The pan-microbiome profiling system Taxa4Meta identifies clinical dysbiotic features and classifies diarrheal disease.

Targeted metagenomic sequencing is an emerging strategy to survey disease-specific microbiome biomarkers for clinical diagnosis and prognosis. However, this approach often yields inconsistent or conflicting results owing to inadequate study power and sequencing bias. We introduce Taxa4Meta, a bioinformatics pipeline explicitly designed to compensate for technical and demographic bias. We designed and validated Taxa4Meta for accurate taxonomic profiling of 16S rRNA amplicon data acquired from different sequencing strategies. Taxa4Meta offers significant potential in identifying clinical dysbiotic features that can reliably predict human disease, validated comprehensively via reanalysis of individual patient 16S data sets. We leveraged the power of Taxa4Meta's pan-microbiome profiling to generate 16S-based classifiers that exhibited excellent utility for stratification of diarrheal patients with Clostridioides difficile infection, irritable bowel syndrome, or inflammatory bowel diseases, which represent common misdiagnoses and pose significant challenges for clinical management. We believe that Taxa4Meta represents a new "best practices" approach to individual microbiome surveys that can be used to define gut dysbiosis at a population-scale level.

Sex-Dependent Efficacy of Dietary Fiber in Pediatric Functional Abdominal Pain.

BACKGROUND & AIMS: Functional abdominal pain disorders (FAPDs) are more prevalent in female patients. Dietary fiber may alleviate FAPD symptoms; however, whether this effect is sex dependent remains unclear. We investigated the sex dependency of dietary fiber benefit on abdominal pain in children with FAPDs and explored the potential involvement of the gut microbiome. METHODS: In 2 cross-sectional cohorts of children with FAPDs (n = 209) and healthy control individuals (n = 105), we correlated dietary fiber intake with abdominal pain symptoms after stratifying by sex. We also performed sex-stratified and sex-interaction analyses on data from a double-blind trial in children with irritable bowel syndrome randomized to psyllium fiber (n = 39) or placebo (n = 49) for 6 weeks. Shotgun metagenomics was used to investigate gut microbiome community changes potentially linking dietary fiber intake with abdominal pain. RESULTS: In the cross-sectional cohorts, fiber intake inversely correlated with pain symptoms in boys (pain episodes: r = -0.24, P = .005; pain days: r = -0.24, P = 0.004) but not in girls. Similarly, in the randomized trial, psyllium fiber reduced the number of pain episodes in boys (P = .012) but not in girls. Generalized linear regression models confirmed that boys treated with psyllium fiber had greater reduction in pain episodes than girls (P = .007 for fiber × sex × time interaction). Age, sexual development, irritable bowel syndrome subtype, stool form, and microbiome composition were not significant determinants in the dietary fiber effects on pain reduction. CONCLUSIONS: Dietary fiber preferentially reduces abdominal pain frequency in boys, highlighting the importance of considering sex in future dietary intervention studies for FAPDs. (ClincialTrials.gov, Number NCT00526903).

Donor-recipient specificity and age-dependency in fecal microbiota therapy and probiotic resolution of gastrointestinal symptoms.

Fecal microbiota transplantation (FMT) has proven to be an effective treatment for recurrent Clostridioides difficile infection (rCDI) in both adult and pediatric patients. However, as microbiome development is a critical factor in children, it remains unclear whether adult fecal donors can provide age-appropriate functional restoration in pediatric patients. To address this issue, we conducted an integrated systems approach and found that concordant donor strain engraftment, along with metabolite restoration, are associated with FMT outcomes in both adult and pediatric rCDI patients. Although functional restoration after FMT is not strain-specific, specialized metabolic functions are retained in pediatric patients when adult fecal donors are used. Furthermore, we demonstrated broad utility of high-resolution variant-calling by linking probiotic-strain engraftment with improved gastrointestinal symptoms in adults with irritable bowel syndrome and in children with autism spectrum disorder. Our findings emphasize the importance of strain-level identification when assessing the efficacy of probiotics and microbiota-based therapeutics.

Common Variable Immunodeficiency Patient Fecal Microbiota Transplant Recapitulates Gut Dysbiosis.

