Metagenomic, metabolomic, and lipidomic shifts associated with fecal microbiota transplantation for recurrent Clostridioides difficile infection.

Recurrent C. difficile infection (rCDI) is an urgent public health threat for which the last resort and lifesaving treatment is a fecal microbiota transplant (FMT). However, the exact mechanisms which mediate a successful FMT are not well understood. Here we use longitudinal stool samples collected from patients undergoing FMT to evaluate changes in the microbiome, metabolome, and lipidome after successful FMTs. We show changes in the abundance of many lipids, specifically acylcarnitines and bile acids, in response to FMT. These changes correlate with Enterobacteriaceae, which encode carnitine metabolism genes, and Lachnospiraceae, which encode bile salt hydrolases and baiA genes. LC-IMS-MS revealed a shift from microbial conjugation of primary bile acids pre-FMT to secondary bile acids post-FMT. Here we define the structural and functional changes in successful FMTs. This information will help guide targeted Live Biotherapeutic Product development for the treatment of rCDI and other intestinal diseases.

Gastrointestinal-isolated Distress is Common in Alpha-gal Allergic Patients on Mammalian Meat Challenge.

BACKGROUND AND AIMS: Alpha-gal allergy causes a delayed reaction to mammalian meats and has been reported worldwide. Patients with the allergy may present with isolated gastrointestinal (GI) symptoms, but this phenotype is poorly understood. METHODS: We pooled and analyzed symptoms and demographics of patients from two prospective cohorts of patients with a diagnosis of alpha-gal allergy who reacted after eating mammalian meat under observation. We compared the characteristics of patients who demonstrated GI-isolated symptoms on a challenge with those who exhibited symptoms outside the GI tract (skin, respiratory, and circulatory). RESULTS: Among the 91 children and adult alpha-gal allergic patients who exhibited symptoms after oral challenge with mammalian meat, 72.5% experienced GI distress with one or more GI symptoms, which was the most frequent class of symptoms, compared with skin changes in 57.1% and respiratory distress in 5.5%. The most common GI symptoms were abdominal pain (71%) and vomiting (22.0%). GI-isolated symptoms occurred in 37 patients (40.7%) who reacted, and those patients reacted more quickly than patients who exhibited systemic symptoms (median onset of symptoms in GI-isolated group 90 min vs 120 min) and were more likely to be children than adults (relative risk=1.94, 95% CI: 1.04-3.63). CONCLUSIONS: Isolated-GI distress occurred in 4 in every 10 alpha-gal allergic individuals who developed symptoms on oral food challenge with mammalian meat. Alpha-gal allergic patients, particularly children, may exhibit GI distress alone, and adult and pediatric gastroenterologists should be aware of the diagnosis and management of the allergy.

Using Multidimensional Separations to Distinguish Isomeric Amino Acid-Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems.

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid-bile acid (AA-BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome.

Compound identification is an essential task in the workflow of untargeted metabolomics since the interpretation of the data in a biological context depends on the correct assignment of chemical identities to the features it contains. Current techniques fall short of identifying all or even most observable features in untargeted metabolomics data, even after rigorous data cleaning approaches to remove degenerate features are applied. Hence, new strategies are required to annotate the metabolome more deeply and accurately. The human fecal metabolome, which is the focus of substantial biomedical interest, is a more complex, more variable, yet lesser-investigated sample matrix compared to widely studied sample types like human plasma. This manuscript describes a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public, and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach and successfully identified several rare and novel compounds, including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable but not identifiable in the original single-dimension LC-MS data. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with commercially-available instrumentation, and should apply to any dataset requiring deeper annotation of the metabolome.

Exploring pre-implementation perceptions of integrated care in a university setting.

Objective: To examine providers' perceptions of integrated care prior to the merger of a university's student health center and counseling services. Participants: Seventeen providers across student health services (n = 9) and counseling (n = 8) agreed to participate in the qualitative study. Method: Semi-structured individual interviews that focused on the perceived benefits and challenges of the merger were conducted in December 2019. Following the interviews, a thematic analysis was completed. Results: The perceived benefits noted by providers centered on the ability of an interdisciplinary team to improve the coordination of, access to, and quality of care delivered to students. However, more anticipated challenges were reported by providers (e.g., differences in training and care protocols, losing one's autonomy as a provider). Conclusions: This qualitative study provides a more in-depth analysis of providers' perceptions of integrated care prior to implementation in a university setting and may have implications for model adoption.

Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut.

Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.

AGA Clinical Practice Update on Alpha-Gal Syndrome for the GI Clinician: Commentary.

DESCRIPTION: Alpha-gal syndrome is an emerging allergy first described in the early 2000s. The allergy can cause anaphylaxis, gastrointestinal (GI) symptoms, and skin changes one to several hours after ingestion of mammalian products. A GI phenotype that is increasingly recognized manifests with nonspecific symptoms like abdominal pain, diarrhea, nausea or vomiting without predominant skin, respiratory or circulatory symptoms. Though the syndrome has been reported on all continents except Antarctica, in the United States most reports are within the range of the Lone Star tick, extending from New York and Iowa to Texas and Florida. The purpose of this AGA Clinical Practice Update (CPU) Commentary is to increase awareness among gastroenterologists about the presentation and management of alpha-gal syndrome. METHODS: This CPU commentary was commissioned and approved by the AGA Institute Clinical Practice Updates Committee (CPUC) and the AGA Governing Board to provide timely guidance on a topic of high clinical importance to the AGA membership and underwent internal peer review by the CPUC and external peer review through standard procedures of Clinical Gastroenterology and Hepatology. This expert commentary incorporates important as well as recently published studies in this field, and it reflects the experiences of the authors. Formal ratings regarding the quality of evidence or strength of the presented considerations were not included since systematic reviews were not performed.

