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

Abstract

c 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; Na 5 P 3 O 10 ) 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. P revotella intermedia is a black-pigmented anaerobic Gramnegative bacterium which has long been known to be associated with oral diseases, such as chronic periodontitis (1-3), aggressive periodontitis (4-6), puberty-associated gingivitis, acute necrotizing ulcerative gingivitis (7,8), periapical periodontitis (9, 10), and noma (an acute gangrenous disease) (11, 12). Besides being involved in oral diseases, P. intermedia has also been reported to be associated with various systemic diseases, such as cystic fibrosis, chronic bronchitis (13-15), and atherosclerosis (16). Difficulty in controlling the bacterium has been attributed to resistance of P. intermedia to many antibiotics, including penicillins, cephalosporins, and tetracyclines (17, 18). Moreover, P. intermedia cells form a biofilm in which the bacterial cells become more resistant to antibiotics (19). Because biofilm can serve as a reservoir of antibiotic resistance (20), it is of clinical significance to develop alternative antimicrobial approaches for controlling antibiotic-resistant P. intermedia.Inorganic polyphosphate (polyP) is a chain of few or many hundreds of phosphate (P i ) residues linked by high-energy phosphoanhydride (21). Intracellular polyP found in bacteria is considered a virulence factor since it performs various functions, such as serving as an ATP source and substitute, a regulator of the intracellular levels of metal ions, a channel for DNA entry, and a regulator that contributes to bacter...

An In Vitro Study of Bactericidal Effect of Gallium Aluminium Arsenide Laser on Anaerobic Photosensitized Periodontopathics

“…For the reason that biofilm can act as a reservoir of antibiotic resistance, it is of clinical significance to develop alternative antimicrobial approaches for controlling antibioticresistant P. intermedia. 7 In the present study, study group showed reduction in colony count of P. intermedia after being exposed to GaAlAs diode laser for 1 minute from 369 to 229, 134 and 41 after serial dilution of in ThioGlycollate Broth in 1:10, 1:50 and 1:100 ratios which was statistically significant with p value <0.0001.…”

Antibacterial effects of sodium tripolyphosphate againstPorphyromonasspecies associated with periodontitis of companion animals

“…That is, polyP3 exerted bactericidal activity (defined as a ≥ 99.9% killing rate within 24 h [25]) against planktonic cells of all three Porphyromonas species at lower concentrations than the MICs determined by the agar dilution method. Similarly, the MIC of polyP against the human periodontopathic P. gingivalis and P. intermedia was 600–750 mg/L by the agar dilution method, whereas polyP in the liquid medium showed a bactericidal effect against the two bacteria at sub-MIC levels [2223]. Some antimicrobials, including polyP, may exert a higher effect in liquid media than in solid media, presumably because the liquid medium provides an environment favorable for the migration and diffusion of the antimicrobial agents, allowing easy contact with individual bacterial cells.…”

“…Bacterial culture suspension, adjusted to an optical density of 0.1 at 600 nm (OD 600 = 0.1) in B-HK, was dispensed into a polystyrene 96-well plate (100 μL/well) containing B-HK (100 μL/well) with or without polyP3. As dead bacterial cells could attach to the surface of the polystyrene culture plates [2326], heat-killed (100°C for 10 min) bacterial cells were included as controls. After 24 h of incubation, planktonic and loosely bound bacterial cells were removed by aspiration of the spent media.…”

“…PolyP also has the effect of preventing food spoilage because of its antimicrobial action against various Gram-positive bacteria and fungi [2021]. Interestingly, our recent studies have shown that polyP exerts excellent antimicrobial effects against black - pigmented Gram-negative anaerobes associated with human periodontal diseases such as P. gingivalis and P. intermedia [2223].…”

Facile preparation of mussel-inspired antibiotic-decorated titanium surfaces with enhanced antibacterial activity for implant applications

“…Each bacterial strain grown to exponential phase was adjusted to an optical density at 600 nm (OD 600 ) of 0.1 with new broth. Each Ti specimen was placed in each well of 24-well polystyrene plate containing the adjusted bacterial suspension, then the plate was incubated at 37°C aerobically, as described previously [21,22]. After 24 h, the specimens were removed and the bacterial biofilm formed on Ti surface was washed with physiological saline, then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h at room temperature and rinsed three times for 10 min each with distilled water.…”