Purpose: Patients with non-infectious complications have worse clinical outcomes in common variable immunodeficiency (CVID) than those with infections-only. Non-infectious complications are associated with gut microbiome aberrations, but there are no reductionist animal models that emulate CVID. Our aim in this study was to uncover potential microbiome roles in the development of non-infectious complications in CVID. Methods: We examined fecal whole genome shotgun sequencing from patients CVID, and non-infectious complications, infections-only, and their household controls. We also performed Fecal Microbiota transplant from CVID patients to Germ-Free Mice. Results: We found potentially pathogenic microbes Streptococcus parasanguinis and Erysipelatoclostridium ramosum were enriched in gut microbiomes of CVID patients with non-infectious complications. In contrast, Fusicatenibacter saccharivorans and Anaerostipes hadrus, known to suppress inflammation and promote healthy metabolism, were enriched in gut microbiomes of infections-only CVID patients. Fecal microbiota transplant from non-infectious complications, infections-only, and their household controls into germ-free mice revealed gut dysbiosis patterns in recipients from CVID patients with non-infectious complications, but not infections-only CVID, or household controls recipients. Conclusion: Our findings provide a proof of concept that fecal microbiota transplant from CVID patients with non-infectious complications to Germ-Free mice recapitulates microbiome alterations observed in the donors.

Exploration of associations among dietary tryptophan, microbiome composition and function, and symptom severity in irritable bowel syndrome.

BACKGROUND: Imbalance of the tryptophan (TRP) pathway may influence symptoms among patients with irritable bowel syndrome (IBS). This study explored relationships among different components that contribute to TRP metabolism (dietary intake, stool metabolite levels, predicted microbiome metabolic capability) in females with IBS and healthy controls (HCs). Within the IBS group, we also investigated relationships between TRP metabolic determinants, Bifidobacterium abundance, and symptoms of IBS. METHODS: Participants with IBS (Rome III) and HCs completed a 28-day diary of gastrointestinal symptoms and a 3-day food record for TRP intake. They provided a stool sample for shotgun metagenomics, 16 S rRNA analyses, and quantitative measurement of TRP by mass spectrometry. RESULTS: Our cohort included 115 females, 69 with IBS and 46 HCs, with a mean age of 28.5 years (SD 7.4). TRP intake (p = 0.71) and stool TRP level (p = 0.27) did not differ between IBS and HC. Bifidobacterium abundance was lower in the IBS group than in HCs (p = 0.004). Predicted TRP metabolism gene content was higher in IBS than HCs (FDR-corrected q = 0.006), whereas predicted biosynthesis gene content was lower (q = 0.045). Within the IBS group, there was no association between symptom severity and TRP intake or stool TRP, but there was a significant interaction between Bifidobacterium abundance and TRP intake (q = 0.029) in predicting stool character. CONCLUSIONS: Dietary TRP intake, microbiome composition, and differences in TRP metabolism constitute a complex interplay of factors that could modulate IBS symptom severity.

Developing a standardized but extendable framework to increase the findability of infectious disease datasets.

Biomedical datasets are increasing in size, stored in many repositories, and face challenges in FAIRness (findability, accessibility, interoperability, reusability). As a Consortium of infectious disease researchers from 15 Centers, we aim to adopt open science practices to promote transparency, encourage reproducibility, and accelerate research advances through data reuse. To improve FAIRness of our datasets and computational tools, we evaluated metadata standards across established biomedical data repositories. The vast majority do not adhere to a single standard, such as Schema.org, which is widely-adopted by generalist repositories. Consequently, datasets in these repositories are not findable in aggregation projects like Google Dataset Search. We alleviated this gap by creating a reusable metadata schema based on Schema.org and catalogued nearly 400 datasets and computational tools we collected. The approach is easily reusable to create schemas interoperable with community standards, but customized to a particular context. Our approach enabled data discovery, increased the reusability of datasets from a large research consortium, and accelerated research. Lastly, we discuss ongoing challenges with FAIRness beyond discoverability.

Exploring the Role of Vitamin D and the Gut Microbiome: A Cross-Sectional Study of Individuals with Irritable Bowel Syndrome and Healthy Controls.

