before and after Lactobacillus rhamnosus GG intervention. While a specific increase in the L. rhamnosus-related bacteria was observed during the intervention, no other changes in the composition or stability of the microbiota were detected. After the intervention, lactobacilli returned to their initial levels. As previously reported, also the serum lipid profiles remained unaltered during the intervention. Based on a high-resolution microbiota analysis, intake of L. rhamnosus GG did not modify the composition of the intestinal ecosystem in healthy adults, indicating that probiotics confer their health effects by other mechanisms. The most prevailing association between the gut microbiota and lipid profiles was a strong positive correlation between uncultured phylotypes of Ruminococcus gnavus-group and polyunsaturated serum triglycerides of dietary origin. Moreover, a positive correlation was detected between serum cholesterol and Collinsella (Coriobacteriaceae). These associations identified with the spectrometric lipidome profiling were corroborated by enzymatically determined cholesterol and triglyceride levels. Actinomycetaceae correlated negatively with triglycerides of highly unsaturated fatty acids while a set of Proteobacteria showed negative correlation with ether phosphatidylcholines. Our results suggest that several members of the Firmicutes, Actinobacteria and Proteobacteria may be involved in the metabolism of dietary and endogenous lipids, and provide a scientific rationale for further human studies to explore the role of intestinal microbes in host lipid metabolism.
... Bacterial conversion seems to occur via two pathwaysdirect (from cholesterol) and indirect (via coprostanone as an intermediate) (Gérard, 2010;Gérard et al., 2007). Although esterified CoP conjugates can also be formed, the evidence is conflicted as to whether they compose a significant portion of CoP. ...
The promulgation of numeric nutrient criteria for evaluating impairment of waterbodies in Florida is underway. Adherence to the water quality standards needed to meet these criteria will potentially require substantial allocations of public and private resources in order to better control nutrient (i.e., nitrogen and phosphorus) releases from contributing sources. Major sources of nutrients include atmospheric deposition (195-380 mg-N/m(2)/yr, 6 to 16 mg-P/m(2)/yr), reclaimed water irrigation (0.13-29 mg-N/L, 0.02 to 6 mg-P/L), septic systems (3.3 × 10(3)-6.68 × 10(3) g-N/person/yr, 0.49 × 10(3)-0.85 × 10(3) g-P/person/yr) and fertilizer applications (8 × 10(6)-24 × 10(6) mg-N/m(2)/yr). Estimated nitrogen loadings to the Florida environment, as calculated from the above rates are as follows: 5.9 × 10(9)-9.4 × 10(9) g-N/yr from atmospheric deposition, 1.2 × 10(8)-2.6 × 10(10) g-N/yr from reclaimed water, 2.4 × 10(10)-4.9 × 10(10) g-N/year from septic systems, and 1.4 × 10(11) g-N/yr from fertilizer application. Similarly, source specific phosphorus loading calculations are also presented in this paper. A fraction of those nutrient inputs may reach receiving waterbodies depending upon site specific regulation on nutrient control, nutrient management practices, and environmental attenuation. In Florida, the interconnectivity of hydrologic pathways due to the karst landscape and high volumes of rainfall add to the complexity of tracking nutrient loads back to their sources. In addition to source specific nutrient loadings, this review discusses the merits of source specific markers such as elemental isotopes (boron, nitrogen, oxygen, strontium, uranium and carbon) and trace organic compounds (sucralose, gadolinium anomaly, carbamazepine, and galaxolide) in relating nutrient loads back to sources of origin. Although this review is focused in Florida, the development of source specific markers as a tool for tracing nutrient loadings back to sources of origin is applicable and of value to all other geographical locations.
... The human intestine is a complex ecosystem with a large species diversity, of at least 400 different bacterial species [21, 22]. The density varies through the different parts of the GI tract from 10 4 bacteria/mL in the stomach to 10 12 bacteria/g faeces in the distal part of the colon [23, 24]. ...
We assessed horizontal gene transfer between bacteria in the gastrointestinal (GI) tract. During the last decades, the emergence of antibiotic resistant strains and treatment failures of bacterial infections have increased the public awareness of antibiotic usage. The use of broad spectrum antibiotics creates a selective pressure on the bacterial flora, thus increasing the emergence of multiresistant bacteria, which results in a vicious circle of treatments and emergence of new antibiotic resistant bacteria. The human gastrointestinal tract is a massive reservoir of bacteria with a potential for both receiving and transferring antibiotic resistance genes. The increased use of fermented food products and probiotics, as food supplements and health promoting products containing massive amounts of bacteria acting as either donors and/or recipients of antibiotic resistance genes in the human GI tract, also contributes to the emergence of antibiotic resistant strains. This paper deals with the assessment of antibiotic resistance gene transfer occurring in the gut.
