Previous studies have shown that alterations of the bacterial microbiota in the lower female genital tract influence susceptibility to HIV infection and shedding. We assessed geographic differences in types of genital microbiota between HIV-infected and uninfected women from Rwanda and the United States.
Genera of lower genital tract bacterial microbiota were identified by high-throughput pyrosequencing of the 16S rRNA gene from 46 US women (36 HIV-infected, 10 HIV-uninfected) and 40 Rwandan women (18 HIV-infected, 22 HIV-uninfected) with similar proportions of low (0–3) Nugent scores. Species of Lactobacillus were identified by assembling sequences along with reference sequences into phylogenetic trees. Prevalence of genera and Lactobacillus species were compared using Fisher's exact tests.
Overall the seven most prevalent genera were Lactobacillus (74%), Prevotella (56%), Gardnerella (55%), Atopobium (42%), Sneathia (37%), Megasphaera (30%), and Parvimonas (26%), observed at similar prevalences comparing Rwandan to US women, except for Megasphaera (20% vs. 39%, p = 0.06). Additionally, Rwandan women had higher frequencies of Mycoplasma (23% vs. 7%, p = 0.06) and Eggerthella (13% vs. 0%, p = 0.02), and lower frequencies of Lachnobacterium (8% vs. 35%, p<0.01) and Allisonella (5% vs. 30%, p<0.01), compared with US women. The prevalence of Mycoplasma was highest (p<0.05) in HIV-infected Rwandan women (39%), compared to HIV-infected US women (6%), HIV-uninfected Rwandan (9%) and US (10%) women. The most prevalent lactobacillus species in both Rwandan and US women was L. iners (58% vs. 76%, p = 0.11), followed by L. crispatus (28% vs. 30%, p = 0.82), L. jensenii (20% vs. 24%, p = 0.80), L. gasseri (20% vs. 11%, p = 0.37) and L. vaginalis (20% vs. 7%, p = 0.10).
We found similar prevalence of most major bacterial genera and Lactobacillus species in Rwandan and US women. Further work will be needed to establish whether observed differences differentially impact lower genital tract health or susceptibility to genital infections.
Citation: Benning L, Golub ET, Anastos K, French AL, Cohen M, Gilbert D, et al. (2014) Comparison of Lower Genital Tract Microbiota in HIV-Infected and Uninfected Women from Rwanda and the US. PLoS ONE 9(5): e96844. doi:10.1371/journal.pone.0096844
Editor: Sunil K. Ahuja, South Texas Veterans Health Care System and University Health Science Center San Antonio, United States of America
Received: May 29, 2013; Accepted: April 11, 2014; Published: May 9, 2014
Copyright: © 2014 Benning et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The WIHS is funded by the National Institute of Allergy and Infectious Diseases (UO1-AI-35004, UO1-AI-31834, UO1-AI-34994, UO1-AI-34989, UO1-AI-34993, UO1-AI-42590), and by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (UO1-HD-32632). The study is cofunded by the National Cancer Institute, the National Institute on Drug Abuse, and the National Institute on Deafness and Other Communication Disorders. Funding is also provided by the National Center for Research Resources (UCSF-CTSA Grant Number UL1 TR000004). The RWISA was supported by supplements from the National Institute of Allergy and Infectious Diseases to the Bronx/Manhattan Women's Interagency HIV Study (WIHS), which is funded by the National Institute of Allergy and Infectious Diseases (UO1-AI-35004). This work was also supported in part by the AIDS International Training and Research Program (Fogarty International Center, NIH D43-TW001403) and the Center for AIDS Research of the Albert Einstein College of Medicine and Montefiore Medical Center funded by the National Institutes of Health (NIH AI-51519) and by the National Institute of Diabetes and Digestive and Kidney Disease (DK54615). Additional funding for this project was provided by the National Institute of Allergy and Infectious Diseases P01 AI082971 and the Chicago Developmental Center for AIDS Research Grant Number P30 AI082151. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors wish to disclose that Patrick Gillevet has a financial interest in BioSpherex LLC, contracted to do the MTPS sequencing. On 10/21/2013, he was issued a patent for the MTPS process, Multitag Sequencing for Ecogenomics Analysis USPTO 12/515262. Masoumeh Sikaroodi is a consultant for BioSpherex LLC. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.
