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The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization?

The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization?

  • Janneke H. H. M. van de Wijgert, 
  • Hanneke Borgdorff, 
  • Rita Verhelst, 
  • Tania Crucitti, 
  • Suzanna Francis, 
  • Hans Verstraelen, 
  • Vicky Jespers


We conducted a systematic review of the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A) to determine if consistent molecular vaginal microbiota (VMB) composition patterns can be discerned after a decade of molecular testing, and to evaluate demographic, behavioral and clinical determinants of VMB compositions. Studies were eligible when published between 1 January 2008 and 15 November 2013, and if at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization) was used to characterize the VMB. Sixty three eligible studies were identified. These studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment and that bacterial vaginosis (BV) is best described as a polybacterial dysbiosis. The extent of dysbiosis correlates well with Nugent score and vaginal pH but not with the other Amsel criteria. Lactobacillus crispatus is more beneficial than L. iners. Longitudinal studies have shown that a L. crispatus-dominated VMB is more likely to shift to a L. iners-dominated or mixed lactobacilli VMB than to full dysbiosis. Data on VMB determinants are scarce and inconsistent, but dysbiosis is consistently associated with HIV, human papillomavirus (HPV), and Trichomonas vaginalis infection. In contrast, vaginal colonization with Candida spp. is more common in women with a lactobacilli-dominated VMB than in women with dysbiosis. Cervicovaginal mucosal immune responses to molecular VMB compositions have not yet been properly characterized. Molecular techniques have now become more affordable, and we make a case for incorporating them into larger epidemiological studies to address knowledge gaps in etiology and pathogenesis of dysbiosis, associations of different dysbiotic states with clinical outcomes, and to evaluate interventions aimed at restoring and maintaining a lactobacilli-dominated VMB.


It has been known for some time that most vaginal microbiota (VMB) consist predominantly of lactobacilli and that VMB alterations can cause symptomatic conditions [1]. The most familiar condition is bacterial vaginosis (BV), which has traditionally been characterized as a reduction of vaginal lactobacilli and an overgrowth of other (facultative) anaerobic bacteria. In clinical settings, BV is typically diagnosed using Amsel criteria (three of the following four criteria should be present: 1) clue cells on wet mount microscopy; 2) a ‘fishy’ odour after adding 10% KOH to vaginal secretions; 3) vaginal pH>4.5; and 4) thin, homogenous vaginal discharge) [2]. In research settings, BV is also often defined by Gram stain Nugent scoring, which is based on microscopic visualization of three bacterial morphotypes (a Nugent score of 0–3 is considered normal, 4–6 intermediate microbiota, and 7–10 BV) [3]. BV is not highly inflammatory and is therefore often asymptomatic; this is why it is referred to as a vaginosis and not a vaginitis [1]. Two common types of microbiological vaginitis are vaginal candidiasis and trichomoniasis [1]. Vaginal candidiasis is often highly inflammatory and is typically diagnosed by wet mount microscopy and/or culture of Candida species. Trichomoniasis is caused by the sexually transmitted single-celled parasite Trichomonas vaginalis, which can be detected by microscopy, culture or PCR.

Other types of vaginitis have been described (such as aerobic vaginitis, desquamative inflammatory vaginitis, and atrophic vaginitis) but occur less frequently [4][6]. The term ‘aerobic vaginitis’ is used by some clinicians to refer to vaginal inflammation believed to be caused by streptococci, staphylococci and Escherichia coli [4]. While the roles of vaginal Streptococcus agalactiae (also known as Group B streptococcus) and E. coli in invasive maternal and neonatal infections have been well-documented [7], their potential roles in causing a vaginitis syndrome distinct from BV is not universally accepted.

Altered communities of micro-organisms in the vagina are not only implicated in septic postpartum and neonatal infections but also in pelvic inflammatory disease [8], miscarriage and pre-term birth [9], and increased HIV acquisition and onward transmission [10][12]. VMB alterations may therefore be of much greater public health importance than was previously assumed.

In the last decade, phylogenetic analyses of vaginal samples (mostly bacterial 16S ribosomal RNA gene sequencing) have shown that bacterial communities in the vagina are more complex than previously thought. The first study using molecular methods to characterize the VMB was published in 2002 [13]. In a review of studies conducted between 2002 and 2008, Srinivasan and colleagues concluded that BV is indeed associated with a loss of lactobacilli and the introduction and/or overgrowth of other (facultative) anaerobic bacteria, and identified important VMB bacteria that had previously been missed by culture-based methods [14]. These bacteria included species in the Lactobacillus genus (e.g. L. iners) and bacteria associated with BV (e.g. Atopobium vaginae and three bacteria in the Lachnospiraceae family temporarily named BVAB1, 2 and 3) [13], [15], [16].

Since 2008, high throughput molecular techniques have become more affordable and accessible, and many more VMB characterization studies have been performed. We conducted a systematic review of the published literature from 2008 to date, to synthesize current knowledge about the VMB and its determinants, and to identify research gaps.


We conducted a systematic review according to the PRISMA 2009 guidelines [17].

Our first objective was to determine if any consistent VMB composition patterns can be discerned after a decade of molecular testing, despite the fact that different groups have used different molecular techniques and/or operating procedures. Our second objective was to review correlations between molecular compositions, Amsel criteria, and Nugent scoring. Our third objective was to assess which determinants (sociodemographic, physiological, and behavioral risk factors, and the presence of pathogens in the genital tract) have been consistently associated with certain VMB composition patterns in different studies.

Search strategy and selection criteria

Eligible studies included studies that used at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization). We only included PCR and DNA hybridization studies if multiple bacterial species or genera were assessed (either by multiple individual assays or by multiplex assays). We excluded studies that focused on viral, archaeal, fungal, or protozoal diversity, or on development of diagnostic assays. We only considered randomized controlled intervention trials if the baseline data, prior to the intervention, could be used to address one of our objectives. Article selection was based on the first objective; not all articles also addressed the second and third objectives.

We searched the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A.) for articles between 1 January 2008 and 15 November 2013, limiting our search to articles written in English. We started our review in 2008 as opposed to 2002 (when the first molecular VMB data were published) because Srinivasan and Fredricks published a review of the early studies in 2008 [14]. We searched titles and abstracts using the search term ‘vaginal micr*’. Two authors (JvdW and HB) assessed the articles for eligibility, and hand-searched the reference lists of eligible articles to identify additional articles. Five authors (JvdW, HB, RV, VJ, and TC) extracted data from all eligible articles using predefined data extraction tables, which included the data categories presented in tables 1 and 2, a description of the VMB compositions and correlates that the study had identified, and study strengths and weaknesses. Each article was reviewed independently by two authors.

Table 1. Characteristics of molecular vaginal microbiota articles published between 1 January 2008 and 15 November 2013.

Table 2. Vaginal microbiota communities identified by clustering techniques in 17 articles.


Study selection

Our Medline database search yielded 475 results, of which 50 were eligible. We identified 20 additional eligible articles from the reference lists of the initial 50 articles. After data extraction, a further seven articles were rejected because they did not address our objectives appropriately (mostly because they focused on technical laboratory issues or diagnostic assay development). A total of 63 articles are therefore included in this review [18][80].