... The gastrointestinal tract is a complex ecosystem that harbors at least 400–500 different bacterial species with a large variety of physiological properties (Borriello 1986; Cummings and Macfarlane 1997). The intestinal flora in a healthy host reaches densities of up to 10 12 bacteria per gram of contents and it can function as a colonization barrier against invading bacteria (Dubos 1963; Freter 1983; Salminen et al. 1995; van der Waaij et al. 1971 ). ...
We have previously shown that Escherichia coli BJ4 has similar doubling time in mice that are mono-associated (having only the inoculated E. coli BJ4) or streptomycin-treated (having mainly gram-positive bacteria plus the inoculated E. coli BJ4). We also showed that when the mice were conventionalized (fed cecum homogenate from conventional mice or ones with a complete microbial flora), the introduction of complete flora in both cases increased the in vivo doubling time, while decreasing the colony counts in fecal samples. To determine whether the increase in doubling time could explain the decrease in colony counts, we analyzed our previous results by a chemostat model. The analysis shows that the increasing doubling time alone is sufficient to explain the decrease in colony counts in mono-associated mice, but not in the streptomycin-treated mice. The observed decreasing rate in colony counts in streptomycin-treated mice is slower than predicted. Furthermore, whereas the model predicted a decrease to extinction in both mice, the E. coli persist at a frequency 10-80 times higher in streptomycin-treated mice than in mono-associated mice. Thus, while a chemostat model is able to explain some of the population dynamics of intestinal bacteria in mice, additional factors not included in the model are stabilizing the system. Because we find that E. coli declines more slowly and to a higher stabilization frequency in streptomycin-treated mice, which have a more diverse flora before conventionalization, we take these results to suggest that the persistence of E. coli populations is promoted by species diversity. We propose that a mechanism for the persistence may be the presence of new E. coli niches created by keystone species in the more diverse flora.
This study aimed to determine the effect of sodium butyrate (SB) on reproductive tract development and histomorphometric analysis of testes in neonatal kids, as well as on their growth, antioxidant status and some blood metabolites. Thirty-six neonatal Zaraibi kids were divided immediately after 4-5 days from birth into three equal groups (12 kids/ each). The first group (G1) received milk replacer (MR) at a rate of 10% of the body weight until the weaning. The second group (G2) received 9.7% MR supplemented with 0.3% SB. The third group (G3) received whole milk and served as a control. The results revealed that there was significant (p < .001) increase in total and daily gain between the G2 and G1 groups, whereas there was no significant change between G2 and G3 groups. Body condition score was slightly increased (p > .05) in G2 compared with G1. Serum total protein and cholesterol levels were significantly decreased in treated groups compared with the G3 group, on reverse globulin and glucose levels had no significant changes. Also, T3 and testosterone concentrations were significantly (p < .0001 & p < .05) higher in G3 and G2 than G1. Antioxidant status was enhanced through decreasing the oxidative marker and increasing antioxidant enzymes activity in G2. Testis parameters in G3 and G2 kids had the highest values, compared with G1. G1 and G2 had thin basement membrane of seminiferous tubules with few Leydig cells and pyknotic germinal epithelium, while G3 showed thick basement membrane, mild wide interstitial spaces with many Leydig cells. The tubular diameter was also significantly larger in the G3 and G2. It could be concluded that MR supplemented with SB can be used as alternative whole milk in suckling goat kids for maintaining reproductive tract and kids' performance through improving the antioxidant status.
Sodium butyrate (SB) is a novel feed additive in poultry production. This study aimed to evaluate the effects of dietary supplementation of SB on the reproductive performance of the rooster. Three hundred 22-week-old roosters were randomly divided into 2 dietary treatment groups of 6 pen replicates, a basal diet (control) group and a basal diet with SB gruop. The supplementation of SB was carried out through 23 consecutive weeks from 22 to 45 weeks of age. During this period, the live-weight was measured weekly while semen samples were collected every two weeks. Three time-points were chosen for analysis (30, 35 and 45 weeks of age). The results showed that SB improved the semen volume and sperm motility at 30 and 35 weeks of age (P < 0.05), increased the sperm concentration and decreased the abnormal sperm percentage during the whole experimental period (P < 0.05). These improvements were accompanied by increased testosterone levels at 30 and 35 weeks of age (P < 0.05). Moreover, dietary supplementation of SB also increased the enzyme activities of glutathione peroxidase (GPx) at 30 and 35 weeks of age and superoxide dismutase (SOD) at 45 weeks of age in the testes of roosters (P < 0.05). These results suggest that SB may promote testicular growth by increasing the antioxidant capacity and testosterone hormone secretion in adult roosters.
