Stool Composition Research Articles

Gut Bacteria Composition Drives Primary Resistance to Cancer Immunotherapy in Renal Cell Carcinoma Patients

BackgroundThe development of immune checkpoint blockade (ICB) has revolutionized the clinical outcome of renal cell carcinoma (RCC). Nevertheless, improvement of durability and prediction of responses remain unmet medical needs. While it has been recognized that antibiotics (ATBs) decrease the clinical activity of ICB across various malignancies, little is known about the direct impact of distinct intestinal nonpathogenic bacteria (commensals) on therapeutic outcomes of ICB in RCC. ObjectiveTo evaluate the predictive value of stool bacteria composition for ICB efficacy in a cohort of advanced RCC patients. Design, setting, and participantsWe prospectively collected fecal samples from 69 advanced RCC patients treated with nivolumab and enrolled in the GETUG-AFU 26 NIVOREN microbiota translational substudy phase 2 trial (NCT03013335) at Gustave Roussy. We recorded patient characteristics including ATB use, prior systemic therapies, and response criteria. We analyzed 2994 samples of feces from healthy volunteers (HVs). In parallel, preclinical studies performed in RCC-bearing mice that received fecal transplant (FMT) from RCC patients resistant to ICB (NR-FMT) allowed us to draw a cause-effect relationship between gut bacteria composition and clinical outcomes for ICB. The influence of tyrosine kinase inhibitors (TKIs) taken before starting nivolumab on the microbiota composition has also been assessed. Outcome measurements and statistical analysisMetagenomic data (MG) from whole genome sequencing (WGS) were analyzed by multivariate and pairwise comparisons/fold ratio to identify bacterial fingerprints related to ATB or prior TKI exposure and patients’ therapeutic response (overall response and progression-free survival), and compared with the data from cancer-free donors. Results and limitationsRecent ATB use (n = 11; 16%) reduced objective response rates (from 28% to 9%, p < 0.03) and markedly affected the composition of the microbiota, facilitating the dominance of distinct species such as Clostridium hathewayi, which were also preferentially over-represented in stools from RCC patients compared with HVs. Importantly, TKIs taken prior to nivolumab had implications in shifting the microbiota composition. To establish a cause-effect relationship between gut bacteria composition and ICB efficacy, NR-FMT mice were successfully compensated with either FMT from responding RCC patients or beneficial commensals identified by WGS-MG (Akkermansia muciniphila and Bacteroides salyersiae). ConclusionsThe composition of the microbiota is influenced by TKIs and ATBs, and impacts the success of immunotherapy. Future studies will help sharpen the role of these specific bacteria and their potential as new biomarkers. Patient summaryWe used quantitative shotgun DNA sequencing of fecal microbes as well as preclinical models of fecal or bacterial transfer to study the association between stool composition and (pre)clinical outcome to immune checkpoint blockade. Novel insights into the pathophysiological relevance of intestinal dysbiosis in the prognosis of kidney cancer may lead to innovative therapeutic solutions, such as supplementation with probiotics to prevent primary resistance to therapy.

Effectiveness and Tolerance of a Locust Bean Gum-Thickened Formula: A Real-Life Study.

PurposeThickened infant formulas reduce regurgitation frequency and volume. Because the digestive tolerance of locust bean gum-containing formulas is controversial, the effectiveness and tolerance of a locust bean gum-thickened formula in infants presenting with regurgitation was evaluated. No other interventions were allowed during the 1 month follow-up period.MethodsWe conducted an open, prospective, observational study of a locust bean gum-thickened formula administered to infants presenting with moderate to severe regurgitation according to parents during 1 month. Effectiveness and tolerance were assessed by evaluating gastrointestinal symptoms and quality of life indicators.ResultsA total of 2,604 infants with an average age of 9.3±4.3 weeks were included in this 1 month trial. Regurgitation frequency and estimated volume decreased significantly (p<0.001) and the episodes were resolved completely in 48% of the infants. A significant decrease in duration of crying and episodes of gas (p<0.001), with improvement in quality of life parameters, was observed. Stool frequency increased and stool consistency softened (p<0.001) to levels within the physiologic range, consistent with the increased fiber load (0.42 g/100 mL).ConclusionLocust bean gum-thickened formula decreased infant regurgitation, was well tolerated, and improved parental quality of life. Stool composition and frequency of the infants remained within the physiologic range.

