Human Fecal Slurries Research Articles

P-1830. The First-in-Class Anti-Staphylococcal Antibiotic Afabicin Desphosphono is Not Associated With Clostridioides difficile Infection in an in vitro Human Gut Model

Abstract Background Antibiotic use is commonly associated with disruption of the healthy human gut microbiota (termed dysbiosis). Dysbiosis can diminish colonisation resistance and increase susceptibility to Clostridioides difficile infection (CDI). Use of the pathogen-specific anti-staphylococcal afabicin was not associated with dysbiosis in animals and healthy volunteers (Nowakowska et al, 2023). Here, we assessed whether this microbiota-sparing property of afabicin would limit CDI in a clinically reflective in vitro human gut model. Methods A well-established gut model was used as described previously (Baines et al, 2005). Four independent models were seeded with human fecal slurry (d0) and inoculated at d14 and d21 with C. difficile spores (107 CFU/mL). Two-week treatments were assessed beginning on d21 including (i) a CDI-positive control antibiotic levofloxacin (149.55 mg/L once daily), (ii) afabicin desphosphono high dose (35.7 mg/L twice daily [BID]), (iii) afabicin desphosphono mid dose (17.9 mg/L BID) and (iv) a vehicle control (1% DMSO BID). Endpoints for the model included facultative and obligate anaerobe counts, total viable and spore C. difficile counts and C. difficile toxin titre and were monitored daily until d63. Results Each of the afabicin desphosphono treated models displayed colonisation resistance and no evidence of C. difficile germination or toxin production for the duration of the study. Further, no relevant changes in major facultative and obligate anaerobe groups were observed, indicating limited dysbiosis, similar to the vehicle control. In contrast, following a pre-treatment period where colonisation resistance was demonstrated (i.e. no germination following C. difficile inoculation), levofloxacin treatment resulted in dysbiosis characterized most notably by reductions (∼5log10 CFU/mL) in facultative anaerobes including lactose fermenting Enterobacteriaceae. Dysbiosis caused by levofloxacin led to CDI including C. difficile germination and toxin production. Conclusion Findings from this study provide further support for the microbiota-sparing property of afabicin and suggest that limited off-target microbiota effects associated with afabicin treatment may reduce incidence of CDI when used clinically. Disclosures Charmaine Normington, PhD, Debiopharm International S.A.: Grant/Research Support Ricardo Chaves, n/a, Debiopharm International S.A.: Salary David Cameron, PhD, Debiopharm International S.A.: Salary Mark H. Wilcox, MD, Astra Zeneca: Advisor/Consultant|Debiopharm International S.A.: Advisor/Consultant|Debiopharm International S.A.: Grant/Research Support|Ferring: Advisor/Consultant|GSK: Advisor/Consultant|GSK: Honoraria|Nestle: Advisor/Consultant|Paion: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: Honoraria|Phico therapeutics: Advisor/Consultant|QPex Biopharma: Advisor/Consultant|Seres: Advisor/Consultant|Seres: Grant/Research Support|Seres: Lecture Fees|Summit: Advisor/Consultant|Summit: Grant/Research Support|The European Tissue Symposium: Advisor/Consultant|The European Tissue Symposium: Grant/Research Support|Tillotts: Advisor/Consultant|Tillotts: Grant/Research Support|Tillotts: Lecture Fees|Vedanta: Advisor/Consultant Caroline Chilton, PhD, Debiopharm International S.A.: Grant/Research Support

Ex vivo biotransformation of lady’s mantle extracts via the human gut microbiota: the formation of phenolic metabolites and their impact on human normal and colon cancer cell lines