Many reproductive-aged women have lower genital tract microbiota that can be classified as either "healthy" microbiota or bacterial vaginosis (BV) –. However, there is considerable heterogeneity within these classifications. For example, while healthy microbiota generally are comprised mostly of Lactobacillus spp., in different women L. crispatus, L. iners, L. jensenii or L. gasseri can be the dominant species –and there are significant differences in the beneficial effects of each –. BV can also vary dramatically between women. For example, Fredricks et al. showed, by sequencing 16S ribosomal RNA genes, that while Gardnerella vaginalis was a dominant bacterium in many cases of BV (comprising as much as 49% of the bacterial sequences), in some cases G. vaginalis was almost absent (1–2% of sequences) . Similarly, while most women with BV had microbiota with substantial proportions of Prevotella sequences (25–30% of sequences), some had no detectable Prevotella sequences. A substantial heterogeneity in BV has been confirmed in other recent studies using molecular-based methods , .
Cross-sectional and longitudinal epidemiologic studies show that BV is associated with increased susceptibility to infection with HIV ,  and other sexually transmitted diseases (STD) , . BV is also associated with increased genital shedding of HIV in women –. While BV is associated with these infections, an unresolved question is whether heterogeneity within BV is associated with HIV.
The 2010 UNAIDS Global Report estimated that in 2009 the prevalence of HIV in Rwandans aged 15–49 was 2.9% (2.5%–3.3%), five times higher than the estimated rate of 0.6% (0.4%–0.8%) in the United States (US) . The rate of 1.9% (1.3%–2.3%) among young Rwandan women (15–24) was 10 times higher than the rate of 0.2% (0.1%–0.3%) among young US women . Differences in types of genital microbiota are potentially among a number of factors that contribute to these large disparities in HIV prevalence between Rwanda and the US. The purpose of the current pilot study was to investigate the heterogeneity of genital bacterial microbiota between HIV-infected and –uninfected Rwandan and US women. Organisms found to be more prevalent in one country, particularly among HIV-infected women compared to HIV-uninfected women, may contribute to HIV susceptibility or shedding. Associations between these organisms and HIV viral load in the female genital tract could be targeted in larger future studies. Although some genital bacteria are easily cultured (e.g. Gardnerella), it has become clear recently that many genital bacteria are either unculturable (e.g. Sneathia) or are difficult to culture since they are anaerobic (e.g. Mobiluncus and Atopobium). Therefore, we used high-throughput pyrosequencing of the 16S rRNA gene to identify the genital microbiota of women in this study. Previous 16S-based high-throughput sequencing studies of microbiota have been performed on US , , , Chinese , and Tanzanian women . We directly compared the genital microbiota between US and Rwandan women, overall and stratified by HIV serostatus.
Materials and Methods
The study population of 86 women came from two parent studies, the Women's Interagency HIV Study (WIHS) and the Rwandan Women's Interassociation Study and Assessment (RWISA). Both the WIHS and the RWISA are observational cohort studies of HIV-infected and uninfected women. Semiannual study visits included extensive interviews, specimen collection, and clinical examinations. The WIHS enrolled a total of 3766 women (2791 HIV-infected, 975 HIV-uninfected) in two waves, from 10/1994 to 11/1995  and 10/2001 to 9/2002 , for the purpose of investigating the progression of HIV infection and associated diseases, such as diabetes and cardiovascular disease, among HIV-infected and demographically similar HIV-uninfected women in the US. The RWISA enrolled 936 women (710 HIV-infected, 226 HIV-uninfected) in 2005 for the purpose of investigating responses to antiretroviral therapy in Rwandan women, including potential impact of their experiences during the 1994 Rwandan genocide . The 46 WIHS participants (36 HIV-infected, 10 HIV-uninfected) included in the current study have been described in two previously conducted substudies , . The 40 RWISA participants were selected for the current study to have Nugent scores similar to the WIHS participants. All data on sexual behavior and STD history were missing for one Rwandan participant.