The impact of storage conditions on human stool 16S rRNA microbiome composition and diversity.

BackgroundMultiple factors can influence stool sample integrity upon sample collection. Preservation of faecal samples for microbiome studies is therefore an important step, particularly in tropical regions where resources are limited and high temperatures may significantly influence microbiota profiles. Freezing is the accepted standard to preserve faecal samples however, cold chain methods are often unfeasible in fieldwork scenarios particularly in low and middle-income countries and alternatives are required. This study therefore aimed to address the impact of different preservative methods, time-to-freezing at ambient tropical temperatures, and stool heterogeneity on stool microbiome diversity and composition under real-life physical environments found in resource-limited fieldwork conditions.MethodsInner and outer stool samples collected from one specimen obtained from three children were stored using different storage preservation methods (raw, ethanol and RNAlater) in a Ugandan field setting. Mixed stool was also stored using these techniques and frozen at different time-to-freezing intervals post-collection from 0–32 h. Metataxonomic profiling was used to profile samples, targeting the V1–V2 regions of 16S rRNA with samples run on a MiSeq platform. Reads were trimmed, combined and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology and R software.ResultsChild donor was the greatest predictor of microbiome variation between the stool samples, with all samples remaining identifiable to their child of origin despite the stool being stored under a variety of conditions. However, significant differences were observed in composition and diversity between preservation techniques, but intra-preservation technique variation was minimal for all preservation methods, and across the time-to-freezing range (0–32 h) used. Stool heterogeneity yielded no apparent microbiome differences.ConclusionsStool collected in a fieldwork setting for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout.

Evaluation of cutaneous, oral and intestinal microbiota in patients affected by pemphigus and bullous pemphigoid: A pilot study

BackgroundSignificant alterations of the cutaneous microbiota (CM) have been recently demonstrated in bullous pemphigoid (BP). Microbiome data of both oral cavity (OM) and gut (GM) from patients affected by bullous disease are not available yet and, further consistent studies focused on the role of such microbial populations are still missing. ObjectiveObjective: In this pilot study we characterized and compared GM, OM and CM of patients affected by pemphigus vulgaris (PV) and BP to investigate a distinctive microbiome composition in this two rare dermatological disorders. MethodsHigh-throughput sequencing of the V1-V3 hyper-variable regions of 16S rRNA was used to compare the bacterial community composition of stool, skin and oral mucosae swabs in a cohort of PV and BP patients. A dedicated bioinformatics software coupled with in-house pipeline was implemented to analyse and compare diseases dataset. ResultsGM samples of both PV and BP patients were principally characterized by Firmicutes and Bacteroidetes phyla. Interestingly, the Firmicutes phylum and Staphylococcus genus were mainly represented in cutaneous samples. The diversity of phyla in oral mucosae was higher than those of gut and skin samples and, Bacteroidetes phylum was significantly underrepresented in all PV samples. ConclusionFirmicutes phylum and Staphilococcus genus were the most represented in OM and CM swabs of PV and BP microbial populations. Moreover, we argue the quantitative imbalance linked to the decrease of Bacteriodetes in the oral cavity of PV patients might be associated to disease typical fetor. To shed light on this peculiar feature further studies are still required.

2567. Effect of Broad vs. Narrow-Spectrum Clostridioides difficile Treatment on Human Stool Bile Acid Composition Over Time

