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Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

Hardy L, Jespers V, Dahchour N, Mwambarangwe L, Musengamana V, Vaneechoutte M, Crucitti T - PLoS ONE (2015)

Bottom Line: Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms.These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment.We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

View Article: PubMed Central - PubMed

Affiliation: Unit of Epidemiology and Control of HIV/STD, Department of Public Health, Institute of Tropical Medicine, Antwerp, Belgium; Laboratory Bacteriology Research, Faculty of Medicine & Health Sciences, University of Ghent, Ghent, Belgium; STI Reference Laboratory, Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

ABSTRACT
Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

No MeSH data available.


Related in: MedlinePlus

Polymicrobial biofilm of A. vaginae and G. vaginalis in different panes.Confocal laser scanning image with 400 x magnification of polymicrobial biofilm in different panes, A: vaginal epithelial cells DAPI in blue, B: all bacteria, BacUni-1 PNA-probe with Alexa Fluor 555 in yellow, C: A. vaginae specific PNA-probe AtoITM1 with Alexa Fluor 488 in green, D: G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red (superimposed image can be seen in Fig 3A).
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pone.0136658.g005: Polymicrobial biofilm of A. vaginae and G. vaginalis in different panes.Confocal laser scanning image with 400 x magnification of polymicrobial biofilm in different panes, A: vaginal epithelial cells DAPI in blue, B: all bacteria, BacUni-1 PNA-probe with Alexa Fluor 555 in yellow, C: A. vaginae specific PNA-probe AtoITM1 with Alexa Fluor 488 in green, D: G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red (superimposed image can be seen in Fig 3A).

Mentions: Using the AtoITM1 PNA-probe, A. vaginae was visualized as dispersed entities, without the presence of biofilm, in 27/119 (22.7%) of the samples. A. vaginae biofilm was present in 26/119 (21.8%) samples. A. vaginae FISH was negative in the remaining 66/119 (55.5%) samples. PNA-FISH using Gard162 detected dispersed-only G. vaginalis in 31/119 (26%) samples, G. vaginalis biofilm in 58/119 (48.7%) samples (e.g., Fig 2) and 30/119 (25.3%) samples were negative for G. vaginalis. Of the 89 G. vaginalis FISH-positive samples (dispersed or biofilm), 36 samples (41%) were negative for A. vaginae. However, all samples with A. vaginae biofilm showed a G. vaginalis biofilm as well (e.g., Figs 3, 4 and 5).


Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

Hardy L, Jespers V, Dahchour N, Mwambarangwe L, Musengamana V, Vaneechoutte M, Crucitti T - PLoS ONE (2015)

Polymicrobial biofilm of A. vaginae and G. vaginalis in different panes.Confocal laser scanning image with 400 x magnification of polymicrobial biofilm in different panes, A: vaginal epithelial cells DAPI in blue, B: all bacteria, BacUni-1 PNA-probe with Alexa Fluor 555 in yellow, C: A. vaginae specific PNA-probe AtoITM1 with Alexa Fluor 488 in green, D: G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red (superimposed image can be seen in Fig 3A).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548953&req=5

pone.0136658.g005: Polymicrobial biofilm of A. vaginae and G. vaginalis in different panes.Confocal laser scanning image with 400 x magnification of polymicrobial biofilm in different panes, A: vaginal epithelial cells DAPI in blue, B: all bacteria, BacUni-1 PNA-probe with Alexa Fluor 555 in yellow, C: A. vaginae specific PNA-probe AtoITM1 with Alexa Fluor 488 in green, D: G. vaginalis specific PNA-probe Gard162 with Alexa Fluor 647 in red (superimposed image can be seen in Fig 3A).
Mentions: Using the AtoITM1 PNA-probe, A. vaginae was visualized as dispersed entities, without the presence of biofilm, in 27/119 (22.7%) of the samples. A. vaginae biofilm was present in 26/119 (21.8%) samples. A. vaginae FISH was negative in the remaining 66/119 (55.5%) samples. PNA-FISH using Gard162 detected dispersed-only G. vaginalis in 31/119 (26%) samples, G. vaginalis biofilm in 58/119 (48.7%) samples (e.g., Fig 2) and 30/119 (25.3%) samples were negative for G. vaginalis. Of the 89 G. vaginalis FISH-positive samples (dispersed or biofilm), 36 samples (41%) were negative for A. vaginae. However, all samples with A. vaginae biofilm showed a G. vaginalis biofilm as well (e.g., Figs 3, 4 and 5).

Bottom Line: Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms.These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment.We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

View Article: PubMed Central - PubMed

Affiliation: Unit of Epidemiology and Control of HIV/STD, Department of Public Health, Institute of Tropical Medicine, Antwerp, Belgium; Laboratory Bacteriology Research, Faculty of Medicine & Health Sciences, University of Ghent, Ghent, Belgium; STI Reference Laboratory, Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

ABSTRACT
Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

No MeSH data available.


Related in: MedlinePlus