IntroductionFor centuries, various species from the genus Alchemilla have been utilized in traditional medicine worldwide. Among them, Alchemilla vulgaris L. (Rosaceae) stands out as a promising herbal drug candidate due to its phytochemicals displaying anti-inflammatory and antioxidant properties.MethodsIn our study, we investigated the interaction between the human gut microbiota and lady’s mantle herb extract (AV) following the biotransformation of the extract’s constituents and their impact on colorectal cancer cells (HT-29) and normal CCD 841 CoN epithelial cells. The A. vulgaris herb metabolites were obtained by incubating the extract (AV) with human fecal slurries from three healthy donors (D1, D2, and D3).ResultsAfter incubating the AV extract with the human gut microbiota (AVD1-AVD3 samples), thirty-three metabolites were detected and characterized by LC-MS. Among them, one was identified as urolithin C. The AV and AVD1-AVD3 extracts and their metabolites exhibit various levels of antiproliferative and cytotoxic activities against cancer cells. Their biological effect might be linked to the changes and direct activity of bioavailable metabolites. Samples from AVD1, AVD2, and AVD3 increase the lactate dehydrogenase (LDH) released from damaged colon cancer cells in a dose-dependent manner. At 250 μg/mL, AVD1, AVD2, and AVD3 elevated the LDH level by 12.6%, 25.3%, and 30.0%, respectively. The biotransformed samples also showed significantly higher antiproliferative activity than the AV extract. The most active sample from donor 3 (AVD3) reached IC50 = 471 μg/mL.DiscussionThe differences in anticancer effect might be linked to the changes and direct activity of bioavailable metabolites. The non-transformed AV extract affected neither normal nor cancer colon cells, indicating the beneficial effect of the biotransformation procedure on the anticancer properties of the evaluated extracts. The above results clearly indicate that microbial metabolism is a crucial factor that is potent in altering the biological activity of lady’s mantle extract.

Appearance of green tea compounds in plasma following acute green tea consumption is modulated by the gut microbiome in mice.

Studies have suggested that phytochemicals in green tea have systemic anti-inflammatory and neuroprotective effects. However, the mechanisms behind these effects are poorly understood, possibly due to differential metabolism of phytochemicals resulting from variation in gut microbiome composition. To unravel this complex relationship, our team utilized a novel combined microbiome analysis and metabolomics approach applied to low complexity microbiome (LCM) and human colonized (HU) gnotobiotic mice treated with an acute dose of powdered matcha green tea. A total of 20 LCM mice received 10 distinct human fecal slurries for an n=2 mice per human gut microbiome; 9 LCM mice remained un-colonized with human slurries throughout the experiment. We performed untargeted metabolomics on green tea and plasma to identify green tea compounds that were found in plasma of LCM and HU mice that had consumed green tea. 16S ribosomal RNA gene sequencing was performed on feces of all mice at study end to assess microbiome composition. We found multiple green tea compounds in plasma associated with microbiome presence and diversity (including acetylagmatine, lactiflorin, and aspartic acid negatively associated with diversity). Additionally, we detected strong associations between bioactive green tea compounds in plasma and specific gut bacteria, including associations between spiramycin and Gemmiger, and between wildforlide and Anaerorhabdus. Additionally, some of the physiologically relevant green tea compounds are likely derived from plant-associated microbes, highlighting the importance of considering foods and food products as meta-organisms. Overall, we describe a novel workflow for discovering relationships between individual food compounds and composition of the gut microbiome.

Evaluation of Piper nigrum L. as a Prebiotic Ingredient Using In Vitro Colon Model

Black and white pepper of the species Piper nigrum L. is regarded as the king of spices, and Malaysia is the second largest producer of peppercorns, after Indonesia. This spice contains abundant bioactive compounds that are capable of enhancing human health. However, the prebiotic potential of P. nigrum L. as a food ingredient has not yet been explored. Therefore, this research studied P. nigrum L. through in vitro gastrointestinal digestion and colonic fermentation using human faecal slurry. Samples were analysed for the colonic bacterial changes and its metabolites production using HPLC. Both black and white pepper showed prebiotic responses similar to those in inulin, particularly in stimulating the growth of human gastrointestinal microbiota. This study finds that P. nigrum L. promotes the growth of probiotic strains such as Bifidobacterium spp., and Lactobacillus / Enterococcus. Both black and white pepper also showed the ability to suppress colonic pathogen strain like Clostridium histolyticum. In vitro colonic fermentation of P. nigrum L. also significantly stimulate production of health beneficial metabolites. The production of short-chain fatty acids like acetate and propionate were observed to be particularly abundant. This is the contribution of piperine in both black and white pepper. In which, piperine provide both anti-bacterial and anti-inflammatory properties. Overall, P. nigrum L. showed appreciable prebiotic value similar to the commercial prebiotic, inulin. Thus, black and white pepper from Sarawak, Malaysia helps in promoting human gastrointestinal health. This finding may contribute to the value-added of Sarawak pepper as a functional food that can be involved in daily meals as spices.