BackgroundSecondary bile acid production by a diverse commensal flora may be a critical factor in preventing recurrence of Clostridioides difficile infection (CDI). Key enzymes involved are bacterial-encoded bile salt hydrolases (BSHs), felt to be “gatekeepers” to secondary bile acid synthesis. Ridinilazole, a novel narrow-spectrum drug for CDI, demonstrated superior sustained clinical response compared with vancomycin in Phase 2. Longitudinal sampling during this trial allowed for assessment of metabolites differentially present in stools during/after therapy with either broad or narrow-spectrum anti-CDI agent. Previous work characterizing subject’s fecal microbiota in this trial showed that unlike vancomycin, ridinilazole has little effect on commensal flora during and after therapy. We hypothesized that ridinilazole’s microbiota-preserving effect is associated with lack of accumulation of conjugated primary bile acids and/or reaccumulation/persistence of secondary bile acids over the course of CDI treatment, when compared with vancomycin-treated subjects. Furthermore, we hypothesized that we would observe correlations between bile acid profiles and predicted BSH gene abundances.MethodsSequential stool samples were obtained from 44 subjects treated with either ridinilazole or vancomycin (22 in each arm), ranging from time of CDI diagnosis, at end-of-therapy, and up to 40 days after diagnosis. Bile acids were quantitated by liquid chromatography-mass spectrometry. Using the PICRUSt algorithm, metagenomic predictions of BSH gene abundances were performed.ResultsStool bile acid compositions differed between ridinilazole-treated and vancomycin-treated subjects at end-of-therapy. In vancomycin-treated subjects, stool composition became dominated by conjugated primary bile acids and decreased levels of secondary bile acids compared with baseline; the ratio of stool conjugated bile acids to secondary bile acids significantly predicted treatment arm. This ratio was also associated with predicted BSH gene abundance in ridinilazole-treated subjects.ConclusionMicrobiota-preserving CDI treatment with ridinilazole preserves bile acid composition, which may decrease likelihood of recurrence. Disclosures All authors: No reported disclosures.

624. Microbiota and Associations with Treatment Outcome in Fecal Microbiota Transplantation

BackgroundFecal microbiota transplantation (FMT) can be an effective treatment of recurrent Clostridium difficile infection (CDI), although non-responsiveness to treatment remains poorly understood. Here we examine the bacterial composition of stool from FMT recipients using culture-independent methods to identify associations between bacterial community structure, route of FMT administration (colonoscopy or freeze-dried encapsulated FMT (capsules)), treatment outcome and donor. We hypothesized that multiple community structures could be associated with and may better define treatment outcome.MethodsWe tested this hypothesis by analyzing bacterial composition profiles and their relationship with treatment route, outcome and donor using bioinformatics and multivariate statistics on 16S rRNA gene sequences (16S) from 21 individuals (seven male, 14 female, median 68 years) with recurrent CDI prior to and after FMT. Successful endpoint was defined as no relapse of C. difficile associated diarrhea during 12 weeks post-FMT. There were 17 successes (four colonoscopy, 13 capsules) and four failures (all capsules). Analyses of 16S profiles included permutational analysis of variance (PERMANOVA) and linear regression models applied to bacterial abundances and diversity (as responses).ResultsSignificant differences were determined between pre- and post-FMT successes and failures (P < 1e−4, R2 = 0.24). No differences were seen between route (P = 0.15) or donor (P = 0.20). Profiles of failed FMT recipients were more similar to pre-FMT profiles by multidimensional scaling. Analyses of changes in abundance of pre-FMT profiles vs. outcome, controlled for age and sex, identified significant (P < 0.01) differences across 19 of the 25 most abundant taxa. Of the five most abundant taxa, Enterobacteriaceae and Esherichia-Shigella decreased significantly in successful outcomes, while Faecalibacterium, Blautia, and Bacteroides increased. However, variation in individual composition was also significant suggesting that multiple profiles represent successful outcomes.ConclusionIncreases in microbiota diversity are generally achieved in successful FMT regardless of administration route, although more than one bacterial composition profile can be identified.Disclosures All authors: No reported disclosures.

