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Background Exposure to persistent organic pollutants (POPs) and gastrointestinal microbial disruption positively corelate with a predisposition to factors including obesity, metabolic syndrome, and type 2 diabetes; however, it is unclear if and how the microbiome contributes to this relationship. Results Here, we show that early-life exposure to a potent aryl hydrocarbon receptor (AHR) agonist in mice resulted in persistent microbiota disruptions associated with impaired glucose homeostasis later in life. 2,3,7,8-tetrachlorodibenzofuran (TCDF)-exposed mice exhibited a profound disruption in the gut microbiome characterized by decreased abundances of Akkermansia muciniphila (A. muciniphila), decreased levels of cecal short chain fatty acids (SCFAs) and indole-3-lactic acid (ILA), and reduction of gut hormones GLP-1 and PYY. Importantly, microbial and metabolic phenotypes associated with early-life POP exposure were transferable to germ-free recipients in the absence of POP carry-over. Consistent with these in vivo studies, we reveal a direct, AHR-independent, POP-microbiota interaction that significantly affected the growth, physiology, gene expression, and metabolic activity of A. muciniphila, resulting in suppressed activity along the ILA pathway. Conclusions These data point to a complex effect of POPs on the host and microbiota providing strong evidence that early-life, short-term, and self-limiting POP exposure can adversely impact the microbiome which persists into later life with associated health implications.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient–derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients.Significance:Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis.See related commentary by Jain and Dudeja, p. 1838.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Table from Human Colon Cancer–Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice

Supplementary Figure from Human Colon Cancer–Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that plays an integral role in homeostatic maintenance by regulating cellular functions such as cellular differentiation, metabolism, barrier function, and immune response. An important but poorly understood class of AHR activators are compounds derived from host and bacterial metabolism of tryptophan. The commensal bacteria of the gut microbiome are major producers of tryptophan metabolites known to activate the AHR, while the host also produces AHR activators through tryptophan metabolism. We used targeted mass spectrometry-based metabolite profiling to determine the presence and metabolic source of these metabolites in the sera of conventional mice, germ-free mice, and humans. Surprisingly, sera concentrations of many tryptophan metabolites are comparable between germ-free and conventional mice. Therefore, many major AHR-activating tryptophan metabolites in mouse sera are produced by the host, despite their presence in feces and mouse cecal contents. AHR activation is rarely studied in the context of a mixture at relevant concentrations, as we present here. The AHR activation potentials of individual and pooled metabolites were explored using cell-based assays, while ligand binding competition assays and ligand docking simulations were used to assess the detected metabolites as AHR agonists. The physiological and biomedical relevance of the identified metabolites was investigated in the context of cell-based models for cancer and rheumatoid arthritis. We present data here that reframe AHR biology to include the presence of ubiquitous tryptophan metabolites, improving our understanding of homeostatic AHR activity and models of AHR-linked diseases.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient–derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. Significance: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825

Background Dyslipidemia is a feature of impaired metabolic health in conjunction with impaired glucose metabolism and central obesity. However, the contribution of factors to postprandial lipemia in healthy but metabolically at-risk adults is not well understood. We investigated the collective contribution of several physiologic and lifestyle factors to postprandial triglyceride (TG) response to a high-fat meal in healthy, overweight and obese adults. Methods Overweight and obese adults (n = 35) underwent a high-fat meal challenge with blood sampled at fasting and hourly in the 4-hour postprandial period after a breakfast containing 50 g fat. Incremental area under the curve (iAUC) and postprandial magnitude for TG were calculated and data analyzed using a linear model with physiologic and lifestyle characteristics as explanatory variables. Model reduction was used to assess which explanatory variables contributed most to the postprandial TG response. Results TG responses to a high-fat meal were variable between individuals, with approximately 57 % of participants exceeded the nonfasting threshold for hypertriglyceridemia. Visceral adiposity was the strongest predictor of TG iAUC (β = 0.53, p = 0.01), followed by aerobic exercise frequency (β = 0.31, p = 0.05), insulin resistance based on HOMA-IR (β = 0.30, p = 0.04), and relative exercise intensity at which substrate utilization crossover occurred (β = 0.05, p = 0.04). For postprandial TG magnitude, visceral adiposity was a strong predictor (β = 0.43, p p = 0.01), and exercise intensity for substrate utilization crossover (β = 0.53, p = 0.01). Conclusions Postprandial TG responses to a high-fat meal was partially explained by several physiologic and lifestyle characteristics, including visceral adiposity, insulin resistance, aerobic exercise frequency, and relative substrate utilization crossover during exercise. Trial Registration ClinicalTrials.gov, NCT04128839, Registered 16 October 2019 – Retrospectively registered.

