Stool Homogenates Research Articles

Human metabolome variation along the upper intestinal tract

Most processing of the human diet occurs in the small intestine. Metabolites in the small intestine originate from host secretions, plus the ingested exposome1 and microbial transformations. Here we probe the spatiotemporal variation of upper intestinal luminal contents during routine daily digestion in 15 healthy male and female participants. For this, we use a non-invasive, ingestible sampling device to collect and analyse 274 intestinal samples and 60 corresponding stool homogenates by combining five mass spectrometry assays2,3 and 16S rRNA sequencing. We identify 1,909 metabolites, including sulfonolipids and fatty acid esters of hydroxy fatty acids (FAHFA) lipids. We observe that stool and intestinal metabolomes differ dramatically. Food metabolites display trends in dietary biomarkers, unexpected increases in dicarboxylic acids along the intestinal tract and a positive association between luminal keto acids and fruit intake. Diet-derived and microbially linked metabolites account for the largest inter-individual differences. Notably, two individuals who had taken antibiotics within 6 months before sampling show large variation in levels of bioactive FAHFAs and sulfonolipids and other microbially related metabolites. From inter-individual variation, we identify Blautia species as a candidate to be involved in FAHFA metabolism. In conclusion, non-invasive, in vivo sampling of the human small intestine and ascending colon under physiological conditions reveals links between diet, host and microbial metabolism.

SARS CoV-2 detected in neonatal stool remote from maternal COVID-19 during pregnancy.

BackgroundIn utero transmission of SARS coronavirus 2 (SARS-CoV-2) has not been fully investigated. We investigated whether newborns of mothers with COVID-19 during pregnancy might harbor SARS-CoV-2 in the gastrointestinal tract.MethodsThis cohort study investigated stool from 14 newborns born at 25–41 weeks admitted at delivery to our urban academic hospital whose mothers had COVID-19 during pregnancy. Eleven mothers had COVID-19 resolved more than 10 weeks before delivery. Newborn stool was evaluated for SARS-CoV-2 RNA, Spike protein, and induction of inflammatory cytokines interleukin-6 (IL-6) and interferon-γ (IFN-γ) in macrophages.ResultsDespite negative SARS CoV-2 nasal PCRs from all newborns, viral RNAs and Spike protein were detected in the stool of 11 out of 14 newborns as early as the first day of life and increased over time in 6. Stool homogenates from all 14 newborns elicited elevated inflammatory IL-6 and IFN-γ from macrophages. Most newborns were clinically well except for one death from gestational autoimmune liver disease and another who developed necrotizing enterocolitis.ConclusionsThese findings suggest in utero transmission of SARS-CoV-2 and possible persistent intestinal viral reservoirs in the newborns. Further investigation is required to understand the mechanisms and their clinical implications.Impact SARS-CoV-2 RNAs or Spike protein was detected in the stool of 11 out of 14 preterm newborns born to mothers with resolved COVID-19 weeks prior to delivery despite negative newborn nasal PCR swabs.These novel findings suggest risk of in utero SARS-CoV-2 transmission to the fetal intestine during gestation.The presence of SARS-CoV-2 RNAs and Spike protein in the intestines of newborns may potentially impact the development of the gut microbiome and the immune system; the long-term health impact on the preterm infants should be further investigated.

Atg16l1 in dendritic cells is required for antibacterial defense and autophagy in murine colitis.

Autophagy-related 16-like 1 (Atg16l1) contributes to the susceptibility to ulcerative colitis (UC). The functional consequences of Atg16l1 in UC pathogenesis are poorly understood. We aimed to confirm how Atg16l1 deficiency in dendritic cells (DCs) affects murine colitis development. Atg16l1f/f mice and mice with Atg16l1 deficiency in CD11c+ DCs (Atg16l1ΔDC ) were generated for colitis models induction. Disease activity index, weight loss, colon score/length, and histopathological analysis were assessed for colitis severity. Mononuclear cells from mesenteric lymph node (MLN) were extracted for CD44/CD69 measurement by flow cytometry. Bacterial cultures of MLN and stool homogenates were used to evaluate the bacterial translocation. Bone marrow-derived dendritic cells (BMDCs) were isolated and cultured for immunofluorescence of autophagy-related proteins. Atg16l1 knockout in CD11c+ DCs exacerbated intestinal inflammation of dextran sulfate sodium (DSS)-induced colitis in vivo. Atg16l1 deficiency in CD11c+ DCs had no effect on the expression of CD44 and CD69. Bacterial translocation showed that bacteria amount in MLN and stool of DSS-induced colitis with Atg16l1 deficiency significantly higher than that of control. Immunofluorescence revealed that Atg16l1 deficiency obviously inhibited co-expression of LC3 and Lamp1 with S. typhimurium, enhanced co-expression of rab5 and rab7 with S. typhimurium, while did not affect Beclin1. We confirmed that Atg16l1 deficiency in DCs exacerbated the intestinal inflammation of DSS-induced colitis. Atg16l1 deficiency in DCs promotes the bacterial translocation of DSS-induced colitis in vivo and regulates autophagy and phagocytosis in BMDCs. Findings provided a novel perspective to study UC pathogenesis.

