In Vitro Effects of Polyphosphate against Prevotella intermedia in Planktonic Phase and Biofilm

Abstract

Polyphosphate (polyP) has gained a wide interest in the food industry due to its potential as a decontaminating agent. In this study, we examined the effect of sodium tripolyphosphate (polyP3; Na5P3O10) against planktonic and biofilm cells of Prevotella intermedia, a major oral pathogen. The MIC of polyP3 against P. intermedia ATCC 49046 determined by agar dilution method was 0.075%, while 0.05% polyP3 was bactericidal against P. intermedia in time-kill analysis performed using liquid medium. A crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that sub-MICs of polyP3 significantly decreased biofilm formation. Under the scanning electron microscope, decreased numbers of P. intermedia cells forming the biofilms were observed when the bacterial cells were incubated with 0.025% or higher concentrations of polyP3. Assessment of biofilm viability with LIVE/DEAD staining and viable cell count methods showed that 0.05% or higher concentrations of polyP3 significantly decreased the viability of the preformed biofilms in a concentration-dependent manner. The zone sizes of alpha-hemolysis formed on horse blood agar produced by P. intermedia were decreased in the presence of polyP3. The expression of the genes encoding hemolysins and the genes of the hemin uptake (hmu) locus was downregulated by polyP3. Collectively, our results show that polyP is an effective antimicrobial agent against P. intermedia in biofilms as well as planktonic phase, interfering with the process of hemin acquisition by the bacterium.

FIG 1

Antibacterial effect of polyP3 against P. intermedia ATCC 4906. (A) MIC determination of polyP3 by agar dilution method. The bacterial cells were spot inoculated (approximately 10⁵ to 10⁶ cells/spot) onto brucella blood agar plates containing polyP3 at various concentrations and incubated at 37°C for 3 days anaerobically. The MIC was defined as the lowest concentration that inhibited the bacterial growth on the plate. (B) Time-kill curve of polyP3 against P. intermedia ATCC 49046 in liquid medium. Results are presented as the means ± SDs from three independent experiments. In the presence of 0.05 and 0.075% polyP3, complete killing of P. intermedia cells was observed at 16 h and 8 h, respectively.

FIG 2

Effect of polyP3 on biofilm formation and ultrastructure of P. intermedia ATCC 4906. A P. intermedia culture was diluted to an OD₆₀₀ of approximately 0.1 in B-HK broth and then further incubated in 24-well plates with polyP3 at the indicated concentrations for 24 h. (A) Analyses of the biofilm biomass of P. intermedia ATCC 49046. The biofilm biomass was quantitated by crystal violet staining. Heat-killed bacterial cells that were initially killed by exposure to 100°C for 10 min were included as controls. Data are means ± SDs from two independent experiments performed in triplicate. *, P < 0.05, versus value for control. (B) SEM images of P. intermedia biofilm cells. Scale bars, 20 μm (upper) and 5 μm (lower).

FIG 3

Effect of polyP3 on viability of preformed P. intermedia biofilm. Preestablished biofilms of P. intermedia ATCC 49046 were treated with polyP3 at the indicated concentrations for 24 h. (A) Biofilm biomass was quantitated by crystal violet staining. The viable biofilm bacteria were enumerated by detachment of cells, followed by agar plating count. The results are expressed as the means ± SDs from two independent experiments performed in triplicate. *, P < 0.05, versus value for control. (B) CLSM images of the P. intermedia biofilms. Magnification is ×600.

FIG 4

Effect of polyP3 on beta-hemolysis produced by P. intermedia ATCC 49046. Various concentrations of polyP3 were prepared and added to 5% horse blood BHIA in the absence of hemin and vitamin K₁. P. intermedia was incubated on the agar plates for 5 days at 37°C. (A) Images of beta-hemolysis zone produced after 3, 4, and 5 days. (B) Area of the beta-hemolysis zone quantified using ImageJ version 1.49j (National Institutes of Health, Bethesda, MD). Results are expressed as means ± SDs from two independent experiments.

FIG 5

Expression levels of the genes related to hemin uptake and hemolysis in the presence of 0.05% polyP3. The expression of the genes was measured by qRT-PCR and normalized to that of the 16S rRNA gene. The expression level of each gene in the absence of polyP3 was set as 1-fold. The results are expressed as the means ± SDs from three independent experiments.