No changes in levels of bone formation and resorption markers following a broad-spectrum antibiotic course

BackgroundIntestinal bacteria influence bone remodeling in rodents, and antibiotic manipulation of the rodent gut microbiota increases bone formation and prevents ovariectomy-induced bone loss. In theory, these effects may be mediated by changes in sex hormone biotransformation in the gut, gut serotonin secretion or nutrition-induced secretion of glucagon-like peptide 2 (GLP-2) and glucose-dependent insulinotropic hormone (GIP). Antibiotics change the human gut microbiota, but the effect of antibiotic treatment on human bone turnover is unknown.MethodsWe analyzed serum levels of bone turnover markers, serotonin, GLP-2 and sex hormones before, immediately after, and eight, 42 and 180 days after a 4-day per oral antibiotic cocktail (vancomycin 500 mg, gentamycin 40 mg and meropenem 500 mg once-daily) in twelve healthy adult males. Fasting and meal-stimulated procollagen type I amino-terminal propeptide (P1NP), C-telopeptide of type I collagen (CTX) and osteocalcin levels were measured.ResultsWhile the antibiotic course reduced the stool abundance and composition of anaerobic bacteria as confirmed by cultivation studies, neither short nor long-term alterations in serum P1NP, CTX and osteocalcin were observed. Furthermore, we did not observe any changes in levels of serum GLP-2, serotonin or sex hormones.ConclusionEradication of anaerobic bacteria from healthy adult males had no effect on serum bone turnover markers.

Third-party fecal microbiota transplantation following allo-HCT reconstitutes microbiome diversity

AbstractWe hypothesized that third-party fecal microbiota transplantation (FMT) may restore intestinal microbiome diversity after allogeneic hematopoietic cell transplantation (allo-HCT). In this open-label single-group pilot study, 18 subjects were enrolled before allo-HCT and planned to receive third-party FMT capsules. FMT capsules were administered no later than 4 weeks after neutrophil engraftment, and antibiotics were not allowed within 48 hours before FMT. Five patients did not receive FMT because of the development of early acute gastrointestinal (GI) graft-versus-host disease (GVHD) before FMT (n = 3), persistent HCT-associated GI toxicity (n = 1), or patient decision (n = 1). Thirteen patients received FMT at a median of 27 days (range, 19-45 days) after HCT. Participants were able to swallow and tolerate all FMT capsules, meeting the primary study endpoint of feasibility. FMT was tolerated well, with 1 treatment-related significant adverse event (abdominal pain). Two patients subsequently developed acute GI GVHD, with 1 patient also having concurrent bacteremia. No additional cases of bacteremia occurred. Median follow-up for survivors is 15 months (range, 13-20 months). The Kaplan-Meier estimates for 12-month overall survival and progression-free survival after FMT were 85% (95% confidence interval, 51%-96%) and 85% (95% confidence interval, 51%-96%), respectively. There was 1 nonrelapse death resulting from acute GI GVHD (12-month nonrelapse mortality, 8%; 95% confidence interval, 0%-30%). Analysis of stool composition and urine 3-indoxyl sulfate concentration indicated improvement in intestinal microbiome diversity after FMT that was associated with expansion of stool-donor taxa. These results indicate that empiric third-party FMT after allo-HCT appears to be feasible, safe, and associated with expansion of recipient microbiome diversity. This trial was registered at www.clinicaltrials.gov as #NCT02733744.

A Synbiotic Infant Formula with High Magnesium Content Improves Constipation and Quality of Life.

PurposeTo evaluate the efficacy of synbiotic formula with partial whey hydrolysate and high magnesium content in infants presenting with functional constipation.MethodsSixty-five infants with functional constipation were included. Forty infants were treated during one month with parental reassurance and the intervention formula and were compared to a control group of 25 infants treated with parental reassurance only. Parents completed a quality of life (QoL) questionnaire at baseline and during the last week of the study.ResultsAt inclusion, stool characteristics and QoL were similar in both groups. The control group was slightly older than the intervention group (7.5±3.9 vs. 6.2±3.6 weeks). At onset, stool composition was “hard and tight” (Bristol stool scale 1 and 2) in all infants. After one month, stool composition remained unchanged in the control group except in two infants that developed “creamy” stools (Bristol stool scale type 3 and 4). In the intervention group, stools remained “hard and tight” in 27.5%, and became “creamy” in 47.5%, “loose” (Bristol stool scale type 5) in 22.5% and “watery” (Bristol stool type 6 and 7) in 2.5%. The benefit of the intervention formula was estimated to be “very important” in 70%. The median scores for QoL improved significantly in the intervention group for all parameters and for one in the control group.ConclusionThe intervention formula significantly improved functional constipation resulting in a better QoL of the parents and infants.