Stool Immune Profiles Evince Gastrointestinal Inflammation in Parkinson's Disease.

Gastrointestinal symptoms are common in Parkinson's disease and frequently precede the development of motor impairments. Intestinal inflammation has been proposed as a driver of disease pathology, and evaluation of inflammatory mediators in stool could possibly identify valuable early-stage biomarkers. We measured immune- and angiogenesis-related proteins in human stool to examine inflammatory profiles associated with Parkinson's disease. Stool samples and subjects' self-reported metadata were obtained from 156 individuals with Parkinson's disease and 110 without, including spouse and nonhousehold controls. Metadata were probed for disease-associated differences, and levels of 37 immune and angiogenesis factors in stool homogenates were measured by multiplexed immunoassay and compared across experimental groups. Parkinson's disease patients reported greater incidence of intestinal disease and digestive problems than controls. Direct comparison of levels of stool analytes in patients and controls revealed elevated vascular endothelial growth factor receptor 1, interleukin-1α, and CXCL8 in patients' stool. Paired comparison of patients and spouses suggested higher levels of multiple factors in patients, but this was complicated by sex differences. Sex, body mass index, a history of smoking, and use of probiotics were found to strongly influence levels of stool analytes. Multivariate analysis accounting for these and other potential confounders confirmed elevated levels of interleukin-1α and CXCL8 and also revealed increased interleukin-1β and C-reactive protein in stool in Parkinson's disease. These differences were not dependent on subject age or disease duration. Levels of stool immune factors indicate that intestinal inflammation is present in patients with Parkinson's disease. © 2018 International Parkinson and Movement Disorder Society.

Systematic Bias Introduced by Genomic DNA Template Dilution in 16S rRNA Gene-Targeted Microbiota Profiling in Human Stool Homogenates.

Variability in representation of microbial communities can be caused by differences in microbial composition or artifacts introduced at sample collection or processing. Alterations in community representation introduced by variations in starting DNA concentrations have not been systematically investigated in stool samples. The goal of this study was to evaluate the effect of the genomic DNA (gDNA) concentration in the resulting 16S rRNA gene library composition and compare its effect to other sample processing variables in homogenized human fecal material. Compared to a gDNA input of 1ng/μl, inputs of ≤1.6 × 10-3ng/μl resulted in a marked decrease in the concentration of the 16S rRNA gene amplicon (P < 0.001). Low gDNA concentrations (≤1.6 × 10-3ng/μl) were also associated with a decrease (P < 0.001) in the number of operational taxonomic units and significant divergence in β-diversity profiles (unweighted UniFrac distance, P < 0.001), as characterized by an overestimation of Proteobacteria and underestimation of Firmicutes. Even a gDNA concentration of 4 × 10-2ng/μl showed a significant impact on the β-diversity profile (unweighted UniFrac distance, P = 0.03). Overall, the gDNA concentration explained 22.4% to 38.1% of the microbiota variation based on various β-diversity measures (P < 0.001). By comparison, the DNA extraction methods and PCR volumes tested did not significantly affect the microbial composition profile, and the PCR cycling method explained less than 3.7% of the microbiota variation (weighted UniFrac distance, P = 0.03). The 16S rRNA gene yield and the microbial community representation of human homogenized stool samples are significantly altered by gDNA template concentrations of ≤1.6 × 10-3ng/μl. In addition, data from studies with a gDNA input of ≤4 × 10-2ng/μl should be interpreted with caution. IMPORTANCE The genomic DNA input for stool samples utilized for microbiome composition has not been determined. In this study, we determined the reliable threshold level under which conclusions drawn from the data may be compromised. We also determined the type of microbial bias introduced by less-than-ideal genomic input.

