Fecal Suspension Research Articles

Impact of enrofloxacin on the human intestinal microbiota revealed by comparative molecular analysis

The indigenous human intestinal microbiota could be disrupted by residues of antibiotics in foods as well as therapeutically administered antibiotics to humans. These disruptions may lead to adverse health outcomes. To observe the possible impact of residues of antibiotics at concentrations below therapeutic levels on human intestinal microbiota, we performed studies using in vitro cultures of fecal suspensions from three individuals with 10 different concentrations (0, 0.1, 0.5, 1, 5, 10, 15, 25, 50 and 150 μg/ml) of the fluoroquinolone, enrofloxacin. The bacterial communities of the control and enrofloxacin dosed fecal samples were analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. In addition, changes of functional gene expression were analyzed by a pyrosequencing-based random whole-community mRNA sequencing method. Although each individual had a unique microbial composition, the communities of all individuals were affected by enrofloxacin. The proportions of two phyla, namely, Bacteroidetes and Proteobacteria, were significantly reduced with increasing concentrations of enrofloxacin exposure, while the proportion of Firmicutes increased. Principal Coordinate Analysis (PCoA) using the Fast UniFrac indicated that the community structures of intestinal microbiota were shifted by enrofloxacin. Most of the mRNA transcripts and the anti-microbial drug resistance genes increased with increasing concentrations of enrofloxacin. 16S rRNA gene pyrosequencing of control and enrofloxacin treated fecal suspensions provided valuable information of affected bacterial taxa down to the species level, and the community transcriptomic analyses using mRNA revealed the functional gene expression responses of the changed bacterial communities by enrofloxacin.

In vitro Analysis of the Impact of Enrofloxacin Residues on the Human Intestinal Microbiota Using 1H-NMR Spectroscopy

Exposure of humans to antimicrobial residues in food-producing animals may alter the intestinal microbiota which could result in a potential risk to human health. To determine the effect of enrofloxacin on the human intestinal microbiota, fecal suspensions (25%) were cultured in the presence of 0.06–5 µg/ml enrofloxacin. The bacterial community was analyzed by plating on selective culture media, pyrosequencing and nuclear magnetic resonance (NMR) spectroscopy. Pyrosequencing analysis of 16S rRNA genes and viable counts on Bacteroides sp., Enterococcus sp., and Bifidobacterium sp. selection medium indicated that there were no significant changes in the bacteria numbers at the selected enrofloxacin concentrations (0.06, 0.1, and 1 µg/ml) relative to the control samples after a 48 h incubation. NMR analysis showed remarkably similar spectra in cultures treated with 0.06, 0.1, and 1 µg/ml enrofloxacin, with some slight differences in peak heights. However, hierarchical clustering analysis indicated significant differences in metabolite concentrations between the control and those samples treated with 1 µg/ml enrofloxacin. Leucine, phenylalanine, proline, and 2-oxovalerate were positively correlated with the concentration of enrofloxacin. NMR analysis is a potentially useful tool to monitor changes of the human intestinal microbiota, in addition to traditional culture methods and pyrosequencing.

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.

Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions

Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong. Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water. Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity. In this study, we examined coronavirus stability on lettuce surfaces. A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 104 to 1.7 × 106 throughout the experimental period of 30 days. Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce. UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated. Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans.

In vitro enrofloxacin binding in human fecal slurries

Most antibiotic inactivation studies have been conducted through in vitro incubations of human use aminoglycosides, beta-lactams, and fluoroquinolones, usually at fecal concentrations expected with therapeutic dose regimens in humans and animals. Less is known about the inactivation of these molecules when ingested at concentrations consistent with residue levels present in animal-derived foods from antibiotic treated animals. In this investigation, we used the fluoroquinolone, enrofloxacin which is specifically marketed for veterinary medicine as test compound. Fecal suspensions at 10%, 25%, and 50% (w/v) were subjected to physicochemical and molecular characterization and used in the drug binding studies. The fecal binding of enrofloxacin added at concentrations of 0.06, 0.1, 1, 5, 15, 50, and 150mg/L was determined in various fecal slurry suspensions using analytical chemistry and microbiological assay methods. There was consistent correlation between both assay methods. By the analytical chemistry assay, the 10%, 25% and 50% diluted autoclaved fecal samples dosed with enrofloxacin showed binding of 50±4.6%, 54±6.5% and 56±6.8% of the enrofloxacin, respectively. Binding of enrofloxacin to fecal contents occurred rapidly within 10min and remained constant over the incubation period. Denaturing gradient gel electrophoreses and pyrosequencing analysis showed varied profiles of the bacterial composition of the human intestinal microbiota for fecal samples from different individuals. This study provided information on methodological questions that have concerned regulatory authorities on in vitro testing to determine if concentrations of veterinary antimicrobial agent residues entering the human colon remain microbiologically active.

