Human Fecal Microorganisms Research Articles

The effects of high-fat foods on gut microbiota and small molecule intestinal gases: release kinetics and distribution in vitro colon model

Profiling intestinal gases and their responses to dietary changes can reveal the products and functions of the gut microbiota and their influence on human health. High-fat foods (HFF) can alter the gut microbiota and its metabolites, posing a potential health risk. However, little is known about the effects of HFF on intestinal gas distribution. Therefore, in this study, we used human fecal microorganisms as strains, an in vitro three-chamber colon model and an intestinal gas array sensor as tools. We performed in vitro fermentation using HFF as the fermentation substrate to reveal the effects of HFF on the kinetics of intestinal gas production and changes in the gut microbiota and its metabolites. We found that dietary fatty acids stimulated the production of H2S and volatile organic compounds in the colon, promoted Firmicutes abundance, and decreased Bacteroidetes abundance. These results highlight the potential role of HFF in altering the gut microbiota and intestinal gas, which can lead to health hazards.

Fecal Microbiota and Human Intestinal Fluid Transplantation: Methodologies and Outlook.

Fecal microbiota transplantation (FMT) is a therapy that involves the transplantation of healthy human fecal microorganisms into the gut of patients to rebuild or consolidate the intestinal microecology. It has been utilized in many diseases. However, FMT had a limited effect on patients with small intestinal diseases because of the unique ecological characteristics of the microorganisms. Thus, we proposed a new microecology transplantation therapy called human intestinal fluid transplantation (HIFT). Human intestinal fluid can be collected through a nasojejunal tube and be made into capsules using the freeze-dried powder method. In addition, strict standards for donor screening and management have been established. We are currently developing a high-standard HIFT preparation system and conducting high-quality clinical studies to validate the safety and efficacy of HIFT combined with FMT.

Investigations into the stability of 17 psychoactive drugs in a "simulated postmortem blood" model.

In the postmortem environment, some drugs and metabolites may degrade due to microbial activity, even forming degradation products that are not produced in humans. Consequently, underestimation or overestimation of perimortem drug concentrations or even false negatives are possible when analyzing postmortem specimens. Therefore, understanding whether medications may be susceptible to microbial degradation is critical in order to ensure that reliable detection and quantitation of drugs and their degradation products is achieved in toxicology screening methods. In this study, a "simulated postmortem blood" model constructed of antemortem human whole blood inoculated with a broad population of human fecal microorganisms was used to investigate the stability of 17 antidepressant and antipsychotic drugs. Microbial communities present in the experiments were determined to be relevant to postmortem blood microorganisms by 16S rRNA sequencing analyses. After 7days of exposure to the community at 37°C, drug stability was evaluated using liquid chromatography coupled with diode array detection (LC-DAD) and with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Most of the investigated drugs were found to be stable in inoculated samples and noninoculated controls. However, the 1,2-benzisothiazole antipsychotics, ziprasidone and lurasidone, were found to degrade at a rate comparable with the known labile control, risperidone. In longer experiments (7 to 12 months), where specimens were stored at -20°C, 4°C, and ambient temperature, N-dealkylation degradation products were detected for many of the drugs, with greater formation in specimens stored at -20°C than at 4°C.

Differentiating Sources of Fecal Contamination to Wilderness Waters Using Droplet Digital PCR and Fecal Indicator Bacteria Methods.

Human activity in wilderness areas has the potential to affect aquatic ecosystems, including through the introduction of microorganisms associated with fecal contamination. We examined fecal microorganism contamination in water sources (lake outlets, snowmelt streams) in the popular Absaroka Beartooth Wilderness in the United States. Although the region is remote, increasing human visitation has the potential to negatively affect water quality, with particular concern about human-derived microorganism fecal contaminants. We used standard fecal indicator bacterial assays that quantified total coliform bacteria and Escherichia coli concentrations, together with more specific polymerase chain reaction-based microbial assays that identified possible human sources of fecal microorganisms in these waters. Total coliforms were detected at all lake outlets (21 of 21 sites), and E coli was detected at 11 of 21 sites. Droplet digital polymerase chain reaction assays revealed the presence of human feces-derived microorganisms, albeit at abundances below the limit of detection (<10 gene copies per milliliter of water) at all but 1 of the sampling sites. Our results suggest low prevalence of water-borne pathogens (specifically E coli and human-derived Bacteroides) in this popular wilderness area. However, widespread detection of total coliforms, Bacteroides, and E coli highlight the importance of purifying water sources in wilderness areas before consumption. Specific sources of total coliforms and E coli in these waters remain unknown but could derive from wild or domesticated animals that inhabit or visit the Absaroka Beartooth Wilderness. Hence, although contamination by human fecal microorganisms appears minimal, human visitation could indirectly influence fecal contamination through domesticated animals.

