Antibiotic susceptibility and molecular diagnosis of Atopobium vaginae, a new pathogen in bacterial vaginosis

The vaginal microflora is a complex ecosystem. A healthy vaginal microflora consists mainly of lactobacilli, which, by the production of lactic acid, bacteriocines and hydrogen peroxide and their adherence to the vaginal epithelial wall, confer colonization resistance of the vagina against pathogenic agents. Disturbance of this ecosystem can lead to bacterial vaginosis (BV), a condition whereby the lactobacilli are overgrown by anaerobic bacteria such as Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Mycoplasma spp., Peptostreptococcus spp. and Prevotella spp. The presence of BV can lead to serious sequelae: women are more prone to HIV infection or other sexually transmitted infections such as gonorrhea and the presence of BV in the first trimester of the pregnancy increases the chance for preterm birth sevenfold. Furthermore, after treatment with metronidazole or clindamycine, relapse is very frequent. Therefore, a better characterization of the vaginal microflora might make it possible to identify a group of women at a higher risk for preterm birth or to find a treatment that is more effective in the long run. It is not possible to characterize the vaginal microbiome with conventional techniques such as culture, therefore in the last decade the development of molecular techniques has lead to new discoveries and understandings of the vaginal ecosystem. A. vaginae was only recently associated with BV and a high load of this bacterium is an important marker for BV. Possibly, A. vaginae is responsible for the frequent recurrence after treatment, as it has been described as resistant to metronidazole. We determined the antibiotic susceptibility of A. vaginae for sixteen antimicrobial agents, including clindamycin and metronidazole, the two recommended agents for treatment of BV. Not all A. vaginae isolates are metronidazole resistant, as seemed to be a straightforward conclusion from a previous study on a more limited number of strains. After addition of antimicrobial agents to a rich culture medium, to create a more selective medium enhancing growth of A. vaginae, we could isolate some more strains for which we evaluated the in vitro susceptibility for secnidazole, as a possible treatment agent, compared to metronidazole. Both agents are equally effective in vitro against A. vaginae. We developed a modification of the classification system of Ison and Hay, based on Gram stain, for the normal vaginal microflora and introduced new categories such as grade Ia, Ib and grade I-like, based on the presence of different Lactobacillus cell types and diphteroid cell types, as observed on Gram stain. For the further characterization with molecular methods, we developed and/or validated six real-time PCR assays. The four most important vaginal Lactobacillus species, i.e. L. crispatus, L. gasseri, L. iners and L. jensenii were chosen, together with the presently known two most important markers of a disturbed vaginal microflora, i.e. A. vaginae and G. vaginalis. In grade II samples, we found almost no L. iners but a high concentration of L. gasseri. By contrast, in grade III samples we established a high concentration of L. iners and a low concentration of L. gasseri. The results show a negative association between L. gasseri and L. iners and between L. gasseri and A. vaginae indicating that grade II and III are separate entities as has already been suggested by treatment studies during pregnancy. The results of this study were partially confirmed by a second study with tRFLP on vaginal samples of pregnant women, where we found that the presence of L. gasseri and L. iners in the vagina indicate a microflora that is less stable than with the presence of L. crispatus and L. jensenii. Further efforts for a better characterization of the different grades and search for new (bacterial) markers can give better results for the prediction of preterm birth. The developed real-time PCRs will be used in further studies, i.e. in a longitudinal study vaginal microflora dynamics during the menstrual cycle of different women and to assess the effect of the administration of probiotics on the vaginal microflora. Molecular methods based on DNA detection have shed a new light on the vaginal microflora and combination of different methods can bring a more complete understanding about the etiology of the BV syndrome