Potential for aerosolization of Clostridium difficile after flushing toilets: the role of toilet lids in reducing environmental contamination risk

Toilet facilities in healthcare settings vary widely, but patient toilets are commonly shared and do not have lids. When a toilet is flushed without the lid closed, aerosol production may lead to surface contamination within the toilet environment. To substantiate the risks of airborne dissemination of C.difficile following flushing a toilet, in particular when lids are not fitted. We performed in-situ testing, using faecal suspensions of C.difficile to simulate the bacterial burden found during disease, to measure C.difficile aerosolization. We also measured the extent of splashing occurring during flushing of two different toilet types commonly used in hospitals. C.difficile was recoverable from air sampled at heights up to 25 cm above the toilet seat. The highest numbers of C.difficile were recovered from air sampled immediately following flushing, and then declined 8-fold after 60 min and a further 3-fold after 90 min. Surface contamination with C.difficile occurred within 90 min after flushing, demonstrating that relatively large droplets are released which then contaminate the immediate environment. The mean numbers of droplets emitted upon flushing by the lidless toilets in clinical areas were 15-47, depending on design. C.difficile aerosolization and surrounding environmental contamination occur when a lidless toilet is flushed. Lidless conventional toilets increase the risk of C.difficile environmental contamination, and we suggest that their use is discouraged, particularly in settings where CDI is common.

Erythropoietin Improves Skeletal Muscle Microcirculation Through the Activation of eNOS in a Mouse Sepsis Model

Sepsis and septic shock remain the major causes of morbidity and mortality in intensive care units. One mechanism that leads to organ failure is microcirculatory dysfunction. Erythropoietin (EPO) is a glycoprotein produced by the kidney that primarily regulates erythropoiesis, but it also can exert hemodynamic, anti-inflammatory, and tissue protective effects. We previously reported that administration of EPO to septic mice improves mouse skeletal muscle capillary perfusion and tissue bioenergetics. The objective of this study was to explore the potential mechanism(s) involved. Sepsis was induced by intraperitoneal (i.p.) injection of a fecal suspension (12.5 g in 0.5 saline/mouse) in mice. At 18 hours after sepsis induction, a single dose of rHuEPO (400 U/kg) was given to the mice. Mouse capillary perfusion density and nicotinamide adenine dinucleotide (NADH) fluorescence in skeletal muscle were observed using intravital microscopy. Endothelial cells derived from the skeletal muscle were treated with rHuEPO (5 U/mL) and endothelial nitric oxide synthase (eNOS) activation and activity were assessed. Septic mice had decreased capillary perfusion density and increased tissue NADH fluorescence indicating impaired tissue bioenergetics, whereas animals treated with rHuEPO demonstrated an improvement in capillary perfusion density and decreased skeletal muscle NADH fluorescence. The beneficial effect of rHuEPO did not occur in septic mice treated with l-NAME (an NOS inhibitor, 20 mg/kg) or mice genetically deficient in eNOS. Treatment of endothelial cells with rHuEPO resulted in activation of eNOS as indicated by increased eNOS phosphorylation and NO production. Our results suggest that eNOS plays an important role in mediating the beneficial effect of rHuEPO on microcirculation in this septic mouse model.