In Vitro Prebiotic Effects of Malto-Oligosaccharides Containing Water-Soluble Dietary Fiber.

This study measured the proliferative activity of malto-oligosaccharide (MOS) as a prebiotic against Bifidobacteria, resistance to digestion in vitro, and changes during in vitro fermentation by human fecal microorganisms. It consisted of 21.74%, 18.84%, and 11.76% of maltotriose, maltotetraose, and maltopentaose produced by amylase (HATT), respectively. When 1% of MOS was added to a modified PYF medium as the carbon source, proliferation of Bifidobacterium breve was increased significantly. During the in vitro digestion test, MOS was partially degraded by intestinal enzymes. Fermentation characteristics by human fecal microorganisms were evaluated by adding 1% galacto-oligosaccharide (GOS), as well as 1% and 2% MOS as carbon sources to the basal medium, respectively. In comparison with the addition of 1% of MOS and GOS, the total short chain fatty acid (SCFA) content increased over time when 2% of MOS was added. The species diversity and richness of intestinal microbiota increased significantly with 2% MOS compared to those with 1% GOS. In addition, the 2% addition of MOS reduced intestinal pathobiont microorganisms and increased commensal microorganisms including Bifidobacterium genus. Collectively, MOS produced by amylase increased the SCFA production and enhanced the growth of beneficial bacteria during in vitro fermentation by human fecal microbiota.

Potential for gulls to transport bacteria from human waste sites to beaches

Contamination of recreational beaches due to fecal waste from gulls complicates beach monitoring and may pose a risk to public health. Gulls that feed at human waste sites may ingest human fecal microorganisms associated with that waste. If these gulls also visit beaches, they may serve as vectors, transporting fecal microorganisms to the beach where they may subsequently contaminate sand and water. In this study, samples collected from landfills, treated wastewater storage lagoons, and public beaches demonstrated a spatial and temporal overlap of markers for gull and human-associated microorganisms. In addition, markers for gull, fecal indicator bacteria, and the human-associated marker, HF183, were detected in gull feces and cloacae samples. Further, HF183 was detected in cloacae samples from gulls that were documented by radio-telemetry traveling between human waste sites and public beaches. This study highlights the potential for gulls that visit human waste sites to disperse human-associated microorganisms in the beach landscape.

Comparison of the Microbial Community Structures of Untreated Wastewaters from Different Geographic Locales

Microbial sewage communities consist of a combination of human fecal microorganisms and nonfecal microorganisms, which may be residents of urban sewer infrastructure or flowthrough originating from gray water or rainwater inputs. Together, these different microorganism sources form an identifiable community structure that may serve as a signature for sewage discharges and as candidates for alternative indicators specific for human fecal pollution. However, the structure and variability of this community across geographic space remains uncharacterized. We used massively parallel 454 pyrosequencing of the V6 region in 16S rRNA genes to profile microbial communities from 13 untreated sewage influent samples collected from a wide range of geographic locations in the United States. We obtained a total of 380,175 high-quality sequences for sequence-based clustering, taxonomic analyses, and profile comparisons. The sewage profile included a discernible core human fecal signature made up of several abundant taxonomic groups within Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. DNA sequences were also classified into fecal, sewage infrastructure (i.e., nonfecal), and transient groups based on data comparisons with fecal samples. Across all sewage samples, an estimated 12.1% of sequences were fecal in origin, while 81.4% were consistently associated with the sewage infrastructure. The composition of feces-derived operational taxonomic units remained congruent across all sewage samples regardless of geographic locale; however, the sewage infrastructure community composition varied among cities, with city latitude best explaining this variation. Together, these results suggest that untreated sewage microbial communities harbor a core group of fecal bacteria across geographically dispersed wastewater sewage lines and that ambient water quality indicators targeting these select core microorganisms may perform well across the United States.

