Exploring the Bacterial Diversity of the Male Urethra During Idiopathic Urethritis

Abstract

Idiopathic urethritis (IU) comprises up to 50% of symptomatic cases of male urethritis in clinical settings. The syndrome is of an unknown etiology but may be due to an as yet unidentified bacterial pathogen(s). We were interested in identifying pathogens that could cause IU using multiple methods. Shotgun metagenomic sequencing or 16S rRNA sequencing methods can provide rich datasets but are limited by the completeness of the corresponding sequence reference databases. We generated metagenomic and 16S datasets from DNA extracted from urethral swabs of men with IU to determine the composition of their urethral microbiome. In order to enrich the corresponding reference databases used to identify the reads in the sequence datasets, I cultivated bacteria from the first void urine (FVU) of men with IU. My goal was to grow and whole genome sequence bacterial isolates that are not currently represented in the reference databases. Of the 216 men we enrolled at the Bell Flower STD clinic in Indianapolis, IN, 59 men had IU. I grew a total of 802 isolates from the FVU of the IU patients and identified those isolates using colony-based 16S rRNA PCR. Based on % sequence similarity to the nearest type strain, I sorted the 16S alleles into four categories: Species (≥98 % identity) (N=264), Genus (≥95 % identity) (N=407), Closest Match (<95 % identity) (N=95), and No Hit (0 % identity) (N=22). There were 24 genera represented in the isolate collection. Of these, the six most abundant genera were Streptococcus, Staphylococcus, Corynebacterium, Haemophilus, Gardnerella, and Prevotella. These six genera composed nearly 80% of all IU-associated isolates. All sequences below 98% sequence similarity represent potentially novel strains of bacteria. We will proceed with whole genome sequencing of bacterial isolates with the goal of improving genome database coverage of bacterial diversity in the male urethra.