The microbiome in PTEN hamartoma tumor syndrome.

Germline PTEN mutations defining PTEN hamartoma tumor syndrome (PHTS) confer heritable predisposition to breast, endometrial, thyroid and other cancers with known age-related risks, but it remains impossible to predict if any individual will develop cancer. In the general population, gut microbial dysbiosis has been linked to cancer, yet is unclear whether these are associated in PHTS patients. In this pilot study, we aimed to characterize microbial composition of stool, urine, and oral wash from 32 PTEN mutation-positive individuals using 16S rRNA gene sequencing. PCoA revealed clustering of the fecal microbiome by cancer history (P = 0.03, R 2 = 0.04). Fecal samples from PHTS cancer patients had relatively more abundant operational taxonomic units (OTUs) from family Rikenellaceae and unclassified members of Clostridia compared to those from non-cancer patients, whereas families Peptostreptococcaceae, Enterobacteriaceae, and Bifidobacteriaceae represented relatively more abundant OTUs among fecal samples from PHTS non-cancer patients. Functional metagenomic prediction revealed enrichment of the folate biosynthesis, genetic information processing and cell growth and death pathways among fecal samples from PHTS cancer patients compared to non-cancer patients. We found no major shifts in overall diversity and no clustering by cancer history among oral wash or urine samples. Our observations suggest the utility of an expanded study to interrogate gut dysbiosis as a potential cancer risk modifier in PHTS patients.

Metabolites and microbial composition of stool of women with fecal incontinence: Study design and methods.

This paper aims to report the rationale, design, and the specific methodology of an ongoing nested observational study that will determine the association of the metabolite and microbial composition of stool with fecal incontinence (FI). Eligible cases are participants with FI enrolled in the Controlling Anal Incontinence in women by Performing Anal Exercises with Biofeedback or Loperamide (CAPABLe) trial, a Pelvic Floor Disorders Network trial across eight clinical centers in the United States. Women of similar age without FI in the last year served as controls. All subject collected stool samples at the baseline and 24-week visit at home using a standardized collection method. Samples were shipped to and stored at centralized laboratories. Specimen collection commenced December 2014 and was completed in May 2016. Fecal water and DNA has been extracted and is currently being analyzed by targeted metabolic profiling for stool metabolites and 16S rRNA gene sequencing for stool microbiota. This article describes the rationale and design of a study that could provide a paradigm shift for the treatment of FI in women.

Innovative Dietary Intervention Answers to Baby Colic.

PurposeThe purpose of this paper is to evaluate the efficacy of a lactose- reduced synbiotic partial whey hydrolysate in formula fed infants presenting with colic and the impact of this dietary intervention in mean crying time and quality of life.MethodsForty infants with infantile colic were treated during one month with parental reassurance and the intervention formula (partial whey hydrolysate, reduced lactose, Bifidobacterium lactis BB12 and galacto-oligosaccharides) and were compared to a control group of 20 infants with infantile colic treated with parental reassurance and a standard infant formula. Parents completed a quality of life (QoL) questionnaire assessing the burden of infantile colic. Wilcoxon test, t-test and Mann-Whitney test were used to compare QoL scores before and after intervention as well as between the intervention and control group.ResultsAt inclusion, duration of crying did not differ between both groups. Crying duration decreased with 2.7 hours (from 3.2 to 0.5 hours) in the intervention group while duration of crying decreased only with 1.2 hours in the control group (p<0.001). Stool composition became looser in the intervention group, but defecation frequency did not change. The median scores of the QoL questionnaire improved significantly in the intervention group for all parameters. In the control group, parameters improved significantly also but not for the parent-child and social interaction. The score changes were significantly greater in the intervention than in the control group.ConclusionThe intervention formula (partial whey hydrolysate, synbiotic, reduced lactose) significantly reduced the duration of crying and improved QoL of the parents and infants.

