This work was supported by NIH R01AI141439 to J.-X.C and M.S, and R35GM136223 to J.-X.C.

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)&#160;office at Boston University.
1. Preparation of bacteria and antibiotics stock solution
<ol>
	<li>Prepare the antibiotics (gentamicin sulfate or amoxicillin) stock solution at ...

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...

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 &#65279;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 possible clinical outcome, it is crucial to introduce effective therapy within 24 h. However, the current gold standard method, like disk diffusion or broth dilution method, usually requires at least 24 h for the preincubation procedure for clinical samples and an additional 16-24 h to obtain the minimal inhibitory concentration (MIC) results. Overall, these methods are too time-consuming to guide an immediate decision for infectious disease treatment in the clinic, which leads to the emergence and spread of antimicrobial resistance4.
Genotypic AST methods, such as polymerase chain reaction (PCR)-based techniques5, have been developed for rapid detection. Such techniques measure the specific resistance genetic sequences in order to provide rapid AST results. They do not rely on time-consuming cell culture; however, only specific known genetic sequences with resistance are tested. Therefore, its application is limited to various bacterial species or different mechanisms of resistance. Also, they cannot provide MIC results for therapy decisions6,7. Besides, novel phenotypic methods for rapid AST are under development to overcome these limitations8, including microfluidic devices9,10,11,12,13, optical devices14,15,16, phenotypic AST quantifying the nucleic acids copy number17,18, and Raman spectroscopic methods19,20,21,22,23,24. These methods reduce time to guide AST results, however, most of them are only applicable to bacterial isolates, not directly to clinical specimens, and still require long-time preincubation.
In this work, we present a method for rapid determination of the susceptibility of bacteria in the urine and whole blood via monitoring of the cellular metabolic activity by SRS imaging. Water (H2O) takes part in the vast majority of essential biomolecular synthesis processes in living cells. As an isotopologue of water, through enzyme-catalyzed H/D exchange reaction between the redox-active hydrogen atom in NADPH and the D atom in D2O, deuterium can be incorporated into biomass inside a cell25,26. A deuterated fatty acid synthesis reaction is mediated by the deuterium labeled NADPH. The D2O incorporation into reactions of amino acids (AAs) results in the deuterated protein production26 (Figure 1). In this way, the newly synthesized C-D bond-containing biomolecules in single microbial cells can be employed as a general metabolic activity marker to be detected. To read out de novo synthesized C-D bonds, Raman spectroscopy, a versatile analytical tool providing specific and quantitative chemical information of biomolecules, is widely used to determine antimicrobial susceptibility and significantly reduce the testing time to a few hours27,28,29,30. However, due to the inherent low efficiency of the Raman scattering process, the spontaneous Raman spectroscopy is of low detection sensitivity. Therefore, it is challenging to obtain real-time image results using spontaneous Raman spectroscopy. Coherent Raman scattering (CRS), including coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has reached high detection sensitivity because of the coherent light field to generate orders of magnitude larger than that of spontaneous Raman spectroscopy, thereby rendering high-speed, specific, and &#65279;quantitative chemical imaging at the single cell level31,32,33,34,35,36,37,38,39.
Here, based on our most recent work40, we present a protocol for rapid determination of the metabolic activity and antimicrobial susceptibility by femtosecond SRS C-D imaging of D2O incorporation of bacteria in the normal medium, urine, and whole blood environment at the single-cell level. Femtosecond SRS imaging facilitates monitoring single cell metabolism inactivation concentration (SC-MIC) against antibiotics at the single bacterium level within 2.5 h. The SC-MIC results are validated by standard MIC test via broth microdilution. Our method is applicable for determining antimicrobial susceptibility of bacteria urinary tract infection (UTI) and bloodstream infection (BSI) pathogens with a much reduced assay time compared to the conventional method, which opens the opportunity for rapid phenotypic AST in the clinic at the single-cell level.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acousto-optic modulation</td>