The study was conducted in accordance with protocols approved by the Rwandan National Ethics Committee and the Institutional Review Board (IRB) of Montefiore Medical Center, Bronx, NY, USA for RWISA participants and guidelines of the US Department of Health and Human Services and the IRBs of each of the WIHS institutions and the WIHS Executive Committee for WIHS participants. Written informed consent was obtained for all participants from both cohorts.
Genital tract samples for both studies were collected by cervicovaginal lavage (CVL) performed by irrigation of the cervix with 10 mL of nonbacteriostatic sterile saline, followed by aspiration from the posterior fornix. All CVL samples were gently vortexed to evenly distribute cells before they were aliquotted; RWISA CVL samples were processed within 2 hours of collection and WIHS CVL samples were held on ice until processing within 6 hours of collection. CVL samples from both studies were stored at −70°C. Specimens from HIV-infected WIHS participants were collected between 1/1995 and 4/1997, from HIV-uninfected WIHS participants between 10/2005 and 3/2006, and from all RWISA participants between 5/2005 and 11/2005. The methods used for DNA isolation and multitag pyrosequencing have been described elsewhere , . Briefly, bar-coded primer sets each containing the 27F and 355R 16S rRNA gene primers were used. Only forward reads were used to identify bacteria using the Bayesian Classifier provided by the Ribosomal Database II Project (RDP 10). For each subject, analysis was performed on bacterial taxa found at ≥1% of the total sequences with the premise that the most abundant taxa contribute most significantly to the functionality of the microbial community. The average number of sequences obtained per sample was 3857 (range 895–7866) for HIV-uninfected Rwandan women, 3563 (range 491–7016) for HIV-infected Rwandan women, 1412 (range 224–2707) for HIV-uninfected US women, and 579 (249–1683) for HIV-infected US women. Species of Lactobacillus were identified by assembling sequences along with reference sequences into phylogenetic trees with a 96% overlap identity and 80% confidence threshold using Geneious Pro 4.6.1 software (Auckland, New Zealand). Reference sequences used included AF257097 (L. crispatus), M58820 (L. gasseri), AF243176 (L. jensenii), AF243177 (L. vaginalis), and AY526083 (L. iners).
Figure 1 shows the study selection and analytic design. Rwandan and US women were selected and frequency matched on the basis of Nugent score. Women were subsequently stratified by Lactobacillus-predominance versus non-Lactobacillus predominance.
There were two analytic aims, to investigate the heterogeneity of microbiota associated with BV (Aim 1) and to investigate the heterogeneity of healthy microbiota (Aim 2). For each aim we conducted two separate comparisons. First we compared all Rwandan and US women. Secondly, we compared by country and HIV status (four groups): HIV-infected Rwandan (RW+), HIV-uninfected Rwandan (RW-), HIV-infected US (US+), and HIV-uninfected US (US-) women. We compared prevalence, defined as the proportion of women in whom a given genus or species was present. A genus or species was considered present in a woman if at least 1% of all her sequences corresponded to it.
Fisher's exact tests were used to determine differences in the prevalence of individual genera and Lactobacillus spp. and to compare categorical demographic and behavioral characteristics. Mann-Whitney U tests were used to compare continuous demographic and behavioral characteristics. P values less than 0.05 were considered to be statistically significant and P values between 0.05 and 0.10, marginally significant. All analyses were conducted in SAS version 9 (SAS, Cary, North Carolina, USA).
Table 1 shows a comparison of demographic and behavioral characteristics between Rwandan and US women, stratified by Lactobacillus level. In general, US women had more sexual experience and higher proportions of STDs than Rwandan women. Differences between countries tended to be larger and more statistically significant among women with low Lactobacillus, especially for genital warts, syphilis, and PID, than among women with high Lactobacillus. The higher prevalence of HIV infection among US women versus Rwandan women overall (78% vs. 45%, p = 0.002) was reflected in each stratum and, again, was more pronounced among women with low Lactobacillus. None of the 18 HIV-infected Rwandan women had used antiretroviral medication (ART) compared to 19 (53%) of the 36 HIV-infected US women who were naïve to ART. Specimens from Rwandan women were collected before wide ART availability in Rwanda and between January 1995 and April 1997 for US women, when ART use was not yet optimal. CD4+ cell count was similar between RW+ and US+ women (218 vs. 254, p = 0.54). Log10 HIV RNA viral load was also similar (4.77 vs. 4.61, p = 0.76).
