Characterization of the vaginal microbiota among sexual risk behavior groups of women with bacterial vaginosis

Christina A Muzny, Imran R Sunesara, Ranjit Kumar, Leandro A Mena, Michael E Griswold, David H Martin, Elliot J Lefkowitz, Jane R Schwebke, Edwin Swiatlo, Christina A Muzny, Imran R Sunesara, Ranjit Kumar, Leandro A Mena, Michael E Griswold, David H Martin, Elliot J Lefkowitz, Jane R Schwebke, Edwin Swiatlo

Abstract

Background: The pathogenesis of bacterial vaginosis (BV) remains elusive. BV may be more common among women who have sex with women (WSW). The objective of this study was to use 454 pyrosequencing to investigate the vaginal microbiome of WSW, women who have sex with women and men (WSWM), and women who have sex with men (WSM) with BV to determine if there are differences in organism composition between groups that may inform new hypotheses regarding the pathogenesis of BV.

Methods: Vaginal swab specimens from eligible women with BV at the Mississippi State Department of Health STD Clinic were used. After DNA extraction, 454 pyrosequencing of PCR-amplified 16S rRNA gene sequences was performed. Sequence data was classified using the Ribosomal Database Program classifer. Complete linkage clustering analysis was performed to compare bacterial community composition among samples. Differences in operational taxonomic units with an abundance of ≥ 2% between risk behavior groups were determined. Alpha and beta diversity were measured using Shannon's Index implemented in QIIME and Unifrac analysis, respectively.

Results: 33 WSW, 35 WSWM, and 44 WSM were included. The vaginal bacterial communities of all women clustered into four taxonomic groups with the dominant taxonomic group in each being Lactobacillus, Lachnospiraceae, Prevotella, and Sneathia. Regarding differences in organism composition between risk behavior groups, the abundance of Atopobium (relative ratio (RR)=0.24; 95%CI 0.11-0.54) and Parvimonas (RR=0.33; 95%CI 0.11-0.93) were significantly lower in WSW than WSM, the abundance of Prevotella was significantly higher in WSW than WSWM (RR=1.77; 95%CI 1.10-2.86), and the abundance of Atopobium (RR=0.41; 95%CI 0.18-0.88) was significantly lower in WSWM than WSM. Overall, WSM had the highest diversity of bacterial taxa.

Conclusion: The microbiology of BV among women in different risk behavior groups is heterogeneous. WSM in this study had the highest diversity of bacterial taxa. Additional studies are needed to better understand these differences.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Bar graph of the most…
Figure 1. Bar graph of the most abundant OTUs within the total sample and within each sexual risk behavior group.
The bacteria group represents sequences which cannot be classified beyond the kingdom (bacteria) level.
Figure 2. Heatmap with colors showing the…
Figure 2. Heatmap with colors showing the abundance of microbial taxa (in proportion) found in the vaginal bacterial communities of 112 women with BV (color key is indicated to the left of the heatmap diagram).
(A) Complete linkage clustering of samples based on the species composition and abundance of vaginal bacterial communities that define clusters I-IV. (B) Nugent score, pH measurement, and sexual risk behavior group for each of the 112 vaginal bacterial community samples (color keys for Nugent scores, pH measurements, and sexual risk behavior groups are to the right and the left of the heatmap diagram). (C) Complete linkage clustering of taxa based on Spearman’s correlation coefficient profiles. (D) Shannon diversity indices calculated from the 112 vaginal bacterial communities.
Figure 3. Pie chart representation of the…
Figure 3. Pie chart representation of the vaginal bacterial communities comprising clusters I-IV within each sexual risk behavior group of women.
The number of women from each sexual risk behavior group is indicated in parentheses. Note that 3 samples from the WSWM group contained taxonomic groups that were considered as outliers (i.e., had a major proportion of unidentified bacteria, Enterobacteriaceae, or Serratia) and were not included in this analysis.
Figure 4. Forest plot depicting the differences…
Figure 4. Forest plot depicting the differences in abundance estimates (blue dot) and 95% confidence intervals (CIs) (solid red line) among sexual risk behavior groups of each of the taxonomic groups with an abundance of ≥2% across the entire sequence library.
A relative ratio of 1 (dotted vertical line) signifies that both comparison groups have the same mean abundance. If the solid red line for sexual risk behavior group comparisons does not overlap with a relative ratio of 1, a statistically significant difference between the two comparison groups is supported.
Figure 5. Box plot of Shannon’s diversity…
Figure 5. Box plot of Shannon’s diversity for each sexual risk behavior group.
The red plus signs depict the average value of Shannon’s diversity for each sexual risk behavior group; blue dots represent outliers. The WSM group had the highest diversity compared to the other sexual risk behavior groups.
Figure 6. Unweighted beta-diversity Unifrac analysis.
Figure 6. Unweighted beta-diversity Unifrac analysis.
This figure depicts a distinct cluster within the WSM group (indicated in green) that is not present within the WSW or WSWM groups (indicated in blue and red, respectively).