Irritable bowel syndrome (IBS) is a common disorder of gut-brain interaction with multifaceted pathophysiology. Prior studies have demonstrated higher rates of vitamin D deficiency in individuals with IBS compared to healthy controls (HC), as well as associations of vitamin D concentration with IBS symptoms. A systematic review of 10 mouse and 14 human studies reported a positive association between vitamin D (serum levels and supplementation) and beta diversity of gut microbiome in a variety of conditions. The present retrospective case-control study aimed to compare vitamin D (25(OH)D) plasma concentrations and gut microbiome composition in adult women with IBS (n=99) and HC (n=62). Plasma concentrations of 25(OH)D were assessed using the Endocrine Society Guidelines definition of vitamin D deficiency (25(OH)D <20 ng/ml) and insufficiency (25(OH)D >20-<30 ng/ml). 16S rRNA microbiome gene sequencing data was available for 39 HC and 62 participants with IBS. Genus-level Bifidobacterium and Lactobacillus and phylum-level Firmicutes and Bacteroidetes relative abundances were extracted from microbiome profiles. Results showed vitamin D deficiency in 40.3% (n=25) vs. 41.4% (n=41), and insufficiency 33.9% (n=21) vs. 34.3% (n=34) in the HCs vs. IBS groups, respectively. The odds of IBS did not differ depending on 25(OH)D status (p=0.75 for deficient, p=0.78 for insufficient), and the average plasma vitamin D concentration did not differ between IBS (mean 24.8 ng/ml) and HCs (mean 25.1 ng/ml; p=0.57). We did not find evidence of an association between plasma 25(OH)D concentration and richness, Shannon index, Simpson index or specific bacterial abundances in either HCs or the IBS group.

Multisite Detection of Tn1549-Mediated vanB Vancomycin Resistance in Multidrug-Resistant Enterococcus faecalis ST6 in Texas and Florida.

In the United States, vanB-mediated resistance in enterococci is rare. We characterized three sequence type (ST) 6, vancomycin-resistant Enterococcus faecalis isolates causing bacteremia in unique patients in spatiotemporally distinct settings. Isolates were recovered between 2018 and 2020 in two cities in the United States (Houston, TX; Miami, FL). The isolates harbored the vanB operon on a chromosomally located Tn1549 transposon, and epidemiological data suggested multiple introductions of the vanB gene cluster into ST6 E. faecalis.

Randomized placebo-controlled trial of oral tannin supplementation on COVID-19 symptoms, gut dysbiosis and cytokine response.

The clinical study aim was to investigate whether a tannin-based dietary supplementation could improve the efficacy of standard-of-care treatment of hospitalized COVID-19 patients by restoring gut microbiota function. Adverse events and immunomodulation post-tannin supplementation were also investigated. A total of 124 patients receiving standard-of-care treatment were randomized to oral tannin-based supplement or placebo for a total of 14 days. Longitudinal blood and stool samples were collected for cytokine and 16S rDNA microbiome profiling, and results were compared with 53 healthy controls. Although oral tannin supplementation did not result in clinical improvement or significant gut microbiome shifts after 14-days, a reduction in the inflammatory state was evident and significantly correlated with microbiota modulation. Among cytokines measured, MIP-1α was significantly decreased with tannin treatment (p = 0.03) where it correlated positively with IL-1ß and TNF- α, and negatively with stool Bifidobacterium abundance.

Matrix metalloproteinase 7 contributes to intestinal barrier dysfunction by degrading tight junction protein Claudin-7.

