DNase inhibits Gardnerella vaginalis biofilms in vitro and in vivo

Abstract

Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities.

Figure 1.

Gardnerella vaginalis forms 3-dimensional biofilms that are inhibited by DNase. G. vaginalis strain 49 145 was grown on glass supports for 24 hours and treated with vehicle control (A and C) or with DNase (B and D). Two-dimensional images of DAPI-stained biofilms (A and B) or 3-dimensional reconstruction from serial z-stack images or propidium iodide–stained biofilms (C and D) demonstrate both bacteria-associated and extracellular DNA. Similar results were obtained with G. vaginalis strain ARG3 (data not shown).

Figure 2.

Gardnerella vaginalis releases extracellular DNA (eDNA) in early stationary-growth phase. Strain 49 145 was incubated for 18 hours, with OD₅₉₅ recorded as a measure of bacterial growth (▪; left axis). eDNA was quantified by spectroscopy from the cell-free supernatant after extraction and precipitation (•; right axis). Trend lines are plotted for the OD curve (dashed line) and eDNA concentrations (solid line). The experiment was repeated 3 times; a representative experiment is shown.

Figure 3.

DNase inhibits the de novo formation of Gardnerella vaginalis biofilms. A, Strain 49 145 was incubated with DNase at the indicated concentrations or in vehicle control for 24 hours, and biofilm biomass was quantified by the safranin assay. Normalization (100%) was to control-treated G. vaginalis. B, Heat inactivation of DNase abrogates its biofilm inhibiting properties. C, DNase at various concentrations (▪, vehicle control; ♦, 8 µg/mL; ▴, 200 µg/mL) does not inhibit planktonic growth of G. vaginalis over 24 hours. ***P < .001; ****P < .0001.

Figure 4.

DNase disrupts previously established biofilms. A, Gardnerella vaginalis biofilms were grown for 24 hours prior to treatment with DNase or vehicle control. B, A total of 100 µg/mL of DNase was active on biofilms within 15 minutes. C, G. vaginalis density in biofilm fraction (white) and supernatant fraction (black). Statistical comparison is for supernatant organisms. Total organisms were not statistically significantly different. **P < .01; ***P < .001.

Figure 5.

Interaction of DNase and metronidazole on preformed Gardnerella vaginalis biofilms. DNase was added at 167 µg/mL final concentration (bottom row), with 10-fold dilutions in each subsequent row above (top row, vehicle control). Metronidazole was added at 1.67 mg/mL final concentration (right column), with 5-fold dilutions in each subsequent column to the left (left column, vehicle control). The remaining percentage of the maximum biofilm was calculated as in prior figures, and a heat map (red, >75% remaining; green, 50%–75%; blue, <50%) was constructed.

Figure 6.

DNase decreases vaginal Gardnerella vaginalis colonization in a murine model. Two groups of 5 C57/BL6 mice were colonized with strain ARG37 and 100 µg/mL DNase or vehicle control. Quantitative cultures were obtained 48 hours following the initial inoculation. **P < .01.