References

    1. Fleury FJ (1981) Adult vaginitis. Clin Obstet Gynecol 24: 407-438. doi:10.1097/00003081-198106000-00008. PubMed: .
    1. 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. PubMed: .
    1. Rein MF, Holmes KK (1983) Non-specific vaginitis, vulvovaginal candidiasis, and trichomoniasis. In: Remington JS, Swartz MN. Current Clinical Topics in Infectious Diseases. New York: McGraw-Hill; pp. 281-315.
    1. 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: 1863-1868. doi:10.1086/315127. PubMed: .
    1. Sweet RL (2000) Gynecologic conditions and bacterial vaginosis: implications for the non-pregnant patient. Infect Dis Obstet Gynecol 8: 184-190. doi:10.1155/S1064744900000260. PubMed: .
    1. Leitich H, Bodner-Adler B, Brunbauer M, Kaider A, Egarter C et al. (2003) Bacterial vaginosis as a risk factor for preterm delivery: a meta-analysis. Am J Obstet Gynecol 189: 139-147. doi:10.1016/j.ajog.2003.10.281. PubMed: .
    1. Cherpes TL, Meyn LA, Krohn MA, Lurie JG, Hillier SL (2003) Association between acquisition of herpes simplex virus type 2 in women and bacterial vaginosis. Clin Infect Dis 37: 319-325. doi:10.1086/375819. PubMed: .
    1. Myer L, Denny L, Telerant R, Souza Md, Wright TC Jr. et al. (2005) Bacterial vaginosis and susceptibility to HIV infection in South African women: a nested case-control study. J Infect Dis 192: 1372-1380. doi:10.1086/462427. PubMed: .
    1. Peipert JF, Lapane KL, Allsworth JE, Redding CA, Blume JD et al. (2008) Bacterial vaginosis, race, and sexually transmitted infections: does race modify the association? Sex Transm Dis 35: 363-367. doi:10.1097/OLQ.0b013e31815e4179. PubMed: .
    1. Brotman RM, Klebanoff MA, Nansel TR, Yu KF, Andrews WW et al. (2010) Bacterial vaginosis assessed by gram stain and diminished colonization resistance to incident gonococcal, chlamydial, and trichomonal genital infection. J Infect Dis 202: 1907-1915. doi:10.1086/657320. PubMed: .
    1. Hillier SL, Marrazzo J, Holmes KK (2008) Bacterial vaginosis. In: Holmes KK, Sparling PF, Mardh PA. Sexually transmitted diseases. 4th edition. New York: McGraw-Hill; pp. 737-768.
    1. Srinivasan S, Fredricks DN (2008) The human vaginal bacterial biota and bacterial vaginosis. Interdiscip Perspect. Infect Dis 2008: 750479.
    1. Swidsinski A, Doerffel Y, Loening-Baucke V, Swidsinski S, Verstraelen H et al. (2010) Gardnerella biofilm involves females and males and is transmitted sexually. Gynecol Obstet Invest 70: 256-263. doi:10.1159/000314015. PubMed: .
    1. Muzny CA, Schwebke JR (2013) Gardnerella vaginalis: Still a prime suspect in the pathogenesis of bacterial vaginosis. Curr Infect Dis Rep 15: 130-135. doi:10.1007/s11908-013-0318-4. PubMed: .
    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. PubMed: .
    1. Cherpes TL, Hillier SL, Meyn LA, Busch JL, Krohn MA (2008) A delicate balance: risk factors for acquisition of bacterial vaginosis include sexual activity, absence of hydrogen peroxide-producing lactobacilli, black race, and positive herpes simplex virus type 2 serology. Sex Transm Dis 35: 78-83. doi:10.1097/OLQ.0b013e318156a5d0. PubMed: .
    1. Moi H (1990) Prevalence of bacterial vaginosis and its association with genital infections, inflammation, and contraceptive methods in women attending sexually transmitted disease and primary health clinics. Int J STD AIDS 1: 86-94. PubMed: .
    1. Barbone F, Austin H, Louv WC, Alexander WJ (1990) A follow-up study of methods of contraception, sexual activity, and rates of trichomoniasis, candidiasis, and bacterial vaginosis. Am J Obstet Gynecol 163: 510-514. doi:10.1016/0002-9378(90)91186-G. PubMed: .