micewas determined. Single E.coli cells were identified inthinfrozen sections fromthelarge intestines bytheuse ofa probespecific forE.coli 23SrRNA.Furthermore, thetotal bacterial population was visualized withan rRNA probetargeting thedomainBacteria. Bythistechnique, allE.coli cells were seenembeddedinthemucosal material overlying theepithelial cells ofthelarge intestine, andno direct attachment totheepithelium was observed. Thelarge intestine isthemostheavily colonized partofthe gastrointestinal tract ofmammals. Atleast 400to500different bacterial species arethought tobepresent atanytimeinthe healthy humanintestinal tract, andup to1012bacteria are foundineveryg offeces (2,5).Mostworkperformed on the bacterial flora oftheguthasconcentrated on theanalysis of fecal specimens. Muchless information abouttheflora ofthe cecum or thatassociated withtheintestinal mucosa isfound, andinsitu investigations ofthephysiology ofintestinal bacte- riaandinformation aboutspatial distribution aretherefore of greatinterest. Recentdevelopments within molecular microbiology and molecular evolution haveprovided new perspectives andtools
The human intestine harbours a complex and diverse bacterial community called the gut microbiota. This microbiota, stable during the lifetime, is specific of each individual despite the existence of a phylogenetic core shared by the majority of adults. The influence of the gut microbiota on host’s physiology has been largely studied using germfree animals and recently it has been proposed that the gut microbiota affects nutrient acquisition, energy regulation and fat storage. Indeed, germfree animals are resistant to diet induced obesity and display low levels of blood and liver lipids.
In humans, several grams of lipids reach the colon each day. These lipids have an impact on the gut microbiota composition characterized by an increase of the Firmicutes/Bacteroides ratio. Concurrently, the gut microbiota is able to convert lipids, including fatty acids or cholesterol, leading to the production of metabolites with potential health effects.
A new approach is conceptualized for measuring small-area human populations by using biomarkers in sewage. The basis for the concept (SCIM: Sewage Chemical-Information Mining) is supported by a comprehensive examination and synthesis of data published across several disciplines, including medicine, microbiology, clinical chemistry, and environmental science. Accurate measures of human populations are fundamental to numerous disciplines, including economics, marketing, politics, sociology, public health and safety (e.g., disease management; assessment of natural hazards; disaster prevention and response), quality of life, and the environment. Knowing the size, distribution, and flow of a small-area (local) population facilitates understanding the numerous and complex linkages and interactions between humans and the environment. Examples include material-flow (substance-flow) analysis, determining the magnitude of per capita contribution of pollutant loadings to watersheds, or forecasting future impacts of local populations on the environment or a population's demands on resources. While no definitive approach exists for measuring small-area populations, census-taking is a long-established convention. No approach exists, however, for gauging small-area populations in real-time, as none is able to capture population dynamics, which involve transient changes (e.g., daily influx and efflux) and lasting changes (e.g., births, deaths, change in residence). Accurate measurement of small-area populations in real time has never been possible but is essential for facilitating the design of more sustainable communities. Real-time measurement would provide communities the capability of testing what-if scenarios in design and policy decisions.
Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static.
Fluorescent oligonucleotide probes targeting rRNA were used to develop an in situ hybridization technique by which the spatial distribution of Escherichia coli in the large intestines of streptomycin-treated mice was determined. Single E. coli cells were identified in thin frozen sections from the large intestines by the use of a probe specific for E. coli 23S rRNA. Furthermore, the total bacterial population was visualized with an rRNA probe targeting the domain Bacteria. By this technique, all E. coli cells were seen embedded in the mucosal material overlying the epithelial cells of the large intestine, and no direct attachment to the epithelium was observed.
Escherichia coli BJ4, a rat isolate, was used to examine the growth and differentiation of the microorganism in its natural habitat, the intestine. Growth of E. coli BJ4 in the large intestine of streptomycin-treated mice was compared with its growth in laboratory media. By a number of methods, it was shown that E. coli BJ4 differentiates, during growth in the intestine, into two distinct populations, one that has the characteristics of the laboratory-grown strain and one that appears as a coccoid cell. Furthermore, it was shown that there is a natural selection for the coccoid-type cell in the intestine, while in laboratory media growth of rod-shaped E. coli BJ4 is enhanced.
