Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods

Abstract

The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases. INTRODUCTIONPrevious studies on the microbial flora of the human vagina indicate that micro-organisms normally present in the human vagina play a key role in preventing successful colonization by 'undesirable' organisms, including those responsible for bacterial vaginosis, yeast infections, sexually transmitted diseases and urinary tract infections (Donders et al., 2000;Gupta et al., 1998;Sobel, 1999;van De Wijgert et al., 2000). Moreover, epidemiologic studies have clearly established that abnormal vaginal microbial communities and lower genital tract infections are significantly associated with an increased risk of HIV infection (Cohen et al., 1995;Martin et al., 1999;Sewankambo et al., 1997). Based on this, researchers have speculated that the normal vaginal microbial ecosystem may play a pivotal role in lo...

Preliminary characterization of the normal microbiota of the human vulva using cultivation-independent methods

“…Vulvar scrape samples were stored at 220 u C and community bacterial DNA was isolated using the techniques described by Zhou et al (2004). PCR amplification of an internal region of the 16S rRNA genes from each vulvar microbial community was accomplished using the bacteria-specific primers 8Fm (59-AGAGTTTGATCMTGGCTCAG-39) and 926R (59-CCGTCAATTCCTTTRAGTTT-39) (Weisburg et al, 1991).…”

“…The most abundant phylotypes in the vulvas of three of the four women were most similar to either Lactobacillus crispatus or Lactobacillus iners, whilst the community of the fourth (W5) was dominated almost equally by phylotypes similar to L. iners, Atopobium vaginae and a phylotype most similar to Megasphaera elsdenii (13-14 % sequence divergence; Table 2). Differences among women did not appear to relate to either their age or the day Aquabacterium citratiphilum (1.0, 1.9 %), Brevibacillus formosus (1.9, 2.5 %), Eubacterium tarantellae ( Acinetobacter johnsonii (1.8, 1.6 %), Bacillus jeotgali (1.8, 2.8 %), Bacillus psychrosaccharolyticus (0.9, 5.3 %), Brevundimonas variabilis (1.8, 1.7 %), 'Corynebacterium genitalium' (7.9, 1.6 %), Corynebacterium thomssenii (9.6, 0.0 %), Delftia tsuruhatensis (0.9, 0.0 %), Eubacterium desmolans (0.9, 10.6 %), Lactococcus lactis (0.9, 0.0 %), Legionella pneumophila (0.9, 6.6 %), Methylobacterium extorquens (0.9, 1.5 %), Methylomicrobium album ( in the menstrual cycle that the samples were taken (Tables 1-3), although the sample size of this study was too small to make conclusive statements.The vaginal communities of the women in this study were sampled at the same time that the vulvar samples were taken and their composition has been reported previously (Coolen et al, 2005;Zhou et al, 2004). The dominant phylotypes from the vulva were also dominant members of communities in the corresponding vagina.…”

“…1; Zhou et al, 2004). Analysis of these vaginal samples using terminal restriction fragment length polymorphisms have indicated that the vaginal community composition is consistent over the menstrual cycle and over a period of at least 2 months (Coolen et al, 2005).The vaginal communities of the five women in the study by Zhou et al (2004) were also commonly encountered among women in a larger study (Forney et al, 2006) that was a subsample of more than 3000 women from across North America (Parsonnet et al, 2005). We expect that a larger study of microbial communities of the vulva will show that vulvar communities are heavily influenced by the vaginal microbiota, and that the communities identified here will reflect healthy vulvar and vaginal communities in general.…”

Effect of iron on the probiotic properties of the vaginal isolate Lactobacillus jensenii CECT 4306

“…Since the first microbiological study of the human vagina, lactobacilli have been considered the dominant members of the microbiota of this organ (Doderlein, 1982), a finding that has been further confirmed by several recent metagenomic studies (Biagi et al, 2009;Fettweis et al, 2012;Martín et al, 2014;Pavlova et al, 2002;Song et al, 1999;Verhelst et al, 2004;Vitali et al, 2007;Wilks et al, 2004;Zhou et al, 2004). In total, around 20 different species of lactobacilli have been consistently isolated from the vagina, although only one or two species predominate at a time in a given individual.…”

“…In total, around 20 different species of lactobacilli have been consistently isolated from the vagina, although only one or two species predominate at a time in a given individual. Dominant strains vary as a function of race and lifestyle, but strains of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri are the most frequent (Biagi et al, 2009;Pavlova et al, 2002;Song et al, 1999;Verhelst et al, 2004;Vitali et al, 2007;Wilks et al, 2004;Zhou et al, 2004).…”