Background: Previous studies implicated matrix metalloproteinases (MMPs), such as MMP-7, in inflammatory bowel diseases (IBD) by showing increased activity during inflammation of the gut. However, the pathophysiological roles of MMP-7 have not been clearly elucidated. Methods: The expression of MMP-7 was assessed in colonic biopsies of patients with ulcerative colitis (UC), in rodents with experimental colitis, and in cell-based assays with cytokines. Wild-type and MMP-7-null mice treated with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid were used for determining the pro-inflammatory function(s) of MMP-7 in vivo. Results: MMP-7 was highly expressed in patients with UC and in rodents with experimental colitis. IL-1ß, IL-4, IL-13, TNFα, or lipopolysaccharide enhanced MMP-7 expression in human colonic epithelial cells, rat colonic smooth muscle cells, and THP-1-derived macrophages. Active MMP-7 degraded tight junction protein Claudin-7 in epithelial cells, cleaved recombinant Claudin-7 in cell-free system, and increased Caco-2 monolayer permeability. Immunostaining of colon biopsies revealed up-regulation of MMP-7 and reduction of Claudin-7 in UC patients. Compared to wild-type mice, Mmp7 -/- mice had significantly less inflammation in the colon upon DSS insult. DSS-induced alterations in junction proteins were mitigated in Mmp7 -/- mice, suggesting that MMP-7 disrupts the intestinal barrier. MMP-7 antibody significantly ameliorated colonic inflammation and Claudin-7 reduction in 2 different rodent models of colitis. Summary: MMP-7 impairs intestinal epithelial barrier by cleavage of Claudin-7, and thus aggravating inflammation. These studies uncovered Claudin-7 as a novel substrate of MMP-7 in the intestinal epithelium and reinforced MMP-7 as a potential therapeutic target for IBD.

Neonatal antibiotics have long term sex-dependent effects on the enteric nervous system.

Infants and young children receive the highest exposures to antibiotics globally. Although there is building evidence that early life exposure to antibiotics increases susceptibility to various diseases including gut disorders later in life, the lasting impact of early life antibiotics on the physiology of the gut and its enteric nervous system (ENS) remains unclear. We treated neonatal mice with the antibiotic vancomycin during their first 10 postnatal days, then examined potential lasting effects of the antibiotic treatment on their colons during young adulthood (6 weeks old). We found that neonatal vancomycin treatment disrupted the gut functions of young adult female and male mice differently. Antibiotic-exposed females had significantly longer whole gut transit while antibiotic-treated males had significantly lower faecal weights compared to controls. Both male and female antibiotic-treated mice had greater percentages of faecal water content. Neonatal vancomycin treatment also had sexually dimorphic impacts on the neurochemistry and Ca2+ activity of young adult myenteric and submucosal neurons. Myenteric neurons of male mice were more disrupted than those of females, while opposing changes in submucosal neurons were seen in each sex. Neonatal vancomycin also induced sustained changes in colonic microbiota and lasting depletion of mucosal serotonin (5-HT) levels. Antibiotic impacts on microbiota and mucosal 5-HT were not sex-dependent, but we propose that the responses of the host to these changes are sex-specific. This first demonstration of long-term impacts of neonatal antibiotics on the ENS, gut microbiota and mucosal 5-HT has important implications for gut function and other physiological systems of the host. KEY POINTS: Early life exposure to antibiotics can increase susceptibility to diseases including functional gastrointestinal (GI) disorders later in life. Yet, the lasting impact of this common therapy on the gut and its enteric nervous system (ENS) remains unclear. We investigated the long-term impact of neonatal antibiotic treatment by treating mice with the antibiotic vancomycin during their neonatal period, then examining their colons during young adulthood. Adolescent female mice given neonatal vancomycin treatment had significantly longer whole gut transit times, while adolescent male and female mice treated with neonatal antibiotics had significantly wetter stools. Effects of neonatal vancomycin treatment on the neurochemistry and Ca2+ activity of myenteric and submucosal neurons were sexually dimorphic. Neonatal vancomycin also had lasting effects on the colonic microbiome and mucosal serotonin biosynthesis that were not sex-dependent. Different male and female responses to antibiotic-induced disruptions of the ENS, microbiota and mucosal serotonin biosynthesis can lead to sex-specific impacts on gut function.

Key difference between transition state stabilization and ground state destabilization: increasing atomic charge densities before or during enzyme-substrate binding.