An experimental study of postmortem decomposition of methomyl in blood.

Methomyl (S-methyl-1-N-[(methylcarbamoyl)oxy]thioacetimidate) is a carbamate pesticide. It has been noted that in some cases of methomyl poisoning, methomyl is either not detected or detected only in low concentrations in the blood of the victims. However, in such cases, methomyl is detected at higher concentrations in the vitreous humor than in the blood. This indicates that methomyl in the blood is possibly decomposed after death. However, the reasons for this phenomenon have been unclear. We have previously reported that methomyl is decomposed to dimethyl disulfide (DMDS) in the livers and kidneys of pigs but not in their blood. In addition, in the field of forensic toxicology, it is known that some compounds are decomposed or produced by internal bacteria in biological samples after death. This indicates that there is a possibility that methomyl in blood may be decomposed by bacteria after death. The aim of this study was therefore to investigate whether methomyl in blood is decomposed by bacteria isolated from human stool. Our findings demonstrated that methomyl was decomposed in human stool homogenates, resulting in the generation of DMDS. In addition, it was observed that three bacterial species isolated from the stool homogenates, Bacillus cereus, Pseudomonas aeruginosa, and Bacillus sp., showed methomyl-decomposing activity. The results therefore indicated that one reason for the difficulty in detecting methomyl in postmortem blood from methomyl-poisoning victims is the decomposition of methomyl by internal bacteria such as B. cereus, P. aeruginosa, and Bacillus sp.

Lovastatin Lactone Inhibits Methane Production in Human Stool Homogenates

Introduction: Research has linked an altered gut microbiome to symptoms of irritable bowel syndrome (IBS). In constipation-predominant IBS (IBS-C), methane production appears important as methane levels on breath are proportional to the degree of constipation, and antibiotic elimination of methane significantly improves constipation. The predominant methane-producing organism in humans is Methanobrevibacter smithii. Recent data suggests certain HMG-CoA reductase inhibitors (statins) may reduce methane production by inhibiting methanogenesis. This study examined the effects of various statins on methane production in fresh human stool homogenates. Methods: Five female subjects recruited based on high breath methane production (average=69.6ppm) provided a total of 8 fresh stool samples each. Samples were homogenized in 1XPBS (2g stool/3ml PBS) in an anaerobic chamber at 37°C and divided into 5 stoppered flasks. To assess statin effects, head gas withdrawn through a stopcock was collected at baseline, then every 30min for a total of 270min, and analyzed on a Quintron Model SC gas chromatograph for methane levels. Nine statins were initially assessed for methane inhibition at a concentration of 5mg/g stool: lovastatin lactone and hydroxyacid, pravastatin lactone and hydroxyacid, simvastatin lactone, mevastatin lactone, rosuvastatin lactone, and atorvastatin lactone and hydroxyacid. Once the ideal statin was determined, the effects of concentrations of 0.04, 0.12, 0.48, 1, 5, and 10mg/g stool were tested by sampling head gas every 90min after statin addition, up to 720min.Further assessments were then performed comparing three forms of lovastatin (lactone, diol and hydroxyacid). Results: Lovastatin lactone was identified as the only effective methane inhibitor (Figure 1), significantly inhibiting methane levels by -65% of the control stool. Lovastatin lactone at 5mg/g produced the maximum inhibiting effect resulting in an average methane level of 3% of the control over time (Figure 2). In a final validation comparison of 3 forms of lovastatin (5mg/g), both the lactone and diol forms proved to be effective. In all assessments, hydroxyacid forms were least able to inhibit methane production in fresh stool samples.Figure 1Figure 2Conclusion: Lovastatin lactone and lovastatin diol at concentrations of 5mg/g stool significantly reduce methane production in human stool homogenates. Future studies are needed to assess the effect of this statin on methane production and IBS-C symptoms in humans.

