Antibiotic Susceptibility Determination within One Cell Cycle at Single-Bacterium Level by Stimulated Raman Metabolic Imaging

Abstract

The widespread use of antibiotics has significantly increased the number of resistant bacteria, which has also increased the urgency of rapid bacterial detection and profiling their antibiotic response. Current clinical methods for antibiotic susceptibility testing (AST) rely on culture and require at least 16 to 24 h to conduct. Therefore, there is an urgent need for a rapid method that can test the susceptibility of bacteria in a culture-free manner. Here we demonstrate a rapid AST method by monitoring the glucose metabolic activity of live bacteria at the single-cell level with hyperspectral stimulated Raman scattering (SRS) imaging. Using vancomycin-susceptible and -resistant enterococci E. faecalis as models, we demonstrate that the metabolic uptake of deuterated glucose in a single living bacterium can be quantitatively monitored via hyperspectral SRS imaging. Remarkably, the metabolic activity of susceptible bacteria responds differently to antibiotics from the resistant strain within only 0.5 h from the addition of antibiotics. Therefore, bacterial susceptibility and the minimum inhibitory concentration (MIC) of antibiotics can be determined within one cell cycle. Our metabolic imaging method is applicable to other bacteria species including E. coli, K. Pneumoniae, and S. aureus as well as different antibiotics, regardless of their mechanisms of inhibiting or killing bacteria.

Spectral tracing of deuterium for imaging glucose metabolism

“…This conceptual feature also distinguishes STRIDE from earlier attempts of single-channel glucose metabolic imaging (mostly reporting lipid synthesis) in bacteria or cultured cells. 24, 25, 48-50…”

Digital, Rapid, Accurate, and Label-Free Enumeration of Viable Microorganisms Enabled by Custom-Built On-Glass-Slide Culturing Device and Microscopic Scanning

“…However, the opaque appearance of agar gel determines that it is not a good choice for imaging application [ 22 ]. As an alternative, agarose gel has been extensively employed for imaging due to better optical clarity [ 12 , 15 , 23 , 24 , 25 , 26 , 27 , 28 ]. Notably, the flatness of the gel surface is critical for acquirement of high quality scanning image and the accuracy of micro-colonies counting.…”

Imaging the invisible—Bioorthogonal Raman probes for imaging of cells and tissues

“…The conventional testing assay takes 2 days to 1 week to provide results about the bacteria susceptibility. Recently, single-cell Raman deuterium labeling was introduced as a potential approach for a rapid antibiotic susceptibility testing [56]. In this approach clinical samples can be directly measured after incubation with heavy water.…”