Effects of Electroacupuncture on the Evolution of Fecal Peritonitis in Mice

Abstract Background: There are few observations on the impact of electroacupuncture on the evolution of infectious diseases. Objective: The aim of this trial was to study the effect of electroacupuncture on the evolution of fecal peritonitis in mice. Subjects: Ten mice were submitted to acupuncture and electroacupuncture treatment immediately after induction of fecal peritonitis, and the evolution of infection was compared to mice receiving the same acupuncture but no electrostimulation. Setting Experiments were developed at the Infectious Diseases Unit, of the Federal University of Espirito Santo, in Vitora, ES, Brazil. Intervention: Twenty Swiss male mice received an intraperitoneal injection of a suspension of homologous feces (1:9 dilution). Needles were inserted in ST 36 and GB 30 acupoints in all animals. One group (EA group, 10 mice) received electrostimulation (30 minutes, 2Hz, 3 mA) and the other group of mice (control group, 10 mice) did not receive electrostimulation. Main Outcome Measures: Leu...

DETECÇÃO E ISOLAMENTO DE ROTAVÍRUS CAPRINO DO GRUPO A

RESUMO Rotavírus é a principal causa de diarreia em animais jovens de várias espécies. Os rotavirus estão classificados na família Reoviridae e contêm um genoma formado por 11 segmentos de RNA de cadeia dupla, envolvidos em três camadas proteicas. Neste estudo, foi detectado rotavírus pela eletroforese em gel de poliacrilamida (PAGE) em amostras de fezes de caprinos, com 80 dias de idade, durante surto de diarreia em um rebanho leiteiro. O perfil eletroforético das amostras, denominadas cap01/2007 e cap02/2007, foi caracterizado no PAGE como sendo de rotavírus do grupo A. Para o isolamento viral, suspensão fecal a 20% em tampão tris 0,1M pH 7,3 foi filtrada e tratada com tripsina cristalina na concentração de 5mg/mL. A amostra caprina cap01/2007 foi inoculada em cultura de células MA104 (Rim de Macaco Rhesus). Foram testados dois tratamentos (T1 e T2): após adsorção (estufa 37º C por uma hora) adicionou-se meio de manutenção (Dulbecco-MEM) sem tripsina, mantendo o inóculo (T1). No tratamento 2 (T2), foi realizado o mesmo pro cedimento anterior, porém o inóculo foi dispensado. O efeito citopático foi observado na primeira passagem para ambos os tratamentos (T1 e T2) com 48 horas de inoculação e o isolamento viral foi confirmado no PAGE. O perfil de migração manteve-se inalterado após três passagens sucessivas. Este é o primeiro relato de isolamento de rotavírus em caprinos no Brasil.

Effects of short term fasting on the evolution of fecal peritonitis in mice

To investigate the effect of 72 hours food suppression on the evolution of fecal peritonitis in mice evaluating the mortality and measuring the number and size of abscesses formed into the peritoneal cavity. Mice receiving commercial diet and water ad libitum (control group, N=35) and mice fasted during 72 h (N=35), receiving only water ad libitum, were inoculated by i.p. route, with 4uL/g body weight of a fecal suspension diluted 1:6 or 1:9 in 0.15M NaCl solution (1:6 dilution, 22 controls and 18 fasted; 1:9 dilution, 13 controls and 17 fasted). Animals were followed up until two weeks after fecal inoculation, when the survivors were euthanized for evaluation of the number and size of intra-peritoneal abscesses. Mortality was evaluated by Kaplan Meyer curves. Mortality was significantly higher in fasted groups than in controls. However the number and size of abscesses were significantly less in fasted groups than in controls. Seventy two hours food suppression increased the susceptibility to endotoxic shock (high mortality after peritonitis induction) and the resistance to infection with fecal microorganisms (less number and size of intra-peritoneal abscesses).