Immunosurveillance of the gastrointestinal epithelium by mononuclear phagocytes (MNPs) is essential for maintaining gut health. However, studying the complex interplay between the human gastrointestinal epithelium and MNPs such as dendritic cells (DCs) is difficult, since traditional cell culture systems lack complexity, and animal models may not adequately represent human tissues. Microphysiological systems, or tissue chips, are an attractive alternative for these investigations, because they model functional features of specific tissues or organs using microscale culture platforms that recreate physiological tissue microenvironments. However, successful integration of multiple of tissue types on a tissue chip platform to reproduce physiological cell-cell interactions remains a challenge. We previously developed a tissue chip system, the gut organoid flow chip (GOFlowChip), for long term culture of 3-D pluripotent stem cell-derived human intestinal organoids. Here, we optimized the GOFlowChip platform to build a complex microphysiological immune-cell-epithelial cell co-culture model in order to study DC-epithelial interactions in human stomach. We first tested different tubing materials and chip configurations to optimize DC loading onto the GOFlowChip and demonstrated that DC culture on the GOFlowChip for up to 20 h did not impact DC activation status or viability. However, Transwell chemotaxis assays and live confocal imaging revealed that Matrigel, the extracellular matrix (ECM) material commonly used for organoid culture, prevented DC migration towards the organoids and the establishment of direct MNP-epithelial contacts. Therefore, we next evaluated DC chemotaxis through alternative ECM materials including Matrigel-collagen mixtures and synthetic hydrogels. A polysaccharide-based synthetic hydrogel, VitroGel®-ORGANOID-3 (V-ORG-3), enabled significantly increased DC chemotaxis through the matrix, supported organoid survival and growth, and did not significantly alter DC activation or viability. On the GOFlowChip, DCs that were flowed into the chip migrated rapidly through the V-ORG matrix and reached organoids embedded deep within the chip, with increased interactions between DCs and gastric organoids. The successful integration of DCs and V-ORG-3 embedded gastric organoids into the GOFlowChip platform now permits real-time imaging of MNP-epithelial interactions and other investigations of the complex interplay between gastrointestinal MNPs and epithelial cells in their response to pathogens, candidate drugs and mucosal vaccines.

The material properties of bone tissue depend on the activity of remodeling bone cells, but the impact of bone cell metabolism on bone tissue is uncertain. To date, the metabolome of bone has not been evaluated for cortical bone, bone marrow, or whole bone including both tissue types. Furthermore, it is of particular interest whether the cortical bone metabolome reflects the sexual dimorphism observed in cortical bone material properties. We hypothesized that the metabolome of cortical bone differs from that of bone marrow, and that the cortical bone metabolome is sexually dimorphic. We first evaluated the metabolic profiles of isolated cortical bone, bone marrow, and whole bone for 20-week female C57Bl/6 mice (n = 10). We then compared metabolic profiles for isolated cortical bone from a separate group of 20-week female and male C57Bl6/mice (n = 10 / sex). Femurs from the same mice were evaluated for flexural material properties. Strength groupings (high strength males, high strength females, low strength males) were utilized to inform comparisons in the isolated humerus cortical bone metabolome. The metabolome of isolated cortical bone, bone marrow, and whole bone are distinct. The isolated cortical bone metabolome is also distinct between males and females. The female mice show evidence of lipid metabolism, whereas male mice show evidence of amino acid metabolism. Finally, 12 metabolic pathways were differentially regulated between bones that differed in strength. High-strength bones from both male and female mice included metabolites associated with tryptophan and purine metabolism. Taken together, these data demonstrate the power of metabolomics to provide insight into the effects of metabolism on bone physiology. These data add to an intricate picture of bone as an organ that is sexually dimorphic both in material and metabolomic profiles.

