Numerous studies show that the vaginal microbiota of healthy women is dominated by Lactobacillus [1, 2, 3, 4]. Lactobacilli are thought to ensure colonization resistance of the vaginal microbial community. The vaginal epithelium can be colonized by other microbes, but they are less abundant in healthy women.

Many vaginal microorganisms are not so readily culturable or completely unculturable [5, 6, 7, 8], including some Lactobacillus species that refuse to grow on standard media. It was shown that one of the most prevalent species, Lactobacillus iners, cannot grow on Sharpe (MRS) and Rogosa agars used to culture lactobacilli [9]. Therefore, culture-based studies provide very scarce data on the diversity of species constituting the vaginal microbial community. To date, the most comprehensive results can be achieved using methods of molecular genetics.

Health of the vaginal microbiota is determined by the abundance of lactobacilli (no less than 80 % of all species isolated from the sample) measured by real-time polymerase chain reaction assays [10]. If lactobacilli constitute 20 to 80 % of the whole microbial community, the vaginal microbiota is considered moderately dysbiotic. Moderate dysbiosis is very often asymptomatic; therefore, it presents a particular interest for researchers and health professionals and raises the question of whether it is necessary to treat this condition in the absence of signs of vaginal inflammation.

Of importance is identification of microbiological markers associated with clinical signs of vaginal inflammation in patients with moderate dysbiosis. Studies of the diversity of lactobacilli in the vaginal microbiota of reproductive-age women showed that prevalence of some lactobacilli varies in patients with different types of vaginal flora [11]. Perhaps, there is an association between the diversity of lactobacilli in patients with moderate dysbiosis and the presence of subjective symptoms and objective signs of vaginal inflammation. We cannot rule out the possibility that clinical manifestations of pathology in moderate dysbiosis depend on the diversity and abundance of opportunistic bacteria inhabiting the vagina.

The aim of this study was to estimate the correlation between some microbiological characteristics and the presence of clinical signs of vaginal infection in patients with moderate vaginal dysbiosis.

METHODS

The study was carried out in 135 women with moderate vaginal dysbiosis aged 18 to 53 (mean age was 26.9 ± 6.9 years), outpatients of the Medical Center Harmony (Yekaterinburg) in 2011–2016. Exclusion criteria were HIV, parenteral hepatitis, sexually transmitted infections, namely Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis, and antimicrobial therapy started as early as 4 weeks before the study.

Samples (posterolateral vaginal wall swabs) were collected into Eppendorf tubes containing 1 ml sodium chloride solution. DNA was extracted using the Proba-GS reagent kit (R&P DNA-Technology, Russia). Abundance and diversity of species in the samples were evaluated by real-time PCR and the Femoflor reagent kit (R&P DNA-Technology). Identification and quantification of 6 Lactobacillus species (Lactobacillus crispatus, L. iners, L. jensenii, L. gasseri, L. johnsonii, L. vaginalis) was done by real-time PCR with reagent kits for scientific research (R&P DNA-Technology) and the DT-96 PCR detection system by the same vendor.

Patients were questioned about their complaints and examined to identify clinical signs of infection-induced inflammation of the lower genital tract.

Statistical analysis was performed using Microsoft Office Excel 2007. Significance of differences was estimated by the two-tailed Fisher’s test using WinPepi software.

The study was approved by the Ethics Committee of the Ural State Medical University (Protocol No. 4 dated May 05, 2015). All patients gave their informed consent.

RESULTS 

All patients were divided into two groups depending on the presence of clinical signs of an inflammatory infection (II) in the lower genital tract. Group 1 consisted of 91 patients with clinical signs of II, group 2 included 44 healthy women. We attempted to establish associations between the proportion of lactobacilli in the microbiota, the dominant species of lactobacilli, the dominant species of opportunistic microorganisms (OMs), and II.

Based on the proportion of lactobacilli (20–40 %, 40–60 % and 60–80 %), all patients were divided into 3 subgroups. Then the relative share of each subgroup in groups 1 and 2 was estimated (fig. 1). The difference between the groups was insignificant.

Prevalence of dominant Lactobacillus species in groups 1 and 2 was different (fig. 2). L. iners was significantly more common in group 1 (patients with II) than in group 2: 45 women (49.5 %) vs. 9 (20.5 %), respectively (p = 0.002). L. gasseri, on the contrary, was significantly more common in group 2 (healthy women) than in group 1: 23 patients (52.3 %) vs. 21 (23.1 %), respectively (p = 0.001). Prevalence of L. crispatus, L. jensenii and L. vaginalis in both groups was comparable.

Opportunistic pathogens were represented by dominant Gardnerella vaginalis/ Prevotella bivia/Porphyromonas spp. (GPP) in every second woman with or without clinical signs of II. Other OMs were far less common. No significant difference was revealed between OM prevalence in groups 1 and 2 (see the table).

DISCUSSION

Study results demonstrate that lactobacilli inhabiting the vagina of reproductive-age women are represented mainly by L. crispatus, L. iners, L. gasseri, and L. jensenii, which is consistent with the results of other studies [1, 2, 12, 13]. It is noteworthy that L. iners and L. gasseri dominate the Lactobacillus community in patients with moderate dysbiosis. A number of researchers have demonstrated that the presence of these lactobacilli is associated with an increased risk of bacterial vaginosis and poor pregnancy outcome [14, 15, 16]. Previously, we showed that L. gasseri can dominate the vaginal microbiota of patients with moderate dysbiosis [11]. Frequent detection of L. gasseri as a dominant species in patients without clinical signs of II, whose microbiota can be described as moderately dysbiotic, prompts us to assume that it can be a normal variant of the healthy vaginal flora and does not require any treatment. At the same time, moderate dysbiosis characterized by dominant L. iners is very often accompanied by clinical signs of II. Moreover, L. iners dominance is associated with an increased risk of marked vaginal dysbiosis [11]. Recent studies show that L. iners are higly adaptable and can survive in the presence of abundant Oms [17, 18]. Therefore, dominance of L. iners is a very unfavorable factor and requires medical correction.

We were unable to identify an association between the proportion of lactobacilli in the microbiota and the presence of II in patients with moderate vaginal dysbiosis. However, the obtained results may have been influenced by a small patient sample size, which means that such an association remains a possibility.

In more than half of patients with moderate vaginal dysbiosis, opportunistic bacteria were represented by GPP. In the studies in vitro Gardnerella vaginalis, a member of the GPP group, was shown to have a high adhesion capacity [19, 20] and stimulate growth of other OMs, including Prevotella bivia, also a GPP representative [19]. It was hypothesized that G.vaginalis could be the first microorganism that colonizes the vagina and prepares the environment for other pathogens [21, 22]. This can explain high prevalence of GPP as dominant opportunistic pathogens in patients with moderate dysbiosis. It is possible that as dysbiosis progresses, the contribution of other OMs to pathology increases; this may be true for Atopobium vaginae, a microorganism associated with vaginal dysbiosis [23, 24, 25, 26]. In our study A. vaginae was twice more common in patients with II than in healthy women. However, the difference was not statistically significant. We assume that the lack of significance was due to the small number of healthy women in group 2.

CONCLUSIONS 

Dominance of Lactobacillus iners in the Lactobacillus community of the vaginal microbiota of women with moderate dysbiosis is associated with clinical signs of the infection of the lower genital tract, while dominance of L. gasseri is typical for clinically healthy women with moderate dysbiosis. Thus, these microorganisms can be used as microbiological markers when it is unclear whether dysbiosis requires treatment.