The majority of microbes most probably exist in nature in close association with particular surfaces. The adhesive properties of microorganisms were first recognized at the beginning of this century. Since then it has been shown that bacterial adhesion is important in plant and animal hosts, pathogenesis, medical devices, aquatic and soil ecosystems, biodegradation, and industrial processes.
The growth physiology of Escherichia coli during colonization of the intestinal tract was studied with four animal models: the streptomycin-treated mouse carrying a reduced microflora, the monoassociated mouse with no other microflora than the introduced strain, the conventionalized streptomycin-treated mouse, and the conventionalized monoassociated mouse harboring a full microflora. A 23S rRNA fluorescent oligonucleotide probe was used for hybridization to whole E. coli cells fixed directly after being taken from the animals, and the respective growth rates of E. coli BJ4 in the four animal models were estimated by correlating the cellular concentrations of ribosomes with the growth rate of the strain. The growth rates thus estimated from the ribosomal content of E. coli BJ4 in vivo did not differ in the streptomycin-treated and the monoassociated mice. After conventionalization there was a slight decrease of the bacterial growth rates in both animal models.
It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.
Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial
groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no
information about the specific mechanisms of interaction with the host is available. In order to provide some insight into
the molecular mechanisms involved in the interaction with the host, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human
plasminogen-binding activity for four strains belonging to three bifidobacterial species: Bifidobacterium lactis, B. bifidum, and B. longum. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative
plasminogen-binding proteins in the cell wall fraction of the model strain B. lactis BI07. The data suggest that plasminogen binding to B. lactis is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved
cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding
the mechanisms involved in the Bifidobacterium-host interaction.
Steroids are a family of organic compounds that have a five-ring perhydrocyclo-pentanophenanthrene nucleus. The numbering system and skeletal structures for steroids are shown in Figure 13.1. Steroids vary in the number and location of double bonds; in the type, number, and position of functional groups; and in the stereochemical configuration of substituents below (α) or above (ß) the plane of the nucleus. Most steroids have 18ß- and 19 ß-methyl groups bonded to C-10 and C-13. The hydrogen atom in steroids saturated at at C-5 can be ß-oriented or α-oriented (allo). Alkyl side chains with various functional groups can be present at C-17, usually in the 17 ß orientation.
Ursodeoxycholic acid (UDCA)-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. UDCA is the 7β-OH epimer of the primary (i.e. synthesized by the liver) bile acid chenodeoxycholic acid (CDCA). Epimerization proceeds in two subsequent and reversible steps, catalysed by a 7α- and a 7β-hydroxysteroid dehydrogenase (7α- and 7β-HSDH), with 7oxo-lithocholic acid (7oxo-LCA) as the intermediate product. The aim of this study was to test the 7α- and 7β-HSDH activities of anaerobic whole cell cultures of a number of lactic acid bacteria and human intestinal isolates, using CDCA, UDCA and 7oxo-LCA as the substrates. Among 140 strains tested, 21 exhibited at least one of both 7-HSDH activities. 7α-HSDH activity was detected in six strains, 7β-HSDH in nine strains, and both activities in six other strains. All active strains were isolated from normal human and infant faeces. They belonged to the genera Clostridium, Eubacterium and Ruminococcus, whereas no strain of Lactobacillus, Bifidobacterium or Streptococcus was found to be active under our study conditions. The present study therefore revealed, for the first time, a number of normal human intestinal isolates supporting the epimerization of CDCA to UDCA, and further extended our knowledge of those intestinal bacteria which are responsible for 7α- or 7β-HSDH activity. Key words: screening, bile acids, epimerization, intestinal microflora, lactic acid bacteria, probiotics.