Colonic Catabolism of Ellagitannins, Ellagic Acid, and Raspberry Anthocyanins: In Vivo and In Vitro Studies

Red raspberries contain principally anthocyanins and ellagitannins. After ingestion of raspberries by humans, trace levels of anthocyanins, absorbed in the upper gastrointestinal tract, are excreted in urine in amounts corresponding to <0.1% of intake. Urine also contains urolithin-O-glucuronides derived from colonic metabolism of the ellagitannins. Raspberry feedings with ileostomists show that substantial amounts of the anthocyanin and ellagitannin intake are excreted in ileal fluid. In subjects with an intact functioning colon, these compounds would pass to the large intestine. The aim of this study was to identify raspberry-derived phenolic acid catabolites that form in the colon and those that are subsequently excreted in urine. In vitro anaerobic incubation of ellagitannins with fecal suspensions demonstrated conversion to ellagic acid and several urolithins. Fecal suspensions converted 80% of added ellagic acid to urolithins. In vivo, urolithins are excreted in urine as O-glucuronides, not aglycones, indicating that the colonic microflora convert ellagitannins to urolithins, whereas glucuronidation occurs in the wall of the large intestine and/or postabsorption in the liver. Unlike ellagitannins, raspberry anthocyanins were converted in vitro to phenolic acids by anaerobic fecal suspensions. Urinary excretion of phenolic acids after ingestion of raspberries indicates that after formation in the colon some phenolic acids undergo phase II metabolism, resulting in the formation of products that do not accumulate when anthocyanins are degraded in fecal suspensions. There is a growing realization that colonic catabolites such as phenolic acids and urolithins may have important roles in the protective effects of a fruit- and vegetable-rich diet.

Isolation of catechin-converting human intestinal bacteria

To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. Two bacterial strains, rK3 and aK2, were isolated from an epicatechin-converting human faecal suspension. The isolates catalysed individual steps in the degradation of ⁻-epicatechin and ⁺-catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C-ring of both ⁻-epicatechin and ⁺-catechin giving rise to 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol to 5-(3,4-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin-converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections—augmented with local observations and land-use patterns—indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination.

Lactobacillus rhamnosus JCM 2771: Impact on Metabolism of Isoflavonoids in the Fecal Flora from a Male Equol Producer

Many beneficial effects of probiotics have been reported; however, few have focussed on the effects of Lactobacillus, a probiotic, on the bioconversion of isoflavonoids. We hypothesized that Lactobacillus rhamnosus will modify the metabolism of isoflavone. In an in vitro incubation, L. rhamnosus JCM 2771 produced daidzein from daidzin along with genistein. However, daidzin and genistein were not detected in the incubation solution of daidzein with L. rhamnosus. In the fecal suspension from a male equol producer with daidzein, equol was detected in the presence of a low or high concentration of L. rhamnosus. In the fecal incubation with daidzin, the equol concentration increased with an increasing concentration of L. rhamnosus JCM 2771. L. rhamnosus affected the equol production in the in vitro incubation of daidzein with fecal flora from a male equol producer. We demonstrated for the first time that L. rhamnosus JCM 2771 could produce genistein from daidzin and affect the equol production of fecal flora from a male equol producer in vitro.

Molecular characterization of G and P rotavirus genotypes, G9 strain, from children with acute gastroenteritis in the Metropolitan Region of Belém, Pará State, from 1999 to 2007

