Comparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is classified as one of the priority pathogens that threaten human health. Resistance detection with conventional microbiological methods takes several days, forcing physicians to administer empirical antimicrobial treatment that is not always appropriate. A need exists for a rapid, accurate, and cost-effective method that allows targeted antimicrobial therapy in limited time. In this pilot study, we investigate the efficacy of three different label-free Raman spectroscopic approaches to differentiate methicillin-resistant and -susceptible clinical isolates of S. aureus (MSSA). Single-cell analysis using 532 nm excitation was shown to be the most suitable approach since it captures information on the overall biochemical composition of the bacteria, predicting 87.5% of the strains correctly. UV resonance Raman microspectroscopy provided a balanced accuracy of 62.5% and was not sensitive enough in discriminating MRSA from MSSA. Excitation of 785 nm directly on the petri dish provided a balanced accuracy of 87.5%. However, the difference between the strains was derived from the dominant staphyloxanthin bands in the MRSA, a cell component not associated with the presence of methicillin resistance. This is the first step toward the development of label-free Raman spectroscopy for the discrimination of MRSA and MSSA using single-cell analysis with 532 nm excitation. IMPORTANCE Label-free Raman spectra capture the high chemical complexity of bacterial cells. Many different Raman approaches have been developed using different excitation wavelength and cell analysis methods. This study highlights the major importance of selecting the most suitable Raman approach, capable of providing spectral features that can be associated with the cell mechanism under investigation. It is shown that the approach of choice for differentiating MRSA from MSSA should be single-cell analysis with 532 nm excitation since it captures the difference in the overall biochemical composition. These results should be taken into consideration in future studies aiming for the development of label-free Raman spectroscopy as a clinical analytical tool for antimicrobial resistance determination.

Keywords: 532-nm single-cell analysis; 785 nm fiber probe; MRSA; Raman microscopy; Raman spectroscopy; UV resonance Raman spectroscopy; single-cell analysis.

FIG 1

Mean Raman spectrum (A) and difference spectrum (B) of MRSA and MSSA strains by using 785 nm excitation on bacterial colonies directly on the petri dish. arb. u, arbitrary units. Spectra are shifted vertically for clarity.

FIG 2

Mean Raman spectrum (A) and difference spectrum (B) of MRSA and MSSA strains by using UVRR. arb. u, arbitrary units. Spectra are shifted vertically for clarity.

FIG 3

Mean Raman spectrum (A) and difference spectrum (B) of MRSA and MSSA strains by using single-cell analysis with 532 nm excitation. arb. u, arbitrary units. Spectra are shifted vertically for clarity.