Types of genital microbiota are similar between Rwandan and US women, overall
To compare the types of genital microbiota found in Rwandan and US women, genital tract samples obtained from each of the 86 women were used to identify bacterial genera by pyrosequencing of the 16S rRNA gene. Of the 86 prevalent genera identified across all 86 women (Table S1), 17 were unique to Rwandan women, 33 were unique to US women, and 36 were common to both groups. These 86 genera accounted for a median of 97% of all sequences (range 90–100%) among Rwandan women and 96% (90–100%) among US women (p = 0.12). Among Rwandan women, the median number of genera was 4.5 (range 1–15) compared to a median of 7.5 (range 1–23) for US women (p = 0.33).
Figures 2 and 3 show the distributions of the most dominant genera in Rwandan and US women, respectively. Eleven genera comprised at least 10% of sequences in at least one Rwandan and at least one US woman: Lactobacillus, Prevotella, Gardnerella, Atopobium, Sneathia, Parvimonas, Megasphaera, Chryseobacterium, Gemella, Lachnobacterium, and Syntrophococcus (Figures 2 and 3). Proportions of these genera did not differ between Rwandan and US women, except for Lachnobacterium and Megasphaera (data not shown).
Stacked columns for each of the 40 individual women show the distributions of the proportion of sequences corresponding to a given genus. All 22 genera with proportions of at least 0.1 in at least one woman were included. Numbers in parentheses in the legend indicate the number of women in whom the genus was prevalent.
Stacked columns for each of the 46 individual women show the distributions of the proportion of sequences corresponding to a given genus. All 17 genera with proportions of at least 0.1 in at least one woman were included. Numbers in parentheses in the legend indicate the number of women in whom the genus was prevalent.
Overall, the seven most prevalent genera were Lactobacillus (74%), Prevotella (56%), Gardnerella (55%), Atopobium (42%), Sneathia (37%), Megasphaera (30%), and Parvimonas (26%) (Table S1). Prevalences were similar between countries for all except Megasphaera (20% Rwanda vs. 39% US, p = 0.06). The most prevalent Lactobacillus species in both Rwandan and US women was L. iners (58% vs. 76%, p = 0.11), followed by L. crispatus (28% vs. 30%, p = 0.82), L. jensenii (20% vs. 24%, p = 0.80), L. gasseri (20% vs. 11%, p = 0.37) and L. vaginalis (20% vs. 7%, p = 0.10) (Table S2).
Prevalence of some microbiota associated with BV higher among US women
Table 2 shows the 28 most prevalent genera (of 83) observed among the 48 women with low Lactobacillus. The table is grouped by high (≥40%), moderate (16–39%), and low (10–15%) overall prevalence, and further stratifies by geography and HIV status. Rwandan women had lower prevalence of 10 genera (Megasphaera, Lachnobacterium, Allisonella, Mobiluncus, Propionibacterium, Citrobacter, Achromobacter, Escherichia/Shigella, Enterobacter, and Phocaeicola) and higher prevalence of Mycoplasma and Eggerthella, compared to US women. Prevalences for the other 16 genera were not significantly different between the two countries. Results were similar for the four group comparison (RW+, RW−, US+, US−). Of genera observed in both countries, Megasphaera, Lachnobacterium and Allisonella were higher in US+ and US−; Asaccharobacter was higher in RW+ and US+; Mycoplasma was highest in RW+; Mobiluncus and Dialister were highest in US−; and Propionibacterium was highest in US+.