Human fecal metabolism of soyasaponin I.

The metabolism of soyasaponin I (3-O-[alpha-L-rhamnopyranosyl-beta-D-galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24-triol) by human fecal microorganisms was investigated. Fresh feces were collected from 15 healthy women and incubated anaerobically with 10 mmol soyasaponin I/g feces at 37 degrees C for 48 h. The disappearance of soyasaponin I in this in vitro fermentation system displayed apparent first-order rate loss kinetics. Two distinct soyasaponin I degradation phenotypes were observed among the subjects: rapid soyasaponin degraders with a rate constant k = 0.24 +/- 0.04 h(-)(1) and slow degraders with a k = 0.07 +/- 0.02 h(-)(1). There were no significant differences in the body mass index, fecal moisture, gut transit time, and soy consumption frequency between the two soyasaponin degradation phenotypes. Two primary gut microbial metabolites of soyasaponin I were identified as soyasaponin III (3-O-[beta-D-galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24-triol) and soyasapogenol B (olean-12-ene-3beta,22beta,24-triol) by NMR and electrospray ionized mass spectroscopy. Soyasaponin III appeared within the first 24 h and disappeared by 48 h. Soyasapogenol B seemed to be the final metabolic product during the 48 h anaerobic incubation. These results indicate that dietary soyasaponins can be metabolized by human gut microorganisms. The sugar moieties of soyasaponins seem to be hydrolyzed sequentially to yield smaller and more hydrophobic metabolites.

Linoleic Acid Conjugation by Human Intestinal Microorganisms Is Inhibited by Glucose and Other Substrates In Vitro and in Gnotobiotic Rats

The anticarcinogen conjugated linoleic acid (CLA) is a product of bacterial activity that isomerizes linoleic acid (LA) in the rumen of herbivores. Therefore, fatty dairy products in the human diet are enriched with CLA. Although bacteria capable of in vitro LA conjugation were detected in the human intestinal tract, CLA synthesis from dietary sunflower seed oil was not observed in gnotobiotic rats associated with these intestinal bacteria. The objective of the study was to investigate variables that affect LA conjugation. In vitro, LA conjugation was strongly inhibited by glucose and other substrates. Concentrations of 1.5 mmol glucose/L inhibited LA conjugation by 50%. Methyl-alpha-D-glucoside was a less effective inhibitor than glucose, and 2-deoxy-D-glucose did not inhibit LA conjugation at all. To analyze the concentration of carbohydrates in intestinal contents, the LA-conjugating bacterial mixed culture and human fecal microorganisms were introduced into germ-free rats. Samples of feces and cecum and colon contents of both groups exhibited in vitro LA-conjugating activity. Rats associated with human intestinal microorganisms contained 5.7 +/- 1. 3 mmol glucose/L in the cecal contents and 6.6 +/- 1.0 mmol glucose/L in the colonic contents. Rats associated with CLA-producing bacterial culture contained 3.4 +/- 1.3 mmol glucose/L in the cecal contents and 4.2 +/- 1.0 mmol glucose/L in the colonic contents. These values are within a range that may explain the observed inhibition of LA conjugation in vivo.

Epimerization of the four 3,7-dihydroxy bile acid epimers by human fecal microorganisms in anaerobic mixed cultures and in feces.

