The clinical effectiveness of Fidaxomicin compared to Vancomycin in the treatment of Clostridioides difficile infection, a single center real-world experience.

BACKGROUND: The use of fidaxomicin is recommended as first line therapy for all patients with Clostridioides difficile infection (CDI). However, real-world studies have shown conflicting evidence of superiority. METHODS: We conducted a retrospective single center study of patients diagnosed with CDI between 2011-2021. A primary composite outcome of clinical failure, 30-day relapse or CDI-related death was used. A multivariable cause specific Cox proportional hazards model was used to evaluate fidaxomicin compared to vancomycin in preventing the composite outcome. A separate model was fit on a subset of patients with C. difficile ribotypes adjusting for ribotype. RESULTS: There were 598 patients included, of whom 84 received fidaxomicin. The primary outcome occurred in 8 (9.5%) in the fidaxomicin group compared to 111 (21.6%) in the vancomycin group. The adjusted multivariable model showed fidaxomicin was associated with 63% reduction in the risk of the composite outcome compared to vancomycin (HR = 0.37, 95% CI 0.17-0.80). In the 337 patients with ribotype data after adjusting for ribotype 027, the results showing superiority of fidaxomicin were maintained (HR = 0.19, 95% CI 0.05-0.77). CONCLUSION: In the treatment of CDI, we showed that real-world use of fidaxomicin is associated with lower risk of a composite endpoint of treatment failure.

The impact of 30-day antecedent antibiotic exposure on Clostridioidesdifficile ribotype patterns and the relationship with clinical outcomes: A single center study.

BACKGROUND: Antibiotic exposure is a known risk factor for Clostridioides difficile infection (CDI) and recurrence and can lead to infection with specific C. difficile strains. In this study, we sought to explore the relationship between antecedent antibiotic exposure and C. difficile antimicrobial resistance, and the impact of resistance on clinical outcomes. METHODS: This was a single center retrospective study evaluating patients with CDI between 2011 and 2021. A logistic regression model was used to evaluate the relationship between antecedent antibiotics in the 30 days prior to CDI and resistance among isolates. In addition, an exploratory analysis using a cause-specific Cox proportional hazards model evaluated the association between resistance and a composite outcome of clinical failure, relapse at 30 days or CDI-related death. RESULTS: we analyzed one isolate from 510 patients; resistance was noted in 339 (66.5 %) of the isolates. Exposure to fluoroquinolones and macrolides was associated with 2.4 (95 % CI 1.4-4.4) and 4.7 (95 % CI 1.1-20.5) increased odds of having resistance compared to other antibiotic class exposure, respectively. There were 58 (17.0 %) patients in the resistance group who developed the composite outcome and 24 (14.2 %) patients who lacked resistance who developed the composite outcome (HR 1.32, 95 % CI 0.81-2.14). CONCLUSION: These findings suggest that fluoroquinolone and macrolide exposure were significantly associated with isolating a resistant strain, but we did not find significant differences in clinical outcomes based on the presence of antimicrobial resistance.

Assessing the role of the gut microbiome in methylmercury demethylation and elimination in humans and gnotobiotic mice.

The risk of methylmercury (MeHg) toxicity following ingestion of contaminated foodstuffs (e.g., fish) is directly related to the kinetics of MeHg elimination among individuals. Yet, the factors driving the wide range of inter-individual variability in MeHg elimination within a population are poorly understood. Here, we investigated the relationship between MeHg elimination, gut microbiome demethylation activity, and gut microbiome composition using a coordinated human clinical trial and gnotobiotic mouse modeling approach together with metagenomic sequence analysis. We first observed MeHg elimination half-lives (t1/2) ranging from 28 to 90 days across 27 volunteers. Subsequently, we found that ingestion of a prebiotic induced changes in the gut microbiome and mixed effects (increased, decrease, and no effect) on elimination in these same individuals. Nonetheless, elimination rates were found to correlate with MeHg demethylation activity in cultured stool samples. In mice, attempts to remove the microbiome via generation of germ-free (GF) animals or through antibiotic (Abx) treatment both diminished MeHg demethylation to a similar extent. While both conditions substantially slowed elimination, Abx treatment resulted in significantly slower elimination than the GF condition, indicating an additional role for host-derived factors in supporting elimination. Human fecal microbiomes transplanted to GF mice restored elimination rates to that seen in control mice. Metagenomic sequence analysis of human fecal DNA did not identify genes encoding proteins typically involved in demethylation (e.g., merB, organomercury lyase). However, the abundance of several anaerobic taxa, notably Alistipes onderdonkii, were positively correlated with MeHg elimination. Surprisingly, mono-colonization of GF free mice with A. onderdonkii did not restore MeHg elimination to control levels. Collectively, our findings indicate the human gut microbiome uses a non-conventional pathway of demethylation to increase MeHg elimination that relies on yet to be resolved functions encoded by the gut microbes and the hostClinical Trial NCT04060212, prospectively registered 10/1/2019.