Summary Chronic low-grade inflammation is a subclinical condition directly and indirectly linked to the development of a wide range of diseases responsible for the vast majority of morbidity. To examine mechanisms coupled to chronic disease, a group of overweight and obese human subjects without known inflammatory diseases participated in a high-fat meal challenge as an acute inflammation stimulus. Analysis of serum metabolites grouped by baseline cytokine levels revealed that single samples had little power in differentiating groups. However, an analysis that incorporated temporal response separated inflammatory response phenotypes and allowed us to create a metabolic signature of inflammation which revealed metabolic components that are crucial to a cytokine-mediated inflammation response. The use of temporal response, rather than a single time point, improved metabolomic prediction of high postprandial inflammation responses and led to the development of a dynamic biosignature as a potential tool for stratifying risk to a wide range of diseases., Graphical abstract, Highlights • Dynamic responses often provide insight into disease pathology • Temporal metabolic responses to acute inflammation were explored in obese people • Temporal metabolite levels differentiated low, mid, and high inflammation groups • Inflammation-linked metabolites were shown to be predictors of cytokine responses, Pathophysiology; Systems biology; Metabolomics

ObjectiveTo compare the early responses to joint injury in conventional and germ-free mice.DesignPost-traumatic osteoarthritis (PTOA) was induced using a non-invasive anterior cruciate ligament rupture model in 20-week old germ-free (GF) and conventional C57BL/6 mice. Injury was induced in the left knees of n=8 GF and n=10 conventional mice. To examine the effects of injury, n=5 GF and n=9 conventional naïve control mice were used. Mice were euthanized 7 days post-injury, followed by synovial fluid recovery for global metabolomic profiling and analysis of epiphyseal trabecular bone by micro-computed tomography (μCT). Global metabolomic profiling assessed metabolic differences in the joint response to injury between GF and conventional mice. Magnitude of trabecular bone volume loss measured using μCT assessed early OA progression in GF and conventional mice.ResultsμCT found that GF mice had significantly less trabecular bone loss compared to conventional mice, indicating that the GF status was protective against early OA changes in bone structure. Global metabolomic profiling showed that conventional mice had greater variability in their metabolic response to injury, and a more distinct joint metabolome compared to their corresponding controls. Furthermore, differences in the response to injury in GF compared to conventional mice were linked to mouse metabolic pathways that regulate inflammation associated with the innate immune system.ConclusionsThese results suggest that the gut microbiota promote the development of PTOA during the acute phase following joint trauma possibly through the regulation of the innate immune system.

Purpose of ReviewArsenic exposure is a public health concern of global proportions with a high degree of interindividual variability in pathologic outcomes. Arsenic metabolism is a key factor underlying toxicity, and the primary purpose of this review is to summarize recent discoveries concerning the influence of the human gut microbiome on the metabolism, bioavailability, and toxicity of ingested arsenic. We review and discuss the current state of knowledge along with relevant methodologies for studying these phenomena.Recent FindingsBacteria in the human gut can biochemically transform arsenic-containing compounds (arsenicals). Recent publications utilizing culture-based approaches combined with analytical biochemistry and molecular genetics have helped identify several arsenical transformations by bacteria that are at least possible in the human gut and are likely to mediate arsenic toxicity to the host. Other studies that directly incubate stool samples in vitro also demonstrate the gut microbiome’s potential to alter arsenic speciation and bioavailability. In vivo disruption or elimination of the microbiome has been shown to influence toxicity and body burden of arsenic through altered excretion and biotransformation of arsenicals. Currently, few clinical or epidemiological studies have investigated relationships between the gut microbiome and arsenic-related health outcomes in humans, although current evidence provides strong rationale for this research in the future.SummaryThe human gut microbiome can metabolize arsenic and influence arsenical oxidation state, methylation status, thiolation status, bioavailability, and excretion. We discuss the strength of current evidence and propose that the microbiome be considered in future epidemiologic and toxicologic studies of human arsenic exposure.