Dietary-Fibre-Rich Fractions Isolated from Broccoli Stalks as a Potential Functional Ingredient with Phenolic Compounds and Glucosinolates.

The Brassica oleracea industry generates large amounts of by-products to which value could be added because of the characteristics of their composition. The aim was to extract different fibre fractions from broccoli stalks to obtain potential new added-value ingredients. Using an ethanol and water extraction procedure, two fibre-rich fractions (total fibre fraction, TFB, and insoluble fibre fraction, IFB) were obtained. These fractions were analysed to determine the nutritional, (poly)phenols and glucosinolates composition and physicochemical properties, comparing the results with those of freeze-dried broccoli stalks (DBS). Although TFB showed a higher content of total dietary fibre, IFB had the same content of insoluble dietary fibre as TFB (54%), better hydration properties, higher content of glucosinolates (100 mg/100 g d.w.) and (poly)phenols (74.7 mg/100 g d.w.). The prebiotic effect was evaluated in IFB and compared with DBS by in vitro fermentation with human faecal slurries. After 48 h, the short-chain fatty acid (SCFA) production was higher with IFB than with DBS because of the greater presence of both uronic acids, the main component of pectin, and (poly)phenols. These results reveal that novel fibre-rich ingredients-with antioxidant, technological and physiological effects-could be obtained from broccoli stalks by using green extraction methods.

Inter- and Intraindividual Differences in the Capacity of the Human Intestinal Microbiome in Fecal Slurries to Metabolize Fructoselysine and Carboxymethyllysine.

The advanced glycation endproduct carboxymethyllysine and its precursor fructoselysine are present in heated, processed food products and are considered potentially hazardous for human health. Upon dietary exposure, they can be degraded by human colonic gut microbiota, reducing internal exposure. Pronounced interindividual and intraindividual differences in these metabolic degradations were found in anaerobic incubations with human fecal slurries in vitro. The average capacity to degrade fructoselysine was 27.7-fold higher than that for carboxymethyllysine, and degradation capacities for these two compounds were not correlated (R2 = 0.08). Analysis of the bacterial composition revealed that interindividual differences outweighed intraindividual differences, and multiple genera were correlated with the individuals’ carboxymethyllysine and fructoselysine degradation capacities (e.g., Akkermansia, Alistipes).

An in vitro study: prebiotic effects of edible palm hearts in batch human fecal fermentation system.

Edible palm hearts (EPH), known as palmito, chonta or swamp cabbage in America or umbut in Malaysia, is a type of vegetable harvested from palm tree species. EPH is firm and smooth and described as having a flavor resembling artichoke. It has underlying prebiotic potential that selectively stimulates the growth and activity of beneficial colonic microbiota, thus enhancing the host's health. This study is the first to present results of EPH from local species such as oil palm (Elaeis guineensis), sago palm (Metroxylon sagu) and coconut (Cocos nucifera) using in vitro colonic fermentation with human fecal slurry. Samples obtained at 0, 6, 12 and 24 h were evaluated by bacterial enumeration using fluorescent in situ hybridization (FISH), and short-chain fatty acids (SCFA) were analyzed by high-performance liquid chromatography (HPLC). All EPH samples revealed induction effects towards Bifidobacterium spp., Lactobacillus-Enterococcus and Bacteroidaceae/Prevotellaceae populations similar to those in inulin fermentation. A significant decrease (P ≤ 0.05) in pathogenic Clostridium histolyticum group was observed in the response of raw sago palm hearts. In general, all samples stimulate the production of SCFA. Particularly in the colonic fermentation of sago palm heart, acetate and propionate revealed the highest concentrations of 286.18 and 284.83 mmol L-1 in raw and cooked form, respectively. This study concluded that edible palm hearts can be a potential prebiotic ingredient that promotes human gastrointestinal health, as well as discovering a new direction towards an alternative source of functional foods. © 2022 Society of Chemical Industry.