Loss of interleukin-10 receptor disrupts intestinal epithelial cell proliferation and skews differentiation towards the goblet cell fate.

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 ß-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.

Dynamic Gut Microbiome Changes in Response to Low-Iron Challenge.

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.

Determinants of the postprandial triglyceride response to a high-fat meal in healthy overweight and obese adults.

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 (ß = 0.53, p = 0.01), followed by aerobic exercise frequency (ß = 0.31, p = 0.05), insulin resistance based on HOMA-IR (ß = 0.30, p = 0.04), and relative exercise intensity at which substrate utilization crossover occurred (ß = 0.05, p = 0.04). For postprandial TG magnitude, visceral adiposity was a strong predictor (ß = 0.43, p < 0.001) followed by aerobic exercise frequency (ß = 0.23, p = 0.01), and exercise intensity for substrate utilization crossover (ß = 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.

Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor.

Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease.

Healthy human gut phageome.

The role of bacteriophages in influencing the structure and function of the healthy human gut microbiome is unknown. With few exceptions, previous studies have found a high level of heterogeneity in bacteriophages from healthy individuals. To better estimate and identify the shared phageome of humans, we analyzed a deep DNA sequence dataset of active bacteriophages and available metagenomic datasets of the gut bacteriophage community from healthy individuals. We found 23 shared bacteriophages in more than one-half of 64 healthy individuals from around the world. These shared bacteriophages were found in a significantly smaller percentage of individuals with gastrointestinal/irritable bowel disease. A network analysis identified 44 bacteriophage groups of which 9 (20%) were shared in more than one-half of all 64 individuals. These results provide strong evidence of a healthy gut phageome (HGP) in humans. The bacteriophage community in the human gut is a mixture of three classes: a set of core bacteriophages shared among more than one-half of all people, a common set of bacteriophages found in 20-50% of individuals, and a set of bacteriophages that are either rarely shared or unique to a person. We propose that the core and common bacteriophage communities are globally distributed and comprise the HGP, which plays an important role in maintaining gut microbiome structure/function and thereby contributes significantly to human health.

Antimicrobial susceptibility and ribotypes of Clostridium difficile isolates from a Phase 2 clinical trial of ridinilazole (SMT19969) and vancomycin.

Objectives: We evaluated the antimicrobial susceptibility and ribotypes of Clostridium difficile isolates from participants in a Phase 2 study of ridinilazole, a novel targeted-spectrum agent for treatment of C. difficile infection. Methods: Participants received ridinilazole (200 mg twice daily) or vancomycin (125 mg four times daily) for 10 days (ClinicalTrials.gov: NCT02092935). The MICs of ridinilazole and comparators for C. difficile isolates from stool samples were determined by agar dilution. Toxin gene profiling was performed by multiplex PCR and ribotype identification by capillary electrophoresis. Results: Eighty-nine isolates were recovered from 88/100 participants (one participant had two strains at baseline). The median colony count (cfu/g stool) was 1.9 × 104 (range: 2.5 × 102-7.0 × 106). Twelve participants (three received ridinilazole and nine received vancomycin) experienced recurrence, confirmed by immunoassays for free toxin in stool samples. The ribotype of eight out of nine isolates obtained at recurrence matched those of the initial isolates. All isolates, including those obtained at recurrence, were susceptible to ridinilazole within the expected range [median (range) MIC: 0.12 (0.06-0.5) mg/L]. The median (range) vancomycin MIC was 1 (0.5-4.0) mg/L. At baseline, 13.6% and 13.3% of samples in the ridinilazole and vancomycin groups were positive for VRE, increasing to 23.7% and 29.7% by day 40, respectively. Common ribotypes included 014-20 (14 isolates), 027 (13), 106 (7), 002 (7), 078-126 (4), 001 (4), 087 (3) and 198 (3). Toxin gene profiling of nearly all baseline isolates (98.9%) revealed a binary toxin gene (cdtA/cdtB) prevalence of 35%. Conclusions: Ridinilazole potently inhibited recovered C. difficile isolates. Recurrence was not associated with altered susceptibility.

