Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

Summary

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.

Abstract

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.

Introduction

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 taken^1,2. 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....

Protocol

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

1. Prepare the antibiotics (gentamicin sulfate or amoxicillin) stock solution at a concentration of 1 mg/mL dissolved in sterile1x phosphate-buffered saline (PBS) or dimethyl sulfoxide (DMSO) solvent in 1.5 mL micro tubes. Dissolve....

Results

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 microbial cells.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acousto-optic modulation	Gooch&Housego	R15180-1.06-LTD	Modulating stokes laser beam
Amoxicillin	Sigma Aldrich	A8523-5G
Bandpass filter	Chroma	HQ825/150m	Block the stokes laser beam before the photodiode
Calcium chloride	Sigma Aldrich	C1016-100G	Cation adjustment
Cation-adjusted Mueller-Hinton Broth	Fisher Scientific	B12322	Antimicrobial susceptibility testing of microorganisms by broth dilution methods
Centrifuge	Thermo Scientific	75002542
Cover Glasses	VWR	16004-318
Culture tube with snap cap	Fisher brand	149569B
Daptomycin	Acros	A0386346
Deuterium oxide		151882	Organic solvent to dissolve antibiotics
Deuterium oxide-d6	Sigma Aldrich	156914	Organic solvent as a standard to calibrate SRS imaging system
Escherichia coli BW 25113	The Coli Genetic Stock Center	7636
Eppendorf polypropylene microcentrifuge tubes 1.5 mL	Fisher brand	05-408-129
Gentamicin sulfate	Sigma Aldrich	G4918
Hydrophilic Polyvinylidene Fluoride filters	Millipore-Sigma	SLSV025NB	pore size 5 µm
ImageJ software	NIH	Version: 2.0.0-rc-69/1.52t	Image processing and analysis
Incubating orbital shaker set at 37 °C	VWR	97009-890
Inoculation loop	Sigma	BR452201-1000EA
InSight DeepSee femtosecond pulsed laser	Spectra-Physics	Model: insight X3	Tunable laser source and fixed laser source at 1045 nm for SRS imaging
Lock-in amplifier	Zurich Instrument	HF2LI	Demodulate the SRS signals
Oil condenser	Olympus	U-AAC	NA 1.4
Pseudomonas aeruginosa ATCC 47085 (PAO1)	American Type Culture Collection	ATCC 47085
Photodiode	Hamamatsu	S3994-01	Detector
Polypropylene conical tube 15 mL	Falcon	14-959-53A
Polypropylene filters	Thermo Scientific	726-2520	pore size 0.2 µm
Sterile petri dishes	Corning	07-202-031
Syringe 10 mL	Fisher brand	14955459
UV/Vis Spectrophotometer	Beckman Coulter	Model: DU 530	Measuring optical density at wavelength of 600 nm
Vortex mixer	VWR	97043-562
Water objective	Olympus	UPLANAPO/IR	60×, NA 1.2