Hydrolyzed Ce(IV) salts limit sucrose-dependent biofilm formation by Streptococcus mutans

Abstract

Several studies have focused on the antimicrobial effects of cerium oxide nanoparticles (CeO₂-NP) but few have focused on their effects on bacteria under initial biofilm formation conditions. Streptococcus mutans is a prolific biofilm former contributing to dental caries in the presence of fermentable carbohydrates and is a recognized target for therapeutic intervention. CeO₂-NP derived solely from Ce(IV) salt hydrolysis were found to reduce adherent bacteria by approximately 40% while commercial dispersions of "bare" CeO₂-NP (e.g., 3 nm, 10-20 nm, 30 nm diameter) and Ce(NO₃)₃·6H₂O were either inactive or observed to slightly increase biofilm formation under similar in vitro conditions. Planktonic growth and dispersal assays support a non-bactericidal mode of biofilm inhibition active in the initial phases of S. mutans biofilm production. Human cell proliferation assays suggest only minor effects of hydrolyzed Ce(IV) salts on cellular metabolism at concentrations up to 1 mM Ce, with less observed toxicity compared to equimolar concentrations of AgNO₃. The results presented herein have implications in clinical dentistry.

Keywords: Biofilm; Ce(IV); Dental; Inhibition; Streptococcus mutans.

Figure 1.

The ratio of adherent bacteria (compared to positive control) from the treatment of S. mutans UA159 with Ce-containing agents (125 μM Ce) [CN = Ce(NO₃)₃•6H₂O] and ammonium salts (AN, AS) (1 mM) BHI, 1% sucrose, 37°C, 5% CO₂ for 20 h. *p<0.05, #p<0.0001, student t test.

Figure 2.

Microscopic images (10 x) of adherent S. mutans UA159 grown for 20 h at 37 °C, 5% CO₂ in BHI. The untreated wells (left) displayed significantly more biofilm growth than 250 μM CAS treated wells (right).

Figure 3.

Planktonic growth curves of S. mutans UA159 with 500 μM CAN and CAS treated cells in BHI at 37°C and 5% CO₂ for 10 h. Measurements were run in triplicate. Error bars represent standard deviation of the triplicate measurements.

Figure 4.

MTS cell proliferation assays utilizing (left) TIGK and (right) HGF in the presence of hydrolyzed CAN (red), CAS (blue) and AgNO₃ (gray) for 24 h at 37°C, 5% CO₂. *p<0.05, #p<0.01, t test.

Figure 5.

Distribution of hydrolyzed [Ce(NO₃)₆]^2- (pH 1.8) and CeO₂-NP (3 nm, Strem) (pH 4.5) in 30 mM NO₃⁻.

Figure 6.

Distribution of (left) 5 mM [Ce(NO₃)₆]^2- (pH 1.8) and (right) 5 mM CeO₂-NP (3 nm, Strem) (pH 4.5) in 30 mM NO₃⁻ and with 40 mM KCl.

Figure 7.

Distribution of hydrolyzed [Ce(NO₃)₆]^2- (500 μM Ce) in 50 mM KH₂PO4 (pH 7.4)

Figure 8.

Low-magnification high-angle annular dark field (HAADF) of (a) hydrolyzed [Ce(NO₃)₆]^2- and (b) CeO₂-NP (3nm, Strem) in graphene liquid cells. Inserts show high-resolution images of the nano-particles revealing lattice fringes. Electron energy-loss spectroscopy (EELS) of the Ce M-edges for (c) hydrolyzed [Ce(NO₃)₆]^2- and (d) CeO₂-NP (3nm, Strem) in graphene liquid cells, showing the M₄/M₅ ratio expected for a Ce(IV) valence state.