Unveiling the role of Gardnerella vaginalis in polymicrobial Bacterial Vaginosis biofilms: the impact of other vaginal pathogens living as neighbors

Abstract

Bacterial vaginosis (BV) is characterized by a highly structured polymicrobial biofilm, which is strongly adhered to the vaginal epithelium and primarily consists of the bacterium Gardnerella vaginalis. However, despite the presence of other BV-associated bacteria, little is known regarding the impact of other species on BV development. To gain insight into BV progress, we analyzed the ecological interactions between G. vaginalis and 15 BV-associated microorganisms using a dual-species biofilm model. Bacterial populations were quantified using a validated peptide nucleic acid fluorescence in situ hybridization approach. Furthermore, biofilm structure was analyzed by confocal laser scanning microscopy. In addition, bacterial coaggregation ability was determined as well as the expression of key virulence genes. Remarkably, our results revealed distinct biofilm structures between each bacterial consortium, leading to at least three unique dual-species biofilm morphotypes. Furthermore, our transcriptomic findings seem to indicate that Enterococcus faecalis and Actinomyces neuii had a higher impact on the enhancement of G. vaginalis virulence, while the other tested species had a lower or no impact on G. vaginalis virulence. This study casts a new light on how BV-associated species can modulate the virulence aspects of G. vaginalis, contributing to a better understanding of the development of BV-associated biofilms.

Fig. 1

Coaggregation score of mono- or dual- bacterial species. Coaggregation score was evaluated as following: 0, no aggregation; 1, small aggregates comprising small visible clusters of bacteria; 2, aggregates comprising larger numbers of bacteria, settling to the center of the well; 3, macroscopically visible clumps comprising larger groups of bacteria which settle to the center of the well; 4, maximum score allocated to describe a large, macroscopically visible clump in the center of the well. Auto-aggregation was assessed for each bacterial species, corresponding to the experimental control (CT). Each data point represents the mode

Fig. 2

Biofilm formation profiles for each BV-associated species consortium (10⁷ CFU/mL of BVGv and 10⁷ CFU/mL of BV-associated bacteria) on dual-species biofilms. a Total cells counts by DAPI for mono- (G. vaginalis controls) and dual-species biofilms. b Total percentage of cells detected by PNA FISH for 48 h biofilms. Each data point represents the mean ± s.d.. *, † Values are significantly different between the dual-species consortium and the mono-speciesG. vaginalis biofilm for 24 and 48 h, respectively (independent samples t-test, P < 0.05 for a). * Values are significantly different between the bacterial populations of G. vaginalis and second BV-associated in dual-species biofilms (paired samples t-test for b, P < 0.05)

Fig. 3

An example data set on the organization of the dual-species BV-associated biofilm for 48 h by confocal laser scanning microscopy (CLSM). a G. vaginalis mono-species biofilm labeled with PNA-probe Gard162 and DAPI staining corresponding to an experimental control. b CLSM images of dual-species biofilms for all 15 bacterial consortia. Images were acquired with 512 × 512 resolutions at four different regions of each surface analyzed

Fig. 4

Schematic representation of the distribution of dual-species BV-associated biofilm structure from the bottom to the biofilm top. (Bottom 1—B1) Predominantly G. vaginalis with rare spots of second BV-isolate in the bottom; (Bottom 2—B2) both species in the bottom; (Distribution 1—D1) G. vaginalis exists on clusters in the biofilm; (Distribution 2—D2) G. vaginalis is well distributed in the biofilm; (Top 1—T1) G. vaginalis is reduced from the bottom to the top; (Top 2—T2) G. vaginalis is absence on the top layer of biofilm

Fig. 5

Quantification of the transcription of known virulence genes in G. vaginalis cultured under dual- and mono-species biofilms. a Quantification of vaginolysin (vly) transcription. b Quantification of sialidase (sld) transcription. c Quantification of HMPREF0424_0821 transcript, which encodes type II glycosyl-transferase. d Quantification of HMPREF0424_1122 transcript, which encodes a multidrug ABC transporter. e Quantification of HMPREF0424_0156 transcript, which encodes Bacitracin transport, ATP-binding protein BcrA. f Quantification of HMPREF0424_1196 transcript, which encodes a Rib-protein. The data indicate the fold-change expression of genes in G. vaginalis dual-species compared to mono-species G. vaginalis biofilm cells. For qPCR experiments, the bars represent the mean and the error bars the standard error of the mean (mean ± s.e.m.). * Values are significantly different between the dual-species consortium and the mono-species G. vaginalis biofilm under the same conditions (non-parametric Mann–Whitney U, P < 0.05)

Fig. 6

Hypothetical model of G. vaginalis vaginolysin (vly)-mediated cytotoxicity in different bacterial phenotypes a planktonic cells, b G. vaginalis mono-species biofilm, c dual-species biofilms, corresponding to a pre-formed BVGv biofilm in association with a second BV-associated bacteria