Prevalence of L. crispatus is low among HIV-infected women
Among the 38 women with high Lactobacillus, only 17 genera in addition to Lactobacillus were observed. Of those, only three were observed at prevalences higher than 10% (Gardnerella 21%, Prevotella 16%, and Ureaplasma 11%). Therefore, we restricted analysis to Lactobacillus spp. among these women (Table 3, top), contrasting them to the 26 women with 1–49% of sequences corresponding to Lactobacillus (Table 3, bottom). Both halves of Table 3 show that overall L. iners was the dominant Lactobacillus spp. The diversity of Lactobacillus spp. was similar among the 18 Rwandan women and 20 US women with high Lactobacillus and the 10 Rwandan women with low Lactobacillus. However, there was almost no diversity in Lactobacillus spp. among the 16 US women with low Lactobacillus. Taking HIV into account revealed significantly lower prevalence of L. crispatus among all HIV-infected women with low Lactobacillus (p = 0.03). Among women with high Lactobacillus, the prevalence of L. crispatus was lower among Rwandan HIV-infected women but not US HIV-infected women.
This exploratory pilot study was the first direct comparison of the types of lower genital tract microbiota of US and African women using pyrosequencing of the 16S rRNA gene. The major finding was an overall similarity in the types and prevalences of genital microbiota between US and Rwandan women, despite differences in sexual behaviors and STD history between the groups. This finding is congruous with previous studies. A large epidemiological study of West African women with BV also showed their genital microbiota was similar to that of women in developed countries . Another study comparing phenotypic characteristics of microbiota, including production of acid and H2O2, from East African commercial sex workers and Canadian adolescents also noted similarity, reinforcing what previous work had suggested regarding the most frequently observed vaginal microorganisms across all groups of women studied around the world . This suggests the possibility that the heterogeneity of the most common microbiota associated with BV is not largely impacted by sexual history or geography.
Regarding genera with dissimilar prevalences among US and Rwandan women, the higher frequency of Mycoplasma among HIV-infected Rwandan women could have important implications for HIV transmission. For example, a prospective study of women in the East African countries of Zimbabwe and Uganda found that the risk of HIV acquisition was significantly higher in those with M. genitalium . Likewise in Kenya, women with high M. genitalium organism burdens were more likely to have measurable cervical HIV-1 DNA than were M. genitalium-negative women .
The coliform bacilli Escherichia and closely related Shigella, Enterobacter, and Citrobacter are members of the normal intestinal flora of humans , . A limited number of species, including E. coli, Enterobacter aerogenes, Enterobacter cloacae, S. dysenteriae, and S. flexneri are responsible for most of the infections which cause major health problems around the world. In the vagina, these bacteria are the primary cause of urinary tract infections, particularly E. coli , . Interestingly, these genera were prevalent only in US HIV-infected women in our study. This finding is intriguing, especially given the differences in sexual histories and STD prevalence between US and Rwandan women. Vaginal colonization with coliforms could be a marker of recent sexual activity. Future studies could investigate the association between these coliforms and markers of recent sexual activity such as PSA, RSID, and Y chromosome PCR.
No studies have shown any human disease to be associated with Lachnobacterium, Allisonella, Asaccharobacter, or Phocaeicola. Propionibacterium spp. can cause skin infection . Achromobacter spp. have been associated with cystic fibrosis and immunosuppression . However, the clinical significance, if any, in the human female genital tract for these bacteria is unknown. Studies of the gut microbiome show that individuals can have highly divergent types of microbiota, although the functional profiles are similar . It is possible that in the genital tract, some of the differences we observed in types of microbiota also have similar functions.
Colonization with lactobacilli, particularly the H2O2-producing species L. crispatus and L. jensenii, has a protective effect against the acquisition of BV –. Macklaim et al. showed that L. iners has the ability to survive in the vagina under varying conditions . L. iners was the dominant species among all women in our study, followed by L. crispatus and L. jensenii. The lower prevalence of L. crispatus that we observed among all HIV-infected women from Rwanda, and among some HIV-infected women from the US, could be clinically important if validated in a larger study.
