Title: Biochemical Analysis of Endodontic Bacterial Biofilms by Raman Spectroscopy
Tatiana Ramirez (Presenter)
University of Costa Rica
Grettel Valle-Bourrouet, University of Costa Rica
Objectives: Analyze the biochemical composition of three different endodontic biofilms by means of their Raman spectra.
Methods: Biofilms were grown in cell culture plates, under aerobic and static conditions up to 14 days. A wild strain of Enterococcus faecalis and Actinomyces naeslundii isolated from persistent endodontic infection undergoing retreatment were employed. Also, Pseudomonas aeruginosa strain (PA 01) was used.
Biofilm samples were transferred to a quartz slides with a 10µL loop and dried for 20 min with silica. To record biofilm Raman spectra, on each smear of the quartz slides a central focus point was chosen and around this center, four spectra were collected.
All Raman spectra were recorded with a Raman microspectrometer (ProRaman-L Enwave Optronics). The 50x objective of the microscope (Leica) was used and the sample was excited using 45-50 mW of the 785 nm diode laser. Raman signal was collected in the spectral interval 600-1800 cm-1, integration time was 60 s, while the spectral resolution was approximately 2cm-1.
For the interpretation of Raman spectra, ID-Expert KnowIt All Informatic System (BioRad), as well as available literature were employed. Multivariate technique principal component analysis (PCA) was used.
Results: : Major peaks, from 669 to 1654 cm-1, were identified, which represent: protein, nucleic acid, lipid, and carbohydrate components. The most distinctive wave numbers were 1004, 1088 cm-1, for nucleic acids. 1242, 1655 cm-1 (Amide III and Amide I) and 1454 cm-1 peak (lipid). The PCA plot of the Raman spectral data of biofilms showed similar composition for the three bacterial strains. Protein and nucleic acid were more abundant in E. faecalis, and P. aeruginosa, compare with A. naeslundii biofilm.
Conclusions: In general, endodontic bacterial biofilms are composed of very similar biomolecules, which are expressed at different levels.