Tissue detection of diphenylchlorin sensitizer (SIM01) by fluorescence and high-performance liquid chromatography

Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the gross national product of many countries. The use of PDT in the treatment of the malignant tumours currently raises great hopes. This physicochemical method is based on the combined action of a nontoxic drug given systematically to the patient and of the visible light delivered locally to the tumour using optical fibres. The radiation will activate the significant substance preferentially fixed on cancerous cells and will cause the death of the tumoral cells while releasing from the toxic ridicalizing species which then will deteriorate vital cellular targets. Tissue distribution and elimination kinetics of the SIM01 were analysed in biological samples from mice tissues by spectrofluorometry and HPLC. Measurements were performed 4, 6, 12, 24 and 48h after an intraperitoneal injection for SIM01 doses of 2, 5 and 15mgkg(-1). Elimination seemed to concern essentially gallbladder, liver and stools, where maximum fluorescence reached, respectively, 20,000, 2800 and 15,000cps for 5mgkg(-1), 6h after injection. Among the tissues examined with HPLC, the highest SIM01 levels were found in stools, urine, liver, gallbladder and spleen. Liver, gallbladder, and stool homogenates from drug-treated animals contained an additional peak (16, 7min) detectable only after injection of at least 15mgkg(-1). Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable kinetic profiles. These techniques should be considered as complementary rather than exclusive for kinetic profiles determination.

Carbon Dioxide Is the Major Metabolite of Quercetin in Humans

A previous study in ileostomy patients indicated that dietary glucosides of the flavonoid quercetin are hydrolyzed efficiently in the intestinal lumen, followed by absorption of a large fraction of the quercetin aglycone. To determine the fate of quercetin, we administered 1.85 MBq (50 microCi) of (14)C-quercetin both orally (100 mg, 330 micromol) and intravenously (iv; 0.3 mg, 1 micromol) to healthy volunteers. Serial plasma samples, urines and stools were collected for 72 h. Total radioactivity was determined by liquid scintillation spectrometry directly in plasma and urine and after repeated methanol extraction of stool homogenate samples. The oral absorption, based on total radioactivity, was surprisingly high, ranging from 36.4 to 53.0%. The biological half-life was very long, ranging from 20 to 72 h. The urinary recovery of total radioactivity ranged from 18.4 to 26.8% after the iv dose and from 3.3 to 5.7% after the oral dose. The corresponding fecal recoveries were only 1.5-5.0% and 1.6-4.6%, respectively. Thus, the total recovery of the (14)C-quercetin doses, in particular after oral administration, was very low. In search for the unaccounted for fraction of the (14)C-quercetin dose, we performed (14)CO(2) recovery studies in three volunteers (3 iv and 3 oral doses). At timed intervals, (14)CO(2) in expired air was trapped in hyamine hydroxide/thymolphthalein and analyzed for radioactivity. As much as 23.0-81.1% of the quercetin dose was recovered as (14)CO(2) in the expired air from these volunteers, after both oral and iv doses. The disposition of quercetin in humans is thus highly complex, requiring further studies.

Fecal short-chain fatty acids in patients with diarrhea-predominant irritable bowel syndrome: in vitro studies of carbohydrate fermentation.

Colonic bacterial production of short-chain fatty acids (SCFA) plays an important role in the salvage of unabsorbed carbohydrate and in colonic absorption of electrolytes and water. The objective of this study was to determine whether patients with diarrhea-predominant irritable bowel syndrome (DP-IBS) have a different pattern and rate of fermentation of carbohydrate and fiber to SCFA compared with controls. Fecal homogenates from 10 patients with DP-IBS and 10 age-matched controls were studied. SCFA were measured by gas chromatography in baseline fecal samples and in fecal homogenates in an in vitro anaerobic fermentation system after incubation with no additional substrate, lactulose, potato starch, citrus pectin, and hemicellulose over a 24-hour period. Net SCFA production rates were calculated for the first 6 h of the incubation period. Patients with DP-IBS had a consistently different pattern of less total SCFA, a lower percentage of acetate (p < 0.05), and a higher proportion of n-butyrate (p < 0.05) than controls. In stool homogenates from both controls and DP-IBS patients, lactulose fermentation resulted in the highest rate of SCFA production followed by pectin, starch, and hemicellulose. However, at all time points, the fecal homogenates from controls generated a higher concentration of total SCFA, acetate, and propionate with all substrates tested. SCFA production rates were higher in controls incubated with lactulose, starch, and hemicellulose. The fecal SCFA profile of patients with DP-IBS is characterized by lower concentrations of total SCFA, acetate, and propionate and a higher concentration and percentage of n-butyrate. Fecal flora from these patients produced less SCFA in an in vitro fermentation system in response to incubations with various carbohydrates and fibers. Differences in SCFA production by colonic bacterial flora in patients with DP-IBS may be related to the development of gastrointestinal symptoms.