The conversion of 3,7-dihydroxy bile acids by anaerobic mixed cultures of intestinal microorganisms was studied in fecal samples from eight healthy adult males. Incubations using substrate chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) were performed simultaneously in separate microbial suspensions from the same fecal samples. A time course study was done on four samples, chosen randomly from the eight. In the incubation of CDCA, substrate CDCA always decreased rapidly in amount; UDCA increased in amount, as did 3 beta, 7 beta-dihydroxy-5 beta-cholanoic acid (3 beta, 7 beta) and 3 beta, 7 alpha-dihydroxy-5 beta-cholanoic acid (3 beta, 7 alpha). In the incubation of UDCA, UDCA gradually decreased in amount; (3 beta, 7 beta), CDCA, and (3 beta, 7 alpha) increased gradually in amount. All reactions involved four epimers. After 48-72 hr UDCA was predominant and the reactions appeared to have reached equilibrium. In cultures from all eight samples, after 72-96 hr, a predominance of beta-hydroxy configurations at 7-position and alpha-hydroxy configurations at 3-position was observed. To compare these bile acid compositions to those in feces, an in vivo study using nine subjects was carried out. Concurrent with the collection of feces, transit time of food through the gut was measured. In samples from five subjects, in which amounts of lithocholic acid (LCA) was small, four 3,7-dihydroxy epimers were found. In samples from the other four, however, CDCA, the predominant epimer in bile, had apparently been converted to LCA by 7-dehydroxylation, and four epimers were not always found. In contrast to the incubation study, UDCA was not always the predominant 3,7-dihydroxy epimer in the fecal study. This may have been due to the transit times, which averaged 26.4 +/- 8.9 SD hr, being much shorter than the time it took for the incubation reactions to reach equilibrium.

Transformation of bile acids by mixed microbial cultures from human feces and bile acid transforming activities of isolated bacterial strains.

Microbiol transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7 alpha-hydroxy group in both primary bile acids and epimerization of the 3 alpha-hydroxy group in all metabolic bile acids. Extensive epimerization of the 7 alpha-hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7 alpha-Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7 alpha-dehydroxylase activity was revealed in one of the isolates, and unidentified gram-positive nonsporeforming anaerobic bacterium. 3 alpha-Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3 alpha- and 7 alpha-dehydrogenations. No microorganism accounting for 7 alpha-epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.

Metabolism of androgens and estrogens by human fecal microorganisms

Modifications of androgens and estrogens were observed in incubations with mixed human fecal cultures. Both oxidative and reductive reactions have been shown to occur. Moreover, the fecal concentration determines the types of metabolites formed. Analysis of reactions of high fecal concentration resulted in the identification of products formed by reduction. 4-ene-3-ketosteroids were reduced in ring A. 5β-3-ketosteroids were reduced to 5β-3-hydroxysteroids. 17-keto-androgens and estrogens were reduced to 17β-hydroxy products. Analysis of low fecal concentration incubations resulted in products formed by oxidation and by cleavage of conjugated steroids. 5β-androstane-3,17-dione was found to be converted to 4-androstene-3,17-dione. And 17β-hydroxysteroids were converted to 17-oxosteroids. Furthermore the estrogen conjugates estrone-3-sulfate and estradiol-3-glucuronide were hydrolyzed to form estrone and estradiol respectively. A number of the reductive reactions occurred under aerobic conditions. This may reflect the ability of the fecal extract to lower the oxidation-reduction potential of the reaction mixture. The androgen and estrogen metabolites varied depending on the concentration of feces and the availability of oxidizing agents, such as phenazine methosulfate or menadione

The biosynthesis of ethyl esters of lithocholic acid and isolithocholic acid by rat intestinal microflora

Recent reports concerning the tumor-promoting action of lithocholic acid in the colon and liver suggest that the metabolism of this major fecal bile acid may be important in carcinogenesis at various target sites. The metabolism of [ 14COOH]-lithocholic acid by rat intestinal microflora derived from standard laboratory chow-fed animals produced slightly more non-polar metabolites than those incubations which utilized flora from animals on a high lean-beef regimen. Purification of the crude bacterial extracts by Sephadex LH-20 chromatography and analysis of the radioactive peaks by glass fiber paper chromatography resulted in the identification of two neutral metabolites. Confirmation of their identity as ethyl lithocholate and ethyl isolithocholate was achieved by gas-liquid chromatography and combined gas-liquid chromatography-chemical ionization mass spectrometry. The formation of ethyl esters of lithocholic acid and isolithocholic acid by the intestinal microflora requires the presence of ethanol and anaerobic incubation conditions. These data support results obtained previously with single human fecal microorganisms. Since the formation of these derivatives in vitro occurs under anaerobic conditions only, it is possible that such derivatives may form physiologically in the colon. The carcinogenic potential of these derivatives is under investigation.