The origin of the enormous catalytic power of enzymes has been extensively studied through experimental and computational approaches. Although precise mechanisms are still subject to much debate, enzymes are thought to catalyze reactions by stabilizing transition states (TSs) or destabilizing ground states (GSs). By exploring the catalysis of various types of enzyme-substrate noncovalent interactions, we found that catalysis by TS stabilization and the catalysis by GS destabilization share common features by reducing the free energy barriers (ΔG ‡s) of reactions, but are different in attaining the requirement for ΔG ‡ reduction. Irrespective of whether enzymes catalyze reactions by TS stabilization or GS destabilization, they reduce ΔG ‡s by enhancing the charge densities of catalytic atoms that experience a reduction in charge density between GSs and TSs. Notably, in TS stabilization, the charge density of catalytic atoms is enhanced prior to enzyme-substrate binding; whereas in GS destabilization, the charge density of catalytic atoms is enhanced during the enzyme-substrate binding. Results show that TS stabilization and GS destabilization are not contradictory to each other and are consistent in reducing the ΔG ‡s of reactions. The full mechanism of enzyme catalysis includes the mechanism of reducing ΔG ‡ and the mechanism of enhancing atomic charge densities. Our findings may help resolve the debate between TS stabilization and GS destabilization and assist our understanding of catalysis and the design of artificial enzymes.

The role of gut microbial ß-glucuronidase in drug disposition and development.

Gut microbial ß-glucuronidase (gmGUS) is involved in the disposition of many endogenous and exogenous compounds. Preclinical studies have shown that inhibiting gmGUS activity affects drug disposition, resulting in reduced toxicity in the gastrointestinal tract (GIT) and enhanced systemic efficacy. Additionally, manipulating gmGUS activity is expected to be effective in preventing/treating local or systemic diseases. Although results from animal studies are promising, challenges remain in developing drugs by targeting gmGUS. Here, we review the role of gmGUS in host health under physiological and pathological conditions, the impact of gmGUS on the disposition of phenolic compounds, models used to study gmGUS activity, and the perspectives and challenges in developing drugs by targeting gmGUS.

KOMB: K-core based de novo characterization of copy number variation in microbiomes.

Characterizing metagenomes via kmer-based, database-dependent taxonomic classification has yielded key insights into underlying microbiome dynamics. However, novel approaches are needed to track community dynamics and genomic flux within metagenomes, particularly in response to perturbations. We describe KOMB, a novel method for tracking genome level dynamics within microbiomes. KOMB utilizes K-core decomposition to identify Structural variations (SVs), specifically, population-level Copy Number Variation (CNV) within microbiomes. K-core decomposition partitions the graph into shells containing nodes of induced degree at least K, yielding reduced computational complexity compared to prior approaches. Through validation on a synthetic community, we show that KOMB recovers and profiles repetitive genomic regions in the sample. KOMB is shown to identify functionally-important regions in Human Microbiome Project datasets, and was used to analyze longitudinal data and identify keystone taxa in Fecal Microbiota Transplantation (FMT) samples. In summary, KOMB represents a novel graph-based, taxonomy-oblivious, and reference-free approach for tracking CNV within microbiomes. KOMB is open source and available for download at https://gitlab.com/treangenlab/komb.

Intestinal Predictors of Whole Blood Serotonin Levels in Children With or Without Autism.

Hyperserotonemia, or elevated levels of whole blood serotonin (WB5-HT), was the first biomarker linked to autism spectrum disorder (ASD). Despite numerous studies investigating the etiology of hyperserotonemia, results have been inconsistent. Recent findings suggest a relationship between the immune system and hyperserotonemia. The current study investigated whether intestinal 5-HT levels, 5-HT gene expression, or intestinal cell types predict WB5-HT. Participants included thirty-one males aged 3-18 who were classified into one of three groups: ASD and functional GI issues, typically developing with GI issues, and typically developing without GI issues. Samples from a lower endoscopy were analyzed to examine the pathways in predicting WB-5HT. Results demonstrated an association between T-Lymphocytes and WB5-HT.

Emu: species-level microbial community profiling of full-length 16S rRNA Oxford Nanopore sequencing data.