Study characteristics

Table 1 shows characteristics of the 63 articles (references are included in the table and are not repeated here). It should be noted that one article could include data from more than one data extraction category, which is why some column totals exceed 63. Most of the articles reported data from North America (31 articles), followed by Europe (13), Africa (10), Asia (9), and Central America (3). Most sample sizes were small, with only 19 articles reporting a sample size larger than 100. Most of the study populations were non-pregnant adult women of reproductive age, with or without BV as the only diagnosed condition (28 articles). Other study populations included adolescents/virgins (3 articles), pregnant women (7), postmenopausal women (5), women attending a sexual health clinic (7) or with confirmed HIV, HPV or other infections (16), female sex workers (2), women who have sex with women (WSW; 3), and women undergoing in-vitro fertilization (IVF) (2). Sequencing was the most commonly employed molecular technique; earlier studies tended to sequence DNA isolated from individual colonies obtained by bacterial culture (15 articles), whereas later studies extracted DNA directly from genital samples followed by next generation sequencing (18 studies used 454 sequencing (454 Life Sciences Corporation, Branford, CT, US) and four used other platforms). PCR was also commonly used with 19 studies using quantitative PCR (qPCR) for individual species/genera, and four studies using qualitative multiplex PCR. Other molecular techniques included DNA fingerprinting techniques (13), phylogenetic DNA microarrays (4), and hybridization to oligonucleotide probes coupled to beads (1). One of the main aims in all articles was to describe the VMB in a particular study population, and 20 articles included longitudinal data (indicated in Table 1 as longitudinal VMB changes); three articles also described cervical and/or endometrial microbiota and three rectal and/or oral reservoirs of vaginal bacteria; and 17 employed a clustering technique to characterize bacterial communities.

Definitions used for a healthy VMB and for BV

Most of the 63 articles that we reviewed used a Nugent score of 0–3 to define a healthy VMB (26 articles), with an additional 12 using a Nugent score of 0–3 plus the presence of fewer than three Amsel criteria, and nine using a Nugent score of 0–6. Fourteen articles did not provide a definition, and the remaining 27 articles used a variety of definitions based on microscopy, vaginal pH, and clinical symptoms. In the 37 articles that described a comparison between a healthy VMB and BV, BV was mostly defined as a Nugent score of 7–10 (20 articles), with an additional nine using a Nugent score of 7–10 plus the presence of three or more Amsel criteria, and the remaining eight using a variety of other definitions based on microscopy and clinical symptoms.

Vaginal bacterial communities by clustering

The 17 studies that used a clustering technique to characterize the composition of VMB bacterial communities can be subdivided into those that used comprehensive data based on next generation sequencing of DNA extracted from vaginal samples (10 articles) [36], [40], [42], [48], [56], [58], [60], [65], [71], [72] and those that used less comprehensive data by sequencing DNA of bacterial culture colonies (2) [28], [50], fingerprinting (3) [24], [31], [35], or qPCR (2) [62], [79] (Table 2). The articles included different study populations and employed a variety of molecular and clustering procedures, but consistent clustering patterns can be discerned nonetheless. A total of three to nine clusters were described. The majority of studies found one cluster dominated by L. iners (15 articles) and one dominated by L. crispatus (11) (Table 2). Clusters dominated by L. jensenii (2 articles), L. gasseri (5), or G. vaginalis (4) were less common. Clusters containing high proportions of multiple Lactobacillus spp., or Lactobacillus spp. combined with G. vaginalis, were described in eight articles.

All studies identified at least one cluster that was not dominated by a single taxon, but contained mixtures of anaerobes with or without Lactobacillus spp. (Table 2). These clusters typically contained L. iners (sometimes L. gasseri) and G. vaginalis, plus mixtures of other strict or facultative anaerobic bacteria. In the ten studies that employed next generation sequencing of DNA extracted from vaginal samples, the 25 most abundant taxa consistently (in at least 50% of studies) included the following additional taxa: A. vaginae, Eggerthella spp., Mobiluncus spp., Lachnospiraceae (including the species BVAB1-3), Dialister spp., Megasphaera spp., Parvimonas (formerly Peptostreptococcus) spp., Veillonella spp., Streptococcus spp., Staphylococcus spp., Gemella spp., Prevotella spp., Porphyromonas spp., Bacteroides spp., Sneathia spp., Leptotrichia spp., Mycoplasma spp., Ureaplasma spp., and Escherichia/Shigella spp. Other bacterial taxa were often found but either in low abundance or not consistently. Most articles identified more than one cluster with mixed taxa; these clusters typically did not differ significantly in the total number or types of bacterial taxa present but they did differ in their relative proportions. We were not able to discern consistent patterns across studies. Only three of the 17 articles reported clusters dominated by streptococci, staphylococci, Proteus spp., or Escherichia/Shigella spp. (Table 2).

Longitudinal VMB patterns

One of the conclusions of the Human Microbiome Project was that within-subject microbiota variation over time was lower than between-subject variation for all habitats, including the vagina [67]. Similarly, several longitudinal VMB studies showed that a majority of women have a stable VMB microbiome [23], [26], [54], [59], [62]. A study in post-menopausal women showed that the VMB is usually stable in that group as well [49]. However, while one study suggests that an increased VMB diversity is associated with a decreased stability [23], others suggest that this is not necessarily the case: for example, a BV-associated VMB can be stable and persist for a long time [59]. The few articles that describe VMB transitions in molecular detail agree that women who have a L. crispatus-dominated VMB at baseline are less likely to transition to a BV-associated VMB than women who have a L. iners-dominated VMB [33], [30], [59]. The study that evaluated VMB transitions in the greatest detail found that a L. crispatus VMB more often transitions to a L. iners-dominated or mixed lactobacilli VMB than to a BV-associated VMB, and that a L. iners-dominated VMB compared to a L. crispatus-dominated VMB is twice as likely to transition to a BV-associated VMB [59]. Data on transitions related to L. jensenii, L. gasseri, and L. vaginalis-dominated VMBs are rare [33], [59]. One study in women who had clinical BV showed that BV is more likely to be persistent when BVAB1-3, Peptoniphilus lacrimalis, or Megasphaera type 2 are present in the VMB [21].

Extravaginal reservoirs of VMB bacteria

The Human Microbiome Project also concluded that vaginal microbial communities are relatively ‘simple’ at genus-level compared to oral and gut communities, but have a higher diversity of Lactobacillus spp. [67]. Solt et al. identified 673 genera in the rectum, 275 in the mouth, and 112 in the vagina [81]. Three studies assessed the presence of lactobacilli and other VMB taxa in rectal and oral specimens, as well as vaginal specimens, to test the hypothesis that the gut and mouth act as extravaginal reservoirs of VMB bacteria [34], [55], [61]. Lactobacilli and various BV-associated bacteria were indeed often found in the rectum, while lactobacilli were sometimes, and G. vaginalis consistently, found in the mouth. In one study, women who had high quantities of G. vaginalis in the mouth or rectum, or Megasphaera, Leptotrichia, or Sneathia spp. in the rectum, were more likely to develop clinical BV during follow-up; in contrast, women who had L. crispatus in the rectum were less likely to develop clinical BV [61].