Tiliae flos metabolites and their beneficial influence on human gut microbiota biodiversity ex vivo

Ethnopharmacological relevanceThe linden flower (Tiliae flos) has been used for centuries to treat and relieve symptoms of the common cold, throat irritation, and upper respiratory tract disturbances. Traditionally, this herb is administered orally, and thus it undergoes intestinal metabolism. Although it is pharmacopeial plant material, there are no reports about its interaction with human gut microbiota. Aim of the studyThe study aimed to determine the interaction between human gut microbiota and the linden flower extracts, resulting in the biotransformation of the extract's constituents and changes in the microbiota composition. Material and methodsThe linden flower metabolites were obtained by incubation of extract with human faecal slurries from 5 healthy donors. The UHPLC-DAD-MSn analysis determined the composition of raw extract and analysis of microbial metabolites. The intestinal microbiota isolation and sequencing were used to determine changes in microbiota composition. The anti-inflammatory activity was tested using the LPS-stimulated human neutrophils model and ELISA test. ResultsAfter incubation of linden flower extract with human gut microbiota, twenty metabolites were detected and characterized, and three among them were identified. The extract changed human gut microbiota composition but did not cause dysbiosis (change in the abundance of forty-three genera). Raw extract and their metabolites exhibit different levels of inhibition of cytokines production by LPS-stimulated neutrophils, but the reduction of TNF-α production was observed. ConclusionsThe linden flower extract has a beneficial influence on human gut microbiota because it promotes increasing the abundance of bacteria responsible for SCFAs production. The anti-inflammatory effect might be linked to both microbiota composition changes and direct activity of bioavailable metabolites. Increased abundance of SCFAs producers may inhibit the production of pro-inflammatory cytokines. A low concentration of phenolic compounds in metabolized linden flower extract and responsible for anti-inflammatory properties, and the multitude of biological and chemical particles and their interactions may weaken these properties.

The use of first-generation cephalosporin antibiotics, cefalexin and cefradine, is not associated with induction of simulated Clostridioides difficile infection.

ObjectivesThe use of broad-spectrum cephalosporins is associated with induction of Clostridioides difficile infection (CDI). Recent knowledge on the importance of the healthy microbiota in preventing pathogen colonization/outgrowth highlights the caution needed when prescribing broad-spectrum antibiotics. The use of historical narrow-spectrum antibiotics, such as first-generation cephalosporins, is gaining increased attention once more as they have a reduced impact on the microbiota whilst treating infections. Here, the effects of two first-generation cephalosporins, compared with a third-generation cephalosporin, on the human microbiota were investigated and their propensity to induce simulated CDI.MethodsThree in vitro chemostat models, which simulate the physiochemical conditions of the human colon, were seeded with a human faecal slurry and instilled with either narrow-spectrum cephalosporins, cefalexin and cefradine, or a broad-spectrum cephalosporin, ceftriaxone, at concentrations reflective of colonic levels.ResultsInstillation of cefalexin was associated with reduced recoveries of Bifidobacterium and Enterobacteriaceae; however, Clostridium spp. recoveries remained unaffected. Cefradine exposure was associated with decreased recoveries of Bifidobacterium spp., Bacteroides spp. and Enterobacteriaceae. These changes were not associated with induction of CDI, as we observed a lack of C. difficile spore germination/proliferation, thus no toxin was detected. This is in contrast to a model exposed to ceftriaxone, where CDI was observed.ConclusionsThese model data suggest that the minimal impact of first-generation cephalosporins, namely cefalexin and cefradine, on the intestinal microbiota results in a low propensity to induce CDI.

A Gut-Restricted Lithocholic Acid Analog as an Inhibitor of Gut Bacterial Bile Salt Hydrolases.

Bile acids play crucial roles in host physiology by acting both as detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. Structure-activity relationship (SAR) studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in mouse and human fecal slurry, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.

