Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome.

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.

Malnourished Microbes: Host-Microbiome Interactions in Child Undernutrition.

Childhood undernutrition is a major global health burden that is only partially resolved by nutritional interventions. Both chronic and acute forms of child undernutrition are characterized by derangements in multiple biological systems including metabolism, immunity, and endocrine systems. A growing body of evidence supports a role of the gut microbiome in mediating these pathways influencing early life growth. Observational studies report alterations in the gut microbiome of undernourished children, while preclinical studies suggest that this can trigger intestinal enteropathy, alter host metabolism, and disrupt immune-mediated resistance against enteropathogens, each of which contribute to poor early life growth. Here, we compile evidence from preclinical and clinical studies and describe the emerging pathophysiological pathways by which the early life gut microbiome influences host metabolism, immunity, intestinal function, endocrine regulation, and other pathways contributing to child undernutrition. We discuss emerging microbiome-directed therapies and consider future research directions to identify and target microbiome-sensitive pathways in child undernutrition.

Mind over matter: the microbial mindscapes of psychedelics and the gut-brain axis.

Psychedelics have emerged as promising therapeutics for several psychiatric disorders. Hypotheses around their mechanisms have revolved around their partial agonism at the serotonin 2 A receptor, leading to enhanced neuroplasticity and brain connectivity changes that underlie positive mindset shifts. However, these accounts fail to recognise that the gut microbiota, acting via the gut-brain axis, may also have a role in mediating the positive effects of psychedelics on behaviour. In this review, we present existing evidence that the composition of the gut microbiota may be responsive to psychedelic drugs, and in turn, that the effect of psychedelics could be modulated by microbial metabolism. We discuss various alternative mechanistic models and emphasize the importance of incorporating hypotheses that address the contributions of the microbiome in future research. Awareness of the microbial contribution to psychedelic action has the potential to significantly shape clinical practice, for example, by allowing personalised psychedelic therapies based on the heterogeneity of the gut microbiota.

The effect of oral probiotics on response to vaccination in older adults: a systematic review of randomised controlled trials.

This systematic review evaluated the impact of oral probiotics on the immune response to vaccination in older people. A literature search was performed in three electronic databases up to January 2023. Randomised controlled trials (RCTs) conducted in older people (age ≥ 60 years) investigating oral probiotics and vaccine response outcomes were included. Characteristics and outcome data of the included studies were extracted and analysed and study quality was assessed using the Cochrane Risk of Bias Tool for randomised trials. Ten RCTs involving 1,560 participants, reported in 9 papers, were included. Nine studies involved the seasonal influenza vaccine and one a COVID-19 vaccine. All studies used lactobacilli, some in combination with bifidobacteria. Studies reported outcomes including anti-vaccine antibody titres or concentrations, seroconversion and seroprotection. When comparing antibody titres, seroprotection rate and seroconversion rate between probiotic and placebo groups expressed as a response ratio, the weighted mean values were 1.29, 1.16 and 2.00, respectively. Meta-analysis showed that probiotics increase seroconversion rates to all three strains of the seasonal influenza vaccine: odds ratio (95% confidence interval) 2.74 (1.31, 5.70; P = 0.007) for the H1N1 strain; 1.90 (1.04, 3.44; P = 0.04) for the H3N2 strain; 1.72 (1.05, 2.80; P = 0.03) for the B strain. There was a low level of heterogeneity in these findings. Several studies were at high risk of bias due to missing outcome data. Lactobacilli may improve the vaccine response, but further research is needed to be more certain of this.

Targeted inhibition of gut bacterial ß-glucuronidase activity enhances anticancer drug efficacy.

Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.

Microbiomes in physiology: insights into 21st-century global medical challenges.

