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The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at -80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or -80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or -80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Resident microbiota activate regulatory cells that modulate intestinal inflammation and promote and maintain intestinal homeostasis. IL-10 is a key mediator of immune regulatory function. Our studies described the functional importance and mechanisms by which gut microbiota and specific microbial components influenced the development of intestinal IL-10-producing B cells. We used fecal transplant to germ-free (GF) Il10+/EGFP reporter and Il10-/- mice to demonstrate that microbiota from specific pathogen-free mice primarily stimulated IL-10-producing colon-specific B cells and T regulatory-1 cells in ex-GF mice. IL-10 in turn down-regulated microbiota-activated mucosal inflammatory cytokines. TLR2/9 ligands and enteric bacterial lysates preferentially induced IL-10 production and regulatory capacity of intestinal B cells. Analysis of Il10+/EGFP mice crossed with additional gene-deficient strains and B cell co-transfer studies demonstrated that microbiota-induced IL-10-producing intestinal B cells ameliorated chronic T cell-mediated colitis in a TLR2, MyD88 and PI3K-dependent fashion. In vitro studies implicated PI3Kp110δ and AKT downstream signaling. These studies demonstrated that resident enteric bacteria activated intestinal IL-10-producing B cells through TLR2, MyD88 and PI3K pathways. These B cells reduced colonic T cell activation and maintained mucosal homeostasis in response to intestinal microbiota.

The second-generation antipsychotic olanzapine is effective in reducing psychotic symptoms but can cause extreme weight gain in human patients. We investigated the role of the gut microbiota in this adverse drug effect using a mouse model. First, we used germ-free C57BL/6J mice to demonstrate that gut bacteria are necessary and sufficient for weight gain caused by oral delivery of olanzapine. Second, we surveyed fecal microbiota before, during, and after treatment and found that olanzapine potentiated a shift towards an \"obesogenic\" bacterial profile. Finally, we demonstrated that olanzapine has antimicrobial activity in vitro against resident enteric bacterial strains. These results collectively provide strong evidence for a mechanism underlying olanzapine-induced weight gain in mouse and a hypothesis for clinical translation in human patients.

Giardia lamblia (Giardia) is among the most common intestinal pathogens in children in low- and middle-income countries (LMICs). Although Giardia associates with early-life linear growth restriction, mechanistic explanations for Giardia- associated growth impairments remain elusive. Unlike other intestinal pathogens associated with constrained linear growth that cause intestinal or systemic inflammation or both, Giardia seldom associates with chronic inflammation in these children. Here we leverage the MAL-ED longitudinal birth cohort and a model of Giardia mono-association in gnotobiotic and immunodeficient mice to propose an alternative pathogenesis of this parasite. In children, Giardia results in linear growth deficits and gut permeability that are dose-dependent and independent of intestinal markers of inflammation. The estimates of these findings vary between children in different MAL-ED sites. In a representative site, where Giardia associates with growth restriction, infected children demonstrate broad amino acid deficiencies, and overproduction of specific phenolic acids, byproducts of intestinal bacterial amino acid metabolism. Gnotobiotic mice require specific nutritional and environmental conditions to recapitulate these findings, and immunodeficient mice confirm a pathway independent of chronic T/B cell inflammation. Taken together, we propose a new paradigm that Giardia -mediated growth faltering is contingent upon a convergence of this intestinal protozoa with nutritional and intestinal bacterial factors. Giardia lamblia intestinal infection is independently associated with faltering linear growth in children in low-middle income countries, yet the mechanistic pathway has not been clearly identified. Authors utilise the MAL-ED cohort, and a gnotobiotic murine model, to explain Giardia -induced effects on childhood growth.

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.

Perinatal perceived stress can contribute to worse health outcomes for the parent–child dyad. Given the emerging relationship between the microbiota-gut-brain axis and stress, this study sought to elucidate connections between bowel symptoms and the gut microbiome in relation to perceived stress at three time points in the perinatal period: two during pregnancy and one postpartum. Ninety-five pregnant individuals participated in a prospective cohort study from April 2017 to November 2019. Researchers assessed Perceived Stress Scale-10 (PSS); bowel symptoms (according to the IBS Questionnaire); psychiatrist assessment of new onset or exacerbated depression and anxiety; and fecal samples analyzed for alpha diversity (measures of gut microbiome diversity utilizing Shannon, Observed OTUs, and Faith’s PD) at each timepoint. Covariates included weeks of gestation and weeks postpartum. PSS scores were divided into “Perceived Self-Efficacy” and “Perceived Helplessness.” Increased gut microbial diversity was associated with decreased bowel symptoms, decreased overall perceived stress, increased ability to cope with adversity, and decreased distress in the postpartum period. This study found a significant association between a less diverse microbial community, lower self-efficacy early in pregnancy, and greater bowel symptoms and perceived helplessness later in the perinatal period, relationships that may ultimately point to novel diagnostic methods and interventions for perceived stress based on the microbiota-gut-brain axis.

