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

Abstract

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 <i>Staphylococcus aureus</i> 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 <i>S. aureus</i> 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 <i>S. aureus</i> 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 <i>S. aureus</i> case and further clarify potential limitations and development requirements of this approach in the move toward industrial applications.