Title: A New Look at An Ancient Practice: The Impact of Male

Title: A New Look at An Ancient Practice: The Impact of Male Circumcision on
the Genital Microbiota
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.