We previously reported that the 7 alpha-dehydroxylation of cholic acid appears to be carried out by a multi-step pathway in intestinal anaerobic bacteria both in vitro and in vivo. The pathway is hypothesized to involve an initial oxidation of the 3 alpha-hydroxy group and the introduction of a double bond at C4-C5 generating a 3-oxo-4-cholenoic bile acid intermediate. The loss of water generates a 3-oxo-4,6-choldienoic bile acid which is reduced (three steps) yielding deoxycholic acid. We synthesized, in radiolabel, the following putative bile acid intermediates of this pathway 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, 12 alpha-dihydroxy-3-oxo-4,6-choldienoic acid, and 12 alpha-hydroxy-3-oxo-4-cholenoic acid and showed that they could be converted to 3 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid (deoxycholic acid) by whole cells or cell extracts of Eubacterium sp. VPI 12708. During studies of this pathway, we discovered the accumulation of two unidentified bile acid intermediates formed from cholic acid. These bile acids were purified by thin-layer chromatography and identified by gas-liquid chromatography-mass spectrometry as 12 alpha-hydroxy-3-oxo-5 alpha-cholanoic acid and 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic (allo-deoxycholic acid). Allo-deoxycholic acid was formed only in cell extracts prepared from bacteria induced by cholic acid, suggesting that their formation may be a branch of the cholic acid 7 alpha-dehydroxylation pathway in this bacterium.
The amount and composition of fecal neutral sterols and bile acids excreted by adult male germfree and conventional rats have been determined. The amounts of neutral sterols excreted were 12.8 (germfree) and 19.5 (conventional) mg/kg of body wt per day. The germfree rats excreted cholesterol and lathosterol (methostenol was not assayed); the conventional rats excreted coprostanol and coprostanone in addition. The amounts of bile acids excreted were 11.3 (germfree) and 21.4 (conventional) mg/kg of body wt per day. The bile acids excreted by the rats were tentatively identified as tauro-β-muricholate, tauro-α-muricholate, and tauro-cholate, besides an unidentified component. The conventional rats excreted the corresponding unconjugated acids as well as many other unconjugated bile acids.
No significant correlation was found between the amount of coprosterols and the total amount of neutral sterols excreted by the conventional rats. This suggests that bacterial reduction of cholesterol is not an important mechanism of increasing neutral sterol excretion of conventional rats as compared to germfree rats. Evidence is presented that suggests that this difference in neutral sterol excretion is due to changes in intestinal secretion and sloughing between the two types of animal. The factors reponsible for the differences in bile acid excretion have not been identified.
We have described a number of microbial transformations of the three classes of steroids occurring in the human intestine. These include: hydrolysis, dehydrogenation (hydroxyl and nuclear), dehydroxylation, epimerization, and side chain cleavage reactions all leaving the steroid nucleus intact. In many cases the responsible organism has been isolated and the enzyme(s) has been purified and studied. Areas of paucity in information are nonetheless evident. Isolation of unknown organisms, details of the mechanism of action of most of the described enzymes, and a thorough understanding of the biological significance of the reaction products to the host must await further research. New applications of these enzymes remain in the future.
The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.
A small, anaerobic, gram-positive coccobacillus that reduces cholesterol to coprostanol was isolated from a hog sewage lagoon. This isolate, strain HLT (T = type strain) does not require cholesterol for growth, but it requires lecithin and has phospholipase activity. Much acid is produced by the fermentation of amygdalin, lactose, and salicin. Arabinose, cellobiose, fructose, glucose, mannose, and melibiose are fermented weakly. Acetic, formic, and succinic acids are produced, as is hydrogen. The isolate does not reduce nitrate, produce indole, or hydrolyze starch and gelatin. Esculin is hydrolyzed. The properties of strain HLT are most similar to those of members of the genus Eubacterium. Because strain HL (= ATCC 51222) has unique morphological and physiological properties, we propose that it should be the type strain of a new species in the genus Eubacterium, Eubacterium coprostanoligenes.
Bacterial bile salt hydrolases catalyze the degradation of conjugated bile acids in the mammalian gut. The crystal structures of conjugated bile acid hydrolase (CBAH) from Clostridium perfringens as apoenzyme and in complex with taurodeoxycholate that was hydrolyzed to the reaction products taurine and deoxycholate are described here at 2.1 and 1.7 A resolution, respectively. The crystal structures reveal close relationship between CBAH and penicillin V acylase from Bacillus sphaericus. This similarity together with the N-terminal cysteine classifies CBAH as a member of the N-terminal nucleophile (Ntn) hydrolase superfamily. Both crystal structures show an identical homotetrameric organization with dihedral (D(2) or 222) point group symmetry. The structure analysis of C. perfringens CBAH identifies critical residues in catalysis, substrate recognition, and tetramer formation which may serve in further biochemical characterization of bile acid hydrolases.