Metabolic derangements identified through untargeted metabolomics in a cross-sectional study of Nigerian children with severe acute malnutrition

Severe acute malnutrition (SAM) is a major cause of child mortality worldwide, however the pathogenesis of SAM remains poorly understood. Recent studies have uncovered an altered gut microbiota composition in children with SAM, suggesting a role for microbes in the pathogenesis of malnutrition. To elucidate the metabolic consequences of SAM and whether these changes are associated with changes in gut microbiota composition. We applied an untargeted multi-platform metabolomics approach [gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS)] to stool and plasma samples from 47 Nigerian children with SAM and 11 control children. The composition of the stool microbiota was assessed by 16S rRNA gene sequencing. The plasma metabolome discriminated children with SAM from controls, while no significant differences were observed in the microbial or small molecule composition of stool. The abundance of 585 features in plasma were significantly altered in malnourished children (Wilcoxon test, FDR corrected P < 0.1), representing approximately 15% of the metabolome. Consistent with previous studies, children with SAM exhibited a marked reduction in amino acids/dipeptides and phospholipids, and an increase in acylcarnitines. We also identified numerous metabolic perturbations which have not been reported previously, including increased disaccharides, truncated fibrinopeptides, angiotensin I, dihydroxybutyrate, lactate, and heme, and decreased bioactive lipids belonging to the eicosanoid and docosanoid family. Our findings provide a deeper understanding of the metabolic consequences of malnutrition. Further research is required to determine if specific metabolites may guide improved management, and/or act as novel biomarkers for assessing response to treatment.

Shifts in Lachnospira and Clostridium sp. in the 3-month stool microbiome are associated with preschool age asthma.

Asthma is a chronic disease of the airwaysaffectingone in ten children in Westernized countries.Recently, our group showed that specific bacterial genera in early life are associated with atopy and wheezing in 1-year-old children. However, little is known about the link between the early life gut microbiome and the diagnosis of asthma in preschool age children. To determine the role of the gut microbiota in preschool age asthma, children up to 4years of age enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) study were classified as asthmatic (n=39) or matched healthy controls (n=37). 16S rRNA sequencing and quantitative PCR (qPCR) were used to analyse the composition of the 3-month and 1-year gut microbiome of these children.At 3 months the abundance of the genus,Lachnospira(L), was decreased (P=0.008), whereas the abundance of the species,Clostridium neonatale(C), was increased (P=0.07) in asthmatics. Quartile analysis of stool composition at 3-months revealed a negative association between the ratio of these two bacteria (L/C) and asthma risk by 4 years of age [quartile 1: odds ratio (OR)=15, P=0.02, CI (confidence interval)= 1.8-124.7; quartile 2: OR=1.0, ns; quartile 3: OR=0.37, ns]. We conclude that opposing shifts in the relative abundances ofLachnospiraandC. neonatalein the first 3 months of life are associated with preschool age asthma, and that the L/C ratio may serve as a potential early life biomarker to predict asthma development.

Comparison of the Microbiome Composition of Rectal Stool vs Laxative-purged Liquid Stool in Patients Undergoing Screening Colonoscopy

Comparison of the Microbiome Composition of Rectal Stool vs Laxative-purged Liquid Stool in Patients Undergoing Screening Colonoscopy

Nutritional Composition of Stool Donorsʼ Diets Relative to That of the U.S. Population: Results from 44 Donors from an International Stool Bank for Fecal Microbiota transplantation

Nutritional Composition of Stool Donorsʼ Diets Relative to That of the U.S. Population: Results from 44 Donors from an International Stool Bank for Fecal Microbiota transplantation

Differences of microbiota in small bowel and faeces between irritable bowel syndrome patients and healthy subjects

