Differences in Vaginal Bacterial Communities of Women in North America: Implications for disease diagnosis and prevention

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1 Differences in Vaginal Bacterial Communities of Women in North America: Implications for disease diagnosis and prevention Larry J. Forney, Pawel Gajer, Christopher J. Williams, Maria G. Schneider, Stacey L. McCulle, Shara Karlebach, Alison A. Dormer, Reshma Gorle, Jennifer Russell, Catherine C. Davis, Carol O. Tacket, Ligia Peralta, Kevin A. Ault, and Jacques Ravel

2 Microbial cells in and on the human body (10 12 ) outnumber human cells by a factor of ten to one. Dethlefsen et al. An ecological and evolutionary perspective on human microbe mutualism and disease. Nature (2007) vol. 449 (7164) pp

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4 Birth Sterile Puberty Increased estrogen; colonization and succession Reproductive age Glycogen metabolism to produce lactic acid Menopause Decreased estrogen; less lactic acid

5 Host Environment Microbial Communities

6 The bacterial communities normally found in the vagina represent a first line of defense against infectious diseases affecting the female reproductive tract. These include: Yeast infections (candidiasis) Sexually transmitted diseases (syphilis, gonorrhea, chlamydia, trichomoniasis, HIV) Pelvic inflammatory disease (PID)

7 An ecological community is defined as a group of actually or potentially interacting species living in the same place. A community is bound together by the network of influences that species have on one another. Inherent in this view is the notion that whatever affects one species also affects many others -- the "balance of nature".

8 Can not discriminate between normal and abnormal conditions until normal conditions have been accurately defined. Questions Are there different kinds of communities in different healthy women? If so, are the communities functionally equivalent? Are there differences among racial groups? Do these differences have important consequences for women s health?

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10 Disturbed ecosystem (1) Increased risk to disease (2) May account for BV symptoms Intensity Normal: dynamic equilibrium Disturbances to the microbiome are often imposed by human actions: - Hygiene - Diet - Sexual practices Frequency or Duration But also include natural conditions: - Menstruation - Parturition - Hormone variation

11 Resilience is the amount of disturbance that an ecosystem can withstand without changing its self-organizing processes. 1. Not all disturbances have equal intensity, occur at the same frequency, or endure for the same length of time. Therefore, not all disturbances have the same consequences. 2. Communities with fundamental differences in species composition and structure will differ in resilience. Holling, C.S Resilience and stability of ecological systems. Annual Review of Ecology and Systematics. 4:1-23.! Disturbed communities are more readily invaded by weedy species (opportunistic pathogens).

12 AIMS STUDY DESIGN Are there differences in the kinds of bacterial communities found in women of different ethnic groups? Are there associations between kinds of bacterial communities, vaginal ph, and Nugent scores? Cross sectional study of >400 asymptomatic women Four ethnic groups: Caucasian, Black, Hispanic, Asian Self-collected mid-vaginal swabs Determine bacterial community composition Vaginal ph measurements Nugent scores

13 Pyrosequencing of V1-V2 region of bacterial 16S rrna genes Vaginal swab Total genomic DNA PCR with universal 16S rrna gene primers Pyrosequence amplicons [~2,200 sequences per sample] Classification of gene sequences

14 Percentage of community

15 Cluster size Major taxa Not dominated by Lactobacillus, higher proportions of non-lactobacillus species Lactobacillus iners and others Lactobacillus crispatus Lactobacillus iners Lactobacillus gasseri Lactobacillus iners and Lactobacillus crispatus Lactobacillus jensenii All include significant proportion of lactic acid bacteria, though not necessarily species of Lactobacillus. These populations constitute ecological guilds organisms that exploit environmental resources in a similar way, and so competition can be expected.

16 L. iners & L. crispatus L. jensenii L. iners & others mixed community L. iners L. gasseri Asian Black Hispanic White L. crispatus

17 Several community states are evident: Dominated by species of Lactobacillus (>90% of community) 1. Lactobacillus iners dominated 2. Lactobacillus crispatus dominated 3. Lactobacillus jensenii dominated 4. Lactobacillus gasseri dominated Not dominated by species of Lactobacillus 5. Diverse: One or more lactobacilli and other taxa

18 A plot of principle coordinate analysis (PCoA) shows distribution of states in 3-D space. Community states dominated by Lactobacillus spp. are found on the four outer vertices. 45% of communities are found at vertices. 25% of the communities are intermediate forms and found along the edges of the tetrahedron. Community states having multiple taxa are found at an inner vertex within the tetrahedron. 30% of the communities

19 Vaginal microbial communities are hypothesized to be in a dynamic equilibrium in health, and substantial deviations can occur in response to one or more factors leading to many observed states for each microbial community. Host factors that affect the vaginal environment (e.g., quantity and composition of vaginal secretions, immune system, others) Habits and practices that alter biotic or abiotic characteristics of the environment (douching, poor hygiene, others)

20 Race Mean ph ± SD Median ph ± MAD a Caucasian 4.5 ± ± 0.59 Black 4.7 ± ± 1.00 Hispanic 4.8 ± ± 0.74 Asian 4.6 ± ± 0.59 Overall 4.7 ± ± 0.59 a Median absolute deviation (MAD) For a significant proportion of women (>50%) the vaginal ph is >4.5

21 Lowest ph values are associated with community states dominated by L. iners and L. crispatus. Highest ph values are associated with community states not dominated by species of Lactobacillus. Occasionally community states dominated by species of Lactobacillus have a high ph

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23 BV is a polymicrobial clinical syndrome resulting from replacement of the normal H2O2 producing Lactobacillus sp. in the vagina with high concentrations of anaerobic bacteria (e.g., Prevotella sp. and Mobiluncus sp.), G. vaginalis, and Mycoplasma hominis. BV is the most prevalent cause of vaginal discharge or malodor; however, more than 50% of women with BV are asymptomatic. The cause of the microbial alteration is not fully understood. BV is associated with having multiple sex partners, a new sex partner, douching, and lack of vaginal lactobacilli; whether BV results from acquisition of a sexually transmitted pathogen is unclear. Reference: Treatment Guidelines 2006,

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27 Race Mean score ± SD Median score ± MAD a Caucasian 1.8 ± ± 0.0 Black 4.4 ± ± 5.2 Hispanic 3.7 ± ± 4.4 Asian 2.2 ± ± 1.5 Overall 3.0 ± ± 1.5 a Median absolute deviation (MAD) A significant proportion (>50%) of Black and Hispanic women do not have normal vaginal microbiota based on Nugent scores.

28 1. Multiple kinds of bacterial communities are found in human vaginas These communities differ in terms of bacterial species composition All are dominated by lactic acid bacteria The frequency of each community differs among women in different ethnic groups 2. Communities that differ in terms of species composition and structure are likely to differ in terms of their resilience. Establish risk for disease in the context of community states Longitudinal studies are needed to assess variation in community composition over time 3. The median vaginal ph is 4.4 This value varies among ethnic groups: the median vaginal ph in Hispanic and Black women is 5.0 and 4.7, respectively 4. A significant proportion of Black and Hispanic women (>50%) do not have normal vaginal microbiota based on Nugent scores. This may largely be due to the difficulty of distinguishing morphotypes of lactic acid bacteria