NEW FINDINGS: What is the topic of this review? The role of the gut microbiome in physiology and how it can be targeted as an effective strategy against two of the most important global medical challenges of our time, namely, metabolic diseases and antibacterial resistance. What advances does it highlight? The critical roles of the microbiome in regulating host physiology and how microbiome analysis is useful for disease stratification to enable informed clinical decisions and develop interventions such as faecal microbiota transplantation, prebiotics and probiotics. Also, the limitations of microbiome modulation, including the potential for probiotics to enhance antimicrobial resistance gene reservoirs, and that currently a 'healthy microbiome' that can be used as a biobank for transplantation is yet to be defined. ABSTRACT: The human gut microbiome is a key factor in the development of metabolic diseases and antimicrobial resistance, which are among the greatest global medical challenges of the 21st century. A recent symposium aimed to highlight state-of-the-art evidence for the role of the gut microbiome in physiology, from childhood to adulthood, and the impact this has on global disease outcomes, ageing and antimicrobial resistance. Although the gut microbiome is established early in life, over time the microbiome and its components including metabolites can become perturbed due to changes such as dietary habits, use of antibiotics and age. As gut microbial metabolites, including short-chain fatty acids, secondary bile acids and trimethylamine-N-oxide, can interact with host receptors including G protein-coupled receptors and can alter host metabolic fluxes, they can significantly affect physiological homoeostasis leading to metabolic diseases. These metabolites can be used to stratify disease phenotypes such as irritable bowel syndrome and adverse events after heart failure and allow informed decisions on clinical management and treatment. While strategies such as use of probiotics, prebiotics and faecal microbiota transplantation have been proposed as interventions to treat and prevent metabolic diseases and antimicrobial resistance, caution must be exercised, first due to the potential of probiotics to enhance antimicrobial resistance gene reservoirs, and second, a 'healthy gut microbiome' that can be used as a biobank for transplantation is yet to be defined. We highlight that sampling other parts of the gastrointestinal tract may produce more representative data than the faecal microbiome alone.

Microbe-Immune Crosstalk: Evidence That T Cells Influence the Development of the Brain Metabolome.

Cross-talk between the immune system and the brain is essential to neuronal development, neuronal excitability, neuroplasticity, and neurotransmission. Gut microbiota are essential to immune system development and immune function; hence, it is essential to consider more broadly the microbiota-immune-brain axis in neurodevelopment. The gut, brain, and microbial metabolomes obtained from C57Bl/6 and T-cell-deficient mice across four developmental timepoints (postnatal day 17, 24, 28, and 84) were studied by 1H NMR spectroscopy. 16S rRNA gene sequencing was performed on cecal and fecal samples. In the absence of T-cells, the developmental trajectory of the gut microbiota and of the host's metabolic profile was altered. The novel insights from this work include (1) the requirement of functional T-cells for the normal trajectory of microbiotal development and the metabolic maturation of the supra-organism, (2) the potential role for Muribaculaceae taxa in modulating the cecal availability of metabolites previously implicated with a role in the gut-brain axis in T-cell deficient mice, and (3) the impact of T-cell-deficiency on central levels of neuroactive metabolites.

Effect of scheduled antimicrobial and nicotinamide treatment on linear growth in children in rural Tanzania: A factorial randomized, double-blind, placebo-controlled trial.