Longitudinal human gut microbiome datasets generated using community-level, sequence-based approaches often report a sub-set of long-lived “resident” taxa that rarely, if ever, are lost. This result contrasts with population-level turnover of resident clones on the order of months to years. We hypothesized that the disconnect between these results is due to a relative lack of simultaneous discrimination of the human gut microbiome at both the community and population-levels. Here, we present results of a small, longitudinal cohort study (n = 8 participants) of healthy human adults that identifies static and dynamic members of the gut microbiome at the clone level based on cultivation/genetic discrimination and at the operational taxonomic unit/amplified sequence variant levels based on 16S rRNA sequencing. We provide evidence that there is little “stability” within resident clonal populations of the common gut microbiome bacterial family, Enterobacteriaceae. Given that clones can vary substantially in genome content and that evolutionary processes operate on the population level, these results question the biological relevance of apparent stability at higher taxonomic levels.

Arsenic is a human carcinogen and is linked to diverse pathologies in many organ systems. Arsenic exposure primarily occurs by ingestion of inorganic arsenic (iAs). Post-ingestion metabolism of iAs involves sequential cellular transformations by the microbiome and then by the host. Methylation is particularly critical for detoxification in both bacterial and mammalian cells, and is performed by a conserved mechanism in both phyla that involves alternating oxidations and reductions of the arsenic. Methylation-independent redox state modifications, glutathionylation, and thiolation reactions can also occur both in the gut-lumen and in host cells and, in combination, these can result in host cells being exposed to diverse inorganic and organic arsenicals with vastly different toxicological impacts. The toxicity of arsenate (AsV) stems from it being a phosphate analog whereas inorganic arsenite (iAsIII) and trivalent-methylated arsenic toxicities stem from their being electrophiles that bind to reactive cysteines or selenocysteines, in particular in the active sites of critical redox-active enzymes. In general, toxicities of inorganic or organic pentavalent forms are lower than those of the corresponding trivalent compounds. Metabolism of arsenic in either prokaryotes or eukaryotes has been studied in depth; however, few studies have assessed the interplay between the two. In particular, little is known about how metabolism by the microbiome impacts host exposure, metabolism, and outcomes. Understanding microbiome-host arsenic-interactions will foster development of novel targeted strategies to relieve or prevent arsenic-associated pathologies.

Arsenic poisons an estimated 200 million people worldwide through contaminated food and drinking water. Confusingly, the gut microbiome has been suggested to both mitigate and exacerbate arsenic toxicity. Here, we show that the microbiome protects mice from arsenic-induced mortality. Both antibiotic-treated and germ-free mice excrete less arsenic in stool and accumulate more arsenic in organs compared to control mice. Mice lacking the primary arsenic detoxification enzyme (As3mt) are hypersensitive to arsenic after antibiotic treatment or when derived germ-free, compared to wild-type and/or conventional counterparts. Human microbiome (stool) transplants protect germ-free As3mt-KO mice from arsenic-induced mortality, but protection depends on microbiome stability and the presence of specific bacteria, including Faecalibacterium. Our results demonstrate that both a functional As3mt and specific microbiome members are required for protection against acute arsenic toxicity in mouse models. We anticipate that the gut microbiome will become an important explanatory factor of disease (arsenicosis) penetrance in humans, and a novel target for prevention and treatment strategies., It is unclear whether the gut microbiome can mitigate or exacerbate arsenic toxicity. Here, Coryell et al. show that the human gut microbiome protects mice from arsenic-induced mortality, with protection levels correlating with the relative abundance of the human commensal Faecalibacterium.

Rapid testing methods can identify outbreaks and trigger preventive strategies for slowing the spread of SARS-CoV-2, the virus that causes COVID-19. The “gold-standard” detection method for SARS-CoV-2 is reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed on samples collected using a nasopharyngeal (NP) swab. While NP RT-qPCR provides high sensitivity, it requires trained personnel to administer and suffers from lengthy time-to-result. Recently, the testing community has turned to rapid saliva-based screening methods including saliva-to-RT-qPCR and/or saliva-to-RT-LAMP (reverse transcription loop-mediated isothermal amplification) to identify infected individuals regardless of symptomatic presentation. Here, we report a simple and rapid RT-LAMP fluorometric assay performed directly on heat-inactivated saliva, without the addition of buffers or proteinase K treatments we call saliva LAMP (SLAMP). Over the course of two days, a total of 243 individuals were tested using NP RT-qPCR, saliva-based qPCR, and saliva-based RT-LAMP. Of the 243 NP RT-qPCR tests, 65 were positive, 178 were negative, and SLAMP demonstrated a 91% sensitivity and 98% specificity. SLAMP sensitivity becomes 95% when samples negative in saliva tests while positive in NP RT-qPCR are excluded from evaluation, potentially indicating significant differences in viral titer between collection sites on the body. SLAMP is performed in triplicates and takes 45 min to run in the laboratory, requiring less technician time and instrument run time than NP RT-qPCR. These results demonstrate that saliva-based RT-LAMP can enable frequent and rapid screening of large numbers of people to identify pre-symptomatic and asymptomatic individuals thereby controlling outbreaks.

Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about how cytokines shape these processes. Given that the anti-inflammatory cytokine interleukin (IL)-10 promotes intestinal barrier function, and insufficient IL-10 signaling increases susceptibility to intestinal diseases like inflammatory bowel disease, we hypothesized that IL-10 signaling modulates processes underlying IEC proliferation and differentiation. This was tested using in vivo and in vitro IEC-specific IL-10 receptor 1 (IL-10R1) depletion under homeostatic conditions. Our findings revealed that loss of IL-10R1 drove lineage commitment toward a dominant goblet cell phenotype while decreasing absorptive cell-related features. Diminished IL-10 signaling also significantly elevated IEC proliferation with relatively minor changes to apoptosis. Characterization of signaling pathways upstream of proliferation demonstrated a significant reduction in the Wnt inhibitor, DKK1, increased nuclear localization of β-catenin, and increased transcripts of the proliferation marker, OLFM4, with IL-10R1 depletion. Phosphorylated STAT3 was nearly completely absent in IL-10R1 knockdown cells and may provide a mechanistic link between our observations and the regulation of these cellular processes. Our results demonstrate a novel role for IL-10 signaling in intestinal mucosal homeostasis by regulating proper balance of proliferation and IEC lineage fate.

The microbial ars operon encodes the primary bacterial defense response to the environmental toxicant, arsenic. An important component of this operon is the arsR gene, which encodes ArsR, a member of the family of proteins categorized as DNA-binding transcriptional repressors. As currently documented, ArsR regulates its own expression as well as other genes in the same ars operon. This study examined the roles of four ArsR proteins in the well-developed model Gram-negative bacterium Agrobacterium tumefaciens 5A. RNASeq was used to compare and characterize gene expression profiles in ± arsenite-treated cells of the wild-type strain and in four different arsR mutants. We report that ArsR-controlled transcription regulation is truly global, extending well beyond the current ars operon model, and includes both repression as well as apparent activation effects. Many cellular functions are significantly influenced, including arsenic resistance, phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, iron homeostasis, and many others. While there is evidence of some regulatory overlap, each ArsR exhibits its own regulatory profile. Furthermore, evidence of a regulatory hierarchy was observed; i.e. ArsR1 represses arsR4, ArsR4 activates arsR2, and ArsR2 represses arsR3. Additionally and unexpectedly, aioB (arsenite oxidase small subunit) expression was shown to be under partial positive control by ArsR2 and ArsR4. Summarizing, this study demonstrates the regulatory portfolio of arsenite-activated ArsR proteins and includes essentially all major cellular functions. The broad bandwidth of arsenic effects on microbial metabolism assists in explaining and understanding the full impact of arsenic in natural ecosystems, including the mammalian gut.

Environmental toxicant exposure contributes to morbidity and mortality of many human diseases. With respect to arsenic, microbially driven chemical transformations dictate its toxicity and mobility in virtually every environment yet studied, so a general hypothesis is that the human gut microbiome determines disease outcome following exposure. However, the complex nature of the gut microbiome and the myriad of potential interactions with human cells/tissues make it challenging to quantify the influence of specific arsenic-active functions—a requisite step in developing effective disease prevention and/or clinical intervention strategies. To control both mammalian and microbial function during toxicant exposure, we genetically defined the gut microbiome of mice using only Escherichia coli strain, AW3110 (▵arsRBC), or the same strain carrying a single genome copy of the Fucus vesiculosus metallothionein gene (AW3110::fmt); a cysteine-rich peptide that complexes with arsenite, facilitating bioaccumulation and reducing its toxic effects. AW3110::fmt bioaccumulated significantly more arsenic and gnotobiotic mice colonized by this strain excreted significantly more arsenic in stool and accumulated significantly less arsenic in organs. Moreover, AW3110::fmt gnotobiotic mice were protected from acute toxicity exposure (20 ppm AsIII) relative to controls. This study demonstrates—in a highly controlled fashion—that a single microbiome function (arsenic bioaccumulation) encoded by a single gene in a single human gut microbiome bacterium significantly alters mammalian host arsenic exposure. The experimental model described herein allows for a highly controlled and directed assessment of microbiome functions, and is useful to quantify the influence of specific microbiome-arsenic interactions that help mitigate human disease.

