The ISME Journal (2007) 1, 121–133 & 2007 International Society for Microbial Ecology All rights reserved 1751-7362/07 $30.00 www.nature.com/ismej ORIGINAL ARTICLE Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women Xia Zhou1, Celeste J Brown1, Zaid Abdo2, Catherine C Davis3, Melanie A Hansmann3, Paul Joyce2, James A Foster1 and Larry J Forney1 1Department of Biological Sciences, University of Idaho, Moscow, ID, USA; 2Departments of Mathematics and Statistics, University of Idaho, Moscow, ID, USA and 3FemCare Product Safety and Regulatory Affairs, Procter & Gamble Company, Cincinnati, OH, USA The maintenance of a low pH in the vagina through the microbial production of lactic acid is known to be an important defense against infectious disease in reproductive age women. Previous studies have shown that this is largely accomplished through the metabolism of lactic acid bacteria, primarily species of Lactobacillus. Despite the importance of this defense mechanism to women’s health, differences in the species composition of vaginal bacterial communities among women have not been well defined, nor is it known if and how these differences might be linked to differences in the risk of infection. In this study, we defined and compared the species composition of vaginal bacterial communities in 144 Caucasian and black women in North America. This was carried out based on the profiles of terminal restriction fragments of 16S rRNA genes, and phylogenetic analysis of 16S rRNA gene sequences of the numerically dominant microbial populations. Among all the women sampled, there were eight major kinds of vaginal communities (‘supergroups’) that occurred in the general populace at a frequency of at least 0.05 (P ¼ 0.99). From the distribution of these supergroups among women, it was possible to draw several conclusions. First, there were striking, statistically significant differences (P ¼ 0.0) in the rank abundance of community types among women in these racial groups. Second, the incidence of vaginal communities in which lactobacilli were not dominant was higher in black women (33%) as compared to Caucasian women (7%). Communities not dominated by lactobacilli had Atopobium and a diverse array of phylotypes from the order Clostridiales. Third, communities dominated by roughly equal numbers of more than one species of Lactobacillus were rare in black women, but common in Caucasian women. We postulate that because of these differences in composition, not all vaginal communities are equally resilient, and that differences in the vaginal microbiota of Caucasian and black women may at least partly account for known disparities in the susceptibility of women in these racial groups to bacterial vaginosis and sexually transmitted diseases. The ISME Journal (2007) 1, 121–133; doi:10.1038/ismej.2007.12; published online 10 May 2007 Subject Category: microbial population and community ecology Keywords: vagina; communities; 16S rRNA; bacteria; T-RFLP Introduction precludes the growth of many pathogenic organ- isms. Over the years, a large number of different The high levels of estrogen in reproductive age species of lactic acid bacteria, primarily species of women cause large amounts of glycogen to be Lactobacillus, have been demonstrated to reside deposited in the vaginal epithelium (Paavonen, in the human vagina (Redondo-Lopez et al., 1990; 1983), and it is subsequently metabolized primarily Antonio et al., 1999) and be key players in this by bacterial populations to produce organic acids process. These species effectively constitute an (Boskey et al., 1999). The resulting low pH (4–4.5) of ecological guild – a group of species that have the vagina creates a restrictive environment that similar requirements and play a similar role within a community – and maintaining high numbers of these populations is a hallmark of healthy condi- Correspondence: Dr LJ Forney, Department of Biological Sciences, tions. Events that lead to decreased numbers of Life Sciences South, Rm. 252, University of Idaho, Moscow, lactic acid bacteria in the vagina and the concomi- ID 83844, USA. E-mail: [email protected] tant increase in the abundances of other bacteria are Received 7 December 2006; revised 15 February 2007; accepted 20 collectively known as bacterial vaginosis (BV; February 2007; published online 10 May 2007 Thorsen et al., 1998; Koumans and Kendrick, Vaginal microbiota of Caucasian and black woman X Zhou et al 122 2001). During BV, there is an increase in vaginal pH 1999; Taha et al., 1999). Myer et al. (2005) argue that (44.5) that is often accompanied by the production interventions to reduce the occurrence of BV may of odor, and a thin, grayish white vaginal discharge have an impact on the spread of HIV, and that