BACKGROUND: Stunting among children in low-resource settings is associated with enteric pathogen carriage and micronutrient deficiencies. Our goal was to test whether administration of scheduled antimicrobials and daily nicotinamide improved linear growth in a region with a high prevalence of stunting and enteric pathogen carriage. METHODS AND FINDINGS: We performed a randomized, 2 × 2 factorial, double-blind, placebo-controlled trial in the area around Haydom, Tanzania. Mother-child dyads were enrolled by age 14 days and followed with monthly home visits and every 3-month anthropometry assessments through 18 months. Those randomized to the antimicrobial arm received 2 medications (versus corresponding placebos): azithromycin (single dose of 20 mg/kg) at months 6, 9, 12, and 15 and nitazoxanide (3-day course of 100 mg twice daily) at months 12 and 15. Those randomized to nicotinamide arm received daily nicotinamide to the mother (250 mg pills months 0 to 6) and to the child (100 mg sachets months 6 to 18). Primary outcome was length-for-age z-score (LAZ) at 18 months in the modified intention-to-treat group. Between September 5, 2017 and August 31, 2018, 1,188 children were randomized, of whom 1,084 (n = 277 placebo/placebo, 273 antimicrobial/placebo, 274 placebo/nicotinamide, and 260 antimicrobial/nicotinamide) were included in the modified intention-to-treat analysis. The study was suspended for a 3-month period by the Tanzanian National Institute for Medical Research (NIMR) because of concerns related to the timing of laboratory testing and the total number of serious adverse events (SAEs); this resulted in some participants receiving their final study assessment late. There was a high prevalence of stunting overall (533/1,084, 49.2%). Mean 18-month LAZ did not differ between groups for either intervention (mean LAZ with 95% confidence interval [CI]: antimicrobial: -2.05 CI -2.13, -1.96, placebo: -2.05 CI -2.14, -1.97; mean difference: 0.01 CI -0.13, 0.11, p = 0.91; nicotinamide: -2.06 CI -2.13, -1.95, placebo: -2.04 CI -2.14, -1.98, mean difference 0.03 CI -0.15, 0.09, p = 0.66). There was no difference in LAZ for either intervention after adjusting for possible confounders (baseline LAZ, age in days at 18-month measurement, ward, hospital birth, birth month, years of maternal education, socioeconomic status (SES) quartile category, sex, whether the mother was a member of the Datoga tribe, and mother's height). Adverse events (AEs) and SAEs were overall similar between treatment groups for both the nicotinamide and antimicrobial interventions. Key limitations include the absence of laboratory measures of pathogen carriage and nicotinamide metabolism to provide context for the negative findings. CONCLUSIONS: In this study, we observed that neither scheduled administration of azithromycin and nitazoxanide nor daily provision of nicotinamide was associated with improved growth in this resource-poor setting with a high force of enteric infections. Further research remains critical to identify interventions toward improved early childhood growth in challenging conditions. TRIAL REGISTRATION: ClinicalTrials.gov NCT03268902.

An In Vitro Pilot Fermentation Study on the Impact of Chlorella pyrenoidosa on Gut Microbiome Composition and Metabolites in Healthy and Coeliac Subjects.

The response of a coeliac and a healthy gut microbiota to the green algae Chlorella pyrenoidosa was evaluated using an in vitro continuous, pH controlled, gut model system, which simulated the human colon. The effect of C. pyrenoidosa on the microbial structure was determined by 16S rRNA gene sequencing and inferred metagenomics, whereas the metabolic activitywas determined by1H-nuclear magnetic resonancespectroscopic analysis. The addition of C. pyrenoidosa significantly increased the abundance of the genera Prevotella, Ruminococcus and Faecalibacterium in the healthy donor, while an increase in Faecalibacterium, Bifidobacterium and Megasphaera and a decrease in Enterobacteriaceae were observed in the coeliac donor. C. pyrenoidosa also altered several microbial pathways including those involved in short-chain fatty acid (SCFA) production. At the metabolic level, a significant increase from baseline was seen in butyrate and propionate (p < 0.0001) in the healthy donor, especially in vessels 2 and 3. While acetate was significantly higher in the healthy donor at baseline in vessel 3 (p < 0.001) compared to the coeliac donor, this was markedly decreased after in vitro fermentation with C. pyrenoidosa. This is the first in vitro fermentation study of C. pyrenoidosa and human gut microbiota, however, further in vivo studies are needed to prove its efficacy.

Consumer Safety Considerations of Skin and Oral Microbiome Perturbation.

Microbiomes associated with human skin and the oral cavity are uniquely exposed to personal care regimes. Changes in the composition and activities of the microbial communities in these environments can be utilized to promote consumer health benefits, for example, by reducing the numbers, composition, or activities of microbes implicated in conditions such as acne, axillary odor, dandruff, and oral diseases. It is, however, important to ensure that innovative approaches for microbiome manipulation do not unsafely disrupt the microbiome or compromise health, and where major changes in the composition or activities of the microbiome may occur, these require evaluation to ensure that critical biological functions are unaffected. This article is based on a 2-day workshop held at SEAC Unilever, Sharnbrook, United Kingdom, involving 31 specialists in microbial risk assessment, skin and oral microbiome research, microbial ecology, bioinformatics, mathematical modeling, and immunology. The first day focused on understanding the potential implications of skin and oral microbiome perturbation, while approaches to characterize those perturbations were discussed during the second day. This article discusses the factors that the panel recommends be considered for personal care products that target the microbiomes of the skin and the oral cavity.

