Characterization of the Acinetobacter baumannii Secretome Using Size-Exclusion Chromatography and Raman Spectroscopy

Acinetobacter baumannii, a multidrug-resistant pathogen of critical priority within the ESKAPE group, poses a significant threat to global healthcare, particularly in the context of nosocomial infections. Its pathogenesis is mediated not only by antibiotic resistance determinants but also by a complex repertoire of secreted virulence factors. However, comprehensive characterization of the A. baumannii secretome remains methodologically challenging due to spectral overlap in complex biological matrices. In this study, we applied a hybrid approach integrating size-exclusion chromatography with Raman spectroscopy to deconvolute the cell-free supernatant of A. baumannii. Chromatographic fractionation into seven fractions reduced spectral complexity and enabled the identification of unique metabolic profiles. Fraction 3 exhibited a distinct composition, containing specific markers for phosphatidylserine (~1724 cm⁻¹), cysteine, phosphatidylinositol, and DNA (~770–806 cm⁻¹), as well as CH₂ groups of lipids and amino acids (~1450–1456 cm⁻¹), while lacking signals corresponding to methionine-containing compounds, nucleic acid backbones, and polypeptide backbones characteristic of other fractions. Analysis revealed distinct biochemical specialization across fractions: Fraction 2 was enriched in glutamine/asparagine-associated signals (~990, ~998 cm⁻¹), Fraction 4 contained a unique carotenoid marker (~1154 cm⁻¹), Fraction 6 exhibited a phenylalanine-specific peak (~1104 cm⁻¹), and Fraction 7 demonstrated the highest intensity of cysteine-containing protein, nucleotide, and phospholipid signals. These findings open new avenues for the discovery of biomarkers associated with virulence and antibiotic resistance in A. baumannii.