Antibacterial and Antibiofilm Effects of L-Carnitine-Fumarate on Oral Streptococcal Strains Streptococcus mutans and Streptococcus sobrinus

Abstract

Streptococcus mutans is a major pathogenic habitant of oral caries. Owing to its physiological and biochemical features, it prevails in the form of plaque biofilm together with another important mutans streptococci species, Streptococcus sobrinus. Both species are considered as initiators of cavity lesions, and biofilm is essential to the dental caries process. Compared with the planktonic populations, the biofilm form has higher resistance to environmental conditions and antibiotics. Dental plaques also secure the long-term survival of microorganisms and protection from any stress conditions. To address the need for new antibiofilm agents, we have focused on L-carnitine-fumarate, a fumarate-conjugated quaternary ammonium compound. Using the macro-broth susceptibility testing method, we established its MIC value as 6.0 mg/mL. The MBC value, determined from the broth dilution minimum inhibitory concentration test by sub-culturing it to BHI agar plates, was established as 7.0 mg/mL. Antibiofilm efficacy was tested in 96-well plates coated with saliva using BHI broth supplemented with 1% sucrose as a standard approach. The obtained results allowed us to assess the MIBC as 7.5 mg/mL and the MBBC value as 10.0 mg/mL. The latter concentration also caused approximately 20% eradication of pre-existing biofilm. EPS-rich matrix, forming the core of the biofilm and enabling a confined acidic microenvironment, was also examined and confirmed the effectiveness of 10.0 mg/mL L-carnitine-fumarate concentration in inhibiting EPS formation. Furthermore, the anti-adherent and anti-aciduric impacts of L-carnitine-fumarate were investigated and revealed significant inhibitory effects at sub-MIC concentrations. The influence of L-carnitine-fumarate on the phosphotransferase system was investigated as well. Our results provide a new insight into the antibacterial potential of L-carnitine-fumarate as a valuable compound to be considered for alternative or adjunct anti-caries and antibiofilm preventive approaches.

Keywords: L-carnitine; Streptococcus mutans; biofilm.

Figure 1

Bacteriostatic and bactericidal efficacy of L-carnitine-fumarate on the planktonic form of Streptococcus mutans UA159 and Streptococcus sobrinus SL1. Dose-dependent effect of different concentrations of LC evaluated by macro-dilution method at 24 h (A). Time-dependent effect of sub-MIC and MIC values of LC assessed by macro-dilution method up to 24 h (B). Time-dependent effect of sub-MBC and MBC values of LC assessed by macro-dilution method up to 24 h (C). MBC values were determined from broth macro-dilution minimum inhibitory concentration test by sub-culturing it on BHI agar plates that did not contain LC. Control—10% 1 × PBS; LC—L-carnitine-fumarate; # p < 0.05, ∆ p ≤ 0.01, * p < 0.001 compared to control.

Figure 2

Effect of L-carnitine-fumarate on the biofilm growth of Streptococcus mutans UA159 and Streptococcus sobrinus SL1. Dose-dependent effect of different concentrations of LC evaluated at 24 h. MBIC values were determined on 96-well plates coated with human saliva after 24 h incubation with LC and assessed by alamarBlue (A) and crystal violet (B) staining methods. Insert: representative images of biofilms of S. mutans formed on artificial teeth coated with human saliva after 24 h incubation period with 7.5 mg/mL LC. Time-dependent effect of sub-MBIC and MBIC values of LC evaluated up to 24 h. MBIC values were determined on 96-well plates coated with human saliva after 24 h incubation with LC and assessed by alamarBlue (C) and crystal violet (D) staining methods. Control—10% 1 × PBS; LC—L-carnitine-fumarate; # p < 0.05, ∆ p ≤ 0.01, * p < 0.001 compared to control.

Figure 3

Biocidal effect of L-carnitine-fumarate against the pre-existing biofilm of Streptococcus mutans UA159 and Streptococcus sobrinus SL1. Dose-dependent effect of different concentrations of LC evaluated at 24 h. MBBC values were determined on 96-well plates coated with human saliva after 24 h incubation with LC and assessed by alamarBlue staining methods (A). Time-dependent effect of sub-MBBC and MBBC values of LC evaluated up to 24 h. MBBC values were determined on 96-well plates coated with human saliva after 24 h incubation with LC and assessed by alamarBlue (B). Time-dependent effect of MBBC value of LC evaluated after 24 h incubation period in BHI broth supplemented with 1% sucrose only (C). Control—10% 1 × PBS; LC—L-carnitine-fumarate;—∆ p ≤ 0.01, * p < 0.001 compared to control.

Figure 4

Effect of L-carnitine-fumarate on the detachment of the pre-existing biofilm of Streptococcus mutans UA159 and Streptococcus sobrinus SL1. Dose-dependent effect of different concentrations of LC evaluated at 24 h. MBEC values were determined on 96-well plates coated with human saliva after 24 h incubation with LC and assessed by crystal violet staining methods (A). Effect of 7.5 mg/mL and 10 mg/mL concentrations of LC evaluated at 24 h and at 48 h assessed by crystal violet staining methods (B). Insert: representative images of biofilms of S. mutans on artificial teeth coated with human saliva after 24 h incubation period with 10 mg/mL LC. Representative images of EPS presence on pre-existing biofilm of S. mutans after 24 h incubation period with 10.0 mg/mL LC stained with Alexa Fluor 633-conjugated concanavalin A (C). Control—10% 1 × PBS; LC—L-carnitine-fumarate; # p < 0.05, ∆ p ≤ 0.01, compared to control.

Figure 5

Effect of L-carnitine-fumarate on the adherence of Streptococcus mutans UA159. Glass adherence in the absence (sucrose-independent) and presence of 5% sucrose (sucrose-dependent) was evaluated by measuring changes in OD₆₀₀ nm after adding 0.5 M NaOH as described in the Section 2 (A). Plastic adherence in the presence of 5% sucrose (sucrose-dependent) was assessed at different time points on 96-well plates not coated with human saliva, by measuring changes in OD₅₉₅ nm (B). Control—10% 1 × PBS; LC—L-carnitine-fumarate; # p < 0.05, * p < 0.001 compared with control.

Figure 6

Effect of L-carnitine-fumarate on the aciduricity and PTS of Streptococcus mutans UA159. Acid tolerance was determined by measuring the survival of S. mutans UA159 at pH = 5.0 assessed on BHI agar plates incubated for 24 h at 37 °C (A). PEP level was assessed at mid-logarithmic (6 h) and late logarithmic/early stationary phase (12 h) as described in the Section 2 (B). Control—10% 1 × PBS; LC—L-carnitine-fumarate; PEP—phosphoenolpyruvate; # p < 0.05, ∆ p ≤ 0.01, * p < 0.001 compared with control. N₀ and N—CFU counts before (N₀, time = 0 h) and after 2 h (N_, time = 2 h) treatment in pH = 5.0 culture, respectively.