Secondary bile acids, produced solely by intestinal bacteria, can accumulate to high levels in the enterohepatic circulation of some individuals and may contribute to the pathogenesis of colon cancer, gallstones, and other gastrointestinal (GI) diseases. Bile salt hydrolysis and hydroxy group dehydrogenation reactions are carried out by a broad spectrum of intestinal anaerobic bacteria, whereas bile acid 7-dehydroxylation appears restricted to a limited number of intestinal anaerobes representing a small fraction of the total colonic flora. Microbial enzymes modifying bile salts differ between species with respect to pH optima, enzyme kinetics, substrate specificity, cellular location, and possibly physiological function. Crystallization, site-directed mutagenesis, and comparisons of protein secondary structure have provided insight into the mechanisms of several bile acid-biotransforming enzymatic reactions. Molecular cloning of genes encoding bile salt-modifying enzymes has facilitated the understanding of the genetic organization of these pathways and is a means of developing probes for the detection of bile salt-modifying bacteria. The potential exists for altering the bile acid pool by targeting key enzymes in the 7alpha/beta-dehydroxylation pathway through the development of pharmaceuticals or sequestering bile acids biologically in probiotic bacteria, which may result in their effective removal from the host after excretion.
The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible
for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing
bacterium of human intestinal origin. Strain D8 was isolated from a 10−8 dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to
coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase,
and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed,
suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could
reduce 1.5 μmol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism
identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%)
to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a
band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly
responsible for cholesterol conversion in the human population.
The establishment of the conversion of cholesterol to coprostanol by the intestinal microflora was followed in 25 healthy Swedish children by gas‐chromatographic analysis of fecal samples taken at 0, 1, 3, 6, 9, 12, 15, 18, 21, and 24 months of age. In 15 children, the microbial conversion of cholesterol to coprostanol started during the second half of the first year of life. During the second year of life, a sequestered distribution of the conversion rate could be distinguished. At the end of the study, 13 of the children were stable high converters, 3 were stable low converters, and 6 were unstable converters while the conversion pattern of three children could not be determined. The initiation of the establishment of conversion was delayed by breast‐feeding, and only one child had coprostanol in feces while still being breast‐fed. Those children exclusively breast‐fed for more than 4 months or weaned after 6 months of age had significantly lower conversion rates at 12 months of age than did the other children (p < 0.05). Once conversion started, previous breast‐feeding tended to result in a stable, later high rate of conversion of cholesterol. Between 6 and 15 months of age, the conversion rate increased with age, but after standardization for the period of exposure to foods other than breast milk, no age factor could be identified.
Biohydrogenation of Δ ⁵ ‐steroids was studied in vitro by incubating various steroids with a pure culture of Eubacterium 21,408 in a brain‐thioglycollate medium under anaerobic conditions.
Eubacterium 21,408, a strictly anaerobic bacterium, was isolated from rat cecal contents. It differed from any previously described Eubacterium species by its requirements for a Δ ⁵ ‐3β‐hydroxy steroid.
Eubacterium 21,408 reduced the 5,6‐double bond of cholesterol, campesterol, β‐sitosterol and stigmasterol, exclusively yielding the corresponding 5β‐saturated derivatives. Similarly, the 4,5‐double bond of allocholesterol was reduced to yield coprostanol. Neither the 7,8‐double bond in 7‐dehydrocholesterol or in lathosterol, nor the 22,23‐double bond in the side chain of stigmasterol were reduced.
In the absence of a 3‐hydroxyl function ( e.g. in cholest‐5‐ene), when the 3‐hydroxyl function was in the 3α‐position ( e.g. in epicholesterol) or was substituted ( e.g. in 3β‐chloro‐cholest‐5‐ene and in cholesteryl esters), no saturation of the 5,6‐double bond was observed.
When incubated with Eubacterium 21,408, the carbonyl group of 4‐cholesten‐3‐one, was reduced to the corresponding 3β‐hydroxyl group along with saturation of the 4,5‐double bond. The bacterium also reduced the 3‐oxo group of cholestanone and coprostanone to a 3β‐hydroxyl group.
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.
The handling of cholesterol by the intestine involves a balance between absorption, excretion and metabolism by gut microflora. Between 34-57% of dietary cholesterol is absorbed from the human intestine. Variables effecting the efficiency of cholesterol absorption include the absolute amount of cholesterol consumed, the presence of plant sterols, the fiber content of the diet, transit time and possibly the relative proportions of fatty acids in the diet. On average, 150 mg/day of cholesterol is excreted in the feces. Fecal cholesterol derives from biliary secretions into the intestine, sloughing of epithelial cells and unabsorbed dietary cholesterol. The major metabolic products of cholesterol in the gut are coprostanol, coprostanone, cholestanol, cholestanone and epicoprostanol. Bacterial metabolism of cholesterol can be influenced by diet as evidenced by significant variations among different population groups with different dietary habits. Altered patterns of intestinal bacterial metabolism of cholesterol may place persons at a higher risk of developing colonic disorders. Dairy products have been reported to influence the bacterial metabolism of cholesterol and possibly plasma cholesterol levels although the significance of these findings to overall cholesterol balance needs to be further defined.
