Discrimination of MSSA and MRSA strains with Raman spectroscopy using two different wavelengths
Leibniz IPHT // Jena, Germany
Q&A Session III // Friday, October 30 // 8.15 am – 9 am (CET)
Antimicrobial resistance has developed into a global health issue. S. aureus infections are one of the most common causative agents of bacteraemia and sepsis. In the present study we aimed the detection of bacterial resistance determinants using UV-resonance Raman spectroscopy (UVRR) in bulk measurements and 532nm-Raman microspectroscopy in single-cell measurements, in pre-cultured, heat-inactivated MSSA (Methicillin-susceptibleStaphylococcus aureus) and MRSA strains. Four MRSA and MSSA pairs were analyzed that differed only in the presence/absence of the mobile genetic element SCCmec which harbors the resistance gene mecA causing resistance against methicillin. For each strain, three batches were measured on different days, each containing 25×10 spectra in the UVRR and 50 spectra in the visible excitation. Statistical analysis was performed using chemometrics, machine learning and majority voting per strain were applied to make results more suitable to clinical decision making. In the different spectra the Trp, Tyr, Phe band in the MRSA strains was present in both UVRR (1607cm-1) and Raman microspectroscopy (1614cm-1). With UVRR the C, U band 786cm-1 was present in MSSA showing differences in RNA composition compared to MRSA. With the visible excitation the A/G band at 1355cm-1 was present in MRSA and the C band at 779 cm-1 in MSSA. Also, with Raman microspectroscopy the MRSA revealed distinct amino acids and amid bands (1004, 1244, 1673cm-1) while in MSSA only cytochrome (1127cm-1) and carotenoids (1521cm-1) bands were present. The accuracy for differentiation was 64% for the UVRR and 63% for the Raman microspectroscopy. After applying majority voting the accuracy did not change for the UVRR but in the visible excitation it increased to 88% of correctly classified strains. When each pair was analyzed separately, the accuracy for differentiation was up to 86%. It was observed that the band at 780/786 cm-1 (C, U) was present in all MSSA but none MRSA strain, indicating differences in RNA composition. Trp (756/849cm-1), Phe (1002cm-1) and Tyr (1203cm-1) bands were only present in one pair showing differences in protein composition of this pair compared to the others. UVRR and Raman microspectroscopy with an excitation wavelength of 532nm have the potential to be developed as diagnostic tools for the detection of resistant strains in routine clinical laboratories.
Financial support of the MCSA-COFUND Multiply Project (H2020 GA 713694) and the research campus InfectoGnostics (FKZ 13GW0096F) is gratefully acknowledged.