			<td>Gooch&#38;Housego</td>
			<td>R15180-1.06-LTD</td>
			<td>Modulating stokes laser beam</td>
		</tr>
		<tr>
			<td>Amoxicillin</td>
			<td>Sigma Aldrich</td>
			<td>A8523-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandpass filter</td>
			<td>Chroma</td>
			<td>HQ825/150m</td>
			<td>Block the stokes laser beam before the photodiode</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-100G</td>
			<td>Cation adjustment</td>
		</tr>
		<tr>
			<td>Cation-adjusted Mueller-Hinton Broth</td>
			<td>Fisher Scientific</td>
			<td>B12322</td>
			<td>Antimicrobial susceptibility testing of microorganisms by broth dilution methods</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002542</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses</td>
			<td>VWR</td>
			<td>16004-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture tube with snap cap</td>
			<td>Fisher brand</td>
			<td>149569B</td>
			<td></td>
		</tr>
		<tr>
			<td>Daptomycin</td>
			<td>Acros</td>
			<td>A0386346</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td></td>
			<td>151882</td>
			<td>Organic solvent to dissolve antibiotics</td>
		</tr>
		<tr>
			<td>Deuterium oxide-d6</td>
			<td>Sigma Aldrich</td>
			<td>156914</td>
			<td>Organic solvent as a standard to calibrate SRS imaging system</td>
		</tr>
		<tr>
			<td>Escherichia coli BW 25113</td>
			<td>The Coli Genetic Stock Center</td>
			<td>7636</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf polypropylene microcentrifuge tubes 1.5 mL</td>
			<td>Fisher brand</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulfate</td>
			<td>Sigma Aldrich</td>
			<td>G4918</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophilic Polyvinylidene Fluoride filters</td>
			<td>Millipore-Sigma</td>
			<td>SLSV025NB</td>
			<td>pore size 5 &#181;m</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td>Version: 2.0.0-rc-69/1.52t</td>
			<td>Image processing and analysis</td>
		</tr>
		<tr>
			<td>Incubating orbital shaker set at 37 &#176;C</td>
			<td>VWR</td>
			<td>97009-890</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Sigma</td>
			<td>BR452201-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>InSight DeepSee femtosecond pulsed laser</td>
			<td>Spectra-Physics</td>
			<td>Model: insight X3</td>
			<td>Tunable laser source and fixed laser source at 1045 nm for SRS imaging</td>
		</tr>
		<tr>
			<td>Lock-in amplifier</td>
			<td>Zurich Instrument</td>
			<td>HF2LI</td>
			<td>Demodulate the SRS signals</td>
		</tr>
		<tr>
			<td>Oil condenser</td>
			<td>Olympus</td>
			<td>U-AAC</td>
			<td>NA 1.4</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa ATCC 47085 (PAO1)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 47085</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Hamamatsu</td>
			<td>S3994-01</td>
			<td>Detector</td>
		</tr>
		<tr>
			<td>Polypropylene conical tube 15 mL</td>
			<td>Falcon</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene filters</td>
			<td>Thermo Scientific</td>
			<td>726-2520</td>
			<td>pore size 0.2 &#181;m</td>
		</tr>
		<tr>
			<td>Sterile petri dishes</td>
			<td>Corning</td>
			<td>07-202-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL</td>
			<td>Fisher brand</td>
			<td>14955459</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/Vis Spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>Model: DU 530</td>
			<td>Measuring optical density at wavelength of 600 nm</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>Water objective</td>
			<td>Olympus</td>
			<td>UPLANAPO/IR</td>
			<td>60&#215;, NA 1.2</td>
		</tr>
	</tbody>
</table>

rapid antimicrobial susceptibility testing by stimulated raman scattering imaging of deuterium incorporation in a single bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;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 &#65279;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&#65279; 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.

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 &#65279;(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...

Watch this Scientific Journal Video about Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium at JoVE.com

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

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.