This work demonstrates a rapid platform that can determine the antimicrobial susceptibility testing (AST) in cation-adjusted Mueller-Hinton Broth medium, urine and blood by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) incorporation at a single bacterium level. The total AST assay time with the value of the single-cell metabolism inactivation concentration (SC-MIC) obtained is less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples were systematically validated by MIC determined by the Clinical and Laboratory Standards Institute criteria, with a category agreement of 94.6% and 5.4% minor error. Furthermore, SRS imaging of D2O metabolic incorporation can rapidly determine SC-MIC directly in clinical samples for urinary tract infection or septicemia blood infection.
Candida is the single most important cause of fungal bloodstream infections worldwide causing significant mortality as high as 50%. This high mortality rate is, in part, due to the inability to rapidly diagnose and simultaneously initiate an effective antifungal therapy early in the disease process. Current culture-based diagnostics are often slow, requiring several days to complete, and are only 50% sensitive in diagnosing candidemia (Candida bloodstream infection). For every 12 hours of delay in starting correct antifungal therapy, the risk of death for a given patient with candidemia increases by 200%. To address this unmet need, we explored the potential of employing stimulated Raman Scattering (SRS) imaging to diagnose candidemia and probe metabolic differences between resistant and susceptible strain at a single cell level. Metabolism is integral to pathogenicity; microorganism have very short life cycles, and therefore only a few hours are needed to observe a full metabolic cycle. SRS imaging at C-H vibration frequency at 2850 cm-1 revealed a substantial difference in lipogenesis between the susceptible and resistant C. albicans. Treating the C. albicans with fluconazole, an antimicrobial drug that targets ergosterol biosynthesis only affected the lipogenesis in the susceptible strain. Our results show that single-cell metabolic imaging under a SRS microscope can be used for diagnose candidemia and early detection of antimicrobial susceptibility.
Stimulated Raman scattering (SRS) microscopy is a promising technique for label-free imaging of living systems. We demonstrate microsecond-scale SRS spectral imaging by tuning two spectrally focused pulses temporally through a resonant delay-line. Our platform acquired an SRS spectrum within 42 microseconds and formed a spectral image composed of 40,000 pixels in real-time.
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