Title: A New Look at An Ancient Practice: The Impact of Male Circumcision on the Genital Microbiota Abstract: Background: An ancient practice with important cultural, social, and religious implications, male circumcision (MC) has a new role as an effective preventative strategy against HIV acquisition. As numerous challenges make its populationbased implementation unlikely, the search is on for non-surgical alternatives for HIV prevention. In the current study, we assess the impact of male circumcision on the male genital microbiota to better understand one of the potential biological mechanisms through which male circumcision confers its protective effect. Hypothesis: Male circumcision decreases risk for HIV acquisition in HIV-negative men by reducing the load of pro-inflammatory genital anaerobic bacteria, thereby decreasing genital mucosal inflammation and Langerhans cell activation. Study Design: Retrospective analysis of prospectively collected coronal sulci swabs from the Rakai Male Circumcision Randomized-Controlled Trial. A baseline and a follow-up (Year 1) sample were analyzed from 31 individuals each from the Control and the Intervention groups. Methods: DNA was isolated and purified from 250ul of swab eluent using a combined mechanical and enzymatic lysis protocol. Bacterial load was quantified using an in-house pan-bacterial quantitative real-time PCR (qPCR) assay. Bacterial community was characterized using 16S rRNA gene-based pyrosequencing analysis targeting the V3V4 region. Sequences were processed and quality-checked using in-house scripts and ChimeraSlayer and binned according to sample barcodes. Taxonomic classification was performed using the Ribosomal Database Project Naïve Bayesian Classifier using ≥80% bootstrap confidence-level and an abundance-based data matrix was generated at each taxonomic level. Sampling efforts were assessed using species accumulation curve. Family-level microbiota was assessed using heatmap visualization, nonmetric multi-dimensional scaling, and permutational analysis of variance. Indicator species analysis was used to identify bacterial families uniquely present in each state. Assessment of unique OTUs in each indicators species family was performed using maximum-parsimony algorithm in MEGA4. Results: A total of 118,966 V3V4 reads were obtained from 124 samples. Results form the 16S qPCR showed that the bacterial load decreased significantly postcircumcision (one-tailed Wilcoxon Ranked-Sum Test; p = 0.025). Overall, three major coronal sulci microbiota types were identified, with one type found only in the uncircumcised state. Male circumcision significantly changed the overall microbiota in the intervention group (PerMANOVA; p < 0.01) and reduced the inter-individual heterogeneity, while no significantly change in microbiota was observed in the control group between baseline and follow-up. Eight indicator species (bacterial families) were identified for the uncircumcised state and three were identified for the circumcised state. Many of the bacterial OTUs from these indicator families do not have an identifiable genus classification. Discussion: Male circumcision significantly lowers the coronal sulci bacterial load and alters the microbiota. Future directions include additional characterization of the unclassified sequence types, as well as assessing the association of genital microbiota on inflammatory markers, genital mucosal inflammation, and Langerhans cell activation.