Usefulness of a simple photometric determination of chymotrypsin activity in stools — results of a multicentre study

The usefulness of a new photometric test for the determination of chymotrypsin activity in stools was evaluated in a multicentre study. By release of the enzyme from stool particles with a cationic detergent solution after, in most cases, 3 min of homogenization, a photometric measurement of the enzyme activity is possible both in the clear supernatant after centrifugation and in the diluted stool homogenate. The precision of the activity measurement in stool samples with pathologically lowered and normal chymotrypsin activity is good both for in-series (CV = 2.6%, scatter 0.6-5.7%) and for day-to-day determinations (CV = 7.2%, scatter 3.9-13.9%). The results obtained by the photometric determination also exhibit a close correlation with the values measured by pH-stat titrimetry (r = 0.901). The sample-preparation system with a sample-metering chamber is easy to use and gives good agreement with determinations in which the sample metering was done by weighing out the stool (r = 0.961). The photometric test for detection of exocrine pancreatic insufficiency is easy to perform, inexpensive and does not place any undue stress on the patient.

Clinical value of dual-isotope fat absorption test system (FATS) using glycerol [125I]trioleate and glycerol [75Se]triether.

In order to delineate the clinical value of a dual-isotope fat absorption test system (FATS) using glycerol [75Se]triether as lipid-phase marker and glycerol [125I]trioleate as the test lipid, fecal isotope ratios from single stools (and a 72-hr stool homogenate) were compared to quantitative fecal fat excretion. The study included 11 patients without and 24 patients with steatorrhea. With a figure of 0.8% as the upper limit of normal, the test was a reliable indicator of steatorrhea with 87.5% sensitivity and 81.8% specificity; efficiency was 85.7%. Related to a prevalence of steatorrhea of 45.9% as the mean value of 1269 consecutive 72-hr specimens investigated for steatorrhea during 1978-1982, the positive (negative) predictive value of the FATS is 80.3% (87.2%). With 2% as the upper limit of normal, no false positive results ensued. It is concluded that a two-step interpretation of the FATS (0.8% limit and 2% limit) may be regarded a valid qualitative index for steatorrhea. The FATS isotope ratio using single stools correlated well with FATS ratios in the 72-hr stool homogenates (r = 0.97). FATS therefore allows a convenient estimate of steatorrhea from measuring single stools. As a quantitative measure of fecal fat excretion, the FATS is unreliable.

Spectrophotometry of occult blood in feces.

This method for measuring fecal occult blood is based on the heme-catalyzed oxidation of tetramethylbenzidine by H2O2. An aliquot of heated stool homogenate is mixed with acetic acid to chemically separate heme from globin. The heme is extracted into ethyl acetate and reacted with the reagent and H2O2 to produce a green oxidation product. The reaction is followed kinetically for 30 to 60 s at 660 nm. A660 is linearly related to the amount of hemoglobin. The lower limit of detection is 1 to 2 mg of hemoglobin per gram (wet weight) of feces. Within-day precision (CV) of the analysis for hemoglobin added to stool specimens (4 to 30 mg/g) ranged from 2.3 to 7.6%, between-day CV from 2.1 to 8.1%. Analytical recovery of hemoglobin added to fecal specimens (4 to 30 mg/g) ranged from 86.7 to 106.2%. Of the substances known to interfere with conventional dye-oxidation tests for fecal occult blood, only myoglobin and ascorbic acid interfere with hemoglobin quantification by our procedure. The test is fast, inexpensive, and easy to perform, and involves equipment available in hospital laboratories.

Ultrasensitive latex-agglutination-test for the specific immunochemical detection and quantification of faecal occult blood loss.

