Raman Identification of Streptococcus mutans Pathogenic Properties

Methods: S. mutans strain UA159 biofilms were grown in Todd Hewitt broth either with or without 1% sucrose at 37oC, 5% CO2 from overnight cultures for 8 d. Biofilm cells were isolated and evaluated on silica wafers under 633 nm excitation by spontaneous Raman spectroscopy (Renishaw InVia Raman microscope). Spectra were acquired from 800 to 1800 cm-1 with 3 averaged exposure times of at least 100 s each. Images were acquired from multiple locations within the biofilms to account for disparities created by thickness or spatial anomalies.

Results: Detailed fingerprint regions of the chemical bonds were obtained from biofilms grown with or without sucrose supplement. Distinct differences were evident between sucrose-exposed and -unexposed S. mutans biofilm cells at 1580 cm-1 (purine ring stretch for DNA/RNA), 1554 cm-1 (C=C stretch), 1480 cm-1 (R-COO- asymetric carboxylate stretch vibration), and 1103 cm-1 (C-C stretch, C-O-C glycosidic link; symmetrical ring breathing).

Conclusions: The addition of sucrose resulted in Raman peaks that are consistent with the expected metabolic products, e.g., sucrose is first metabolized into alpha-1,3- and alpha-1,6-glucan (which is known to enhance biofilm adhesion and rigidity) followed by lactic acid (which demineralizes tooth hydroxyapatite). The ability to detect these peaks under conditions well documented to stimulate the pathogenic characteristics of a S. mutans biofilm provides preliminary validation that Raman microscopy can be developed for in vivo identification of pathogenic oral biofilms. Investigations monitoring hydroxyapatite demineralization will assess the capacity of Raman spectroscopy to directly monitor S. mutans or other biofilm pathogenesis.

Support: NIDCR/NIST Interagency Agreement Y1-DE-7005-01.