All cells need iron. Both too much and too little iron lead to diseases and unwanted outcomes., Iron is an essential micronutrient for life. In mammals, dietary iron is absorbed primarily in the small intestine. Currently, the impacts of dietary iron on the taxonomic structure and function of the gut microbiome and reciprocal effects on the animal host are not well understood. Here, we establish a mouse model of low-iron challenge in which intestinal biomarkers and reduced fecal iron reveal iron stress while serum iron and mouse behavioral markers indicate maintenance of iron homeostasis. We show that the diversity of the gut microbiome in conventional C57BL/6 mice changes dramatically during 2 weeks on a low-iron diet. We also show the effects of a low-iron diet on microbiome diversity are long lasting and not easily recovered when iron is returned to the diet. Finally, after optimizing taxon association methods, we show that some bacteria are unable to fully recover after the low-iron challenge and appear to be extirpated from the gut entirely. In particular, operational taxonomic units (OTUs) from the Prevotellaceae and Porphyromonadaceae families and Bacteroidales order are highly sensitive to low-iron conditions, while other seemingly insensitive OTUs recover. These results provide new insights into the iron requirements of gut microbiome members and add to the growing understanding of mammalian iron cycling. IMPORTANCE All cells need iron. Both too much and too little iron lead to diseases and unwanted outcomes. Although the impact of dietary iron on human cells and tissues has been well studied, there is currently a lack of understanding about how different levels of iron influence the abundant and diverse members of the human microbiome. This study develops a well-characterized mouse model for studying low-iron levels and identifies key groups of bacteria that are most affected. We found that the microbiome undergoes large changes when iron is removed from the diet but that many individual bacteria are able to rebound when iron levels are changed back to normal. That said, a select few members, referred to as iron-sensitive bacteria, seem to be lost. This study begins to identify individual members of the mammalian microbiome most affected by changes in dietary iron levels.

Clostridioides difficile is an enteric bacterial pathogen that can a cause nosocomial infection leading to debilitating colitis. The development of a murine model of C. difficile infection has led to fundamental discoveries in disease pathogenesis and the host immune response to infection. Recently, C. difficile endogenously present in the microbiota of mice has been reported and was found to complicate interpretation of mouse studies. Here, we report a novel C. difficile strain, named NTCD-035, isolated from the microbiota of our mouse colony. The presence of NTCD-035 in mice prior to challenge with a highly pathogenic C. difficile strain (VPI10463) led to significantly reduced disease severity. Phylogenetic characterization derived from whole genome sequencing and PCR ribotyping identified the isolate as a novel clade 1, ribotype 035 strain that lacks the pathogenicity locus required to produce toxins. Deficiency in toxin production along with sporulation capacity and secondary bile acid sensitivity was confirmed using in vitro assays. Inoculation of germ-free mice with NTCD-035 did not cause morbidity despite the strain readily colonizing the large intestine. Implementation of a culture-based screening procedure enabled the identification of mice harboring C. difficile in their microbiota, the establishment of a C. difficile-free mouse colony, and a monitoring system to prevent future contamination. Taken together, these data provide a framework for screening mice for endogenously harbored C. difficile and support clinical findings that demonstrate the therapeutic potential of non-toxigenic strains in preventing C. difficile associated disease. Abbreviations: PaLoc - Pathogenicity locus, CFUs - Colony forming units, TcdA - toxin-A, TcdB - toxin-B, CdtA - binary toxin A, CdtB - binary toxin B, CdtR - binary toxin R, NTCD - non-toxigenic C. difficile.