ABSTRACT Rotavirus (RV) is the major viral agent associated with gastroenteritis and is responsible for 39% of all diarrheal cases requiring hospitalization, as well as approximately 520 thousand deaths among children under five years of age. Rotavirus belongs to the Reoviridae family, genus Rotavirus and its genome consists of 11 segments of double-stranded RNA which encode six structural viral proteins (VPs) and six non-structural viral proteins (NSPs). VP4 and VP7 proteins make up the outer layer of the RV particle and define the two genotypes, P and G, respectively. To date, at least 23 G genotypes and 31 P genotypes have been identified. G9-type RV strains, which are associated with a more severe disease in humans, have emerged globally. Dual type-specificities have been reported, and G9 strains most often belong to the P[8] genotype. While G9 genotype includes six distinct lineages, four distinct lineages are currently recognized as belonging to the P[8] genotype. The aim of this study was to characterize the VP4 and VP7 genes of the RV G9 genotypes that circulated in the Metropolitan Region of Belem, Para, in northern Brazil from 1999 to 2007. The viral dsRNA of 38 samples was extracted from fecal suspensions and analyzed using polyacrylamide gel electrophoresis to determine electropherotypes, followed by sequencing reactions. Overall, 32 selected G9P[8] samples were analyzed, all of which possessed long electropherotype. Phylogenetic analysis of the VP7 gene showed that all G9 strains belonged to lineage 3 with a high degree of similarity between them; only eight nucleotide changes have been detected in this lineage. However, only three of these amino acid substitutions were observed in our study, occurring at positions 43 (l→V), 66 (A→V) and 73 (Q→R). Of interest, the substitutions at positions 43 and 73 were only found in samples from 2007. Phylogenetic analysis of the VP4 gene revealed that all of the P[8] strains belonged to lineage 3, with 15 nucleotide changes corresponding to four amino acid substitutions at positions 108 (V→l), 172 (R→K), 173 (l→V) and 275 (K→R). The amino acid substitutions at positions 172 and 275 were observed only in RV isolates from 1999 to 2002. Overall, the RV G9 samples showed high homology throughout the entire study period. Interestingly, strains isolated in 2007 were the most divergent with respect to both VP4 and VP7 genes. Our data underscore the need for continuous surveillance of circulating RV strains in our region to detect the emergence of new genetic variants that would pose a challenge to current RV immunization strategies. This should include close monitoring of the genetic diversity of emerging G9 strains.

Dihydrodaidzein-producing Clostridium-like intestinal bacterium, strain TM-40, affects in vitro metabolism of daidzein by fecal microbiota of human male equol producer and non-producers.

Much attention has been focused on the biological effects of equol, a metabolite of daidzein produced by intestinal microbiota. However, little is known about the role of isoflavone metabolizing bacteria in the intestinal microbiota. Recently, we isolated a dihydrodaidzein (DHD)-producing Clostridium-like bacterium, strain TM-40, from human feces. We investigated the effects of strain TM-40 on in vitro daidzein metabolism by human fecal microbiota from a male equol producer and two male equol non-producers. In the fecal suspension from the male equol non-producer and DHD producer, DHD was detected in the in vitro fecal incubation of daidzein after addition of TM-40. The DHD concentration increased as the concentration of strain TM-40 increased. In the fecal suspension from the equol producer, the fecal equol production was increased by the addition of strain TM-40. The occupation ratios of Bifidobacterium and Lactobacillales were higher in the equol non-producers than in the equol producer. Adding isoflavone-metabolizing bacteria to the fecal microbiota should facilitate the estimation of the metabolism of isoflavonoids by fecal microbiota. Studies on the interactions among equol-producing microbiota and DHD-producing bacteria might lead to clarification of some of the mechanisms regulating the production of equol by fecal microbiota.

Influence of coating on the triamcinolone release of alginate chitosan beads for colonic drug delivery

The aim of this study was to evaluate the effect of coating of alginate-chitosan (AL:CS) beads on the colonic drug delivery. The AL:CS systems containing triamcinolone (TC) were coated with the HPMCP and Eudragit? L100 by immersion and by spraying methods. The drug release profile in simulated colonic medium was determined using 5% human fecal content suspension in 0.01 N buffer solution, pH 6.8. The systems coated with HPMCP showed a lower rate of drug delivery in simulated enteric medium. The delivery profile in simulated colonic medium followed zero-order kinetic. The coated systems provided a promising drug-delivery profile for application in colonic drug delivery.

Drug metabolome of the Simvastatin formed by human intestinal microbiota in vitro