Development of a High Throughput In Vitro Fecal Fermentation Method to Screen for TMA Production Inhibitors

ObjectivesCholine is metabolized by gut bacteria to trimethylamine (TMA), which is further metabolized by the host into trimethylamine N-oxide (TMAO). There is significant interest in reducing TMAO formation to reduce atherosclerosis risk. Our objective was to develop an in vitro fermentation methodology to screen for bioactives able to reduce TMA formation. MethodsCholine (5–100 μM) fermentation was optimized under anaerobic conditions at 37°C with the presence of human fecal slurry (OpenBiome) diluted 1:10 in PBS 1X (5–45%) over 36 h. Common dietary phenolics (gallic acid and chlorogenic acid, 0.1–10 mM) were evaluated as TMA production inhibitors under optimal fermentation conditions. 3,3-dimethyl-1-butanol (DMB, 10 mM) was used as a positive control. TMA and choline levels were monitored and analyzed by UPLC-ESI-MS/MS. Cell density (O.D. at 600 nm) was evaluated to account potential cytotoxicity. ResultsTMA kinetic production curves from choline at >25 μM were statistically different from background (no choline added), suggesting its potential to be used to assay inhibition of TMA production. Fecal slurry concentration of 5% did not reach a TMA plateau within 36 h of fermentation, while the kinetics reported by 45% were fast, reaching a plateau at 12 h, suggesting the need for intermediate fecal concentrations. Optimal fermentation conditions were 100 μM choline and 20%. fecal slurry Under those conditions, exogenous choline was consumed within the first 12 h of fermentation, during which TMA formation plateaued. Under optimal conditions plus gallic acid or chlorogenic acid, TMA formation was significantly reduced, reaching 50% of inhibitor-free control at concentrations >5 mM at 8 h. Also, >2 mM reported higher inhibition potential than DMB 10 mM. Of note, no reductions in cell density were reported due to treatment administration, suggesting a lack of cytotoxicity. ConclusionsOur results suggest that gallic acid and chlorogenic acid are promising compounds for in vivo studies. Moreover, our fermentation method can be used to screen for TMA production inhibitors in a high-throughput fashion. Funding SourcesSupported by through startup funding from North Carolina State University and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture.

In vitro (poly)phenol catabolism of unformulated- and phytosome-formulated cranberry (Vaccinium macrocarpon) extracts

Cranberries (Vaccinium macrocarpon) represent an important source of anthocyanins, flavan-3-ols and flavonols. This study aimed at investigating in vitro the human microbial metabolism of (poly)phenols, principally flavan-3-ols, of unformulated- and phytosome-formulated cranberry extracts. After powder characterization, a 24-h fermentation with human faecal slurries was performed, standardizing the concentration of incubated proanthocyanidins. Cranberry (poly)phenol metabolites were quantified by uHPLC-MS2 analyses. The native compounds of both unformulated- and phytosome-formulated cranberry extracts were metabolized under faecal microbiota activity, resulting in twenty-four microbial metabolites. Although some differences appeared when considering different classes of colonic metabolites, no significant differences in the total amount of metabolites were established after 24 h of incubation period. These results suggested that a different formulation had no effect on flavan-3-ol colonic metabolism of cranberry and both unformulated- and phytosome-formulated extract. Both formulations displayed the capability to be a potential source of compounds which could lead to a wide array of gut microbiota metabolites in vitro.

An in vitro model for microbial fructoselysine degradation shows substantial interindividual differences in metabolic capacities of human fecal slurries

Fructoselysine is formed upon heating during processing of food products, and being a key intermediate in advanced glycation end product formation considered to be potentially hazardous to human health. Human gut microbes can degrade fructoselysine to yield the short chain fatty acid butyrate. However, quantitative information on these biochemical reactions is lacking, and interindividual differences therein are not well established. Anaerobic incubations with pooled and individual human fecal slurries were optimized and applied to derive quantitative kinetic information for these biochemical reactions. Of 16 individuals tested, 11 were fructoselysine metabolizers, with Vmax, Km and kcat-values varying up to 14.6-fold, 9.5-fold, and 4.4-fold, respectively. Following fructoselysine exposure, 10 of these 11 metabolizers produced significantly increased butyrate concentrations, varying up to 8.6-fold. Bacterial taxonomic profiling of the fecal samples revealed differential abundant taxa for these reactions (e.g. families Ruminococcaceae, Christenellaceae), and Ruminococcus_1 showed the strongest correlation with fructoselysine degradation and butyrate production (ρ ≥ 0.8).This study highlights substantial interindividual differences in gut microbial degradation of fructoselysine. The presented method allows for quantification of gut microbial degradation kinetics for foodborne xenobiotics, and interindividual differences therein, which can be used to refine prediction of internal exposure.

Eravacycline, a novel tetracycline derivative, does not induce Clostridioides difficile infection in an in vitro human gut model.

