Rapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of D2O Metabolic Incorporation in a Single Bacterium

Abstract

Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic-resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D₂O) metabolism is reported. Metabolic incorporation of D₂O into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% D₂O medium, the fastest among current technologies. Single-cell metabolism inactivation concentration (SC-MIC) is obtained in less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of D₂O metabolic incorporation and SC-MIC determination after 1 h antibiotic treatment and 30 min mixture of D₂O and antibiotics incubation of bacteria in urine or whole blood is demonstrated.

Keywords: antimicrobial susceptibility testing; bacteria; isotope probing; single‐cell imaging; stimulated Raman scattering.

Figure 1

SRS imaging of D₂O metabolic incorporation in a single bacterium. a) Scheme for D₂O labeling of lipid and protein. b) Schematic illustration of SRS setup. AOM: acousto‐optic modulation. DM: dichroic mirror. PD: photodiode. c) SRS and corresponding transmission images of P. aeruginosa after culture in normal and D₂O‐containing medium for 3 h. Scale bar: 20 µm. SRS spectra d) and spontaneous Raman spectra e) of P. aeruginosa after culture in normal and D₂O containing LB medium for 3 h. f) Time‐lapse SRS imaging of P. aeruginosa after culture in D₂O containing medium. g) Average C–D intensity plot over time for bacteria in (f) with N ≥ 10 per group. Hyperspectral SRS (N = 5) h) and spontaneous Raman (N = 20) i) spectra at the C–D signature region, corresponding to the time‐lapse D₂O incorporation of P. aeruginosa after 10, 30, and 45 min culture. Error bars represent the standard error of the mean (SEM).

Figure 2

SRS‐based AST of P. aeruginosa as a function of culture time. Time lapse SRS at C–D and transmission images of P. aeruginosa after culture in D₂O‐containing medium with the addition of 20 µg mL⁻¹ gentamicin (a) or cefotaxime (b). Average C–D intensity plot over time for P. aeruginosa after culture in D₂O‐containing medium with gentamicin (c) or cefotaxime (d) treatment. Number of cells N ≥ 10 per group. Error bars represent the SEM. Scale bar: 20 µm. Histogram plot of the count of bacteria as a function of C–D intensity ratio of antibiotic‐treated group over the control group after 10 min (e) and 30 min (g) treatment. ROC curves of 10 min (f) and 30 min (h) treatment illustrating the ability of the C–D intensity ratio to distinguish susceptible and resistant groups. AUC: area under the curve.

Figure 3

SC‐MIC determination via SRS imaging of D₂O metabolic incorporation in single bacteria. a) Workflow of rapid AST with SC‐MIC determination by SRS imaging of D₂O metabolic incorporation. b) SRS at C–D and corresponding transmission images of P. aeruginosa after culture in D₂O containing medium with the addition of serially diluted gentamicin. c) Statistical analysis of C–D intensity in P. aeruginosa in (b). d–f) SC‐MIC determination for antibiotics with different mechanisms of action. The colored points under different concentration stand for different individual bacterium. The dotted lines indicate the cutoff value at 60% of the control sample. The C–D intensities are normalized to the mean of control without antibiotic treatment. Number of cells N ≥ 10 per group. Error bars represent the SEM. Scale bar: 20 µm.

Figure 4

SC‐MIC determination after 1 h culture of E. coli in urine. a) Bacterial purification protocol for bacteria in urine for rapid AST by SRS imaging of D₂O metabolic incorporation. b) SRS and corresponding transmission images of E. coli in urine after 1 h culture in D₂O‐containing medium with the addition of serially diluted amoxicillin. c) Statistical analysis of C–D intensity in bacteria in (b). Number of cells N ≥ 10 per group. The colored points under different concentration stand for different individual bacterium. The dotted lines indicate the cutoff value at 60% of the control sample. The C–D intensities are normalized to the mean of control without antibiotic treatment. Number of cells N ≥ 10 per group. Error bars represent the SEM. Scale bar: 10 µm. d) The comparison of SC‐MIC and the CLSI susceptibility category for E. coli isolate and E. coli in urine. S: sensitive.

Figure 5

SC‐MIC determination after 1 h culture of P. aeruginosa in blood. a) Bacterial purification protocol for bacteria in blood for rapid AST by SRS imaging of D₂O metabolic incorporation. b) SRS images at C–D, off‐resonance (2407 cm⁻¹), and C–H of bacteria in blood after 1 h culture in D₂O containing medium. c) SRS and corresponding transmission images of P. aeruginosa in blood after 1 h culture in D₂O‐containing medium with the addition of serially diluted gentamicin. d) Statistical analysis of C–D intensity in bacteria in (c). The colored points under different concentration stand for different individual bacterium. The dotted lines indicate the cutoff value at 60% of the control sample. The C–D intensities are normalized to the mean of control without antibiotic treatment. Number of cells N ≥ 10 per group. Error bars represent the SEM. Scale bar: 10 µm. e) Comparison of SC‐MIC and susceptibility category for P. aeruginosa isolate and P. aeruginosa in blood. S: sensitive.