Objective Several studies suggested that colonic microbiota have impacts on irritable bowel syndrome (IBS) patients. However, the knowledge about the association of small intestine (SI) microbiota with IBS is limited. We aimed to investigate the gut microbiota composition of SI and stool in IBS patients. Materials and methods Biopsies of jejunum mucosa by balloon-assisted enteroscopy and faecal samples from 28 IBS patients and 19 healthy controls were analysed by next-generation sequencing method. Results The three major phyla in SI microbiota of case/control groups were Proteobacteria (32.8/47.7%), Bacteroidetes (25.2/15.3%), and Firmicutes (19.8/11.2%), and those of stool were Bacteroidetes (41.3/45.8%), Firmicutes (40.7/38.2%), and Proteobacteria (15.4/7.1%). Analysis based on the family level, IBS patients had a higher proportion of Veillonellaceae (mean proportion 6.49% versus 2.68%, p = 0.046) in stool than controls. Prevotellaceae was more abundant in IBS patients than in control group (14.27% versus 6.13%, p = 0.023), while Mycobacteriaceae (0.06% versus 0.17%, p = 0.024) and Neisseriaceae (6.40% versus 8.94%, p = 0.038) was less abundant in IBS patients’ jejunal mucosa than those in controls. This less abundant jejunal Neisseriaceae was associated with more severe IBS (p = 0.03). The ratio of Firmicutes to Bacteroidetes in the stool of IBS-diarrhoea type patients was approximately three-fold higher, and the ratio of Firmicutes to Actinobacter in SI of IBS-mixed type patients was about nine-fold higher than healthy subjects. Conclusion Higher abundance of colonic Veillonellaceae and SI Prevotellaceae, and lower amount of oral cavity normal flora in proximal SI were found in IBS patients. We may manipulate these bacteria in IBS patients in future studies (ClinicalTrial.gov Number NCT01679730).

Collection media and delayed freezing effects on microbial composition of human stool.

BackgroundDifferent bacteria in stool have markedly varied growth and survival when stored at ambient temperature. It is paramount to develop optimal biostabilization of stool samples during collection and assess long-term storage for clinical specimens and epidemiological microbiome studies. We evaluated the effect of collection media and delayed freezing up to 7 days on microbial composition. Ten participants collected triplicate stool samples each into no media as well as RNAlater® with and without kanamycin or ciprofloxacin. For each set of conditions, triplicate samples were frozen on dry ice immediately (time = 0) or frozen at −80 °C after 3-days and 7-days incubation at 25 °C. Microbiota metrics were estimated from Illumina MiSeq sequences of 16S rRNA gene fragments (V3–V4 region). Intraclass correlation coefficients (ICC) across triplicates, collection media, and incubation time were estimated for taxonomy and alpha and beta diversity metrics.ResultsRNAlater® alone yielded the highest ICCs for diversity metrics at time = 0 [ICC median 0.935 (range 0.89–0.97)], but ICCs varied greatly (range 0.44–1.0) for taxa with relative abundances <1 %. The 3- and 7-day freezing delays were generally associated with stable beta diversity for all three media conditions. Freezing delay caused increased variance for Shannon index (median ICC 0.77) and especially for observed species abundance (median ICC 0.47). Variance in observed species abundance and in phylogenetic distance whole tree was similarly increased with a 7-day delay. Antibiotics did not mitigate variance. No media had inferior ICCs at time 0 and differed markedly from any media in microbiome composition (e.g., P = 0.01 for relative abundance of Bacteroidetes).ConclusionBacterial community composition was stable for 7 days at room temperature in RNAlater® alone. RNAlater® provides some stability for beta diversity analyses, but analyses of rare taxa will be inaccurate if specimens are not frozen immediately. RNAlater® could be used as collection media with minimal change in the microbiota composition.

A Markov Random Field orientation prior for electronic cleansing in CT Colonography.

Tagging of the bowel content with an oral contrast facilitates CT Colonography with limited bowel preparation. Electronic colon cleansing (ECC) reconstructs the colon lumen, devoid of feces from a CT scan acquired with fecal fluid tagging. A popular method to estimate the stool composition in an image (with the purpose of removing it) is the well-established Expectation Maximization (EM) method. The tagged fluid residue appears as a contrast enhanced region with a largely horizontal interface with air above it. One of the issues is the partial volume (PV) effect that creates voxels with attenuations similar to that of the colon wall at the boundary of air and tagged fluid. We present here, a novel orientation prior formulated as a Markov Random Field that is included as part of the EM tissue segmentation framework to mitigate this PV effect at the air and tagged fluid layer. We show quantitative results on a simple synthetic dataset and qualitative results on patient data that highlight improvements due to the inclusion of the orientation prior.

