Published: February 14th, 2022
This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.
To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to quantitatively determine the antimicrobial effects on pathogens. It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.
Antimicrobial resistance (AMR) is a growing global threat to the effective treatment of infectious disease1. It is predicted that AMR will cause an additional 10 million deaths per year and $100 trillion global GDP loss by 2050 if no action for combating antibiotic-resistant bacteria is taken1,2. This stresses the urgent need for rapid and innovative diagnostic methods for antibiotic susceptibility testing (AST) of infectious bacteria to slow down the emergence of antibiotic-resistant bacteria and reduce the related mortality rate3. To ensure the best pos....
The use of human blood specimens is in accordance with the guidelines of the IRB of Boston University and the National Institutes of Health (NIH). Specifically, the specimens are from a bank and are completely deidentified. These specimens are not considered to be human subjects by institutional review board (IRB) office at Boston University.
1. Preparation of bacteria and antibiotics stock solution
The effect of incubation time on deuterium incorporation is measured by spontaneous Raman microspectroscopy at the C-D (2070 to 2250 cm-1) and C-H (2,800 to 3,100 cm-1) region (Figure 4a). The time-lapse single-cell Raman spectra of P. aeruginosa cultured in 70% D2O containing medium show increasing CD/CH intensity over incubation time from 0 to 180 min. (Figure 4b) The increasing C-D abundance in single microbia.......
Rapid AST can be obtained by assessing the response of bacterial metabolic activity to antibiotic treatment using single-cell SRS metabolic imaging within 2.5 h from the sample to SC-MIC results. The response of bacterial metabolic activity and antimicrobial susceptibility can be detected by monitoring the metabolic incorporation of D2O for biomolecule synthesis using SRS imaging of C-D bonds. Since water is ubiquitously used in living cells, SRS metabolic imaging provides a universal method for rapid AST. The.......
|Modulating stokes laser beam
|Block the stokes laser beam before the photodiode
|Cation-adjusted Mueller-Hinton Broth
|Antimicrobial susceptibility testing of microorganisms by broth dilution methods
|Culture tube with snap cap
|Organic solvent to dissolve antibiotics
|Organic solvent as a standard to calibrate SRS imaging system
|Escherichia coli BW 25113
|The Coli Genetic Stock Center
|Eppendorf polypropylene microcentrifuge tubes 1.5 mL
|Hydrophilic Polyvinylidene Fluoride filters
|pore size 5 µm
|Image processing and analysis
|Incubating orbital shaker set at 37 °C
|InSight DeepSee femtosecond pulsed laser
|Model: insight X3
|Tunable laser source and fixed laser source at 1045 nm for SRS imaging
|Demodulate the SRS signals
|Pseudomonas aeruginosa ATCC 47085 (PAO1)
|American Type Culture Collection
|Polypropylene conical tube 15 mL
|pore size 0.2 µm
|Sterile petri dishes
|Syringe 10 mL
|Model: DU 530
|Measuring optical density at wavelength of 600 nm
|60×, NA 1.2
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