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.
Seven healthy volunteers and 3 hospitalized patients were given the same diet for 6 days. The mean intake of cholesterol was 359 mg/day. There was a large difference in the fecal amount of cholesterol and coprostanol among the subjects. The ratio of coprostanol/cholesterol ranged from 0.01 to 4.27. The subjects whose feces contained smaller amount of coprostanol excreted larger amounts of beta-sitosterol and crude fiber, suggesting that the various activities of intestinal flora were lower than the other subjects. The difference in the activity of intestinal flora to convert cholesterol into coprostanol was ascertained by incubating the feces anaerobically with egg yolk in vitro. It was also demonstrated that cholesterol was synthesized de novo and the amount corresponded to 0.1-0.6 g/day. The amounts of sterols in the feces of the other 17 hospitalized patients were also determined and there was an inverse relationship between the serum cholesterol and coprostanol/cholesterol ratio in the feces. These results suggested that the conversion of cholesterol into coprostanol by the intestinal flora might have an important role in the regulation of serum cholesterol level.
The bile acid sulfate sulfatase (BSS) produced by Pseudomonas testosteroni was purified and characterized. Chromatofocusing behavior and amino acid sequence over twelve amino acid residues from N-terminus of the enzyme indicated that BSS was composed of two isoforms of which molecular weights were 125,000 and 103,000. Each isoform was a homodimer of a subunit of which molecular weight was 53,000 or 51,000, respectively. The optimum pH was 8.5 and BSS was stable at pH 5.8-8.0. The thermostability above 32 degrees C was improved by the addition of polyols, such as sorbitol, sucrose, and glycerol. BSS was a Mn(2+)-dependent enzyme and contained 1-2 atoms of manganese in its own protein molecule. All 3 alpha-sulfate esters of the bile acids routinely appearing in human serum were hydrolyzed by BSS to 3 beta-hydroxyl iso-compounds corresponding to each bile acid and sulfuric acid. We tentatively named this novel enzyme BSS (bile acid 3 alpha-sulfate sulfohydrolase).
Recently, a unique bacterium, Eubacterium coprostanoligenes ATCC 51222, that reduces cholesterol to coprostanol was isolated. Because coprostanol is absorbed poorly, it was hypothesized that oral administration of Eu. coprostanoligenes might decrease cholesterol concentration in blood because the micro-organisms will decrease the absorption of endogenous and dietary cholesterol by conversion to coprostanol. To test the hypothesis, three adult New Zealand White rabbits received 4 ml of Eu. coprostanoligenes suspension (ca 2 x 10(7) cells ml-1) daily per os for 10 d; three other adult New Zealand White rabbits received the same dosage of boiled bacterial suspension. Plasma cholesterol concentration of experimental rabbits (183.3 +/- 11.0 mg dl-1, mean +/- S.E.) was significantly lower (P < 0.001) than that of controls (248.8 +/- 12.3 mg dl-1, mean +/- S.E.). The coprostanol-to-cholesterol ratios in contents of digestive tracts of experimental rabbits were greater than those of controls. The data indicate that oral administration of Eu. coprostanoligenes caused a significant hypocholesterolemic effect in rabbits and that this effect can be explained by the conversion of cholesterol to coprostanol in the intestine.
The establishment of the conversion of cholesterol to coprostanol by the intestinal microflora was followed in 25 healthy Swedish children by gas-chromatographic analysis of fecal samples taken at 0, 1, 3, 6, 9, 12, 15, 18, 21, and 24 months of age. In 15 children, the microbial conversion of cholesterol to coprostanol started during the second half of the first year of life. During the second year of life, a sequestered distribution of the conversion rate could be distinguished. At the end of the study, 13 of the children were stable high converters, 3 were stable low converters, and 6 were unstable converters while the conversion pattern of three children could not be determined. The initiation of the establishment of conversion was delayed by breast-feeding, and only one child had coprostanol in feces while still being breast-fed. Those children exclusively breast-fed for more than 4 months or weaned after 6 months of age had significantly lower conversion rates at 12 months of age than did the other children (p < 0.05). Once conversion started, previous breast-feeding tended to result in a stable, later high rate of conversion of cholesterol. Between 6 and 15 months of age, the conversion rate increased with age, but after standardization for the period of exposure to foods other than breast milk, no age factor could be identified.