VMB associations with traditional BV diagnostics

More than half of the articles (37) described molecular VMB data in relation to BV diagnosis by Amsel and/or Nugent criteria [19][23], [25][27], [30], [32], [33], [37], [38], [40][42], [45], [47], [48], [50][54], [56], [59], [60], [62], [63], [65], [67], [72], [73], [75], [76], [79], [80]. Two important areas of consensus emerged. First, almost all women carry vaginal lactobacilli regardless of their BV status by Nugent or Amsel criteria [19], [20], [22], [23], [25], [26], [33], [37][42], [45], [47], [48], [54], [59], [60], [62], [63], [73], [76], [80] but L. crispatus is predominantly found in BV-negative women [20], [22], [23], [25], [32], [33], [37], [38], [40], [42], [48], [50], [54], [59], [60], [62], [63], [65], [73], [75], [76], [80] whereas L. iners (and to a lesser extent L. gasseri) is also found in women with intermediate microbiota or BV [21], [23], [25], [33], [37], [38], [40][42], [47], [50], [54], [59], [60], [62], [63], [65], [73], [76], [79], [80]. This is also reflected in correlation studies, in which L. iners correlates well with BV-associated bacteria but not with L. crispatus [56], [65]. A second area of consensus is that BV diagnosis is characterized by increased bacterial diversity and the presence of the multiple taxa of (facultative) anaerobes that were described in the previous paragraph [19][23], [25][27], [30], [32], [33], [37], [38], [40][42], [45], [47], [48], [50][54], [56], [59], [60], [62], [63], [65], [67], [72], [73], [75], [76], [79], [80]. qPCR studies consistently report a decline in overall Lactobacillus load in BV, and an increase in bacterial loads of BV-associated bacteria [26], [41], [47], [62], [76], [80]. It is not yet clear from the studies we reviewed whether BV is associated with a higher overall bacterial load than healthy lactobacilli-dominated microbiota. Importantly, several studies showed that G. vaginalis and Prevotella spp. are often found regardless of BV status by Nugent or Amsel criteria, but their abundances increase in BV; furthermore, a synergistic effect between them was noted [26], [37], [41], [42], [62], [79]. The role of streptococci, staphylococci, and enterococci is generally not well described; when present, they are usually present in low abundance. One study (using DNA hybridization) reported that their presence in the VMB did not differ by BV status [32], whereas another study (also using DNA hybridization) reported higher levels in women with intermediate microbiota by Nugent score [22].

Higher bacterial diversity and/or higher levels of individual BV-associated bacteria are consistently associated with a higher vaginal pH [30], [42], [60], [67], [72], [80] and Nugent score [42], [47], [51], [80]. Vice versa, increasing abundance of lactobacilli is consistently associated with a lower vaginal pH [30], [40], [67], [80] and Nugent score [80]. Associations with the other Amsel criteria have been less well studied, but one study found that only the Leptotrichia and Eggerthella genera were associated with all four Amsel criteria [60].

VMB associations with other clinical outcomes

Vaginal colonization with Candida spp. seems more common in women with a lactobacilli-dominated VMB than in women with BV [26], [31], [72], as had been noted previously in epidemiological studies using traditional diagnostic methods [11]. In contrast, T. vaginalis has often been strongly associated with BV in the past, and this was confirmed in a study using 454 sequencing [64]. Convincing patterns of associations between bacterial sexually transmitted infections (STIs) and the molecular composition of the VMB did not emerge, most likely due to the fact that women with bacterial STIs were often excluded or the prevalence rates were low.

Eleven studies assessed the VMB by HIV status [20], [30], [40], [44][46], [48], [51], [53], [58], [77] and five studies by HPV status [46], [56], [66], [71], [74]. One study found no relationship between HIV and the VMB [53], but most found trends towards decreased lactobacilli (and particularly L. crispatus) [44], [46], [48] and increased bacterial diversity [45], [51], particularly in women who had both HIV as well as BV by Nugent score [20]. Similar trends were found related to HIV-1 RNA load in the genital tract [30], [77]. One study found an increased prevalence of E. coli-dominated VMB in HIV-positive women [48], and another one found increased HIV transmission from mother to child with increasing VMB diversity in the mother (although this did not reach statistical significance) [58]. HPV also seems to be associated with a reduction in lactobacilli [71] and increased VMB diversity [46], [71], [74]. In one study, Sneathia spp. were strongly associated with the presence of high risk HPV [71].

The cervical microbiota are similar to the VMB, except that bacterial loads are lower [47]. A comprehensive qPCR study showed that cervical bacterial diversity is highest in women with BV, followed by women with cervicitis and healthy women, with only small differences between the latter two; BV was associated with a dramatic reduction in lactobacilli in the vagina and cervix, whereas cervicitis with a reduction in the cervix only [47]. The authors conclude that the VMB does not play a large role in cervicitis. Another study found good agreement between PCR results of five BV-associated species in cervical and endometrial samples of women with pelvic inflammatory disease, although this did not reach statistical significance [27].

BV and gingivitis were also reported to be associated, with counts of P. bivia, P. disiens, M. curtisii, and M. mulieris being particularly high in women with both BV and gingivitis [32]. Finally, a lactobacilli-dominated VMB was associated with a reduced risk of pre-term birth, a higher likelihood of IVF resulting in a live birth, and a reduced risk of vaginal dryness in postmenopausal women in one study each [49], [69], [78].

We found only one study that correlated molecular VMB composition (using both culture and a DNA-DNA checkerboard including L. iners and 12 BV-associated species) with vaginal immune responses [22]. Total viable bacterial counts and the presence of BV-associated bacteria were positively associated with cervicovaginal IL-1α and IL-1β (and BV-associated bacteria also with IL-6 and IL-8), whereas L. iners was negatively associated with IL-1α. The relationships with secretory leukocyte protease inhibitor (SLPI) were the other way around.

VMB associations with demographic and behavioral characteristics

Data on the association between the molecular VMB and age were inconsistent. Six studies did not find an association but four of these only included a narrow age range (exclusively reproductive age or post-menopausal women) [42], [49], [62], [72]; one study did not find a difference between reproductive age and post-menopausal women [76] and another one did not find a difference between adolescents and women of reproductive age [24]. However, three studies that quantified multiple Lactobacillus spp. found lower overall levels, as well as reduced L. crispatus levels and Lactobacillus diversity, in post-menopausal women compared to women of reproductive age [43], [55], [57].

Several articles report that Black African and African-American women compared to Caucasian or Asian women are less likely to carry L. crispatus, L. jensenii, L. gasseri and/or L. vaginalis and more likely to carry L. iners, and are more likely to have a higher bacterial diversity [35], [42], [60], [62], [77], [78]. One study found the same for U.S. Hispanic women [42].

Few studies found significant associations between the VMB at the molecular microbial level and sexual behavior. However, detailed sexual behavior data were mostly not collected, sample sizes were small, or analyses focused on risk factors for BV by Amsel or Nugent criteria even though bacterial molecular data were also available. One study found that the detection of prostate-specific antigen (as a marker of sexual activity within the last 48 hours) was negatively associated with L. crispatus and positively with L. iners and L. gasseri [62]. In another study, the prevalence of various BV-associated bacterial genera was increased with an increasing number of sexual partners [51]. Finally, a comprehensive study found a slight gain of G. vaginalis after sexual debut, but no significant gain of other BV-associated bacteria or loss of lactobacilli [66].