Alpha-Gal Sensitization in a US Screening Population Is Not Associated With a Decreased Meat Intake or Gastrointestinal Symptoms.

INTRODUCTION: Patients with alpha-gal syndrome, a delayed reaction to mammalian meat, can present with isolated gastrointestinal (GI) symptoms. We aimed to estimate the frequency of alpha-gal sensitization in a Southeastern US population and determine the association between sensitization and mammalian product dietary intake or GI symptoms. METHODS: We performed a cross-sectional study of participants who underwent a screening colonoscopy at our center between 2013 and 2015. We quantified serum alpha-gal immunoglobulin E antibodies in participants who were prospectively enrolled at screening colonoscopy and compared diet intake and lower GI symptoms reported in standardized questionnaires among those with elevated versus no alpha-gal IgE antibodies. RESULTS: Alpha-gal IgE antibodies were common-31.4% of screening colonoscopy participants (127 of 404) had elevated serum alpha-gal IgE >0.1 kU/L. Alpha-gal-sensitized participants endorsed similar rates of abdominal pain compared with those without alpha-gal antibodies (33% vs 38%, adjusted odds ratio 0.9, 95% confidence interval 0.7-1.3). Mammalian meat consumption did not differ based on alpha-gal sensitization status (average 1.43 servings/d in sensitized subjects vs 1.50 in alpha-gal IgE-negative subjects, P = 0.9). Alpha-gal-sensitized participants with levels ≥10 (n = 21) were overrepresented in the lowest quartiles of mammalian meat consumption, but not among those with GI symptoms in general. Participants with high alpha-gal antibody levels >2 kU/L (n = 45) or ≥10 U/L (n = 21) did not have a reduced mean daily mammalian meat intake compared with seronegative people. DISCUSSION: Elevated alpha-gal IgE antibodies were common and not associated with a reduced mammalian meat intake, abdominal pain, or diarrhea. Seropositivity did not predict symptomatic alpha-gal sensitization in this general screening population. Other host factors likely contribute to the phenotypic expression of alpha-gal syndrome.

Food Allergies and Alpha-gal Syndrome for the Gastroenterologist.

PURPOSE OF REVIEW: Food allergies are typically not considered as a cause of gastrointestinal (GI) distress without additional allergic symptoms, apart from celiac disease and eosinophilic esophagitis. However, recent reports of patients with alpha-gal syndrome who presented with GI-only symptoms like abdominal pain, vomiting, and diarrhea challenge this paradigm. Alpha-gal syndrome is an IgE-mediated allergy characterized by delayed reactions after eating mammalian meat or mammalian-derived products that contain galactose-alpha-1,3-galactose (alpha-gal). The purpose of this review is to discuss our current understanding of food allergies, GI illness, and the GI manifestations of alpha-gal syndrome. RECENT FINDINGS: Among Southeastern U.S. GI clinic patients who screened positive for serum alpha-gal IgE, a majority of patients reported significant symptom improvement on an alpha-gal-avoidant diet, suggesting that the allergy had played a role in their GI symptoms. Diagnosis of alpha-gal syndrome is typically made with concerning allergic symptoms, elevated alpha-gal specific IgE in the serum, and symptom improvement on an alpha-gal avoidant diet. Alpha-gal syndrome can cause a delayed allergic response that is increasingly recognized worldwide, including among patients with predominant GI symptoms.

Walk before you run: Feasibility challenges and lessons learned from the PROCLAIM study, a multicenter randomized controlled trial of misoprostol for prevention of recurrent Clostridioides difficile during COVID-19.

We analyzed our challenging experience with a randomized controlled trial of misoprostol for prevention of recurrent C. difficile. Despite careful prescreening and thoughtful protocol modifications to facilitate enrollment, we closed the study early after enrolling just 7 participants over 3 years. We share lessons learned, noting the importance of feasibility studies, inclusion of biomarker outcomes, and dissemination of such findings to inform future research design and implementation successes.

RNNSLAM: Reconstructing the 3D colon to visualize missing regions during a colonoscopy.

Colonoscopy is the gold standard for pre-cancerous polyps screening and treatment. The polyp detection rate is highly tied to the percentage of surveyed colonic surface. However, current colonoscopy technique cannot guarantee that all the colonic surface is well examined because of incomplete camera orientations and of occlusions. The missing regions can hardly be noticed in a continuous first-person perspective. Therefore, a useful contribution would be an automatic system that can compute missing regions from an endoscopic video in real-time and alert the endoscopists when a large missing region is detected. We present a novel method that reconstructs dense chunks of a 3D colon in real time, leaving the unsurveyed part unreconstructed. The method combines a standard SLAM system with a depth and pose prediction network to achieve much more robust tracking and less drift. It addresses the difficulties for colonoscopic images of existing simultaneous localization and mapping (SLAM) systems and end-to-end deep learning methods.

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.