Factors Influencing Reconstitution of Mucosal-Associated Invariant T Cells Following Allogeneic Hematopoietic Stem Cell Transplantation

Background: Mucosal-associated invariant T (MAIT) cells are innate-like T cells characterized by high expression of CD161 and a semi-invariant T cell receptor (TCR) comprised of a Vα7.2-Jα33 alpha chain and a limited Vβ repertoire that enables their activation by riboflavin metabolites produced by distinct bacterial and fungal species. MAIT cells are infrequent in cord blood, but undergo TCR-dependent accumulation in neonates in response to gastrointestinal (GI) commensal colonization to comprise approximately 10% of T cells in adult blood. The GI localization of MAIT cells, their capacity to secrete IL-17, and their activation by microbial metabolites suggests a role in mucosal immunity that may be particularly important after allogeneic hematopoietic stem cell transplantation (HCT) when the GI mucosal barrier is compromised and adaptive immunity is impaired. After HCT, the composition of the GI microbiota may be modified by antibiotics, mucositis and immunosuppression, yet its impact on MAIT cell reconstitution, function and post-transplant immunity remain unknown.Aims: To characterize and identify factors influencing MAIT cell reconstitution and function after HCT.Methods: Blood and stool samples were collected from healthy donors and HCT patients prior to and at distinct times after HCT. Absolute counts of MAIT cells, identified as CD3+/CD161hi/Vα7.2+events, were determined in peripheral blood using flow cytometry performed in conjunction with a complete blood count. The bacterial composition of stool was characterized using bacterial 16S rRNA gene PCR with high throughput sequencing and phylogenetic assignment of the amplified fragments. TCR signaling pathway activation in MAIT cells and conventional T cells was evaluated using flow cytometry analysis of phosphoprotein expression after stimulation through the TCR-CD3 complex with anti-CD3/anti-CD28 monoclonal antibodies. TCR Vβ repertoire assessment was performed using high throughput TCRBV gene sequencing.Results: High throughput TCRBV gene sequencing showed that MAIT cells from different donors (n = 3) shared TCRBV sequences, consistent with their capacity to be activated by common GI microbial TCR ligands. Despite GI microbial colonization, MAIT cells from adult donor blood were quiescent and did not proliferate to TCR stimulation. Phosphoprotein flow cytometry established that phosphorylation of proximal TCR signaling pathway molecules (CD3ζ, Lck, and ZAP-70) was diminished and responsible for impaired TCR signaling in adult MAIT cells compared to conventional αβ T cells. MAIT cell proliferation was restored by TCR stimulation in the presence of IL-1β, IL-12, IL-18 and IL-23, raising the possibility that the post-HCT inflammatory environment might be permissive for MAIT cell proliferation driven by GI microbial TCR ligands. We examined the kinetics of MAIT cell reconstitution in HCT patients (n = 163). MAIT cell numbers were lower in patients before conditioning compared to healthy individuals, and were further depleted on the day of stem cell infusion; however, they proliferated in the post-HCT environment in association with induction of Ki67 expression and reached a plateau after day 30 post-HCT (healthy, 56.8/μL; day 30, 6.7/μL). MAIT cell reconstitution after peripheral blood stem cell (PBSC) transplantation was similar comparing myeloablative (MA) and reduced intensity conditioning (RIC) regimens and related compared to unrelated donors, but was highly variable between individuals. Short tandem repeat PCR chimerism studies showed that MAIT cells were of donor origin early after MA and RIC PBSC transplantation. MAIT cell reconstitution was markedly impaired in recipients of cord blood, which contains few MAIT cells, compared to those receiving PBSC, in which MAIT cells are plentiful, suggesting that early MAIT cell reconstitution is primarily derived from mature cells transferred with the HCT graft. Analysis of stool samples from HCT recipients (n = 17) has shown that the relative abundance of distinct gut bacterial species is highly variable between recipients and changed during the course of HCT. Analyses of the relationship between the microbiota and MAIT cell reconstitution will be presented.Conclusions: MAIT cell recovery following HCT varies between different types of transplants and may be influenced by the transferred graft source, the post-HCT environment, and the gut microbiome. DisclosuresNo relevant conflicts of interest to declare.