1. Findings from the Male Urethral Microbiome Project Barbara Van Der Pol, PhD, MPH (HPER) David E Nelson, PhD (Biology)

2. Urethritis Inflammatory response in the urethra causing discharge, pain on urination and burning/itching Leading cause of male healthcare office visits in US ▫69.5% of initial visits in Massachusetts HMO* <20% had identifiable infection *Ratelle, et al STD 2001

3. What causes urethritis? Pathogens?Host Response?

4. Collaboration is Essential Clinical/Epidemiologic questions ▫Why are some men at greater risk of urethritis? ▫How does this impact STI control efforts? ▫Need patient samples and data Molecular tools provide answers ▫What microbial communities are related to these syndromes? ▫How can we identify & quantify them?

5. Methods Enrolled men from STD clinic population ▫Collected paired samples, behavioral data, sexual history, urethral health and prostatitis history ▫Ran STD diagnostic assays Samples sent in nucleic acid preservative to Dave Nelson’s lab ▫DNA isolated/quantified ▫PCR amplified using broad-range 16S primers ▫Deep pyrosequenced specimens at the IU CGB ▫Analysis

7. Bacterial identification: 16S rRNA

8. Nelson et al, 2010 PloS One What we found in adult men

10. What is Urine Measuring? Bladder Urethra External penis Hypothesis: Urine predominately samples the urethra

11. Urine versus Swab Which would YOU choose?

12. PloS One in press Profile of the Communities in Men w/ STI

13. Profile of the Communities in Men w/o STI

14. Urine and Swab samples give similar results on an individual level The closest match of most individual urine or swab specimens was usually the corresponding specimen Of the 22 specimen pairs from STI negative men, only two differed by Kolmogorov-Smirnov test ▫subject#15, p=0.0497 ▫subject #26, p=0.0246

15. Heat Map of Patterns from Men w/ & w/o STI

16. Urine and Swab samples give similar results on an individual level Clustering analysis indicated that the two specimens from subject 15 were more similar to each other than to all other specimens in the group of healthy men

17. “Vagina Monlogues” Redux H+ L. crispatusN. gonnorhoeae

18. Applying what we have learned Have demonstrated utility of urine sampling Have shown differences among men based on STI status Have identified further differences among men WITH urethritis Have begun to identify pathogens similar to those seen in vaginal syndromes Have begun longitudinal analysis of microbiome in young men

19. A good investment? UH3 funded (Aug 2010-2013, $5.4 million TC) Manuscripts: ▫Preliminary exploration of male urethral microbiome (PLoS One Dec, 2010) ▫Comparison of urethral swabs to urine (PloS One in press) ▫Unique composition of microbiome in urethritis related to STI (in prep) ▫Baseline analysis of young men: differences based on sexual behavior & circumcision status (in prep) Presentations: ▫NIH Human Microbiome Symposium 2011 [3 posters, 1 oral presentation] ▫ISSTDR London [1 poster, 1 oral presentation] ▫Midwest Microbial Pathogenesis Conference [1 poster]

20. Summary Through collaboration comes synergy Assistance with experimental design (with analysis in mind) is key Opportunities such as CTSI PDT can open MANY doors Long-term outcome still in-process!

21. Thanks! CTSI PDT JD Fortenberry (IU Adolescent Medicine) Q Dong (UNT Bioinformatics) BP Katz(IU Biostatistics) Evelyn Toh(IU Biology) George Weinstock (WUStL Genomics)