Correction to: Risk Factors for Clostridium difficile Isolation in Inflammatory Bowel Disease: A Prospective Study.

The original version of the article unfortunately contained an error in a percentage value in Results section of Abstract.

Risk Factors for Clostridium difficile Isolation in Inflammatory Bowel Disease: A Prospective Study.

INTRODUCTION: Clostridium difficile is the most commonly isolated stool pathogen in inflammatory bowel disease (IBD). Traditional risk factors for C. difficile may not exist in patients with IBD, and no prior studies have assessed the risk factors for the isolation of C. difficile in both symptomatic and asymptomatic IBD outpatients. METHODS: We prospectively recruited consecutive IBD patients presenting to our outpatient clinic between April 2015 and February 2016. We excluded patients with a diverting ostomy or ileoanal pouch. Demographics, healthcare exposures, medical therapies and disease activity were recorded from medical charts or surveys. A rectal swab was performed from which toxigenic culture and PCR analysis for the presence of toxin and fluorescent PCR ribotyping were performed. The primary outcome of interest was isolation of toxigenic C. difficile. RESULTS: A total of 190 patients were enrolled in this prospective study including 137 (72%) with Crohn's disease and 53 (28%) with ulcerative colitis. At the time of enrollment, 69 (36%) had clinically active disease. Sixteen (8.4%) patients had toxigenic C. difficile isolated on rectal swab at enrollment and four (2.1%) patients had non-toxigenic C. difficile cultured. Mixed infection with more than one toxigenic isolate was present in 5/16 (31.3%) individuals. Patients with CD with a toxin positive isolate were more likely to have a history of CDI in the past 12 months (40 vs. 11.02%, p = 0.027) and an emergency department visit in the past 12 weeks (50 vs. 20.63%, p = 0.048). In UC, individuals with isolation of C. difficile were more likely to be hospitalized within the past 12 months (66.6 vs. 8.51%, p = 0.003). C. difficile isolation at the time of presentation was not associated with a subsequent disease relapse over a 6-month period in CD (p = 0.557) or UC (p = 0.131). CONCLUSION: Healthcare exposures remain a significant risk factor for C. difficile isolation in the IBD population; however, this was not associated with relapse of disease. Further studies assessing the clinical significance of C. difficile isolation is warranted in IBD.

A repeat offender: Recurrent extraintestinal Clostridium difficile infection following fecal microbiota transplantation.

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.

Clostridium difficile ribotype 027: relationship to age, detectability of toxins A or B in stool with rapid testing, severe infection, and mortality.