Even though most studies that evaluated the influence of the menstrual cycle were small, they consistently suggest that high levels of estradiol (assessed by phase in the menstrual cycle or in serum of IVF patients) promote lactobacilli, and particularly L. crispatus [18], [59], [69], [70]. Studies also consistently suggest that menses is the largest disturbing factor during the menstrual cycle, with sometimes large reductions in lactobacilli [38], [59], [62], [63], shifts from L. crispatus to L. iners [38], [70], or the appearance of BV-associated bacteria, streptococci or other Gram-positive cocci [54], [70]. Pregnancy, which is also accompanied by high estradiol levels, is associated with high levels of lactobacilli, particularly L. crispatus, and low bacterial diversity [55], [68]. However, one study found an increasing bacterial diversity in late term pregnancies [68]. In another study, a VMB dominated by L. iners or L. gasseri in the first trimester was more likely to evolve to BV later on during pregnancy; L. crispatus had the opposite effect [33].


Despite the fact that many different molecular techniques and operating procedures with specific advantages and disadvantages have been used (reviewed in [14]) and despite the fact that these technical differences can result in under- or overrepresentation of bacterial species [82], we found several areas of consensus about the VMB composition. Studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment, and that BV is best described as a polybacterial dysbiosis: the Lactobacillus load decreases, and both the diversity and bacterial load of other (facultative) anaerobic bacteria increase [24], [28], [31], [35], [36], [40], [42], [48], [50], [56], [58], [60], [62], [65], [71], [72], [79]. Furthermore, the bacteria associated with this dysbiosis are now well described [24], [28], [31], [35], [36], [40], [42], [48], [50], [56], [58], [60], [62], [65], [71], [72], [79]. Some are consistently found (G. vaginalis, A. vaginae, bacteria in the Lachnospiraceae family (including BVAB1-3), and species in the following genera: Prevotella, Eggerthella, Dialister, Megasphaera, Sneathia, Leptotrichia, Parvimonas (formerly Peptostreptococcus), Veillonella, Bacteroides, Mobiluncus, Porphyromonas, Mycoplasma, Ureaplasma, Streptococcus, Staphylococcus, Gemella, and Escherichia/Shigella) whereas others are not consistently found but can be part of a long tail of minority species. G. vaginalis and Prevotella spp. are also often present in healthy women, but their bacterial loads increase significantly in dysbiosis. Consensus is also emerging about the relative importance of different Lactobacillus species: L. iners is present in almost all women worldwide including those with dysbiosis; L. crispatus is mostly present in healthy women and might be less common in women of African or Hispanic descent; and L. jensenii, L. gasseri, and L. vaginalis are much less common [24], [28], [31], [35], [36], [40], [42], [48], [50], [56], [58], [60], [62], [65], [71], [72], [79]. Furthermore, longitudinal studies have shown that a L. crispatus-dominated VMB might transition to a L. iners-dominated VMB but is less likely to transition directly to a dysbiotic state (and vice versa) [33], [59]. The gut, and to a lesser extent the mouth, serve as extravaginal reservoirs of common VMB bacteria [34], [55], [61].

An increase of bacterial diversity and BV-associated bacteria is consistently associated with an increase in Nugent score and/or vaginal pH, but not with the other three Amsel criteria [30], [42], [47], [51], [60], [67], [72], [80]. This is reassuring because our current knowledge about the epidemiology of vaginal dysbiosis is mostly based on Nugent scoring. A recent study by Srinivasan and colleagues, however, questioned the microbial interpretation of Nugent scoring [83]. This study showed that the Mobiluncus morphotype more likely represents BVAB-1 than Mobiluncus spp., and the Bacteroides morphotype more likely represents Porphyromonas and Prevotella spp than Bacteroides spp. While these are important observations, the clinical relevance is unclear because all of these bacterial species are associated with vaginal dysbiosis. The composition and significance of the intermediate Nugent category remains unclear. One molecular study suggested that this category is a transition state from a lactobacilli-dominated VMB to dysbiosis or vice versa [40], but another study found an association with VMB clusters dominated by the facultative anaerobic bacteria that have been implicated in aerobic vaginitis [4], [22]. While it is important to investigate this further to allow for the proper interpretation of epidemiological studies that have used/are using Nugent scoring to characterize the VMB, it is likely that future studies will replace Nugent scoring by molecular VMB characterization and quantification.

We found much less consensus on VMB associations with sociodemographic, behavioral, and clinical characteristics, mostly because few studies were designed to evaluate these. Three areas of consensus stood out: Vaginal colonization with Candida spp. was consistently more common in women with a lactobacilli-dominated VMB than women with bacterial dysbiosis [26], [31], [72], infection with Trichomonas vaginalis is associated with vaginal dysbiosis [64], and a high level of estradiol is consistently associated with lactobacilli [18], [59], [69], [70]. The latter is also supported by many studies that evaluated the VMB by microscopy (reviewed in [84]). The data on the associations between the VMB and HIV and HPV infection are not entirely consistent but also point in the direction of decreased lactobacilli and increased bacterial diversity when a STI is present. We recently confirmed this in a study in women at high risk of HIV and other STIs in Rwanda [85]. This study showed that women with L. crispatus-dominated VMB had the lowest prevalence of HIV, HPV and herpes simplex type 2 (and had no bacterial STIs), with a slight increase in women with a L. iners-dominated VMB, and a significant increase in women with vaginal dysbiosis. A similar trend was found for HIV-1 RNA shedding in the genital tract of HIV-positive women. Since the study was cross-sectional, the temporality of these relationships remains to be elucidated.

It is worth emphasizing that the molecular studies did not identify large VMB differences between adolescent, reproductive age, and post-menopausal women [24], [79], except in post-menopausal women with vaginal atrophy and dryness [49]. Post-menopausal women have lower estrogen levels, which might lead to less protection from dysbiosis. However, they no longer menstruate, and are therefore protected from the potentially negative effects of menstrual blood and increased vaginal pH on the VMB.

