1. Exploring the Role of the Microbiome in Male Genital Tract Disease Lisa E. Manhart, PhD 17 th IUSTI World Congress - Marrakech 11 May 2016

2. Hologic, Inc. – test kits, reagents, equipment, service on scientific advisory board Qiagen, Inc. – service on scientific advisory board Disclosures

3. What is the microbiome?

4. Which part of the male genital tract? Urethra Penile Shaft and Coronal Sulcus Semen Which clinical entities? Urethritis HIV infection Male Infertility What do we know about the male genital tract microbiome?

5. Urethritis  Most common male STI syndrome 1  Characterized by: Urethral discharge Dysuria Pruritis  Definition: Urethral symptoms, visible discharge and/or ≥5 PMNs/HPF on Gram stained smear of urethral exudates 2 (2015 update to CDC STD Treatment Guidelines 2 recommends lower cutpoint of ≥2 PMNs/HPF) 1 CDC STD Surveillance 2001; 2 Martin, STD 2008; 2 CDC STD Treatment Guidelines

6. Urethritis: Consequences  Clinical sequelae rare Epididymitis (1-2%) 1 Urethral stricture (0.6%) 2  Increased risk of HIV shedding 3 Seminal HIV RNA decreases after successful treatment of urethritis  Transmission of pathogens to sex partners 1 Martin, STD 2008; 2 Santucci, J Urol 2007; 3 Cohen, Lancet 1997

7. Etiology of NGU  Chlamydia trachomatis considered major cause of NGU, but up to half of NGU cases have no identified etiology 1  ~40% men with NGU in PHSKC STD Clinic had no detected pathogen 2 1 Martin, STD 2008; 2 Manhart, CID 2013

8.  Regular PCR targets a known sequence of the bacterial chromosome Specific PCR test for each bacterium  Broad range PCR targets known conserved sequences that bracket regions of the bacterial chromosome that are unique to each bacterium Match unique sequences with database to identify bacteria Identify all the bacteria present in an anatomic site 16S rRNA gene Known “conserved sequences”Unique sequence Broad Range 16S rRNA Gene PCR

9. Bacterial Vaginosis (BV)  Most common female reproductive tract condition 29.9% prevalence in women age 14-49 participating in NHANES 1  Several recently identified bacteria are highly predictive of BV; more common in women with cervicitis 2, 3 1 Koumans STD 2007; 2 Fredricks NEJM 2005; 3 Gorgos, ISSTDR 2009 Bacterium BV 2 AOR (95% CI) More common in women with cervicitis 3 BVAB1 19.0 (2.2 – 910.7)- BVAB2 106.1 (14.3 – 4755.1)++ BVAB3 21.9 (2.5 – 1056.4)++ Atopobium spp 95.0 (14.5 – ∞ )++ Egerthella-like spp 103.8 (13.5 – 4812.8)- Leptotrichia spp 330.6 (23.1 – ∞ )++ Megasphaera α 134.4 (16.6 – 6509.8)++

10. BVABs and NGU Men With (n=157) & Without Urethritis(n=102) OR = 2.9 (1.1-7.4) Manhart et al., Sex Transm Dis 2013;40(12):944-9

11. Etiologies of NGU Manhart et al., Sex Transm Dis 2013;40(12):944-9

12. Urine Microbiome in 19 Asymptomatic Men Using Broad Range PCR Nelson D PLoS One 2010;5(11)e14166 7 bacterial phyla detected Domain Kingdom Phylum Class Order Family Genus Species

13. Urine Microbiome in Asymptomatic Men Nelson D PLoS One 2010;5(11)e14166 25 most common bacterial genera Top 5 (rank abundance) 1.L. iners (12.6%) 2.Sneathia sp. (5.7%) 3.Gemella sp. (5.5%) 4.Aerococcus sp. (52%) 5.Corynebacteria sp. (4.6%)

14. Men with Asymptomatic STI have Different Urine Microbiomes Nelson D PLoS One 2010;5(11)e14166

15. Implications Does a characteristic microbiome enhance susceptibility to STI? OR Does acquisition of STI change the microbiome?

16. Srinivasan PLoS One 2012;7(6):e37818 Microbiota of Women with BV

17. Heterosexual men with NGU have less diverse urethral microbiota Cases Controlsp Median diversity index1.1452.2290.03

18. Penile Microbiota & BV in Partners Penile microbiota of 165 HIV- uncircumcised men yielded 7 community state types (CST) using hierarchical clustering Liu et al. mBio 2015;6(3):e00589-15 Low DensityHigh Density

19. Penile Microbiota & BV in Partners High density CSTs (4 – 7) associated with having a partner with BV PRR = 1.55; 95% CI 1.07 – 2.24 (relative to CST 1-3) High prevalence of BVABs in men with CSTs 4 – 7 Among men with CST 4 – 7, those with a partner with BV had increased bacterial density Liu et al. mBio 2015;6(3):e00589-15 CST 4-7

20. Circumcision and the Penis Microbiome Circumcision was associated with a decrease in anaerobic bacteria Circumcision also associated with decreased diversity of bacterial species Price et al. PLoS One 2010;5(1):e8422 Bacterial Family Oxygen Requirement Pre-circumcision Mean Abundance (SD) Post-circumcision Mean Abundance (SD) Wilcoxon Signed-Rank Test Aerobic236.4 (119.1)466.8 (65.6)p=0.62 Anaerobic71.8 (69.6)4.8 (6.2)p=0.014 Facultative anaerobic22.8 (19.8)78.9 (53.4)p=0.006

21. Semen Microbiome & Infertility More diverse than vaginal microbiota (90 vs. 12 unique phylotypes), but lower bacterial concentrations 1 6 clusters identified in fertile and infertile men 2 Clusters not associated with fertility Only Anaerococcus associated with reduced sperm quality, p=0.0012 More bacteria than sperm 1 Mandar et al, Res Microbiol 2015;166(5):440-447; 2 Hou et al, Fert Steril 2013;100(5):1261-1269

22. Semen Microbiome and Cytokines HIV-uninfected men: no relationship between semen bacterial load and cytokine levels HIV-infected men: For each 10% increase in semen bacterial load, 2% increase in IL-6 IL-17 IP-10 IL1b (3% increase) MIG IL-10 TNF- α Component 1 Component 2 Component 3 Correlated with HIV viral load Liu et al. PLoS Pathog 2014;10(7):e1004262

23. Urine microbiome in men with and without STI differs but…. which came first? Urethritis may be characterized by lower diversity of bacterial species Anaerobes & overall species diversity reduced in circumcised men Female partners with BV associated with a higher density of bacteria detected on penis and BVABs Higher bacterial load in semen associated with increased pro- inflammatory cytokines in HIV+ men Most data on male genital tract microbiome is cross-sectional Longitudinal studies are needed to determine causality What do we know about the male genital tract microbiome?

24. THANKS! David Fredricks Sujatha Srinivasan Tina Fiedler Matt Munch Congzhou Liu M. Sylvan Lowens Jennifer Morgan Matthew Golden Julie Dombrowski Laura Chambers Tashina Robinson Sarah Romano Jeanne Marrazzo Anna Wald Jim Hughes Sean Proll Patricia Totten Olusegun Soge PHSKC STD Clinic Hologic, Inc. donated test kits and reagents NIH/NIAID 1R01AI110666-01 1U19AI113173-01