The approval of new antibiotics is essential to combat infections caused by antimicrobial-resistant pathogens; however, such agents should be tested to determine their effect on the resident microbiota and propensity to select for opportunistic pathogens, such as Clostridioides difficile. Eravacycline is a new antibiotic for the treatment of complicated intra-abdominal infections. Here, we determined the effects of eravacycline compared with moxifloxacin on the microbiota and if these were conducive to induction of C. difficile infection (CDI). We seeded in vitro chemostat models, which simulate the physiological conditions of the human colon, with a human faecal slurry and instilled gut-reflective concentrations of either eravacycline or moxifloxacin. Eravacycline instillation was associated with decreased Bifidobacterium, Lactobacillus and Clostridium species, which recovered 1 week after exposure. However, Bacteroides spp. levels decreased to below the limit of detection and did not recover prior to the end of the experiment. Post-eravacycline, a bloom of aerobic bacterial species occurred, including Enterobacteriaceae, compared with pre-antibiotic, which remained high for the duration of the experiment. These changes in microbiota were not associated with induction of CDI, as we observed a lack of C. difficile spore germination and thus no toxin was detected. Moxifloxacin exposure sufficiently disrupted the microbiota to induce simulated CDI, where C. difficile spore germination, outgrowth and toxin production were seen. These model data suggest that, despite the initial impact of eravacycline on the intestinal microbiota, similar to clinical trial data, this novel tetracycline has a low propensity to induce CDI.

Prebiotic Potential of Taro (Colocasia esculenta) to Modulate Gut Bacteria Composition and Short Chain Fatty Acid Production

Abstract Objectives Taro (Colocasia esculenta) is a high fiber tuber that holds cultural and agricultural importance in the Pacific. The high fiber content offers potential as a dietary prebiotic through the presence of resistant starch (RS). RS aids gut bacteria in the production of short-chain fatty acids (SCFA), which have health benefits to the host. As such, this study aimed to investigate the effect of taro on gut bacteria composition and SCFA production through in vitro human digestion and fecal fermentation methodology. Methods Five taro varieties (Bun Long, Mana Ulu, Moi, Kauai Lehua, Tahitian) were processed. RS concentration of taro was determined using Megazyme Resistant Starch kit. Taro samples were subjected to in vitro human digestion, replicating oral, gastric, small intestinal, and large intestinal phases. Digested taro samples were mixed with fresh human fecal slurries and fermented at 37°C for 24 hours. During fermentation, samples were collected at specific time points for analyzation of SCFA contents using gas chromatography and bacterial composition using 16S rRNA gene targeted amplicon sequencing. Results Tahitian variety had the highest RS concentration of 25.1%, with no significantly difference between Tahitian, Bun Long, and Moi varieties. All taro varieties exhibited statistically similar SCFA concentrations for 3-hydroxybutyric acid, acetic acid and propionic acid. Bun-Long, Tahitian, and Moi varieties exhibited significantly higher butyric acid levels than two other taro varieties. Butyric acid production had the highest correlation coefficient with RS concentration of 0.80. The bacterial community present in all taro varieties after 24 hours of fermentation was markedly different from that of the control communities, including shifts in both the Bacteriodes and Firmicutes phyla. Conclusions Different taro varieties have varying RS concentrations that affect the production of SCFA in an in vitro human digestion and fecal fermentation. The production of health-promoting butyric acid shows a strong positive correlation with the resistant starch concentration of taro. Taro distinctly causes shifts after 24 hours of fecal fermentation in Bacteriodes and Firmicutes phyla. These results provide new insights into the modulation of gut microbiota through dietary interventions. Funding Sources USDA-NIFA Hatch C-Maīki, University of Hawaii.

In vitro fermentation of novel microwave-synthesized non-digestible oligosaccharides and their impact on the composition and metabolites of human gut microbiota

Abstract Three microwave-synthesized non-digestible oligosaccharides (polydextrose, polygalactose and polymannose) were fermented using an in vitro pH-controlled batch incubation inoculated with human fecal slurries, simulating the events in the distal colon. Microbial changes and short-chain fatty acids (SCFAs) were analyzed using 16S rRNA gene sequencing and gas chromatography, respectively. Polydextrose and polygalactose significantly (P

Omadacycline Gut Microbiome Exposure Does Not Induce Clostridium difficile Proliferation or Toxin Production in a Model That Simulates the Proximal, Medial, and Distal Human Colon.

