Dr. Lance Price, Director of Center for Food Microbiology and Environmental Health Translational Genomics Research Institute (TGen) presents his talk: Uncovering the impacts of circumcision on the penis microbiome

1. Uncovering the Impacts of Circumcision
on the Penis Microbiome
Lance B. Price, PhD
Director, Center for Microbiomics and Human
Health

2. Large of number AIDS-associated deaths:
• > 2.0 million people died due to AIDS in 2007.
A trend in decreasing number of new infections:
• 3.0 million in 2001 to 2.7 million in 2007.
• As HIV incidence rate stabilized since 2000, the overall number of
people living with HIV has steadily increased.
UNAIDS

3. The Rakai Uganda Study
• Randomized-controlled trial assessing the effect of
male circumcision on risk of HIV seroconversion in
adult men
– PI: Ron Gray
– Enrolled 4,996 healthy, HIV-negative, uncircumcised
men, aged 15-49
• Randomized into two study arms:
– Intervention arm: Immediate circumcision
– Control arm: Circumcision delayed 24 months
• Collected blood and coronal sulcus swabs every 6
months

4. Coronal sulcus
Circumcised Uncircumcised

5. Outcomes
• Nearly 60% reduction in HIV seroconversion in
circumcised men over the course of 24
months.
– Reduced rates of HPV in men
– Reduced rates of Herpes in men
– Reduced rates of Trichomonas vaginalis &
bacterial vaginosis in female partners

6. Models reduced seroconversion
• Removal of the foreskin = decreased mucosal surface area
• Changes in microbiome = decreased mucosal inflammation
• Keratinization of the inner foreskin = thicker dermal layer
protecting immune cells

8. Microbiome Study Design
• 77 men (control) and 79 men (intervention) from
the Rakai Study
– 312 Swab samples collected from the coronal sulcus
• Baseline
• 1 year
• Bacterial quantification
– 16S qPCR
• Bacterial community characterization
– 16S pyrosequencing

9. Bacterial load

10. -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9
p = 0.048
Control
Intervention
77
76
75
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Control
37
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2
1
156
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Intervention
107
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-5 0 5
Log Response Ratio (16S rRNA gene copy)

11. Community Analysis

12. Our approach: who then what
• Use 16S pyrosequencing to define the
community composition
• Characterize changes in abundance
• Use indicator species analysis to identify key
players
• Evaluate their role in the community and
relationship to the host

13. 454 sequencing of 16S amplicons
V6
V3
Phylogenetic Information V4 V5
0 200 400 600 800 1000 1200 1400 1600
Position in 16S rRNA gene

14. Analysis chain

15. Control Baseline Control Year 1
Campylobacteraceae
Peptostreptococcaceae
Streptococcaceae Prevotellaceae
Actinomycetaceae
Enterobacteriaceae Campylobacteraceae
Peptostreptococcaceae Prevotellaceae
Clostridiales.Family.XIb Streptococcaceae
Actinomycetaceae
Staphylococcaceae Enterobacteriaceae
Clostridiales.Family.XIb
Veillonellaceae Staphylococcaceae
Veillonellaceae
Bifidobacteriaceae Bifidobacteriaceae
Corynebacteriaceae Corynebacteriaceae
Porphyromonadaceae Porphyromonadaceae
Lactobacillaceae
Lactobacillaceae Clostridiales.Family.XIa
Unclassified.Clostridiales
Clostridiales.Family.XIa
Unclassified.Clostridiales
Intervention Baseline Intervention Year 1
Campylobacteraceae
Peptostreptococcaceae
Streptococcaceae
Actinomycetaceae
Enterobacteriaceae
Prevotellaceae
Clostridiales.Family.XIb Campylobacteraceae
Peptostreptococcaceae
Streptococcaceae Prevotellaceae
Staphylococcaceae Actinomycetaceae
Enterobacteriaceae
Veillonellaceae Clostridiales.Family.XIb
Staphylococcaceae
Bifidobacteriaceae Veillonellaceae
Bifidobacteriaceae Corynebacteriaceae
Porphyromonadaceae Corynebacteriaceae Porphyromonadaceae
Lactobacillaceae
Unclassified.Clostridiales Clostridiales.Family.XIa
Lactobacillaceae
Clostridiales.Family.XIa
Unclassified.Clostridiales

16. Control BL
Control Y1
0.6
0.4
0.2
nMDS axis 2
0.0
-0.2
-0.4
-0.6
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
nMDS axis 1

17. Intervention BL
0.6
Intervention Y1 (Post-Circ)
0.4
0.2
nMDS axis 2
0.0
-0.2
-0.4
-0.6
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
nMDS axis 1

18. nMDS plot of Baseline (BL) and Year 1 (Y1 ) Family-level Log-transformed Absolute Abundance Microbiota
Control BL
Control Y1
0.6
0.4
0.2
nMDS axis 2
0.0
-0.2
-0.4
-0.6
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Intervention BL
0.6
Intervention Y1 (Post-Circ)
0.4
0.2
nMDS axis 2
0.0
-0.2
-0.4
-0.6
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
nMDS axis 1

21. Activated Langerhans cells present intact HIV to TCells
Naïve Langerhans cells engulf and destroy HIV

22. Future Directions
• Inflammatory markers
• Langerhans cells recruitment and activation in foreskins
• Cell culture models of Langerhans activation
HIV MC-associated
genital bacterial
changes
Exciting, with low Exciting, with Exciting, with
potential impact potential impact high impact

23. Acknowledgements
• TGen/NAU • Hopkins/Rakai
– Cindy Liu – Ron Gray
– Bruce Hungate – Maria Wawer
– Maliha Aziz – Godfrey Kigozi
– Richard Lester – David Serwadda
– Tania Contente-Cuomo – Aaron Tobian
– Paul Keim
• Umaryland
– Jacques Ravel
Makerere University
School of Public Health