Metabolic fingerprinting of periodontal bacteria: a multi-scale mass spectrometry and vibrational spectroscopy approach

Periodontitis is a prevalent condition characterised by progressive destruction of the supporting periodontium around teeth, resulting from prolonged interactions between the host immune system and bacterial infection. Classifying periodontal bacteria remains challenging due to their high diversity, culturability, strain level variability, biofilm complexity, and methodological constraints. Fourier Transform Infrared (FT-IR) spectroscopy was employed to investigate the biochemical composition of five oral <i>Streptococcus</i> spp., <i>Porphyromonas gingivalis, Actinomyces israelii, Fusobacterium nucleatum</i>, and <i>Parvimonas micra</i> at microbial-community level. In parallel, Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry, the gold standard analytical platform in clinical settings, was used for a comprehensive understanding of cellular protein/peptide molecules, identifying approximately 20 <i>m</i>/<i>z</i> features associated with the clustering pattern. Particularly, <i>P. gingivalis</i> was distinctly clustered within higher <i>m</i>/<i>z</i> range (8000-16 500), primarily driven by features at 9329, 9800, and 11 029 <i>m</i>/<i>z</i>, highlighting the potential of this window for its specific identification. Additionally, Optical Photothermal Infrared (O-PTIR) spectroscopy, combined with chemometrics, was also utilised at single-cell level. Classification patterns across IR techniques and MALDI-MS were comparable, revealing significant variation in protein and lipid profiles among the studied strains. <i>P. gingivalis</i>, <i>F. nucleatum</i> and <i>A. israelii</i> exhibited distinct clustering, while <i>S. anginosus</i> and <i>S. oralis</i> showed significant variation within the <i>Streptococcus</i> spp. The overall clustering pattern was primarily attributed to spectral information within amides (1500-1800 cm^-1) and fatty acids (2800-3050 cm^-1) regions, alongside vibrational bands observed between (900-1200 cm^-1), indicative of polysaccharides and phosphate-containing compounds. The submicron spatial resolution demonstrated by O-PTIR, suggests promising potential for direct application to clinical samples without the need for prolonged culturing.