The mechanism of reduction of cholesterol to coprostanol by Eubacterium coprostanoligenes ATCC 51222 was studied by incubating the bacterium with a mixture of alpha- and beta-isomers of [4-3H,4-14C]cholesterol. Coprostanol, isolated after incubation of [4-3H,4-14C]cholesterol in a growth medium under anaerobic conditions, retained 97% of the tritium originally present in cholesterol. The majority of this tritium (64%), however, was in the C-6 position in coprostanol, which showed that the conversion of cholesterol into coprostanol by E. coprostanoligenes involved the intermediate formation of 4-cholesten-3-one followed by the reduction of the latter to coprostanol. In resting cell assays in which washed bacterial cells were incubated with micellar cholesterol in phosphate buffer at 37 degrees C, both 4-cholesten-3-one and coprostanone were produced in addition to coprostanol. Furthermore, 5-cholesten-3-one, 4-cholesten-3-one, and coprostanone were converted efficiently to coprostanol by E. coprostanoligenes. These results support the hypothesis that the major pathway for reduction of cholesterol by E. coprostanoligenes involves the intermediate formation of 4-cholesten-3-one followed by reduction of the latter to coprostanol through coprostanone as an intermediate.
Listeria monocytogenes is a bacterial pathogen causing severe food-borne infections in humans and animals. It can sense and adapt to a variety of harsh microenvironments outside as well as inside the host. Once ingested by a mammalian host, the bacterial pathogen reaches the intestinal lumen, where it encounters bile salts which, in addition to their role in digestion, have antimicrobial activity. Comparison of the L. monocytogenes and Listeria innocua genomes has revealed the presence of an L. monocytogenes-specific putative gene encoding a bile salt hydrolase (BSH). Here, we show that the bsh gene encodes a functional intracellular enzyme in all pathogenic Listeria species. The bsh gene is positively regulated by PrfA, the transcriptional activator of known L. monocytogenes virulence genes. Moreover, BSH activity increases at low oxygen concentration. Deletion of bsh results in decreased resistance to bile in vitro, reduced bacterial faecal carriage after oral infection of the guinea-pigs, reduced virulence and liver colonization after intravenous inoculation of mice. Taken together, these results demonstrate that BSH is a novel PrfA-regulated L. monocytogenes virulence factor involved in the intestinal and hepatic phases of listeriosis.
Ursodeoxycholic acid-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. This work reports the first isolation of 7-epimerizing bacteria from feces of a healthy volunteer, on the basis of their capacity to epimerize the primary bile acid, chenodeoxycholic acid, to ursodeoxycholic acid. Five isolates were found to be active starting from unconjugated chenodeoxycholic acid and its tauro-conjugated homologue, but none of these strains could epimerize the glyco-conjugated form. Biochemical testing and 16S ribosomal DNA sequencing converged to show that all five isolates were closely related to Clostridium baratii (99% sequence similarity), suggesting that this bacterial species could be responsible at least partially, for this bioconversion in the human gut.
Intensity of the cholesterol-to-coprostanol conversion in the intestine, as assessed by the coprostanol-to-cholesterol ratio in faeces, was found highly variable among 15 human volunteers, ranging from absent to almost complete cholesterol conversion. The number of coprostanoligenic bacteria in the same faecal samples, as estimated by the most probable number method, was found to be less than 10(6) cellsg-1 of fresh stools in the low-to-inefficient converters and at least 10(8) cellsg-1 of fresh stools in the highest converters, indicating that the population level of cultivable faecal coprostanoligenic bacteria correlated with the intensity of cholesterol-to-coprostanol conversion in the human gut. Microbial communities of the samples were profiled by temporal temperature gradient gel electrophoresis (TTGE) of bacterial 16S rRNA gene amplicons. Dendrogram analysis of the TTGE profiles using the Pearson product moment correlation coefficient and a unweighted pair group method with arithmetic averages (UPGMA) algorithm clearly separated banding patterns from low-to-inefficient and high converters in two different clusters suggesting a relationship between TTGE profiles and coprostanoligenic activity. Principal components analysis further demonstrated that a large subset of bands rather than some individual bands contributed to this clustering.