Objective Although polymorphisms of the NOD2 gene predispose to the development of ileal Crohn's disease, the precise mechanisms of this increased susceptibility remain unclear. Previous work has shown that transcript expression of the Paneth cell (PC) antimicrobial peptides (AMPs) α-defensin 4 and α-defensin-related sequence 10 are selectively decreased in Nod2−/− mice. However, the specific mouse background used in this previous study is unclear. In light of recent evidence suggesting that mouse strain strongly influences PC antimicrobial activity, we sought to characterise PC AMP function in commercially available Nod2−/− mice on a C57BL/6 (B6) background. Specifically, we hypothesised that Nod2−/− B6 mice would display reduced AMP expression and activity. Design Wild-type (WT) and Nod2−/− B6 ileal AMP expression was assessed via real-time PCR, acid urea polyacrylamide gel electrophoresis and mass spectrometry. PCs were enumerated using flow cytometry. Functionally, α-defensin bactericidal activity was evaluated using a gel-overlay antimicrobial assay. Faecal microbial composition was determined using 454-sequencing of the bacterial 16S gene in cohoused WT and Nod2−/− littermates. Results WT and Nod2−/− B6 mice displayed similar PC AMP expression patterns, equivalent α-defensin profiles, and identical antimicrobial activity against commensal and pathogenic bacterial strains. Furthermore, minimal differences in gut microbial composition were detected between the two cohoused, littermate mouse groups. Conclusions Our data reveal that Nod2 does not directly regulate PC antimicrobial activity in B6 mice. Moreover, we demonstrate that previously reported Nod2-dependent influences on gut microbial composition may be overcome by environmental factors, such as cohousing with WT littermates.

This study examined associations between the composition and diversity of the intestinal microbiota and measures of depression, anxiety, eating disorder psychopathology, stress, and personality in a group of healthy adult females. Female participants (n = 91) ages 19-50 years with BMI 18.5-25 kg/m2 were recruited from central North Carolina between July 2014 and March 2015. Participants provided a single fecal sample and completed an online psychiatric questionnaire that included five measures: (i) Beck Anxiety Inventory; (ii) Beck Depression Inventory-II; (iii) Eating Disorder Examination-Questionnaire; (iv) Perceived Stress Scale; and (v) Mini International Personality Item Pool. Bacterial composition and diversity were characterized by Illumina sequencing of the 16S rRNA gene, and associations were examined using Kendall's tau-b correlation coefficient, in conjunction with Benjamini and Hochberg's False Discovery Rate procedure. We found no significant associations between microbial markers of gut composition and diversity and scores on psychiatric measures of anxiety, depression, eating-related thoughts and behaviors, stress, or personality in a large cohort of healthy adult females. This study was the first specifically to examine associations between the intestinal microbiota and psychiatric measures in healthy females, and based on 16S rRNA taxonomic abundances and diversity measures, our results do not suggest a strong role for the enteric microbe-gut-brain axis in normal variation on responses to psychiatric measures in this population. However, the role of the intestinal microbiota in the pathophysiology of psychiatric illness may be limited to more severe psychopathology.

Background The Binge Eating Genetics Initiative (BEGIN) is a multipronged investigation examining the interplay of genomic, gut microbiota, and behavioral factors in bulimia nervosa and binge-eating disorder. Methods 1000 individuals who meet current diagnostic criteria for bulimia nervosa or binge-eating disorder are being recruited to collect saliva samples for genotyping, fecal sampling for microbiota characterization, and recording of 30 days of passive data and behavioral phenotyping related to eating disorders using the app Recovery Record adapted for the Apple Watch. Discussion BEGIN examines the interplay of genomic, gut microbiota, and behavioral factors to explore etiology and develop predictors of risk, course of illness, and response to treatment in bulimia nervosa and binge-eating disorder. We will optimize the richness and longitudinal structure of deep passive and active phenotypic data to lay the foundation for a personalized precision medicine approach enabling just-in-time interventions that will allow individuals to disrupt eating disorder behaviors in real time before they occur. Trial registration The ClinicalTrials.gov identifier is NCT04162574 . November 14, 2019, Retrospectively Registered.

Higher stress during pregnancy associates with negative outcomes and elevated inflammation. The gut microbiota, reflecting environment and social interactions, alongside host immune responses have the potential to better understand perceived stress and identify when stress is excessive in pregnancy. Two U.S. cohorts of 84 pregnant individuals, composed of urban women of color and suburban white women, completed the Perceived Stress Scale-10 (PSS-10) and provided fecal and blood samples at two time points. Confirmatory Factor Analysis assessed the robustness of a two-factor PSS-10 model (Emotional Distress/ED and Self-Efficacy/SE). Gut microbiota composition was measured by 16 S rRNA amplicon sequencing and the immune system activity was assessed with a panel of 21 T-cell related cytokines and chemokines. ED levels were higher in the suburban compared to the urban cohort, but levels of SE were similar. ED and SE levels were associated with distinct taxonomical signatures and the gut microbiota data improved the prediction of SE levels compared with models based on socio-demographic characteristics alone. Integration of self-reported symptoms, microbial and immune information revealed a possible mediation effect of Bacteroides uniformis between the immune system (through CXCL11) and SE. The study identified links between distinct taxonomical and immunological signatures with perceived stress. The data are congruent with a model where gut microbiome and immune factors, both impacting and reflecting factors such as close social relationships and dietary fiber, may modulate neural plasticity resulting in increased SE during pregnancy. The predictive value of these peripheral markers merit further study.