Gardnerella vaginalis dominates multi-species biofilms in both pre-conditioned and competitive in vitro biofilm formation models

The online version contains supplementary material available at: https://doi.org/10.1007/s00248-021-0Bacterial vaginosis (BV) is one of the most common bacterial vaginal infections worldwide. Despite its high prevalence, BV etiology is still unknown. Nevertheless, a hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp. and other anaerobic species, of which co-colonization with Fannyhessea vaginae is considered an important diagnostic marker. We previously developed an in vitro biofilm model wherein Gardnerella was first allowed to establish an early biofilm that served as a scaffold for other species to adhere to. To better understand ecological interactions between BV-associated bacteria, we compared triple-species biofilms formed using two distinct models: a pre-conditioned (wherein Gardnerella vaginalis formed the early biofilm) model and a competitive (wherein all three bacteria were co-incubated together) model. Interestingly, synergistic growth interactions were more significant in the competitive model. Furthermore, the biofilm structure and species-specific distribution, as assessed by confocal laser scanning microscopy and using peptide nucleic acid fluorescence in situ hybridization method, revealed two very different triple-species morphotypes, suggesting that different interactions occur in the different models. Interestingly, independent of the model or triple-species consortium tested, we observed that G. vaginalis represented most of the biofilm bacterial composition, further highlighting the relevance of this taxon in BV.This work was supported by the Portuguese Foundation for Science and Technology (FCT) by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI01–0145-FEDER-028271] and by the strategic funding of unit [UID/BIO/04469/2020]. ASR acknowledges the financial support of indi vidual Grant [PD/BD/128037/2016].