It should be noted that this study was exploratory and limited by small sample size. However, although our results may not be generalizable to all Rwandan and US women, our samples were obtained from two established parent studies (WIHS and RWISA), including both HIV-infected and uninfected women, both with sound protocol and infrastructure –. Except for Lactobacillus spp., our analysis was conducted at the level of genera. It is therefore possible that while we observed similarities between countries in the most common genera, there could be important differences between countries at the species level. We have identified several genital bacterial microbiota (Mycoplasma, Escherichia/Shigella, Enterobacter, Citrobacter) which might be targeted in future studies using genus or species-specific PCR to investigate the association between bacterial load and HIV shedding and markers of recent sexual activity. Despite these limitations, this study revealed surprising similarities in the microbiota of US and Rwandan women, as well as some interesting differences that warrant further exploration. Additionally, it would be informative to investigate the clinical significance of these organisms.
Prevalence of all 86 observed genera.
Prevalence of Lactobacillus spp.
The authors would like to thank Dalian Lu for his preparation of the RWISA epidemiologic data. WIHS data in this manuscript were collected by the WIHS Collaborative Study Group with centers (Principal Investigators) at New York City/Bronx Consortium (Kathryn Anastos); Brooklyn, NY (Howard Minkoff, Deborah Gustafson); Washington DC, Metropolitan Consortium (Mary Young); The Connie Wofsy Study Consortium of Northern California (Ruth Greenblatt, Phyllis Tien, Bradley Aouizerat); Los Angeles County/Southern California Consortium (Alexandra Levine); Chicago Consortium (Mardge Cohen); Data Coordinating Center (Stephen Gange, Elizabeth Golub).
Conceived and designed the experiments: GS AL. Performed the experiments: DG PG MS. Analyzed the data: LB EG. Contributed reagents/materials/analysis tools: KA MC AF EM. Wrote the paper: LB GS.
- 1. Nugent RP, Krohn MA, Hillier SL (1991) Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 29: 297–301.
- 2. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, et al. (1983) Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 74: 14–22. doi: 10.1016/0002-9343(83)91112-9
- 3. Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, et al. (2011) The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 118: 533–49. doi: 10.1111/j.1471-0528.2010.02840.x
- 4. Li J, McCormick J, Bocking A, Reid G (2012) Importance of vaginal microbes in reproductive health. Reprod Sci 19: 235–42. doi: 10.1177/1933719111418379
- 5. Brotman RM (2011) Vaginal microbiome and sexually transmitted infections: an epidemiologic perspective. J Clin Invest 121: 4610–7. doi: 10.1172/jci57172
- 6. Marconi C, Donders GG, Bellen G, Brown DR, Parada CM, et al. (2013) Sialidase activity in aerobic vaginitis is equal to levels during bacterial vaginosis. Eur J Obstet Gynecol Reprod Biol 167: 205–9. doi: 10.1016/j.ejogrb.2012.12.003
- 7. Gajer P, Brotman RM, Bai G, Sakamoto J, Schütte UM, et al. (2012) Temporal dynamics of the human vaginal microbiota. Sci Transl Med 4: 132ra52. doi: 10.1126/scitranslmed.3003605
- 8. Fredricks DN, Fiedler TL, Marrazzo MJ (2005) Molecular Identification of Bacteria Associated with Bacterial Vaginosis. N Engl J Med 353: 1899–911. doi: 10.1056/nejmoa043802
- 9. Spear GT, Gilbert D, Landay AL, Zariffard R, French AL, et al. (2011) Pyrosequencing of the genital microbotas of HIV-seropositive and –seronegative women reveals Lactobacillus iners as the predominant Lactobacilllus species. Appl Environ Microbiol 77: 378–381. doi: 10.1128/aem.00973-10
- 10. Hillier SL, Krohn MA, Klebanoff SJ, Eschenbach DA (1992) The relationship of hydrogen peroxide-producing lactobacilli to bacterial vaginosis and genital microflora in pregnant women. Obstet Gynecol 79: 369–73. doi: 10.1016/0020-7292(92)90920-e
- 11. Verstraelen H, Verhelst R, Claeys G, De Backer E, Temmerman M, et al. (2009) Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora. BMC Microbiol 9: 116. doi: 10.1186/1471-2180-9-116
- 12. Eschenbach DA, Davick PR, Williams BL, Klebanoff SJ, Young-Smith K, et al. (1989) Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 27: 251–6.