A clinically reflective model of the human colon was used to investigate the effects of the broad-spectrum antibiotic omadacycline on the gut microbiome and the subsequent potential to induce simulated Clostridium difficile infection (CDI). Triple-stage chemostat gut models were inoculated with pooled human fecal slurry from healthy volunteers (age, ≥60 years). Models were challenged twice with 107 CFU C. difficile spores (PCR ribotype 027). Omadacycline effects were assessed in a single gut model. Observations were confirmed in a parallel study with omadacycline and moxifloxacin. Antibiotic instillation was performed once daily for 7 days. The models were observed for 3 weeks postantibiotic challenge. Gut microbiota populations and C. difficile total viable and spore counts were enumerated daily by culture. Cytotoxin titers and antibiotic concentrations were also measured. Gut microbiota populations were stable before antibiotic challenge. Moxifloxacin instillation caused an ∼4 log10 CFU/ml decline in enterococci and Bacteroides fragilis group populations and an ∼3 log10 CFU/ml decline in bifidobacteria and lactobacilli, followed by simulated CDI (vegetative cell proliferation and detectable toxin). In both models, omadacycline instillation decreased populations of bifidobacteria (∼8 log10 CFU/ml), B. fragilis group populations (7 to 8 log10 CFU/ml), lactobacilli (2 to 6 log10 CFU/ml), and enterococci (4 to 6 log10 CFU/ml). Despite these microbial shifts, there was no evidence of C. difficile bacteria germination or toxin production. In contrast to moxifloxacin, omadacycline exposure did not facilitate simulated CDI, suggesting this antibiotic may have a low propensity to induce CDI in the clinical setting.

Transplanted human fecal microbiota enhanced Guillain Barr\xe9 syndrome autoantibody responses after Campylobacter jejuni infection in C57BL/6 mice

BackgroundCampylobacter jejuni is the leading antecedent infection to the autoimmune neuropathy Guillain-Barré syndrome (GBS), which is accompanied by an autoimmune anti-ganglioside antibody attack on peripheral nerves. Previously, we showed that contrasting immune responses mediate C. jejuni induced colitis and autoimmunity in interleukin-10 (IL-10)-deficient mice, dependent upon the infecting strain. Strains from colitis patients elicited T helper 1 (TH1)-dependent inflammatory responses while strains from GBS patients elicited TH2-dependent autoantibody production. Both syndromes were exacerbated by antibiotic depletion of the microbiota, but other factors controlling susceptibility to GBS are unknown.MethodsUsing 16S rRNA gene high-throughput sequencing, we examined whether structure of the gut microbial community alters host (1) gastrointestinal inflammation or (2) anti-ganglioside antibody responses after infection with C. jejuni strains from colitis or GBS patients. We compared these responses in C57BL/6 mice with either (1) stable human gut microbiota (Humicrobiota) transplants or (2) conventional mouse microbiota (Convmicrobiota).ResultsInoculating germ-free C57BL/6 wild-type (WT) mice with a mixed human fecal slurry provided a murine model that stably passed its microbiota over >20 generations. Mice were housed in specific pathogen-free (SPF) facilities, while extra precautions of having caretakers wear sterile garb along with limited access ensured that no mouse pathogens were acquired. Humicrobiota conferred many changes upon the WT model in contrast to previous results, which showed only colonization with no disease after C. jejuni challenge. When compared to Convmicrobiota mice for susceptibility to C. jejuni enteric or GBS patient strains, infected Humicrobiota mice had (1) 10-100 fold increases in C. jejuni colonization of both strains, (2) pathologic change in draining lymph nodes but only mild changes in colon or cecal lamina propria, (3) significantly lower Th1/Th17-dependent anti-C. jejuni responses, (4) significantly higher IL-4 responses at 5 but not 7 weeks post infection (PI), (5) significantly higher Th2-dependent anti-C. jejuni responses, and (6) significantly elevated anti-ganglioside autoantibodies after C. jejuni infection. These responses in Humicrobiota mice were correlated with a dominant Bacteroidetes and Firmicutes microbiota.ConclusionsThese data demonstrate that Humicrobiota altered host-pathogen interactions in infected mice, increasing colonization and Th-2 and autoimmune responses in a C. jejuni strain-dependent manner. Thus, microbiota composition is another factor controlling susceptibility to GBS.