BACKGROUND: Clostridium difficile infection (CDI) can cause severe disease and death, especially in older adults. A better understanding of risk factors for adverse outcomes is needed. This study tests the hypotheses that infection with specific ribotypes and presence of stool toxins independently associate with severity and constructs predictive models of adverse outcomes. METHODS: Cases of non-recurrent CDI were prospectively included after positive stool tests for toxins A and/or B by enzyme immunoassay (EIA) or tcdB by polymerase chain reaction. Outcomes included severe CDI (intensive care unit admission, colectomy, or death attributable to CDI within 30 days of diagnosis) and 30-day all-cause mortality. Adjusted models were developed to test hypotheses and predict outcomes. RESULTS: In total, 1144 cases were included. The toxin EIA was positive in 37.2% and 35.6% of patients were of age >65 years. One of the 137 unique ribotypes was ribotype 027 (16.2%). Detectable stool toxin did not associate with outcomes. Adjusting for covariates, including age, Ribotype 027 was a significant predictor of severe CDI (90 cases; odds ratio [OR], 1.73; 95% confidence interval [CI], 1.03-2.89; P = .037) and mortality (89 cases; OR, 2.02; 95% CI, 1.19-3.43; P = .009). Concurrent antibiotic use associated with both outcomes. Both multivariable predictive models had excellent performance (area under the curve >0.8). CONCLUSIONS: Detection of stool toxin A and/or B by EIA does not predict severe CDI or mortality. Infection with ribotype 027 independently predicts severe CDI and mortality. Use of concurrent antibiotics is a potentially modifiable risk factor for severe CDI.

Evaluation of portability and cost of a fluorescent PCR ribotyping protocol for Clostridium difficile epidemiology.

Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.

Candidate mediators of chondrocyte mechanotransduction via targeted and untargeted metabolomic measurements.

Chondrocyte mechanotransduction is the process by which cartilage cells transduce mechanical loads into biochemical and biological signals. Previous studies have identified several pathways by which chondrocytes transduce mechanical loads, yet a general understanding of which signals are activated and in what order remains elusive. This study was performed to identify candidate mediators of chondrocyte mechanotransduction using SW1353 chondrocytes embedded in physiologically stiff agarose. Dynamic compression was applied to cell-seeded constructs for 0-30min, followed immediately by whole-cell metabolite extraction. Metabolites were detected via LC-MS, and compounds of interest were identified via database searches. We found several metabolites which were statistically different between the experimental groups, and we report the detection of 5 molecules which are not found in metabolite databases of known compounds indicating potential novel molecules. Targeted studies to quantify the response of central energy metabolites to compression found a transient increase in the ratio of NADP+ to NADPH and a continual decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. These data are consistent with the remodeling of cytoskeletal components by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads.

Genome sequencing of environmental Escherichia coli expands understanding of the ecology and speciation of the model bacterial species.

Defining bacterial species remains a challenging problem even for the model bacterium Escherichia coli and has major practical consequences for reliable diagnosis of infectious disease agents and regulations for transport and possession of organisms of economic importance. E. coli traditionally is thought to live within the gastrointestinal tract of humans and other warm-blooded animals and not to survive for extended periods outside its host; this understanding is the basis for its widespread use as a fecal contamination indicator. Here, we report the genome sequences of nine environmentally adapted strains that are phenotypically and taxonomically indistinguishable from typical E. coli (commensal or pathogenic). We find, however, that the commensal genomes encode for more functions that are important for fitness in the human gut, do not exchange genetic material with their environmental counterparts, and hence do not evolve according to the recently proposed fragmented speciation model. These findings are consistent with a more stringent and ecologic definition for bacterial species than the current definition and provide means to start replacing traditional approaches of defining distinctive phenotypes for new species with omics-based procedures. They also have important implications for reliable diagnosis and regulation of pathogenic E. coli and for the coliform cell-counting test.

Investigation of potentially pathogenic Clostridium difficile contamination in household environs.

As Clostridium difficile spores are resistant to many household cleaning products, the potential for community household contamination is high. The purpose of this study was to assess the prevalence of toxigenic C. difficile from environmental sources from a large urban area. Three to 5 household items or environmental dust was collected from 30 houses in Houston, Texas. A total of 127 environmental samples were collected from shoe bottoms (n = 63), bathroom surfaces (n = 15), house floor dusts (n = 12), or other household surfaces (n = 37). Forty one of 127 samples (32.3%) grew C. difficile. All 41 isolates were positive for toxin A and B genes and no isolate was positive for binary toxin genes. Shoe bottom swab samples had the highest percent of positive samples (25/63; 39.7%) followed by bathroom/toilet surfaces (5/15; 33.3%), house floor dust (4/12; 33.3%), and other surface swabs (7/37; 18.9%). Strains were grouped into 25 different ribotypes, the most prevalent type was 001 (5 strains). In conclusion, a high rate of environmental contamination of C. difficile was observed from community households from a large urban area.