- 13. Macklaim JM, Gloor GB, Anukam KC, Cribby S, Reid G (2011) At the crossroads of vaginal health and disease, the genome sequence of Lactobacillus iners AB-1. Proc Natl Acad Sci U S A 108 Suppl 14688–95. doi: 10.1073/pnas.1000086107
- 14. Martinez RCR, Franceschini SA, Patta MC, Quintana SM, Nunes AC, et al. (2008) Analysis of vaginal lactobacilli from healthy and infected Brazilian women. Appl Environ Microbiol 74: 4539–42. doi: 10.1128/aem.00284-08
- 15. Antonio MAD, Hawes SE, Hillier SL (1999) The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 180: 1950–6. doi: 10.1086/315109
- 16. Hillier SL, Krohn MA, Rabe LK, Klebanoff SJ, Eschenbach DA (1993) The normal vaginal flora, H2O2-producing lactobacilli, and bacterial vaginosis in pregnant women. Clin Infect Dis 16 Suppl 4S273–81. doi: 10.1093/clinids/16.supplement_4.s273
- 17. Hummelen R, Fernandes AD, Macklaim JM, Dickson RJ, Changalucha J, et al. (2010) Deep sequencing of the vaginal microbiota of women with HIV. PLoS One 5: e12078. doi: 10.1371/journal.pone.0012078
- 18. Atashili J, Poole C, Ndumbe PM, Adimora AA, Smith JS (2008) Bacterial vaginosis and HIV acquisition: a meta-analysis of published studies. AIDS 22: 1493–501. doi: 10.1097/qad.0b013e3283021a37
- 19. Mavedzenge SN, Van Der Pol B, Weiss HA, Kwok C, Mambo F, et al. (2012) The association between Mycoplasma genialium and HIV-1 acquisition in African women. AIDS 26: 617–24. doi: 10.1097/qad.0b013e32834ff690
- 20. Martin HL, Richardson BA, Nyange PM, Lavreys L, Hillier SL, et al. (1999) Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis 180: 1863–8. doi: 10.1086/315127
- 21. Wiesenfeld HC, Hillier SL, Krohn MA, Landers DV, Sweet RL (2003) Bacterial vaginosis is a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection. Clin Infect Dis 36: 663–8. doi: 10.1086/367658
- 22. Cu-Uvin S, Hogan JW, Caliendo AM, Harwell J, Mayer KH, et al. (2001) Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract. Clin Infect Dis 33: 894–6. doi: 10.1086/322613
- 23. Coleman JS, Hitti J, Bukusi EA, Mwachari C, Muliro A, et al. (2007) Infectious correlates of HIV-1 shedding in the female upper and lower genital tracts. AIDS 21: 755–9. doi: 10.1097/qad.0b013e328012b838
- 24. Sha BE, Zariffard MR, Wang QJ, Chen HY, Bremer J, et al. (2005) Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis. J Infect Dis 191: 25–32. doi: 10.1086/426394
- 25. Gatski M, Martin DH, Theall K, Amedee A, Clark RA, et al. (2011) Mycoplasma genitalium infection among HIV-positive women: prevalence, risk factors and association with vaginal shedding. Int J STD AIDS 22: 155–9. doi: 10.1258/ijsa.2010.010320
- 26. Annex 1 – HIV and AIDS estimates and data, 2009 and 2001 (2010). In: Global report UNAIDS report on the global AIDS epidemic|2010. Joint United Nations Programme on HIV/AIDS (UNAIDS). pp. 181, 183, 202, 204.
