Fast Antibiotic Susceptibility Testing via Raman Microspectrometry on Single Bacteria: An MRSA Case Study

[Image: see text] Despite recent advances in molecular diagnostics, ultrafast determination of the antibiotic susceptibility phenotype of pathogenic microorganisms is still a major challenge of in vitro diagnostics (IVD) of infectious diseases. Raman microspectroscopy has been proposed as a means to achieve this goal. Previous studies have shown that susceptibility phenotyping could be done through Raman analysis of microbial cells, either in large clusters or down to the single-cell level in the case of Gram-negative rods. Gram-positive cocci such as Staphylococcus aureus pose several challenges due to their size and their different metabolic and chemical characteristics. Using a tailored automated single-cell Raman spectrometer and a previously proposed sample preparation protocol, we acquired and analyzed 9429 S. aureus single cells belonging to three cefoxitin-resistant strains and two susceptible strains during their incubation in the presence of various concentrations of cefoxitin. We observed an effect on S. aureus spectra that is weaker than what was detected on previous bacteria/drug combinations, with a higher cell-to-cell response variability and an important impact of incubation conditions on the phenotypic resistance of a given strain. Overall, the proposed protocol was able to correlate strains’ phenotype with a specific modification of the spectra using majority votes. We, hence, confirm that our previous results on single-cell Raman antibiotic susceptibility testing can be extended to the S. aureus case and further clarify potential limitations and development requirements of this approach in the move toward industrial applications.