Escherichia coli is one of the most well-studied bacterial species, but several significant knowledge gaps remain regarding its ecology and natural history. Specifically, the most important factors influencing its life as a member of the healthy human gut microbiome are either underevaluated or currently unknown. Distinct E. coli population dynamics have been observed over the past century from a handful of temporal studies conducted in healthy human adults. Early studies using serology up to the most recent studies using genotyping and DNA sequencing approaches have all identified long-lived E. coli residents and short-lived transients. This review summarizes these discoveries and other studies that focused on the underlying mechanisms that lead to establishment and maintenance of E. coli residency in healthy human adults. Many fundamental knowledge gaps remain and are highlighted with the hope of facilitating future studies in this exciting research area.

Clostridioides difficile infection (CDI) is the most prevalent healthcare-associated infection in the United States and carries a significant healthcare system burden. As part of an ongoing, active surveillance system of C. difficile throughout Texas, the objective of this study was to assess changes in C. difficile ribotypes of clinical isolates obtained from hospitalized patients in Texas over the past seven years. Fifty hospitals located in Texas, USA sent C. difficile positive stool specimens to a centralized laboratory for PCR ribotyping and toxin characterization between 2011 and 2018. Data collected included specimen collection date, patient age, and sex. Strain genotypes were compiled, and changes in ribotype distribution over time were assessed. Overall, 7796 samples were ribotyped from predominately female patients (58.4%) aged 62 ± 19 years. Samples were obtained from all geographic regions of Texas including Houston/Southwest region (n = 5129; 85%), Dallas/North Texas (n = 579, 9.6%), Central Texas (n = 164; 2.7%), and South Texas (n = 162; 2.6%). The 10 most common ribotypes comprised 73% of all isolates tested during the study period. The most common ribotypes were 027 (17.5%), followed by 014–020 (16.1%), 106 (11.6%), and 002 (9.1%). The prevalence of ribotypes 027, 001, and 078–126 declined significantly over time, while ribotypes 106 and 054 increased in prevalence (P

Background Postprandial lipemia stimulates proinflammatory mediators and is a risk factor for cardiovascular disease. Chronic disease and diet are known to influence the gut microbial community in ways that alter the availability of bioactive compounds capable of influencing the host. The purpose of this study was to identify gut microbiome taxa and inflammatory cytokines differentiating individuals with lower and higher postprandial triglyceridemia. Methods A high-fat meal (43.1% fat) was given to 40 healthy, overweight and obese adults to assess the serum triglyceride response in the immediate four-hour postprandial period. Participants were categorized into two groups (high and low) based on serum triglyceride responses. We measured blood lipids, inflammatory cytokines, fat mass, visceral adiposity and used 16S rRNA target amplicon sequencing to identify microbial taxa in human fecal samples distinguishing the two groups. The gut microbiome was assessed using unconstrained ordination, followed by a high-dimensional class comparison to determine discriminative microbial features of the postprandial triglyceride response (ppTG). ResultsHigh ppTG responders had higher body mass index, visceral adiposity, and fasting serum cholesterol levels than low responders and had a decreased postprandial IL-17 response to the high-fat meal. The overall gut microbiome did not cluster by ppTG response but were found to have four discriminative bacterial features between high and low ppTG. Lower relative abundance of Clostridium Cluster XIVa and higher relative abundance of Pasteurellaceae, Alistipes , and Prevotella was observed in low ppTG relative to high ppTG. Conclusions Our findings suggest that specific gut microbial taxa involved in short-chain fatty acid production can discriminate the postprandial triglyceride response in overweight and obese adults. These findings may have implications in how we develop microbial therapies and choose to monitor and treat individuals with hypertriglyceridemia or who may have an increased risk of chronic disease.

Extraintestinal infection with Clostridium difficile has been reported but remains uncommon. Treatment of this unusual complication is complex given the limitations of current therapeutic options. Here we report a novel case of recurrent extraintestinal C. difficile infection that occurred following fecal microbiota transplantation. Using whole genome sequencing, we confirmed recrudescence rather than reinfection was responsible. The patient ultimately responded to prolonged, targeted antimicrobial therapy informed by susceptibility testing.