Our review also highlighted many research gaps. Most importantly, we still do not sufficiently understand how the VMB is established and maintained, and how bacterial dysbiosis develops and resolves. In particular, the roles of L. crispatus (which seems to inhibit dysbiosis), L. iners (which does not seem to inhibit dysbiosis), and G. vaginalis and Prevotella spp. (which are often present in healthy women in low abundance but greatly increase in abundance in the dysbiotic state) are not well understood. The role of L. iners is particularly controversial [9], [86], [87]. L. iners is well adapted to the vaginal niche, is present in many different types of VMBs, and often persists after antibiotic treatment. This could mean that L. iners easily tolerates the presence of other bacteria (which in turn could lead to dysbiosis), or that it helps to restore a lactobacilli-dominated VMB during and after dysbiosis and/or antibiotic treatment. One appealing hypothesis regarding the development of dysbiosis is the formation of a vaginal biofilm [88]. Current evidence suggests that G. vaginalis can be present in the vagina as dispersed bacteria or as biofilm-associated (cohesive) bacteria, with the former associated with a lower total bacterial load than the latter [89]. In-vitro studies suggest that when the concentration of G. vaginalis increases, it starts to adhere to the vaginal epithelium, providing a scaffolding to which other species adhere [90]. Initial human biopsy studies focused on A. vaginae as another potentially important biofilm member [91], and one study found that L. iners increases G. vaginalis adherence in-vitro (although this did not reach statistical significance) [92]. However, more research is needed to properly evaluate the potential role of all relevant lactobacilli and dysbiosis-associated bacteria in biofilm formation. Furthermore, it is not yet entirely clear whether the dispersed and cohesive forms of G. vaginalis represent different G. vaginalis strains. Recent studies revealed that different G. vaginalis strains have different metabolic and virulence properties [93], encode different types of biofilm-associated proteins [94], and behave differently in in-vitro biofilm experiments [95]. The biofilm hypothesis might explain the high persistence and recurrence rates of dysbiosis because bacteria in the biofilm are dormant and therefore less susceptible to antibiotics [88]. In addition, the extracellular matrix surrounding the bacteria in the biofilm inhibits penetration of lactic acid, natural antimicrobial compounds, and antibiotics [88]. However, other explanations, such as reinfection by sexual partners and spore formation, might also play a role [21]. The biofilm hypothesis might also explain why Candida spp. more commonly overgrow when only dispersed bacteria are present and epithelial cells are exposed: Candida needs to attach to epithelial cells to thrive in the vagina [96].

Whether bacterial dysbiosis is symptomatic or not most likely depends on the degree and nature of the dysbiosis, bacterial loads, type and quantity of virulence factors expressed by bacteria, and the intensity and nature of the host’s immune responses [97]. ‘Thresholds’ (in terms of bacterial loads and diversity) might exist. While most of the above-mentioned dysbiosis-associated bacteria are never pathogenic in immune-competent hosts, streptococci, staphylococci and E. coli can cause invasive disease when present in sufficiently high abundance. S. agalactiae has been particularly well studied in that regard, and studies have indeed shown that the greater the density of colonization, the greater the probability of invasive disease in postpartum women and their neonates [98]. Only three molecular studies included in our review reported VMB clusters dominated by streptococci, staphylococci, and/or E. coli [35], [58], [65] but many additional studies showed presence of these bacteria in low abundance; this is in agreement with studies using selective culture media [7]. In-vitro studies confirm that S. agalactiae only inhibits growth of other bacteria at concentrations higher than 109 colony forming units per ml (but does not inhibit S. aureus and E. coli), and such high concentrations are rarely seen in vivo [98]. If aerobic vaginitis is defined as a VMB composition dominated by these bacteria, we conclude that it does exist but is not common. Future studies of invasive infections by streptococci, staphylococci, and E. coli should determine vaginal concentrations and not just vaginal presence.

We also do not yet sufficiently understand the metabolic synergies and dependencies of the various bacterial communities that are commonly found in the vagina. Recent studies have focused on L. iners, which is present in almost all women worldwide, in healthy and dysbiotic states. These studies suggest that L. iners is highly adapted to the vaginal compartment [9], [86], but it differentially expresses over 10% of its genome in dysbiotic compared to healthy states, with increased expression of a cytolysin, mucin, glycerol transport and related metabolic enzymes [87]. These changes likely result in the production of succinate and other short-chain fatty acids as the end product of metabolism as opposed to lactic acid, leading to an increased vaginal pH. L. iners might also be the first Lactobacillus species to recover after dysbiosis [59], which suggests a bidirectional relationship between L. iners and vaginal pathogens or dysbiosis. Other studies have noted synergistic effects between G. vaginalis and Prevotella spp., perhaps due to metabolic dependencies [91], [98]. At the moment, metagenomic studies of vaginal bacteria are ongoing but difficult to conduct because the public sequence databases do not yet contain all relevant bacterial genomes.

While molecular techniques have significantly improved our understanding of the VMB, some limitations should be noted. Molecular techniques detect viable as well as non-viable organisms, some cannot reliably differentiate species within a genus, some cannot adequately detect minority species, and most are not fully quantitative. We have taken this into account in our data interpretations as much as possible. Furthermore, even when the same molecular techniques were used, different laboratories used different operating procedures. Not all of these are important, but some (such as DNA extraction methods, amplification platform, choice of amplification target or of variable 16S region, choice or design of primers, and the presence or absence of proper negative controls to detect contamination) might result in significant inter-laboratory variation [14]. We were fully aware of these limitations and therefore focused this review on areas of consensus.

Now that the VMB of women with and without dysbiosis in different parts of the world have been well described, and molecular techniques have become more accessible and affordable, we believe that the time has come to incorporate these techniques into larger epidemiological studies with clinical outcomes. These studies should investigate the etiology and pathogenesis research gaps that were outlined above, but also possible transmission patterns of VMB bacteria, and the temporal relationships between the VMB and adverse reproductive health outcomes, such as HIV/STIs, pelvic inflammatory disease, adverse pregnancy outcomes, and invasive infections in pregnant/postpartum women and their neonates. At the moment, most treatment guidelines only advise clinicians to treat symptomatic vaginal dysbiosis, but this might have to be re-evaluated in specific at risk population groups (such as pregnant women or women highly exposed to HIV) if dysbiosis is identified as a strong risk factor for adverse outcomes in sufficiently powered longitudinal studies. In parallel, interventions that prevent dysbiosis, disrupt biofilms, and restore and maintain lactobacilli-dominated microbiota, should continue to be optimized and tested. The VMB studies discussed in this review have provided us with the tools to properly evaluate the safety and efficacy of such interventions. If safe, efficacious and affordable interventions are identified, they could potentially have a significant public health impact.

Supporting Information

Checklist S1.

PRISMA checklist.



Diagram S1.

PRISMA Flow-Diagram.



Author Contributions

Contributed to the writing of the manuscript: JvdW. Conceived the idea for this systematic review: JvdW. Selected the articles: JvdW HB. Extracted the data: JvdW HB VJ RV TC. Reviewed and commented on the manuscript: JvdW HB VJ RV TC SF HV.


