Spectral fingerprinting by FTIR microspectroscopy enables differentiation of Gram status, strain variability and resistance phenotypes among diabetic foot infection isolates.

Diabetic foot infections (DFIs) are serious, often polymicrobial, complicating microbial identification. Fourier-transform infrared (FTIR) microspectroscopy provides rapid, non-destructive molecular fingerprints. This study evaluated the use of FTIR microspectroscopy to differentiate between DFI Gram-positive and Gram-negative bacteria, discriminate among species within each group, and distinguish between reference strains and DFI isolates, as well as between susceptible and resistant phenotypes. The microorganisms analyzed included reference and DFI isolates of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa as well as methicillin resistant S. aureus (MRSA) and extended-spectrum β-lactamase (ESBL)-producing E. coli. FTIR spectra were preprocessed and analyzed. Three spectral ranges of interest corresponding to lipid region, protein (amide I and amide II) vibrations region and DNA/polysaccharides vibrations region were identified. Second derivatives of the spectra were calculated, and multivariate statistical analysis was performed for different sample groups. Spectra were separated into two main clusters consistently corresponding to the Gram-positive and Gram-negative bacteria in all studied spectral ranges: lipid, protein and DNA/polysaccharides regions. Staphylococci genus formed a dense cluster which was subdivided into two sub-clusters in the DNA/carbohydrate region one for S. aureus and the other for S. epidermidis. Gram-negative cluster corresponding to DNA/carbohydrate region was subdivided into three sub-clusters for P. aeruginosa, E. coli and A. baumannii. Moreover, the DNA/polysaccharides FTIR bands demonstrated intensity and frequency shift differences between the compared reference and DFI isolates. FTIR microspectroscopy effectively discriminated between susceptible and resistant bacterial phenotypes. MRSA strains exhibited distinct alterations in lipid-associated C-H stretching bands and phosphate-related vibrational modes relative to their susceptible S. aureus. ESBL-producing E. coli, displayed clear separation from susceptible E. coli in the DNA/polysaccharide region. The findings support the use of FTIR as a diagnostic tool of polymicrobial infections, and demonstrate its ability to differentiate resistant from susceptible phenotypes. These results warrant further validation in larger sample sets to confirm clinical applicability in DFI diagnosis.