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.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Rapid antimicrobial susceptibility testing can be obtained within 2.5 hours in urine and blood, which is considered a tremendous reduction in analysis time compared to the conventional broth microdilution method. It can monitor bacterial metabolic activity in a complex environment, such as whole blood. To begin, check the bacterial concentration from the samples by measuring the optical density with a photometer at 600 nanometer wavelength.To reach a final cell concentration of eight times 10 to the fifth Colony Forming Units, or CFU, per milliliter, dilute the bacterial solution using the normal MHB medium without deuterium. After mixing the bacterial cells by vortex, remove 300 microliter aliquots of the bacterial solution in seven 1.5-milliliter microtubes and a 600 microliter aliquot of the bacterial solution in one 1.5-milliliter microtube. Then add 4.8 microliters of the antibiotic stock solution into the microtube containing 600 microliters of the bacterial solution to achieve the final antibiotic concentration of eight micrograms per milliliter.Add 300 microliters of the bacterial solution with antibiotic to a 300 microliter aliquot of the bacterial solution without antibiotic to achieve a twofold diluted solution with the final antibiotic concentration of four micrograms per milliliter. Repeat the twofold serial dilution of the test antibiotics until the lowest concentration of 0.25 micrograms per milliliter is reached. Discard 300 microliters of the solution from the last microtube.Allot one tube with no antibiotics to the positive control with deuterium treatment, and the negative control with no deuterium. Incubate the bacterial aliquot with the antibiotic containing MHB medium for one hour. Meanwhile, prepare a serial dilution of antibiotics with 100%deuterium containing MHB medium with the same concentration gradients as described before.After one hour of incubation, add 700 microliters of antibiotic serially diluted with 100%deuterium containing MHB medium to the 300 microliters of antibiotic pre-treated bacteria in the same antibiotic concentration. Homogenize the mixture by pipetting up and down several times. Add 700 microliters of antibiotic-free 100%deuterium containing MHB medium to 300 microliters of antibiotic-free bacteria as a positive control.Add 700 microliters of antibiotic-free 0%deuterium containing MHB medium to 300 microliters of antibiotic-free bacteria as a negative control. Incubate all the microtubes at 37 degrees Celsius and 200 rotations per minute for 30 minutes. After incubation, centrifuge the one milliliter of antibiotic and deuterium treated bacterial sample at 6, 200 times g for five minutes at four degrees Celsius.Then wash the pellet twice with purified water. Fix the samples in 10%volume by volume formalin solution, and store them at four degrees Celsius. Check the Escherichia coli concentration from the freshly prepared bacterial sample by measuring the optical density with a photometer at 600 nanometer wavelength.To mimic the clinical urinary tract infection samples, spike the Escherichia coli sample into 10 milliliters of de-identified urine samples to reach a final concentration of 10 to the six CFU per milliliter. Filter the Escherichia coli spiked urine using a five-micron filter, and divide the filtered bacterial solution in 300 microliter aliquots into seven 1.5-milliliter microtubes, and a 600 microliter aliquot in one 1.5-milliliter microtube. Perform deuterium incorporation treatment in the presence of antibiotics as described before.To mimic the clinical bloodstream infection samples, spike Pseudomonas aeruginosa in one milliliter of de-identified human blood to reach a final concentration of 10 to the sixth CFU per milliliter. To lyse the blood, add nine milliliters of sterile purified water. Filter the Pseudomonas aeruginosa spiked blood using a five-micron filter.Then harvest bacteria from the filtered sample to a one milliliter volume by centrifugation at 6, 200 times g for five minutes at four degrees Celsius. After centrifugation, divide the Pseudomonas aeruginosa spiked blood solution in 300 microliter aliquots into seven 1.5-milliliter microtubes and a 600 microliter aliquot of the bacterial solution in one 1.5-milliliter microtube, and perform deuterium incorporation treatment in the presence of antibiotics as described before. For the sample preparation, wash one milliliter of fixed bacteria solution with purified water, and centrifuge the washed bacteria solution at 6, 200 times g for five minutes at four degrees Celsius.Remove the supernatant and enrich the bacterial solution to about 20 microliters