    1. Schwebke JR, Desmond R (2005) Risk factors for bacterial vaginosis in women at high risk for sexually transmitted diseases. Sex Transm Dis 32: 654-658. doi:10.1097/01.olq.0000175396.10304.62. PubMed: .
    1. Gardner HL, Dukes CD (1955) Haemophilus vaginalis vaginitis: a newly defined specific infection previously classified non-specific vaginitis. Am J Obstet Gynecol 69: 962-976. PubMed: .
    1. Bradshaw CS, Morton AN, Hocking J, Garland SM, Morris MB et al. (2006) High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. J Infect Dis 193: 1478-1486. doi:10.1086/503780. PubMed: .
    1. Sanchez S, Garcia PJ, Thomas KK, Catlin M, Holmes KK (2004) Intravaginal metronidazole gel versus metronidazole plus nystatin ovules for bacterial vaginosis: a randomized controlled trial. Am J Obstet Gynecol 191: 1898-1906. doi:10.1016/j.ajog.2004.06.089. PubMed: .
    1. Koumans EH, Sternberg M, Bruce C, McQuillan G, Kendrick J et al. (2007) The prevalence of bacterial vaginosis in the United States, 2001-2004; associations with symptoms, sexual behaviors, and reproductive health. Sex Transm Dis 34: 864-869. doi:10.1097/OLQ.0b013e318074e565. PubMed: .
    1. Evans AL, Scally AJ, Wellard SJ, Wilson JD (2007) Prevalence of bacterial vaginosis in lesbians and heterosexual women in a community setting. Sex Transm Infect 83: 470-475. doi:10.1136/sti.2006.022277. PubMed: .
    1. Marrazzo JM, Koutsky LA, Handsfield HH (2001) Characteristics of female sexually transmitted disease clinic clients who report same-sex behaviour. Int J STD AIDS 12: 41-46. doi:10.1258/0956462011916721. PubMed: .
    1. Marrazzo JM, Koutsky LA, Eschenbach DA, Agnew K, Stine K et al. (2002) Characterization of vaginal flora and bacterial vaginosis in women who have sex with women. J Infect Dis 185: 1307-1313. doi:10.1086/339884. PubMed: .
    1. Bailey JV, Farquhar C, Owen C, Mangtani P (2004) Sexually transmitted infections in women who have sex with women. Sex Transm Infect 80: 244-246. doi:10.1136/sti.2003.007641. PubMed: .
    1. Pinto VM, Tancredi MV, Tancredi Neto A, Buchalla CM (2005) Sexually transmitted disease/HIV risk behaviour among women who have sex with women. AIDS 19 Suppl 4: S64-S69. doi:10.1097/01.aids.0000192072.80572.43. PubMed: .
    1. Evans BA, Kell PD, Bond RA, MacRae KD (1998) Racial origin, sexual lifestyle, and genital infection among women attending a genitourinary medicine clinic in London (1992). Sex Transm Infect 74: 45-49. doi:10.1136/sti.74.1.45. PubMed: .
    1. Edwards A, Thin RN (1990) Sexually transmitted diseases in lesbians. Int J STD AIDS 1: 178-181. PubMed: .
    1. McCaffrey M, Varney P, Evans B, Taylor-Robinson D (1999) Bacterial vaginosis in lesbians: evidence for lack of sexual transmission. Int J STD AIDS 10: 305-308. doi:10.1258/0956462991914168. PubMed: .
    1. Marrazzo JM, Thomas KK, Agnew K, Ringwood K (2010) Prevalence and risks for bacterial vaginosis in women who have sex with women. Sex Transm Dis 37: 335-339. PubMed: .
    1. 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 4: S273-S281. doi:10.1093/clinids/16.Supplement_4.S273. PubMed: .
    1. Eschenbach DA, Hillier S, Critchlow C, Stevens C, DeRouen T et al. (1988) Diagnosis and clinical manifestations of bacterial vaginosis. Am J Obstet Gynecol 158: 819-828. doi:10.1016/0002-9378(88)90078-6. PubMed: .
