Project Summary Over 250 million courses of antibiotics are prescribed annually to out-patients in the United States. The perception that antibiotic use has minimal adverse side effects contributes to their over-use. Since deploying antibiotics seems to be relatively free of toxicity, there are few disincentives to their use even when perceived benefits are marginal. Recent understanding of the human microbiome suggests the relevance of antibiotic exposures to health, with the emerging view that our microbiota are central to human physiology. In animal models, perturbing the microbiota affects metabolic, immune, and cognitive physiology. Antibiotics diffuse into all body compartments, selecting for resistance. We propose to examine the effects of two commonly used antibiotics, a beta-lactam (amoxicillin)and a macrolide (azithromycin) on microbial populations and on metabolic and immune physiology, in healthy volunteers in a prospective randomized clinical trial conducted at the NIH Clinical Center (CC). We hypothesize that in addition to acutely perturbing the human microbiome, these agents will have measurable metabolic and immunologic effects, with effects continuing over the following weeks and months. To test this hypothesis, in Aim 1, we assess the effects of a brief therapeutic antibiotic course on microbiota and metagenome composition. After initial evaluation, antibiotics are given for 5-7 days, with a prolonged post-treatment evaluation. Specimens are obtained from multiple sites at each of 10 time-points and used to estimate bacterial and fungal composition and gene content. In Aim 2, we assess the effects of the antibiotic course on immune physiology. At multiple time points, blood is obtained and used to determine plasma and cellular levels of markers of both innate and adaptive immunity. In Aim 3, we assess the effects of the antibiotic course on metabolic physiology. The specimens obtained are assessed for markers of metabolic and hormonal physiology. A subset of subjects enter the unique CC Metabolic Chamber to quantify 24-hour energy expenditure and carbohydrate and fat utilization. In addition to the primary data analyses, we will build an informatic model integrating the temporal data to provide insight into the complex interdependent physiology between microbiome and host. This project is an opportunity to perform comprehensive and integrated evaluations of two pharmacologic agents prescribed >90 million times annually in the USA. Careful analysis and development of an integrated model to understand the pathophysiology of the perturbations may identify problems below the radar in clinical medicine. The purpose of this renewal is to recruit sufficient numbers of subjects to complete the study after the interruption caused by COVID-19 and the subsequent COVID studies in the Clinical Center. Increasing the number of subjects to ~20 per group will permit robust analyses of the extent and duration of microbiome changes due to the an...