with sterilized water. Deposit the bacterial solution on a poly-L-lysine coated cover glass, sandwich with another cover glass, and seal the sample. In the SRS microscope, a tunable femtosecond laser with an 80 megahertz repetition rate provides the pump and Stokes excitation lasers.The Stokes beam is modulated by an acousto-optical modulator at 2.4 megahertz. The two beams are colinearly combined through a dichroic mirror. Then the pump and Stokes beams are directed into a lab-built laser scanning microscope with a 2D Galvo mirror for laser scanning.A 60x water objective focuses the lasers to the sample and an oil condenser collects the signal from the sample. Two filters are used to filter out the Stokes beam, while the pump beam is detected by a photodiode, after which the stimulated Raman signal is extracted by a lock-in amplifier. Using the control software, input and tune the pump wavelength to 852 nanometer.Adjust the C to D vibrational frequency to 2, 168 wave number to image bacteria using an SRS microscope. Measure the laser power using a power meter. Set the power of pump laser at the sample to eight milliwatts, and the power of Stokes laser at the sample to 50 milliwatts by adjusting the half-wave plate in front of the laser output.Place the standard sample DMSO d6 on the sample stage, and use a 60x water immersion objective to focus the pump and Stokes lasers on the sample. By adjusting the screws of the reflection mirrors, spatially align the pump and Stokes beams and direct the two beams into an upright microscope equipped with 2D Galvo mirror system for laser scanning. In the software control panel, set each SRS image to contain 200 by 200 pixels and the pixel dwell time to 30 microseconds.The total acquisition time for one image is around 1.2 seconds. Set the step size to 150 nanometers, so the image size is about 30 by 30 micrometers squared. After optimizing the system, take the standard sample out, and put the bacterial sample on the sample stage under the 60x water immersion objective.Start the SRS imaging of bacterial samples. Image at least three fields of view for each sample. The effect of incubation time on deuterium incorporation is measured by spontaneous Raman microspectroscopy at the CD and CH regions.The CD over CH intensity ratio plot over deuterium incubation time for single bacteria showed increasing CD over CH intensity over incubation time from zero to 180 minutes. SRS imaging of Pseudomonas aeruginosa was conducted upon incubation with gentamicin and 70%deuterium. The further quantitative statistical analysis showed that CD signals of bacteria were significantly lower at two micrograms per milliliter, or higher gentamicin concentration than without gentamicin treatment.The cutoff intensity threshold at 0.60 concluded that Pseudomonas aeruginosa was metabolically inhibited at two micrograms per milliliter and higher concentrations of gentamicin. The SC-MIC for Pseudomonas aeruginosa against gentamicin in a normal MHB medium was determined to be two micrograms per milliliter, which was within the one-fold difference range with MIC of four micrograms per milliliter determined by the broth microdilution method. Rapid Antimicrobial Susceptibility Testing, or AST, of Escherichia coli spiked urine samples was carried out by SRS imaging.The SC-MIC for the Escherichia coli spiked urine sample against amoxicillin was determined to be four micrograms per milliliter, which has the same susceptibility readout as the MIC of eight micrograms per milliliter by the conventional broth dilution method for pure Escherichia coli in normal MHB medium. The applicability of rapid AST of Pseudomonas aeruginosa spiked in human blood was investigated by SRS imaging. The CD intensity of the SRS image at 2, 168 per centimeter was dominated by bacterial signals originating from the metabolic deuterium incorporation of live bacteria.The SC-MIC for Pseudomonas aeruginosa in blood was determined to be two micrograms per milliliter, which agreed well with the conventional standard MIC result for Pseudomonas aeruginosa in the normal growth medium. The bacterial cell number used for antimicrobial susceptibility testing is kept at about five times 10 to the fifth colony forming units per milliliter, as recommended by the Clinical and Laboratory Standards Institute. Higher bacteria concentration can lead to an increase in the minimal inhibitory concentration.Combining in situ pathogen identification and rapid antimicrobial susceptibility testing diagnosis could be of great potential for translation into a clinic that allows on-time identification of appropriate antimicrobial agents for precise treatment.

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

Abstract