    1. Rifkin SB, Smith MR, Brotman RM, Gindi RM, Erbelding EJ (2009) Hormonal contraception and risk of bacterial vaginosis diagnosis in an observational study of women attending STD clinics in Baltimore, MD. Contraception 80: 63-67. doi:10.1016/j.contraception.2009.01.008. PubMed: .
    1. Marrazzo JM, Martin DH, Watts DH, Schulte J, Sobel JD et al. (2010) Bacterial vaginosis: identifying research gaps proceedings of a workshop sponsored by DHHS/NIH/NIAID. Sex Transm Dis 37: 732-744. doi:10.1097/OLQ.0b013e3181fbbc95. PubMed: .
    1. Srinivasan S, Hoffman NG, Morgan MT, Matsen FA, Fiedler TL et al. (2012) Bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria. PLOS ONE 7: e37818. doi:10.1371/journal.pone.0037818. PubMed: .
    1. Fredricks DN, Fiedler TL, Marrazzo JM (2005) Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 353: 1899-1911. doi:10.1056/NEJMoa043802. PubMed: .
    1. Schwebke JR (2009) Bacterial vaginosis: are we coming full circle? J Infect Dis 200: 1633-1635. doi:10.1086/648093. PubMed: .
    1. Zozaya-Hinchliffe M, Lillis R, Martin DH, Ferris MJ (2010) Quantitative PCR assessments of bacterial species in women with and without bacterial vaginosis. J Clin Microbiol 48: 1812-1819. doi:10.1128/JCM.00851-09. PubMed: .
    1. Eren AM, Zozaya M, Taylor CM, Dowd SE, Martin DH et al. (2011) Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation. PLOS ONE 6: e26732. doi:10.1371/journal.pone.0026732. PubMed: .
    1. 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 1: 4680-4687. doi:10.1073/pnas.1002611107. PubMed: .
    1. Fredricks DN, Fiedler TL, Thomas KK, Oakley BB, Marrazzo JM (2007) Targeted PCR for detection of vaginal bacteria associated with bacterial vaginosis. J Clin Microbiol 45: 3270-3276. doi:10.1128/JCM.01272-07. PubMed: .
    1. Fethers K, Twin J, Fairley CK, Fowkes FJ, Garland SM et al. (2012) Bacterial vaginosis (BV) candidate bacteria: associations with BV and behavioural practices in sexually-experienced and inexperienced women. PLOS ONE 7: e30633. doi:10.1371/journal.pone.0030633. PubMed: .
    1. Muzny CA, Sunesara IR, Martin DH, Mena LA (2011) Sexually transmitted infections and risk behaviors among African American women who have sex with women: does sex with men make a difference? Sex Transm Dis 38: 1118-1125. doi:10.1097/OLQ.0b013e31822e6179. PubMed: .
    1. Workowski KA, Berman SM (2006) Sexually transmitted diseases treatment guidelines, 2006. MMWR Recomm Rep 55: 1-94. PubMed: .
    1. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD et al. (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335-336. doi:10.1038/nmeth.f.303. PubMed: .
    1. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460-2461. doi:10.1093/bioinformatics/btq461. PubMed: .
    1. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rDNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261-5267. doi:10.1128/AEM.00062-07. PubMed: .
    1. Cole JR, Wang Q, Cardenas E, Fish J, Chai B et al. (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37: D141-D145. doi:10.1093/nar/gkp353. PubMed: .
    1. Lozupone C, Hamady M, Knight R (2006) UniFrac - an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7: 371. doi:10.1186/1471-2105-7-371. PubMed: .
    1. Boyles AL, Harris SF, Rooney AA, Thayer KA (2011) Forest Plot Viewer: a new graphing tool. Epidemiology 22: 746-747. doi:10.1097/EDE.0b013e318225ba48. PubMed: .
    1. Falsen E, Pascual C, Sjödén B, Ohlén M, Collins MD (1999) Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp. nov. Int J Syst Bacteriol 49 1: 217-221. doi:10.1099/00207713-49-1-217. PubMed: .