almost (Sobel, 2000). one-third of all new HIV infections might be BV commonly occurs in women of reproductive prevented if all cases of BV could be cured. age. It affects between 20% and 25% of the general However, the existing treatments for BV (oral or population, and up to 50% of women attending vaginal administration of metronidazole) are pla- sexual health clinics (Newton et al., 1997; Morris gued by high failure rates, with symptoms reoccur- et al., 2001; Yen et al., 2003; Sobel, 2005). It is the ring in 54% of women within 3 months following most common cause of vaginal symptoms prompting antibiotic treatment (Bradshaw et al., 2006). Mea- women to seek medical care (Morris et al., 2001; sures that stabilize vaginal microbial communities Marrazzo, 2006), and a significant problem because it and prophylaxis against BV may emerge if we predisposes individuals to sexually transmitted dis- develop an accurate understanding of the species eases including human immunodeficiency virus composition and ecology of vaginal ecosystems in (HIV) (Sewankambo et al., 1997; Taha et al., 1998; normal healthy women. Martin et al., 1999), preterm birth, pelvic inflamma- In this study, we tested the hypothesis that there tory disease, endometritis and infertility (Hillier et al., were fundamental differences in the composition 1995;Sweet,1995;Wiesenfeldet al., 2002; Leitich and structure of bacterial communities in the et al., 2003; Haggerty et al., 2004). The causes of BV vaginas of Caucasian and black women in North are not well understood, and specific infectious America. To do this, we determined the identity and agent(s) have not been identified. However, a number rank abundance of bacterial populations in vaginal of bacterial species are known to be common in bacterial communities of 144 healthy, reproductive women with BV, including Gardnerella vaginalis, age women in these racial groups. The data showed Mobiluncus sp., Mycoplasma hominis and species of that within each racial group there were distinct Clostridiaceae (Thorsen et al., 1998; Koumans and differences in community composition, which al- Kendrick, 2001; Fredricks et al., 2005), but their role lowed us to group women into categories that we in the pathogenesis of BV has not been established. referred to as ‘supergroups’. There were significant The occurrence of BV has been associated with differences in the rank abundance of these commu- behaviors that upset the normal balance of bacteria nities in Caucasian and black women, and we in the vagina including new or multiple sex partners, postulate that these differences in community douching, and the use of an intrauterine device for composition and structure may account for the contraception (http://www.cdc.gov/std/bv/STDFact- differential risk to BV and various vaginal infections Bacterial-Vaginosis). This has led some to suggest of women in these racial groups. that BV is not an infectious disease at all but rather a set of symptoms that reflect an ecological disturbance that is accompanied by changes in the relative Materials and methods abundance of autochthonous organisms in the vagina (http://www.cdc.gov/std/bv/STDFact-Bacterial- Subjects and clinical study design Vaginosis). Curiously, the incidence of BV varies As part of a study on the prevalence of Staphylo- markedly among racial and ethnic groups (Rajama- coccus aureus carriage, 3012 healthy menstruating noharan et al., 1999; Royce et al., 1999), ranging women between the ages of 13 and 40 years were from 6% in Asians and 9% in whites, to 16% in enrolled from five sites in North America (Parsonnet Hispanics and 23% in African Americans. The et al., 2005). The subjects were recruited by Hill Top reasons for differences in the incidence of BV among Research Inc. in Cincinnati, OH, USA, East Bruns- racial groups are unknown, but they cannot be wick, NJ, USA, St Petersburg, FL, USA, Scottsdale, explained by differences in socio-demographics, AZ, USA and Winnipeg, Manitoba, Canada. The sexual activity, health behavior or hygiene alone racial profile of the women corresponded with that (Goldenberg et al., 1996; Royce et al., 1999). of the 1990 USA census: 80% white, 12% black, 5% While there are many reasons for concern about Hispanic and 3% Asian. Subjects were eligible BV, the link between BV and an increased risk for for enrollment if they had regular menstrual cycles acquisition of the HIV (Sewankambo et al., 1997; (21–35 days); used tampons at least occasionally; Taha et al., 1999) is particularly menacing. The were able to read, write and understand English; did number of women with HIV and acquired immuno- not bathe or shower within 2 h of their scheduled deficiency syndrome (AIDS) has steadily increased visit; refrained from