The human colon contains a diverse microbial population which contributes to degradation and metabolism of food components. Drug metabolism in the colon is generally poorly understood. Metabolomics techniques and in vitro colon models are now available which afford detailed characterization of drug metabolites in the context of colon metabolism. The aim of this work was to identify novel drug metabolites of Simvastatin (SV) by using an anaerobic human in vitro colon model at body temperature coupled with systems biology platform, excluding the metabolism of the host liver and intestinal epithelia. Comprehensive two-dimensional gas chromatography with a time-of-flight mass spectrometry (GC×GC-TOFMS) was used for the metabolomic analysis. Metabolites showing the most significant differences in the active faecal suspension were elucidated in reference with SV fragmentation and compared with controls: inactive suspension or buffer with SV, or with active suspension alone. Finally, time courses of selected metabolites were investigated. Our data suggest that SV is degraded by hydrolytic cleavage of methylbutanoic acid from the SV backbone. Metabolism involves demethylation of dimethylbutanoic acid, hydroxylation/dehydroxylation and β-oxidation resulting in the production of 2-hydroxyisovaleric acid (3-methyl-2-hydroxybutanoic acid), 3-hydroxybutanoic acid and lactic acid (2-hydroxypropanoic acid), and finally re-cyclisation of heptanoic acid (possibly de-esterified and cleaved methylpyranyl arm) to produce cyclohexanecarboxylic acid. Our study elucidates a pathway of colonic microbial metabolism of SV as well as demonstrates the applicability of the in vitro colon model and metabolomics to the discovery of novel drug metabolites from drug response profiles.

Metabolism of Isoflavones Found in the Pueraria thomsonii Flower by Human Intestinal Microbiota.

Isoflavones contained in the root and flower of Kudzu (Pueraria lobata and related species) are suggested to be the critical component for its effects. Although metabolism of soy isoflavones has been well studied, the composition of isoflavones found in Kudzu is completely different from that of soy isoflavones. In the present study, we investigated whether isoflavones found in the flower of Pueraria thomsonii, a species of Kudzu, were metabolized by human fecal microbiota and murine small intestinal enzymes. Among 5 glycosidic isoflavones of the Pueraria thomsonii flower, tectorigenin 7-O-xylosylglucoside, tectoridin, genistin and glycitin were completely hydrolyzed by a homogenate of germfree mouse small intestine without contribution of bacteria. Released aglycones were not further metabolized, except that up to half of glycitein disappeared. Mouse small intestinal enzymes did not metabolize 6-hydroxygenistein 6,7-di-O-glucoside. Isoflavone aglycones as well as 6-hydroxygenistein 6,7-di-O-glucoside were highly metabolized by most of the human fecal suspensions. Metabolites were not detected with the present analytical methods in most cases. Although further investigations of the pharmacokinetics of Pueraria thomsonii flower isoflavones are needed, the results of the present study indicate active metabolism of Pueraria thomsonii flower isoflavones in the human intestine.

Deglycosylation of puerarin and other aromatic C‐glucosides by a newly isolated human intestinal bacterium

The human intestinal microbiota may influence the fate of bioactive polyphenols, such as the isoflavone puerarin (daidzein 8-C-glucoside), following their oral intake. Faecal suspensions from 19 healthy subjects were tested for their ability to C-deglycosylate puerarin. Only one of these catalysed this reaction. A rod-shaped Gram-positive bacterium, strain CG19-1, capable of deglycosylating puerarin to daidzein was isolated from the corresponding suspension. However, the strictly anaerobic isolate was unable to utilize puerarin as sole carbon and energy source nor any of the tested carbohydrates. Comparative 16S rRNA gene sequence analysis indicated that strain CG19-1 is a new species of the Lachnospiraceae. Strain CG19-1 also converted other aromatic C-glucosides in addition to puerarin. The xanthone C-glucoside mangiferin was deglycosylated to norathyriol. The flavone C-glucosides homoorientin and vitexin were degraded to 3-(3,4-dihydroxyphenyl)propionic acid via luteolin and 3-(4-hydroxyphenyl)propionic acid respectively. In addition, strain CG19-1 converted flavonoid O-glucosides, but at rates that were lower than those of the C-glucosides tested. The isolate deglycosylated the isoflavone O-glucosides daidzin and genistin to daidzein and genistein respectively. Several O-glucosides of the flavones luteolin and apigenin undergoing deglycosylation were subsequently cleaved to 3-(3,4-dihydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)propionic acid respectively. Moreover, strain CG19-1 cleaved both O-desmethylangolensin and 6'-hydroxy-O-desmethylangolensin to yield 2-(4-dihydroxyphenyl)propionic acid. The corresponding cleavage product, resorcinol, was only observed for O-desmethylangolensin.