Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR - PubMed (original) (raw)

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

Beatrice Vitali et al. Appl Environ Microbiol. 2007 Sep.

Abstract

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

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Figures

FIG. 1.

FIG. 1.

DGGE profiles of healthy women (NI group). A single DGGE profile was reported for each woman in the NI group. The bands correspond to bacterial taxa and GenBank accession numbers as follows: Lactobacillus iners, 3a (AB279893), 6b (AB279894), 8a (AB279895), and 10c (AB279896); Lactobacillus sp., 5a (AB279907) and 26a (AB279908); Lactobacillus acidophilus, 6a (AB279917), 10b (AB279918), 11a (AB279919), 22a (AB279920), 23a (AB279921), and 25b (AB279922); Lactobacillus gasseri, 10a (AB279926), 14a (AB279927), and 25a (AB279928); Lactobacillus vaginalis, 10d (AB279933) and 22b (AB279934); Gardnerella vaginalis, 5b (AB279938), 14c (AB279939), and 25c (AB279940); uncultured Gardnerella sp., 14b (AB279952).

FIG. 2.

FIG. 2.

DGGE profiles of patients developing BV. Three DGGE courses corresponding to the three monthly gynecologic visits were reported. The bands correspond to bacterial taxa and GenBank accession numbers as follows: Lactobacillus iners, 17b (AB279897), 20e (AB279898), and 24b (AB279899); Lactobacillus acidophilus, 17a (AB279923) and 20d (AB279924); Lactobacillus plantarum, 20a (AB279937); Lactobacillus gasseri, 12a (AB279929) and 20b (AB279930); Lactobacillus vaginalis, 17c (AB279935); Atopobium vaginae, 2q (AB279953) and 12g (AB279954); Gardnerella vaginalis, 2m (AB279941), 2o (AB279942), 2p (AB279943), 17d (AB279944), 17e (AB279945), and 24c (AB279946); Leptotrichia amnionii, 2a (AB279955), 2b (AB279956), and 2d (AB279957); Leptotrichia sanguinegens, 24a (AB279960); uncultured Prevotella sp., 2c (AB279962), 2f (AB279963), 2g (AB279964), and 2h (AB279965); Prevotella sp., 12b (AB279968) and 12e (AB279969); uncultured Megasphaera sp., 2l (AB279970), 20f (AB279971), and 20g (AB279972); uncultured Chloroflexi bacterium, 2i (AB279973); uncultured Clostridium sp., 2n (AB279974); Streptococcus sp., 12c (AB279977); Staphylococcus sp., 2e (AB279978) and 20c (AB279979); Veillonella montpellierensis, 12d (AB279980) and 12f (AB279981).

FIG. 3.

FIG. 3.

DGGE profiles of patients developing CA. Three DGGE courses corresponding to the three monthly gynecologic visits were reported. The bands correspond to bacterial taxa and GenBank accession numbers as follows: Lactobacillus sp., 1a (AB279909), 1b (AB279910), 1c (AB279911), 1d (AB279912), 4b (AB279913), 13a (AB279914), and 19a (AB279915); Lactobacillus iners, 4c (AB279900), 16c (AB279901), 18a (AB279902), and 21b (AB279903); Lactobacillus gassseri, 16a (AB279931) and 21a (AB279932); Lactobacillus acidophilus, 16b (AB279925); Lactobacillus vaginalis, 4d (AB279936); Gardnerella vaginalis, 21c (AB279947) and 21d (AB279948); uncultured Ureaplasma sp., 4a (AB279982); uncultured Clostridium sp., 4e (AB279976); Shigella boydii, 18b (AB279983).

FIG. 4.

FIG. 4.

DGGE profiles of patients developing both BV and CV (BV-CA). Three DGGE courses corresponding to the three monthly gynecologic visits were reported. The bands correspond to bacterial taxa and GenBank accession numbers as follows: Lactobacillus iners, 7e (AB279904), 9a (AB279905), and 15b (AB279906); Lactobacillus sp., 15a (AB279916); Gardnerella vaginalis, 7h (AB279949), 7i (AB279950), and 9b (AB279951); Leptotrichia amnionii, 7a (AB279958) and 7b (AB279959); Leptotrichia sanguinegens, 7c (AB279961); uncultured Prevotella sp., 7d (AB279966) and 7f (AB279967); uncultured Clostridium sp., 7g (AB279975).

FIG. 5.

FIG. 5.

Frequency of the occurrence of bacterial species in relation to clinical status of the subject. Frequencies were calculated as the ratios (percentage) of the number of visits characterized by the presence of each species to the total number of visits belonging to each visit group (NI, BV, and CA).

FIG. 6.

FIG. 6.

Real-time PCR evaluation of 16S rrn operons of total eubacteria (A) and lactobacilli (B) and relative abundance of lactobacilli (C) related to each group of visits characterized by a specific clinical status (NI, BV, and CA). The number of operons in 1 ml of vaginal rinsing is expressed as an absolute value. Lactobacillus relative abundance is expressed as the ratio of lactobacillus rrn operons to total eubacterial 16S rrn operons. The box for each group represents the interquartile range (25th to 75th percentile), and the line within this box is the median value. The bottom and top bars indicate the 10th and 90th percentiles, respectively. Outlier values are indicated (black circles).

FIG. 7.

FIG. 7.

Relative abundances of lactobacilli in the vaginal flora of women during NI visits, relative to the clinical history of the women. Lactobacillus relative abundance is expressed as the ratio of lactobacillus 16S rrn operons to total eubacterial 16S rrn operons. The box for each group represents the interquartile range (25th to 75th percentile), and the line within this box is the median value. The bottom and top bars indicate the 10th and 90th percentiles, respectively. Outlier values are indicated (black circles).

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