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

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.