Faecal virome transplantation decreases symptoms of type 2 diabetes and obesity in a murine model.

OBJECTIVE: Development of obesity and type 2 diabetes (T2D) are associated with gut microbiota (GM) changes. The gut viral community is predominated by bacteriophages (phages), which are viruses that attack bacteria in a host-specific manner. The antagonistic behaviour of phages has the potential to alter the GM. As a proof-of-concept, we demonstrate the efficacy of faecal virome transplantation (FVT) from lean donors for shifting the phenotype of obese mice into closer resemblance of lean mice. DESIGN: The FVT consisted of viromes with distinct profiles extracted from the caecal content of mice from different vendors that were fed a low-fat (LF) diet for 14 weeks. Male C57BL/6NTac mice were divided into five groups: LF (as diet control), high-fat (HF) diet, HF+ampicillin (Amp), HF+Amp+FVT and HF+FVT. At weeks 6 and 7 of the study, the HF+FVT and HF+Amp+FVT mice were treated with FVT by oral gavage. The Amp groups were treated with Amp 24 hours prior to first FVT treatment. RESULTS: Six weeks after first FVT, the HF+FVT mice showed a significant decrease in weight gain compared with the HF group. Further, glucose tolerance was comparable between the LF and HF+FVT mice, while the other HF groups all had impaired glucose tolerance. These observations were supported by significant shifts in GM composition, blood plasma metabolome and expression levels of genes associated with obesity and T2D development. CONCLUSIONS: Transfer of caecal viral communities from mice with a lean phenotype into mice with an obese phenotype led to reduced weight gain and normalised blood glucose parameters relative to lean mice. We hypothesise that this effect is mediated via FVT-induced GM changes.

A Two-Way Interaction between Methotrexate and the Gut Microbiota of Male Sprague-Dawley Rats.

Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague-Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity.

A novel mouse model of Campylobacter jejuni enteropathy and diarrhea.

Campylobacter infections are among the leading bacterial causes of diarrhea and of 'environmental enteropathy' (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease with Campylobacter has been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche for Campylobacter infection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to test specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria.

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.

Intervention and Mechanisms of Alanyl-glutamine for Inflammation, Nutrition, and Enteropathy: A Randomized Controlled Trial.

OBJECTIVE: Determine the minimum dosage of alanyl-glutamine (Ala-Gln) required to improve gut integrity and growth in children at risk of environmental enteropathy (EE). METHODS: This was a double-blinded randomized placebo-controlled dose-response trial. We enrolled 140 children residing in a low-income community in Fortaleza, Brazil. Participants were 2 to 60 months old and had weight-for-age (WAZ), height-for-age (HAZ), or weight-for-height (WHZ) z-scores less than -1. We randomized children to 10 days of nutritional supplementation: Ala-Gln at 3 g/day, Ala-Gln at 6 g/day, Ala-Gln at 12 g/day, or an isonitrogenous dose of glycine (Gly) placebo at 12.5 g/day. Our primary outcome was urinary lactulose-mannitol excretion testing. Secondary outcomes were anthropometry, fecal markers of inflammation, urine metabolic profiles, and malabsorption (spot fecal energy). RESULTS: Of 140 children, 103 completed 120 days of follow-up (24% dropout). In the group receiving the highest dose of Ala-Gln, we detected a modest improvement in urinary lactulose excretion from 0.19% on day 1 to 0.17% on day 10 (P = 0.05). We observed significant but transient improvements in WHZ at day 10 in 2 Ala-Gln groups, and in WHZ and WAZ in all Ala-Gln groups at day 30. We detected no effects on fecal inflammatory markers, diarrheal morbidity, or urine metabolic profiles; but did observe modest reductions in fecal energy and fecal lactoferrin in participants receiving Ala-Gln. CONCLUSIONS: Intermediate dose Ala-Gln promotes short-term improvement in gut integrity and ponderal growth in children at risk of EE. Lower doses produced improvements in ponderal growth in the absence of enhanced gut integrity.