Development and Use of a Real-Time Quantitative PCR Method for Detecting and Quantifying Equol-Producing Bacteria in Human Faecal Samples and Slurry Cultures.

This work introduces a novel real-time quantitative PCR (qPCR) protocol for detecting and quantifying equol-producing bacteria. To this end, two sets of primers targeting the dihydrodaidzein reductase (ddr) and tetrahydrodaidzein reductase (tdr) genes, which are involved in the synthesis of equol, were designed. The primers showed high specificity and sensitivity when used to examine DNA from control bacteria, such as Slackia isoflavoniconvertens, Slackia equolifaciens, Asaccharobacter celatus, Adlercreutzia equolifaciens, and Enterorhabdus mucosicola. To demonstrate the validity and reliability of the protocol, it was used to detect and quantify equol-producing bacteria in human faecal samples and their derived slurry cultures. These samples were provided by 18 menopausal women under treatment of menopause symptoms with a soy isoflavone concentrate, among whom three were known to be equol-producers given the prior detection of the molecule in their urine. The tdr gene was detected in the faeces of all these equol-producing women at about 4–5 log10 copies per gram of faeces. In contrast, the ddr gene was only amplified in the faecal samples of two of these three women, suggesting the presence in the non-amplified sample of reductase genes unrelated to those known to be involved in equol formation and used for primer design in this study. When tdr and ddr were present in the same sample, similar copy numbers of the two genes were recorded. However, no significant increase in the copy number of equol-related genes along isoflavone treatment was observed. Surprisingly, positive amplification for both tdr and ddr genes was obtained in faecal samples and derived slurry cultures from two non-equol producing women, suggesting the genes could be non-functional or the daidzein metabolized to other compounds in samples from these two women. This novel qPCR tool provides a technique for monitoring gut microbes that produce equol in humans. Monitoring equol-producing bacteria in the human gut could provide a means of evaluating strategies aimed at increasing the endogenous formation of this bioactive compound.

The role of gut microbiota on the metabolism of black tea theaflavins

The main bioactive polyphenols in black tea are theaflavins, including theaflavin (TF), theaflavin‐3‐gallate (TF3G), theaflavin‐3¢‐gallate (TF3¢G), and theaflavin‐3,3¢‐digallate (TFDG). Theaflavins have been reported as anti‐inflammatory and cancer preventive agents in different in vitro and in vivo models. Because of the poor systemic bioavailability of theaflavins, it is still unclear how these compounds can exert their biological functions. Studies have shown that higher molecular weight polyphenols are metabolized by the microbiota and their metabolites may play an important role in chronic disease prevention. The objective of this study is to identify the microbial metabolites of theaflavins in mice and in humans. Purified TF and TFDG were oral gavaged to specific pathogen free (SPF) mice, germ free (GF) mice, and incubated with human fecal slurries in vitro. Microbial bioconversion was monitored using liquid chromatography/mass spectrometry (LC/MS) by analyzing the MSn (n = 1–3) spectra. Our results indicated that TFDG can be metabolized by gut microbiota to generate gallic acid, pyrogallol, TF, TF3G, and TF3'G, and TF can be further broke down to small molecular metabolites by gut microbiota. Furthermore, we used selective culturing techniques to probe which bacterial groups are responsible for theaflavins metabolism in vitro. Fecal bacteria from SPF‐born 129SvEv mice were anaerobically cultured on blood agar (non‐selective agar), MacConkey agar (selective for gram‐negatives; G−) and Columbia colistin and nalidixic acid agar (gram‐positives; G+). The cultures were then anaerobically incubated in fermentation media containing theaflavins, and the resulting metabolites were analyzed by LC/MS. Lactobacillus plantarum and Escherichia coli NC101 were similarly assessed for theaflavins fermentation abilities. Our results revealed that the G+ bacteria (including L. plantarum on its own) instead of G− bacteria (including E. coli NC101 on its own) were able to produce these metabolites.Support or Funding InformationThis work was supported by NIH R01 grant AT008623.