Latex agglutination was used for the development of a simple and fast immunochemical faecal occult blood test which is ultrasensitive and specific for human haemoglobin. "In vitro" detection limits of 0.0002 and 0.003 ml blood/100 g stool homogenate were obtained for the tube and filter versions of the latex-agglutination test. The quantification of the blood content in stool homogenates is possible by diluting the haemoglobin containing faecal extract until no more agglutination occurs. Considering the nonuniform distribution of blood in faeces "in vivo" sensitivities of greater than 0.002 and greater than 0.03 ml blood loss/day respectively can be expected for the tube and filter versions of the latex agglutination test. This test is about 10,000 times more sensitive than the unspecific chemical guaiac type occult blood tests and at least 1,000 times more sensitive than other immunochemical occult blood tests which use immunofluorescence, radial immunodiffusion or an enzyme immunoassay. Nuclear medical reference methods like 51Cr faecal excretion or 59Fe whole body retention cannot be used for the calibration of the ultrasensitive latex agglutination test since they are not specific for blood losses below 3 (51Cr) and 5 (59Fe) ml/day.

Improved nonradioactive method for routine clinical analysis for long- and medium-chain stool lipids.

We describe a simple, practical method for routine clinical determination of long- and medium-chain stool lipids. Stool lipids were extracted into a hot mixture of benzene/ethanol/water (12/59/29, by vol), saponified in 30 min without formation of ethyl esters, and titrated in a clear, one-phase medium to a sharp color change. CVs for the titration were 2, 1, and 1% for 0.05, 0.25, and 0.5 mmol of fatty acids, respectively. Interference of stool short-chain acids other than fatty acids was not statistically significant. The precision of sampling of stool homogenate was improved by analysis of two aliquots, one from the top and one from the bottom of the suspension. This method of sampling is a practical alternative to the unpleasant homogenization with a blender or to the addition of large quantities of bile salts. The accuracy of the method may be increased, in special cases, by removing potential interferants.

The relationship between serum cholesterol and fecal 7α-dehydroxylase activity in three ethnic groups in South Africa

The ability of fecal bacteria to 7α-dehydroxylate primary bile acids was measured in vitro by incubating stool homogenates with labeled primary bile acids, and was compared to serum cholesterol levels in 4 South African groups: Rural Bantu (50 subjects), Urban White (20), Urban Bantu (17) and Urban Coloured (16). Mean serum cholesterol levels were 137 ± 23, 213 ± 51, 199 ± 62 and 206 ± 46 mg/100 ml, respectively. (Rural Bantu significantly different.) The in vitro conversion of cholic acid by stool homogenates of Rural Bantu was significantly slower than that of the Urban Whites or the Urban Bantu in two-hour incubations. There was no difference of conversion rates in 24-hour incubations of cholic or chenodeoxycholic acids. Gas-liquid chromatographic analysis of stool homogenates showed little difference in the distribution pattern of fecal bile acids or neutral sterols. These data suggest that the activity of the intestinal bacterial flora to convert primary bile acids was significantly reduced in the Rural Bantu as compared to the other groups, corresponding with lower serum cholesterol levels. However, by the time the stools were excreted the degree of conversion was comparable in each group.

Reduced microbial transformation of bile acids in cystic fibrosis.

The microbial transformation of bile acids by incubates of stool homogenates from children with cystic fibrosis is decreased.

Relation of Serum Cholesterol to in vitro 7α-Dehydroxylation of Primary Bile Acids by Fecal Bacteria in Infants and Children

Fresh feces from 104 infants and children (aged 3 days to 16 years) were homogenized and incubated with labeled cholic or chenodeoxycholic acid. After 2 and/or 24 hours' incubation, the percentage of converted (mostly 7α-dehydroxylated) primary bile acids was measured, and the degree of conversion was correlated with serum cholesterol levels. It was found that stool homogenates of patients with low levels of serum cholesterol (&amp;lt; 160 mg/100 ml) converted labeled primary bile acids poorly or not at all, whereas in patients with higher serum cholesterol levels (&amp;gt; l60 mg/100 ml) the conversion process was markedly increased. Thus, highly significant correlations were found between serum cholesterol levels and the capability of the fecal bacterial flora to convert both primary bile acids "in vitro." The possibility is proposed that in man the relatively rapid progressive increase of serum cholesterol level following birth may be related to the colonization of the intestinal tract by 7α-dehydroxylating and/or bile acid degrading bacteria. It is suggested that the prevalence of these bacteria is subject to environmental effects, and it may be one of the important factors regulating cholesterol levels in man.