- 27. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, et al. (2011) Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 108 Suppl 14680–7. doi: 10.1073/pnas.1002611107
- 28. Spear GT, Sikaroodi M, Zariffard MR, Landay AL, French AL, et al. (2008) Comparison of the diversity of the vaginal microbiota in HIV-infected and HIV-uninfected women with or without bacterial vaginosis. J Infect Dis 198: 1131–40. doi: 10.1086/591942
- 29. Ling Z, Kong J, Liu F, Zhu H, Chen X, et al. (2010) Molecular analysis of the diversity of vaginal microbiota associated with bacterial vaginosis. BMC Genomics 11: 488. doi: 10.1186/1471-2164-11-488
- 30. Barkan SE, Melnick SL, Martin-Preston S, Weber K, Kalish LA, et al. (1998) The Women's Interagency HIV Study. WIHS Collaborative Study Group. Epidemiology 9: 117–25. doi: 10.1097/00001648-199803000-00004
- 31. Bacon MC, von Wyl V, Alden C, Sharp G, Robison E, et al. (2005) The Women's Interagency HIV Study: an observational cohort brings clinical sciences to the bench. Clin Diagn Lab Immunol 12: 1013–9. doi: 10.1128/cdli.12.9.1013-1019.2005
- 32. Singh DK, Anastos K, Hoover DR, Burk RD, Shi Q, et al. (2009) Human Papillomavirus Infection and Cervical Cytology in HIV-Infected and HIV-Uninfected Rwandan Women. J Infect Dis 199: 1851–61. doi: 10.1086/599123
- 33. Spear GT, Gilbert D, Sikaroodi M, Doyle L, Green L, et al. (2010) Identification of rhesus macaque genital microbiota by 16S pyrosequencing shows similarities to human bacterial vaginosis: implications for use as an animal model for HIV vaginal infection. AIDS Res Hum Retroviruses 26: 193–200. doi: 10.1089/aid.2009.0166
- 34. Spear GT, Kersh E, Guenthner P, Vishwanathan SA, Gilbert D, et al. (2012) Longitudinal assessment of pigtailed macaque lower genital tract microbiota by pyrosequencing reveals dissimilarity to the genital microbiota of healthy humans. AIDS Res Hum Retroviruses 28: 1244–9. doi: 10.1089/aid.2011.0382
- 35. Pépin J, Deslandes S, Giroux G, Sobéla F, Khonde, et al (2011) The complex vaginal flora of West African women with bacterial vaginosis. PLoS One 9: e25082. doi: 10.1371/journal.pone.0025082
- 36. Schellenberg JJ, Dumonceaux TJ, Hill JE, Kimani J, Jaoko W, et al. (2012) Selection, phenotyping and identification of acid and hydrogen peroxide producing bacteria from vaginal samples of Canadian and East African women. PLoS One 7: e41217. doi: 10.1371/journal.pone.0041217
- 37. Manhart LE, Mostad SB, Baeten JM, Astete SG, Mandaliya K, et al. (2008) High Mycoplasma genitalium organism burden is associated with shedding of HIV-1 DNA from the cervix. J Infect Dis 197: 733–6. doi: 10.1086/526501
- 38. Guentzel MN (1996) Escherichia, Klebsiella, Enterobacter, Serratia, Citrobacter, and Proteus. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston. Chapter 26.
- 39. Hale TL, Keusch GT (1996) Shigella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston. Chapter 22.
- 40. Gupta K, Stapleton AE, Hooton TM, Roberts PL, Fennell CL, et al. (1998) Inverse association of H2O2-producing lactobacilli and vaginal Escherichia coli colonization in women with recurrent urinary tract infections. J Infect Dis 178: 446–50. doi: 10.1086/515635
- 41. Nicolle LE (2013) Urinary tract infection. Crit Care Clin 29: 699–715. doi: 10.1016/j.ccc.2013.03.014
- 42. Perry A, Lambert P (2011) Propionibacterium acnes: Infection beyond the skin. Expert Rev Anti Infect Ther 9: 1149–56. doi: 10.1586/eri.11.137
- 43. Jakobsen TH, Hansen MA, Jensen PØ, Hansen L, Riber L, et al. (2013) Complete genome sequence of the cystic fibrosis pathogen Achromobacter xylosoxidans NH44784–1996 complies with important pathogenic phenotypes. PLoS One 8: e68484. doi: 10.1371/journal.pone.0068484
- 44. Lozupone CA, Stombaugh JI, Gordon JI, Jansson JK, Knight R (2012) Diversity, stability and resilience of the human gut microbiota. Nature 489: 220–30. doi: 10.1038/nature11550