Polyphenol-Rich Aronia melanocarpa Fruit Beneficially Impact Cholesterol, Glucose, and Serum and Gut Metabolites: A Randomized Clinical Trial.

Polyphenol-rich Aronia fruits have great potential as a functional food with anti-inflammatory, hypolipidemic, and hypoglycemic biologic activities. However, clinical intervention trials investigating the impact of Aronia fruit consumption on human health are limited. A randomized, controlled, double-blinded, parallel intervention trial was conducted using 14 human subjects who ingested either 0 mL or 100 mL of Aronia juice daily for 30 days. Anthropometric measurements, fasting, and postprandial measures of glucose and lipid metabolism and inflammation, 16S rRNA fecal microbial composition data, and mass spectrometry-acquired serum and fecal metabolomic data were collected before and after the intervention period. Data were analyzed using general linear models, ANOVA, and t-tests. Daily consumption of Aronia prevented a rise in cholesterol levels (ß = -0.50, p = 0.03) and reduced postprandial glucose (ß = -3.03, p < 0.01). No difference in microbial community composition by condition was identified at any taxonomic level, but a decrease (ß = -18.2, p = 0.04) in microbial richness with Aronia was detected. Serum and fecal metabolomic profiles indicated shifts associated with central carbon and lipid metabolism and decreases in pro-inflammatory metabolites. Our study further informs the development of polyphenol-based dietary strategies to lower metabolic disease risk.

A simplified protocol for deriving sterile, infectious murine Heligmosomoides polygyrus bakeri larvae.

Gastrointestinal helminth infection occurs within a diverse microbiome, complicating the interpretation of whether effects are caused by the parasite versus the microbial community. Here, we present a protocol for deriving sterile larvae of the murine helminth, Heligmosomoides polygyrus bakeri (H. polygyrus), providing experimental control of the microbiome. We describe steps for sterilizing with a bleach solution and developing into infectious larvae using E. coli. We then detail procedures for removing bacterial contaminants before harvesting to ensure the generation of germ-free larvae.

Effects of Early Life Exposures to the Aryl Hydrocarbon Receptor Ligand TCDF on Gut Microbiota and Host Metabolic Homeostasis in C57BL/6J Mice.

BACKGROUND: Exposure to persistent organic pollutants (POPs) and disruptions in the gastrointestinal microbiota have been positively correlated with a predisposition to factors such as obesity, metabolic syndrome, and type 2 diabetes; however, it is unclear how the microbiome contributes to this relationship. OBJECTIVE: This study aimed to explore the association between early life exposure to a potent aryl hydrocarbon receptor (AHR) agonist and persistent disruptions in the microbiota, leading to impaired metabolic homeostasis later in life. METHODS: This study used metagenomics, nuclear magnetic resonance (NMR)- and mass spectrometry (MS)-based metabolomics, and biochemical assays to analyze the gut microbiome composition and function, as well as the physiological and metabolic effects of early life exposure to 2,3,7,8-tetrachlorodibenzofuran (TCDF) in conventional, germ-free (GF), and Ahr-null mice. The impact of TCDF on Akkermansia muciniphila (A. muciniphila) in vitro was assessed using optical density (OD 600), flow cytometry, transcriptomics, and MS-based metabolomics. RESULTS: TCDF-exposed mice exhibited lower abundances of A. muciniphila, lower levels of cecal short-chain fatty acids (SCFAs) and indole-3-lactic acid (ILA), as well as lower levels of the gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), findings suggestive of disruption in the gut microbiome community structure and function. Importantly, microbial and metabolic phenotypes associated with early life POP exposure were transferable to GF recipients in the absence of POP carry-over. In addition, AHR-independent interactions between POPs and the microbiota were observed, and they were significantly associated with growth, physiology, gene expression, and metabolic activity outcomes of A. muciniphila, supporting suppressed activity along the ILA pathway. CONCLUSIONS: These data obtained in a mouse model point to the complex effects 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, with effects persisting into later life with associated health implications. https://doi.org/10.1289/EHP13356.