  1. 1. Holmes KK, Sparling PF, Stamm WE, Piot P, Wasserheit JN, et al. (2008) Sexually Transmitted Diseases. 4th edn New York: McGraw-Hill Medical.
  2. 2. Amsel R, Totten PA, Spiegel CA, Chen KCS, 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. 3. 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.
  4. 4. Donders GGG, Vereecken A, Bosmans E, Dekeersmaecker A, Salembier G, et al. (2002) Definition of a type of abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis. BJOG 109: 34–43. doi: 10.1111/j.1471-0528.2002.00432.x
  5. 5. Sobel JD, Reichman O, Misra D, Yoo W (2011) Prognosis and treatment of desquamative inflammatory vaginitis. Obstet Gynecol 117: 850–855. doi: 10.1097/aog.0b013e3182117c9e
  6. 6. Verstraelen H, Verhelst R, Vaneechoutte M, Temmerman M (2011) Group A streptococcal vaginitis: an unrecognized cause of vaginal symptoms in adult women. Arch Gynecol Obstet 284: 95–98. doi: 10.1007/s00404-011-1861-6
  7. 7. Verani JR, McGee L, Schrag SJ (2010) Prevention of perinatal group B streptococcal disease–revised guidelines from CDC, 2010. MMWR Recomm Rep 59: 1–36.
  8. 8. Taylor BD, Darville T, Haggerty CL (2013) Does bacterial vaginosis cause pelvic inflammatory disease? Sex Transm Dis 40: 117–122. doi: 10.1097/olq.0b013e31827c5a5b
  9. 9. Li J, McCormick J, Bocking A, Reid G (2012) Importance of vaginal microbes in reproductive health. Reprod Sci 19: 235–242. doi: 10.1177/1933719111418379
  10. 10. 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
  11. 11. van de Wijgert J, Morrison C, Cornelisse P, Munjoma M, Moncada J, et al. (2008) Bacterial vaginosis and vaginal yeast, but not vaginal cleansing, increase HIV-1 acquisition in African women. J Acquir Immune Defic Syndr 8: 203–210. doi: 10.1097/qai.0b013e3181743936
  12. 12. Hayes R, Watson-Jones D, Celum C, van de Wijgert J, Wasserheit J (2010) Treatment of sexually transmitted infections for HIV prevention: end of the road or new beginning? AIDS 24: S15–S26. doi: 10.1097/01.aids.0000390704.35642.47
  13. 13. Burton JP, Reid G (2002) Evaluation of the bacterial vaginal flora of 20 postmenopausal women by direct (Nugent score) and molecular (polymerase chain reaction and denaturing gradient gel electrophoresis) techniques. J infect Dis 186: 1770–1780. doi: 10.1086/345761
  14. 14. Srinivasan S, Fredricks DN (2008) The human vaginal bacterial biota and bacterial vaginosis. Interdiscip Perspect Infect Dis 2008: 750479 doi:10.1155/2008/750479.
  15. 15. 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.
  16. 16. 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
  17. 17. Moher D, Liberati A, Tetzlaff J, Altman DG, and the PRISMA Group (2009) Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med 6: e1000097 doi:10.1371/journal.pmed.1000097.
  18. 18. Jakobsson T, Forsum U (2008) Changes in the predominant human lactobacillus flora during in vitro fertilization. Ann Clin Microbiol Antimicrob 7: 14 doi:10.1186/1476-0711-7-14.
  19. 19. Oakley BB, Fiedler TL, Marrazzo JM, Fredricks DN (2008) Diversity of human vaginal bacterial communities and associations with clinically defined bacterial vaginosis. Appl Environ Microbiol 74: 4898–4909. doi: 10.1128/aem.02884-07
  20. 20. 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: 1131–1140. doi: 10.1086/591942
  21. 21. Marrazzo JM, Thomas KK, Fiedler TL, Ringwood K, Fredricks DN (2008) Relationship of specific vaginal bacteria and bacterial vaginosis treatment failure in women who have sex with women. Ann Intern Med 149: 20–8. doi: 10.7326/0003-4819-149-1-200807010-00006
  22. 22. Nikolaitchouk N, Andersch B, Falsen E, Strömbeck L, Mattsby-Baltzer I (2008) The lower genital tract microbiota in relation to cytokine-, SLPI- and endotoxin levels: application of checkerboard DNA-DNA hybridization (CDH). APMIS 116: 263–77. doi: 10.1111/j.1600-0463.2008.00808.x
  23. 23. Wertz J, Isaacs-Cosgrove N, Holzman C, Marsh TL (2008) Temporal shifts in microbial communities in nonpregnant African-American women with and without bacterial vaginosis. Interdiscip Perspect Infect Dis 2008: 181253 doi:10.1155/2008/181253.
  24. 24. Yamamoto T, Zhou X, Williams CJ, Hochwalt A, Forney LJ (2009) Bacterial populations in the vaginas of healthy adolescent women. J Pediatr Adolesc Gynecol 22: 11–18. doi: 10.1016/j.jpag.2008.01.073
  25. 25. Dumonceaux TJ, Schellenberg J, Goleski V, Hill JE, Jaoko W, et al. (2009) Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres. J Clin Microbiol 47: 4067–4077. doi: 10.1128/jcm.00112-09
  26. 26. Biagi E, Vitali B, Pugliese C, Candela M, Donders GG, et al. (2009) Quantitative variations in the vaginal bacterial population associated with asymptomatic infections: a real-time polymerase chain reaction study. Eur J Clin Microbiol Infect Dis 28: 281–285. doi: 10.1007/s10096-008-0617-0
  27. 27. Haggerty CL, Totten PA, Ferris M, Martin DH, Hoferka S, et al. (2009) Clinical characteristics of bacterial vaginosis among women testing positive for fastidious bacteria. Sex Transm Infect 85: 242–248. doi: 10.1136/sti.2008.032821
  28. 28. Kim TK, Thomas SM, Ho M, Sharma S, Reich CI, et al. (2009) Heterogeneity of vaginal microbial communities within individuals. J Clin Microbiol 47: 1181–1189. doi: 10.1128/jcm.00854-08
  29. 29. Shi Y, Chen L, Tong J, Xu C (2009) Preliminary characterization of vaginal microbiota in healthy Chinese women using cultivation-independent methods. J Obstet Gynaecol Res 35: 525–532. doi: 10.1111/j.1447-0756.2008.00971.x
  30. 30. Mitchell C, Moreira C, Fredricks D, Paul K, Caliendo AM, et al. (2009) Detection of fastidious vaginal bacteria in women with HIV infection and bacterial vaginosis. Infect Dis Obstet Gynecol 2009: 236919 doi:10.1155/2009/236919.
  31. 31. Zhou X, Westman R, Hickey R, Hansmann MA, Kennedy C, et al. (2009) Vaginal microbiota of women with frequent vulvovaginal candidiasis. Infect Immun 77: 4130–4135. doi: 10.1128/iai.00436-09
  32. 32. Persson R, Hitti J, Verhelst R, Vaneechoutte M, Persson R, et al. (2009) The vaginal microflora in relation to gingivitis. BMC Infect Dis 9: 6 doi:10.1186/1471-2334-9-6.
  33. 33. 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.
  34. 34. El Aila NA, Tency I, Claeys G, Verstraelen H, Saerens B, et al. (2009) Identification and genotyping of bacteria from paired vaginal and rectal samples from pregnant women indicates similarity between vaginal and rectal microflora. BMC Infect Dis 9: 167 doi:10.1186/1471-2334-9-167.