16S ribosomal RNA-based analysis is the established standard for elucidating the composition of microbial communities. While short-read 16S rRNA analyses are largely confined to genus-level resolution at best, given that only a portion of the gene is sequenced, full-length 16S rRNA gene amplicon sequences have the potential to provide species-level accuracy. However, existing taxonomic identification algorithms are not optimized for the increased read length and error rate often observed in long-read data. Here we present Emu, an approach that uses an expectation-maximization algorithm to generate taxonomic abundance profiles from full-length 16S rRNA reads. Results produced from simulated datasets and mock communities show that Emu is capable of accurate microbial community profiling while obtaining fewer false positives and false negatives than alternative methods. Additionally, we illustrate a real-world application of Emu by comparing clinical sample composition estimates generated by an established whole-genome shotgun sequencing workflow with those returned by full-length 16S rRNA gene sequences processed with Emu.

Mathematical models to study the biology of pathogens and the infectious diseases they cause.

Mathematical models have many applications in infectious diseases: epidemiologists use them to forecast outbreaks and design containment strategies; systems biologists use them to study complex processes sustaining pathogens, from the metabolic networks empowering microbial cells to ecological networks in the microbiome that protects its host. Here, we (1) review important models relevant to infectious diseases, (2) draw parallels among models ranging widely in scale. We end by discussing a minimal set of information for a model to promote its use by others and to enable predictions that help us better fight pathogens and the diseases they cause.

Systems biology approach to functionally assess the Clostridioides difficile pangenome reveals genetic diversity with discriminatory power.

Combatting Clostridioides difficile infections, a dominant cause of hospital-associated infections with incidence and resulting deaths increasing worldwide, is complicated by the frequent emergence of new virulent strains. Here, we employ whole-genome sequencing, high-throughput phenotypic screenings, and genome-scale models of metabolism to evaluate the genetic diversity of 451 strains of C. difficile. Constructing the C. difficile pangenome based on this set revealed 9,924 distinct gene clusters, of which 2,899 (29%) are defined as core, 2,968 (30%) are defined as unique, and the remaining 4,057 (41%) are defined as accessory. We develop a strain typing method, sequence typing by accessory genome (STAG), that identifies 176 genetically distinct groups of strains and allows for explicit interrogation of accessory gene content. Thirty-five strains representative of the overall set were experimentally profiled on 95 different nutrient sources, revealing 26 distinct growth profiles and unique nutrient preferences; 451 strain-specific genome scale models of metabolism were constructed, allowing us to computationally probe phenotypic diversity in 28,864 unique conditions. The models create a mechanistic link between the observed phenotypes and strain-specific genetic differences and exhibit an ability to correctly predict growth in 76% of measured cases. The typing and model predictions are used to identify and contextualize discriminating genetic features and phenotypes that may contribute to the emergence of new problematic strains.

Microbial metabolites: cause or consequence in gastrointestinal disease?

Systems biology studies have established that changes in gastrointestinal microbiome composition and function can adversely impact host physiology. Notable diseases synonymously associated with dysbiosis include inflammatory bowel diseases, cancer, metabolic disorders, and opportunistic and recurrent pathogen infections. However, there is a scarcity of mechanistic data that advances our understanding of taxonomic correlations with pathophysiological host-microbiome interactions. Generally, to survive a hostile gut environment, microbes are highly metabolically active and produce trans-kingdom signaling molecules to interact with competing microorganisms and the host. These specialized metabolites likely play important homeostatic roles, and identifying disease-specific taxa and their effector pathways can provide better strategies for diagnosis, treatment, and prevention, as well as the discovery of innovative therapeutics. The signaling role of microbial biotransformation products such as bile acids, short-chain fatty acids, polysaccharides, and dietary tryptophan is increasingly recognized, but little is known about the identity and function of metabolites that are synthesized by microbial biosynthetic gene clusters, including ribosomally synthesized and posttranslationally modified peptides (RiPPs), nonribosomal peptides (NRPs), polyketides (PKs), PK-NRP hybrids, and terpenes. Here we consider how bioactive natural products directly encoded by the human microbiome can contribute to the pathophysiology of gastrointestinal disease, cancer, autoimmune, antimicrobial-resistant bacterial and viral infections (including COVID-19). We also present strategies used to discover these compounds and the biological activities they exhibit, with consideration of therapeutic interventions that could emerge from understanding molecular causation in gut microbiome research.