Differentiation and classification of bacteria using vancomycin functionalized silver nanorods array based surface-enhanced Raman spectroscopy and chemometric analysis

• High quality and reproducible bacteria SERS spectra can be obtained on AgNR substrates.
• Bacteria species can be differentiated by a first chemometric analysis.
• Bacteria serotypes/strains can be differentiated by a second chemometric analysis.
• Gram+ and Gram− bacteria can be distinguished with high sensitivity and specificity.

Twenty seven different bacteria isolates from 12 species were analyzed using intrinsic surface-enhanced Raman scattering (SERS) spectra with recently developed vancomycin coated silver nanorod (VAN AgNR) substrates. The VAN AgNR substrates could generate reproducible SERS spectra of the bacteria with little to no interference from the environment or bacterial by-products as compared to the pristine substrates. By taking advantage of the structural composition of the cellular wall which varies from species to species, the differentiation of bacterial species is demonstrated by using chemometric analyses on those spectra. A second chemometric analysis step within the species cluster is able to differentiate serotypes and strains. The spectral features used for serotype differentiation arises from the surface proteins, while Raman peaks from adenine dominate the differentiation of strains. In addition, due to the intrinsic structural differences in the cell walls, the SERS spectra can distinguish Gram-positive from Gram-negative bacteria with high sensitivity and specificity, as well as 100% accuracy on predicting test samples. Our results provide important insights for using SERS as a bacterial diagnostic tool and further guide the design of a SERS-based detection platform.

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