  35. 35. Zhou X, Hansmann MA, Davis CC, Suzuki H, Brown CJ, et al. (2010) The vaginal bacterial communities of Japanese women resemble those of women in other racial groups. FEMS Immunol Med Microbiol 58: 169–181. doi: 10.1111/j.1574-695x.2009.00618.x
  36. 36. Forney LJ, Gajer P, Williams CJ, Schneider GM, Koenig SS, et al. (2010) Comparison of self-collected and physician-collected vaginal swabs for microbiome analysis. J Clin Microbiol 48: 1741–1748. doi: 10.1128/jcm.01710-09
  37. 37. 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
  38. 38. Srinivasan S, Liu C, Mitchell CM, Fiedler TL, Thomas KK, et al. (2010) Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis. PLoS One 5: e10197 Doi:10.1371/journal.pone.0010197.
  39. 39. 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.
  40. 40. 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.
  41. 41. 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.
  42. 42. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, et al. (2011) Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci USA (Suppl 1): 4680–4687. doi: 10.1073/pnas.1002611107
  43. 43. Gustafsson RJ, Ahrne S, Jeppsson B, Benoni C, Olsson C, et al. (2011) The Lactobacillus flora in vagina and rectum of fertile and postmenopausal healthy Swedish women. BMC Women’s Health 11: 17 doi:10.1186/1472-6874-11-17.
  44. 44. Spear GT, Gilbert D, Landay AL, Zariffard R, French AL, et al. (2011) Pyrosequencing of the genital microbiotas of HIV-seropositive and -seronegative women reveals Lactobacillus iners as the predominant Lactobacillus species. Appl Environ Microbiol 77: 378–381. doi: 10.1128/aem.00973-10
  45. 45. Dols JA, Smit PW, Kort R, Reid G, Schuren FH, et al. (2011) Microarray-based identification of clinically relevant vaginal bacteria in relation to bacterial vaginosis. Am J Obstet Gynecol 204: 305.e1–7 doi:10.1016/j.ajog.2010.11.012.
  46. 46. Dols JAM, Reid G, Kort R, Reid G, Schuren FH, et al. (2011) PCR-based identification of eight Lactobacillus species and 18 hr-HPV genotypes in fixed cervical samples of South African women at risk of HIV and BV. Diagn Cytopathol 40: 472–477. doi: 10.1002/dc.21786
  47. 47. Ling Z, Liu X, Chen X, Zhu H, Nelson KE, et al. (2011) Diversity of cervicovaginal microbiota associated with female lower genital tract infections. Microb Ecol 61: 704–714. doi: 10.1007/s00248-011-9813-z
  48. 48. Schellenberg JJ, Links MG, Hill JE, Dumonceaux TJ, Kimani J, et al. (2011) Molecular definition of vaginal microbiota in East African commercial sex workers. Appl Environ Microbiol 77: 4066–4074. doi: 10.1128/aem.02943-10
  49. 49. Hummelen R, Macklaim JM, Bisanz JE, Hammond JA, McMillan A, et al. (2011) Vaginal microbiome and epithelial gene array in post-menopausal women with moderate to severe dryness. PLoS One 6: e26602 Doi:10.1371/journal.pone.0026602.
  50. 50. Yoshimura K, Morotomi N, Fukuda K, Nakano M, Kashimura M, et al. (2011) Intravaginal microbial flora by the 16S rRNA gene sequencing. Am J Obstet Gynecol 205: 235.e1–9 doi:10.1016/j.ajog.2011.04.018.
  51. 51. Pépin J, Deslandes S, Giroux G, Sobéla F, Khonde N, et al. (2011) The complex vaginal flora of West African women with bacterial vaginosis. PLoS One 6: e25082 doi:10.1371/journal.pone.0025082.
  52. 52. Hernández-Rodríguez C, Romero-González R, Albani-Campanario M, Figueroa-Damián R, Meraz-Cruz N, et al. (2011) Vaginal microbiota of healthy pregnant Mexican women is constituted by four Lactobacillus species and several vaginosis-associated bacteria. Infect Dis Obstet Gynecol 2011: 851485 doi:10.1155/2011/851485.
  53. 53. Damelin LH, Paximadis M, Mavri-Damelin D, Birkhead M, Lewis DA, et al. (2011) Identification of predominant culturable vaginal Lactobacillus species and associated bacteriophages from women with and without vaginal discharge syndrome in South Africa. J Med Microbiol 60(Pt 2): 180–183 doi:10.1099/jmm.0.024463-0.
  54. 54. Santiago GL, Cools P, Verstraelen H, Trog M, Missine G, et al. (2011) Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles. PLoS One 6: e28180 doi:10.1371/journal.pone.0028180.
  55. 55. Petricevic a L, Domig KJ, Nierscher FJ, Krondorfer I, Janitschek C, et al. (2012) Characterisation of the oral, vaginal and rectal Lactobacillus flora in healthy pregnant and postmenopausal women. Eur J Obstet Gynecol Reprod Health 160: 93–99. doi: 10.1016/j.ejogrb.2011.10.002
  56. 56. Smith BC, McAndrew T, Chen Z, Harari A, Barris DM, et al. (2012) The cervical microbiome over 7 years and a comparison of methodologies for its characterization. PLoS One 7: e40425 doi:10.1371/journal.pone.0040425.
  57. 57. Zhang R, Daroczy K, Xiao B, Yu L, Chen R, et al. (2012) Qualitative and semiquantitative analysis of Lactobacillus species in the vaginas of healthy fertile and postmenopausal Chinese women. J Med Microbiol 61(Pt 5): 729–739. doi: 10.1099/jmm.0.038687-0
  58. 58. Frank DN, Manigart O, Leroy V, Meda N, Valéa D, et al. (2012) Altered vaginal microbiota are associated with perinatal mother-to-child transmission of HIV in African women from Burkina Faso. J Acquir Immune Defic Syndr 60: 299–306. doi: 10.1097/qai.0b013e31824e4bdb
  59. 59. Gajer P, Brotman RM, Gyoyun B, Sakamoto J, Schütte UM, et al. (2012) Temporal dynamics of the human vaginal microbiota. Sci Transl Med 4: 132ra52 doi:10.1126/scitranslmed.3003605.
  60. 60. 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.
  61. 61. 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
  62. 62. Jespers V, Menten J, Smet H, Poradosú S, Abdellati S, et al. (2012) Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests. BMC Microbiol 12: 83 Doi:10.1186/1471-2180-12-83.
  63. 63. Santiago GL, Tency I, Verstraelen H, Verhelst R, Trog M, et al. (2012) Longitudinal qPCR study of the dynamics of L. crispatus, L. iners, A. vaginae, (sialidase positive) G. vaginalis, and P. bivia in the vagina. PLoS One 7: e45281 doi:10.1371/journal.pone.0045281.
  64. 64. Brotman RM, Bradford LL, Conrad M, Gajer P, Ault K, et al. (2012) Association between Trichomonas vaginalis and vaginal bacterial community composition among reproductive-age women. Sex Transm Dis 39: 807–812. doi: 10.1097/olq.0b013e3182631c79
  65. 65. 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.
  66. 66. Mitchell CM, Fredricks DN, Winer RL, Koutsky L (2012) Effect of sexual debut on vaginal microbiota in a cohort of young women. Obstet Gynecol 120: 1306–1313.
  67. 67. Human Microbiome Project Consortium (2012) Structure, function and diversity of the healthy human microbiome. Nature 486: 207–214.
