P4-S4.01 Investigation of the bacterial diversity in urine of urethritis patients and healthy controls using 454 high-throughput-sequencing

Abstract

Background: Non-gonococcal urethritis (NGU) is a common sexually transmitted disease in men but in 30-50% of NGU cases, no known organism is found. We have used 454 high throughput sequencing to analyse the micro flora in urine samples from cases of urethritis and from controls. Methods: Urine samples from men, 10 patients with urethritis and >5 PMNL/hpf and 10 healthy controls with <5 PMNL/hpf, were collected. All samples were tested for Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, U parvum, Trichomonas vaginalis, Herpes Simplex Virus type 1 and 2 and Adenovirus using specific PCR assays. The V3 and V4 regions of the 16S rRNA gene were PCR amplified, tagged and sequenced using the Titanium kit and GS FLX pyrose-quencing system (Roche) according to manufacturer's instructions. Sequences were analysed using the RDP Pyrosequencing Pipeline and CLC Genomics Workbench. Results: From each of the 20 samples, 8150 quality filtered sequences were randomly selected. Sequences were assigned to the genus level using the RDP Classifier. A total of 172 gen-era were identified, 133 in patients and 104 in controls. The median number of genera was 35.5 (19-49) in patients with urethritis compared to 25 (16-43) in the controls. No single genus was present in all samples. Members of the genera Pseudomonas and Sphingomonas were present in all the controls and in most of the patient samples. Also, representatives of the genera Brevundimonas, Micrococcus, Bradyrhizobium and Chryseobacterium were present in 15-18 of the samples. Of the 172 genera, 77 were only found in a single sample and 33 were found in two different samples. Rarefaction analysis at the 5 % level (comparable to genus) suggested that all the controls and most urethritis samples harboured each between 31 and 125 different groups. Four of the urethritis samples apparently contained between 150 and 300 different groups. Conclusions: Urine, even from healthy men, contains a very diverse micro-flora. Though not statistically significant, the total and median number of genera was found higher in patients with urethritis than in controls. Several widespread genera are likely to represent commensals and bacteria present in the environment.