Integrated characterization of extracellular matrix remodeling in Candida parapsilosis biofilms by surface-enhanced raman spectroscopy, protein analysis, ultrastructural imaging and multivariate analysis.

The extracellular matrix (ECM) of Candida parapsilosis biofilms undergoes dynamic changes during maturation, but its temporal molecular and structural organization remains poorly characterized. This study investigated ECM changes in a reference strain (C. parapsilosis ATCC 22019) and a clinical isolate (C. parapsilosis 1799) using Surface-enhanced Raman spectroscopy (SERS), Bradford protein analysis, Scanning transmission electron microscopy (STEM), and Principal component analysis (PCA). SERS profiles showed marked time-associated changes between 72 and 96 h, including attenuation of several nucleic-acid-, glycosidic-, and polysaccharide-associated signals and enhancement of other spectral regions. Soluble protein content decreased in both isolates, whereas selected SERS signals increased, indicating that SERS intensity did not directly reflect bulk protein abundance. STEM revealed increased electron-dense clustered structures at 96 h. PCA demonstrated clear time-associated spectral differentiation, which was retained after exclusion of major citrate-associated spectral regions. The two isolates also displayed distinct temporal SERS profiles, suggesting isolate-dependent ECM maturation changes. The combined findings support dynamic and potentially isolate specific remodeling of the C. parapsilosis ECM during biofilm maturation rather than simple matrix accumulation or degradation. SERS shows potential for monitoring these changes, although tentative spectral assignments, substrate related contributions, and limited biological replication warrant independent molecular validation and further longitudinal studies.