  68. 68. Aagaard K, Riehle K, Ma J, Segata N, Mistretta TA, et al. (2012) A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy. PLoS One 7: e36466 doi:10.1371/journal.pone.0036466.
  69. 69. Hyman RW, Herndon CN, Jiang H (2012) The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer. J Assist Reprod Genet 29: 105–115. doi: 10.1007/s10815-011-9694-6
  70. 70. Hickey RJ, Abdo Z, Zhou X, Nemeth K, Hansmann M, et al. (2013) Effects of tampons and menses on the composition and diversity of vaginal microbial communities over time. BJOG 120: 695–704. doi: 10.1111/1471-0528.12151
  71. 71. Lee JE, Lee S, Lee H, Song YM, Lee K, et al. (2013) Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort. PLoS One 8: e63514 doi:10.1371/journal.pone.0063514.
  72. 72. Drell T, Lillsaar T, Tummeleht L, Simm J, Aaspõllu A, et al. (2013) Characterization of the vaginal micro- and mycobiome in asymptomatic reproductive-age Estonian women. PLoS One 8: e54379 doi:10.1371/journal.pone.0054379.
  73. 73. Pendharkar S, Magopane T, Larsson P-G, de Bruyn G, Gray GE, et al. (2013) Identification and characterisation of vaginal lactobacilli from South African women. BMC Infect Dis 13: 43 doi:10.1186/1471-2334-13-43.
  74. 74. Gao W, Weng J, Gao Y, Chen X (2013) Comparison of the vaginal microbiota diversity of women with and without human papillomavirus infection: a cross-sectional study. BMC Infect Dis 13: 271 doi:10.1186/1471-2334-13-271.
  75. 75. Martínez-Peña MD, Castro-Escarpulli G, Aguilera-Arreola MG (2013) Lactobacillus species isolated from vaginal secretions of healthy and bacterial vaginosis-intermediate Mexican women: a prospective study. BMC Infect Dis 13: 189 doi:10.1186/1471-2334-13-189.
  76. 76. Shipitsyna E, Roos A, Datcu R, Hallén A, Fredlund H, et al. (2013) Composition of the vaginal microbiota in women of reproductive age – sensitive and specific molecular diagnosis of bacterial vaginosis is possible? PLoS One 8: e60670 doi:10.1371/journal.pone.0060670.
  77. 77. Mitchell C, Balkus JE, Fredricks D, Liu C, McKernan-Mullin J, et al. (2013) Interaction between lactobacilli, bacterial vaginosis-associated bacteria, and HIV Type 1 RNA and DNA genital shedding in U.S. and Kenyan women. AIDS Res Hum Retroviruses 29: 13–19. doi: 10.1089/aid.2012.0187
  78. 78. Hyman RW, Fukushima M, Jiang H, Fung E, Rand L, et al. (2013) Diversity of the vaginal microbiome correlates with preterm birth. Reprod Sci 21: 32–40. doi: 10.1177/1933719113488838
  79. 79. Datcu R, Gesink D, Mulvad G, Montgomery-Andersen R, Rink E, et al. (2013) Vaginal microbiome in women from Greenland assessed by microscopy and quantitative PCR. BMC Infect Dis 13: 480. doi: 10.1186/1471-2334-13-480
  80. 80. Ling Z, Liu X, Luo Y, Wu X, Yuan L, et al. (2013) Associations between vaginal pathogenic community and bacterial vaginosis in Chinese reproductive age women. PLoS One 8: e76589 doi:10.1371/journal.pone.0076589.
  81. 81. Solt I, Cohavy O (2012) The great obstetrical syndromes and the human microbiome-a new frontier. Rambam Maimonides Med J 3: e0009 doi:10.5041/RMMJ.10076.
  82. 82. Schellenberg J, Links MG, Hill JE, Dumonceaux TJ, Peters GA, et al. (2009) Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition. Appl Environ Microbiol 75: 2889–2898. doi: 10.1128/aem.01640-08
  83. 83. Srinivisan S, Morgan MT, Liu C, Matsen FA, Hoffman NG, et al. (2013) More than meets the eye: Associations of vaginal bacteria with Gram stain morphotypes using molecular phylogenetic analysis. PLoS One 8: e78633. doi: 10.1371/journal.pone.0078633
  84. 84. van de Wijgert JH, Verwijs MC, Norris Turner A, Morrison CS (2013) Hormonal contraception decreases bacterial vaginosis but oral contraception may increase candidiasis: implications for HIV transmission. AIDS 27: 2141–2153. doi: 10.1097/qad.0b013e32836290b6
  85. 85. Borgdorff H, Tsivtsivadze E, Verhelst R, Marzorati M, Jurriaans S, et al. (2014). Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women. ISME J: doi:10.1038/ismej.2014.26.
  86. 86. 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 1): 4688–4695. doi: 10.1073/pnas.1000086107
  87. 87. Macklaim JM, Fernandes AD, Di Bella JM, Hammond J, Reid G, et al. (2013) Comparative meta-RNA-seq of the vaginal microbiota and differential expression by Lactobacillus iners in health and dysbiosis. Microbiome 1: doi:10.1186/2049-2618-1-12.
  88. 88. Verstraelen H, Swidsinski A (2013) The biofilm in bacterial vaginosis: implications for epidemiology, diagnosis and treatment. Curr Op Infect Dis 26: 86–89. doi: 10.1097/qco.0b013e32835c20cd
  89. 89. 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
  90. 90. Patterson JL, Stull-Lane A, Girerd PH, Jefferson KK (2010) Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial vaginosis-associated anaerobes. Microbiology 156 (Pt 2): 392–399. doi: 10.1099/mic.0.034280-0
  91. 91. Swidsinski A, Mendling W, Loening-Baucke V, Swidsinski S, Verstraelen H, et al. (2005) Adherent biofilms in bacterial vaginosis. Obstet Gynecol 106(5 Pt 1): 1013–1023. doi: 10.1097/01.aog.0000183594.45524.d2
  92. 92. Machado A, Jefferson KK, Cerca N (2013) Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation. Int J Mol Sci 14: 12004–12. doi: 10.3390/ijms140612004
  93. 93. Yeoman CJ, Yildirim S, Thomas SM, Durkin AS, Torralba M, et al. (2010) Comparative genomics of Gardnerella vaginalis strains reveals substantial differences in metabolic and virulence potential. PLoS One 5: e12411 doi:10.1371/journal.pone.0012411.
  94. 94. Harwich MD Jr, Alves JM, Buck GA, Strauss JF 3rd, Patterson JL, et al. (2010) Drawing the line between commensal and pathogenic Gardnerella vaginalis through genome analysis and virulence studies. BMC Genomics 11: 375 doi:10.1186/1471-2164-11-375.
  95. 95. Castro J, Henriques A, Machado A, Henriques M, Jefferson KK, et al. (2013) Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells. Int J Med Sci 10: 1193–1198. doi: 10.7150/ijms.6304
  96. 96. Cotter G, Kavanagh K (2000) Adherence mechanisms of Candida albicans. Br J Biomed Sci. 57: 241–249.
  97. 97. Larsen B, Monif GRG (2001) Understanding the bacterial flora of the female genital tract. Clin Infect Dis 32: e69–77. doi: 10.1086/318710
  98. 98. Pybys V, Onderdonk AB (1997) Evidence for a commensal, symbiotic relationship between Gardnerella vaginalis and Prevotella bivia involving ammonia: potential significance for bacterial vaginosis. J Infect Dis 175: 406–413. doi: 10.1093/infdis/175.2.406