Obesity and Cage Environment Modulate Metabolism in the Zucker Rat: A Multiple Biological Matrix Approach to Characterizing Metabolic Phenomena.

Obesity and its comorbidities are increasing worldwide imposing a heavy socioeconomic burden. The effects of obesity on the metabolic profiles of tissues (liver, kidney, pancreas), urine, and the systemic circulation were investigated in the Zucker rat model using 1H NMR spectroscopy coupled to multivariate statistical analysis. The metabolic profiles of the obese ( fa/ fa) animals were clearly differentiated from the two phenotypically lean phenotypes, ((+/+) and ( fa/+)) within each biological compartment studied, and across all matrices combined. No significant differences were observed between the metabolic profiles of the genotypically distinct lean strains. Obese Zucker rats were characterized by higher relative concentrations of blood lipid species, cross-compartmental amino acids (particularly BCAAs), urinary and liver metabolites relating to the TCA cycle and glucose metabolism; and lower amounts of urinary gut microbial-host cometabolites, and intermatrix metabolites associated with creatine metabolism. Further to this, the obese Zucker rat metabotype was defined by significant metabolic alterations relating to disruptions in the metabolism of choline across all compartments analyzed. The cage environment was found to have a significant effect on urinary metabolites related to gut-microbial metabolism, with additional cage-microenvironment trends also observed in liver, kidney, and pancreas. This study emphasizes the value in metabotyping multiple biological matrices simultaneously to gain a better understanding of systemic perturbations in metabolism, and also underscores the need for control or evaluation of cage environment when designing and interpreting data from metabonomic studies in animal models.

Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry with Electrospray Ionization Quantification of Tryptophan Metabolites and Markers of Gut Health in Serum and Plasma-Application to Clinical and Epidemiology Cohorts.

A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte <20% for the lowest limit of quantification and <15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1-12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeat analysis during a run consisting of 18 plates. The % CV (coefficient of variation) values obtained for each analyte were <15%. Additional biological testing applied the assay to samples collected from healthy control participants and two groups diagnosed with inflammatory bowel disease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylic acid (5-ASA) and one group untreated), with results showing significant differences in the concentrations of picolinic acid, kynurenine, and xanthurenic acid. The short analysis time and 96-well plate format of the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MS metabolomic analysis in large-scale clinical and epidemiological population studies.

Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co-infection with Giardia lamblia and enteroaggregative Escherichia coli.

Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, Giardia lamblia and enteroaggregative Escherichia coli (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent Giardia colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, Giardia-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric Giardia infections. Rather, Giardia perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a Giardia-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to Giardia, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.

Metabolic phenotyping of malnutrition during the first 1000 days of life.

Nutritional restrictions during the first 1000 days of life can impair or delay the physical and cognitive development of the individual and have long-term consequences for their health. Metabolic phenotyping (metabolomics/metabonomics) simultaneously measures a diverse range of low molecular weight metabolites in a sample providing a comprehensive assessment of the individual's biochemical status. There are a growing number of studies applying such approaches to characterize the metabolic derangements induced by various forms of early-life malnutrition. This includes acute and chronic undernutrition and specific micronutrient deficiencies. Collectively, these studies highlight the diverse and dynamic metabolic disruptions resulting from various forms of nutritional deficiencies. Perturbations were observed in many pathways including those involved in energy, amino acid, and bile acid metabolism, the metabolic interactions between the gut microbiota and the host, and changes in metabolites associated with gut health. The information gleaned from such studies provides novel insights into the mechanisms linking malnutrition with developmental impairments and assists in the elucidation of candidate biomarkers to identify individuals at risk of developmental shortfalls. As the metabolic profile represents a snapshot of the biochemical status of an individual at a given time, there is great potential to use this information to tailor interventional strategies specifically to the metabolic needs of the individual.