    1. Zhou X, Brown CJ, Abdo Z, Davis CC, Hansmann MA et al. (2007) Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women. ISME J 1: 121-133. doi:10.1038/ismej.2007.12. PubMed: .
    1. Ferris MJ, Norori J, Zozaya-Hinchliffe M, Martin DH (2007) Cultivation-independent analysis of changes in bacterial vaginosis flora following metronidazole treatment. J Clin Microbiol 45: 1016-1018. doi:10.1128/JCM.02085-06. PubMed: .
    1. Jakobsson T, Forsum U (2007) Lactobacillus iners: a marker of changes in the vaginal flora? J Clin Microbiol 45: 3145. doi:10.1128/JCM.00558-07. PubMed: .
    1. Martin DH, Zozaya M, Lillis R, Miller J, Ferris MJ (2012) The microbiota of the human genitourinary tract: trying to see the forest through the trees. Trans Am Clin Climatol Assoc 123: 242-256 PubMed: .
    1. Flynn TR, Paster BJ, Stokes LN, Susarla SM, Shanti RM (2012) Molecular methods for diagnosis of odontogenic infections. J Oral Maxillofac Surg 70: 1854-1859. doi:10.1016/j.joms.2011.09.009. PubMed: .
    1. Shah HN, Collins DM (1990) Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides . Int J Syst Bacteriol 40: 205-208. doi:10.1099/00207713-40-2-205. PubMed: .
    1. Citron DM, Poxton IR, Jo Baron E (2007) Bacteroides, Porphyromonas, Prevotella, Fusobacterium, and Other Anaerobic Gram-Negative Rods. In: Murray PR, Jo Baron E, Landry ML. Manual of Clinical Microbiology, 9th Edition. Washington, DC: ASM Press; pp. 911-932.
    1. Jousimies-Somer H (1997) Recently described clinically important anaerobic bacteria: taxonomic aspects and update. Clin Infect Dis 25 Suppl 2: S78-S87. doi:10.1086/516227. PubMed: .
    1. Aroutcheva A, Ling Z, Faro S (2008) Prevotella bivia as a source of lipopolysaccharide in the vagina. Anaerobe 14: 256-260. doi:10.1016/j.anaerobe.2008.08.002. PubMed: .
    1. Marrazzo JM, Thomas KK, Fiedler TL, Ringwood K, Fredricks DN (2010) Risks for acquisition of bacterial vaginosis among women who report sex with women: a cohort study. PLOS ONE 5: e11139. doi:10.1371/journal.pone.0011139. PubMed: .
    1. Mitchell C, Manhart LE, Thomas KK, Agnew K, Marrazzo JM (2011) Effect of sexual activity on vaginal colonization with hydrogen peroxide-producing lactobacilli and Gardnerella vaginalis . Sex Transm Dis 38: 1137-1144. doi:10.1097/OLQ.0b013e31822e6121. PubMed: .
    1. Bradshaw CS, Morton AN, Garland SM, Morris MB, Moss LM et al. (2005) Higher-risk behavioral practices associated with bacterial vaginosis compared with vaginal candidiasis. Obstet Gynecol 106: 105-114. doi:10.1097/01.AOG.0000163247.78533.7b. PubMed: .
    1. Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J et al. (2004) Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis. BMC Microbiol 4: 16. doi:10.1186/1471-2180-4-16. PubMed: .
    1. Hill GB (1993) The microbiology of bacterial vaginosis. Am J Obstet Gynecol 169: 450-454. doi:10.1016/0002-9378(93)90339-K. PubMed: .
    1. Catlin BW (1992) Gardnerella vaginalis: characteristics, clinical considerations, and controversies. Clin Microbiol Rev 5: 213-237. PubMed: .
    1. Marrazzo JM, Fiedler TL, Srinivasan S, Thomas KK, Liu C et al. (2012) Extravaginal reservoirs of vaginal bacteria as risk factors for incident bacterial vaginosis. J Infect Dis 205: 1580-1588. doi:10.1093/infdis/jis242. PubMed: .
    1. Ferris MJ, Masztal A, Aldridge KE, Fortenberry JD, Fidel PL Jr. et al. (2004) Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis. BMC Infect Dis 4: 5. doi:10.1186/1471-2334-4-5. PubMed: .

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