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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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 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 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.

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 gentamicin sulfate in sterile PBS solution and amoxicillin in sterile DMSO solvent. Thereafter, store the antibiotics solution at 2-8 °C as suggested.
  2. To make D2O containing cation-adjusted Mueller-Hinton Broth (MHB) media, add 220 mg of MHB broth base to 10 mL of D2O to make 100% D2O containing medium. Sterilize the solution by filtering with filters of 200 nm pore size.
    NOTE: Use this protocol always for making and sterilizing medium solutions in further steps.
  3. To prepare bacterial samples for SRS imaging, add 2 mL of normal MHB media, which does not contain deuterium, to a sterile round-bottom culture tube, and then prewarm it at 37 °C.
  4. Use a sterile loop to select one bacterial (Escherichia coli BW 25113 or Pseudomonas aeruginosa ATCC 47085) colony from the fresh culture on a tryptic soy agar plate. Then suspend it in the prewarmed culture media and gently vortex to prepare the bacteria suspension.
  5. Incubate bacteria at 37 °C in a shaker at 200 revolutions per minute (rpm) until it reaches the logarithmic phase.

2. D2O incorporation treatment in the presence of antibiotics (Figure 2a)

  1. Check the bacterial concentration by measuring the optical density (OD) with a photometer at a wavelength of 600 nm.
  2. Dilute the bacterial solution using the normal MHB medium, which does not contain deuterium, to reach a final cell concentration of 8 x 105 CFU/mL. Vortex gently to mix the bacterial cells.
  3. Prepare 300 µL aliquots of the bacterial solution in seven 1.5 mL micro tubes, and 600 µL aliquots of the bacterial solution in one 1.5 mL micro tubes.
  4. Add 4.8 µL of antibiotic (gentamicin or amoxicillin) stock solution (1 mg/mL) into the micro tube containing 600 µL of the bacterial solution, to make the final antibiotic concentration to 8 µg/mL.
  5. Take 300 µL of solution out of the 8 µg/mL of antibiotic-containing bacteria solution, and add to another 300 µL of bacterial solution, to make two-fold diluted antibiotic- (4 µg/mL) containing bacteria solution.
  6. Repeat the two-fold serial dilution of the test antibiotics, gentamicin, or amoxicillin, until the micro tube with the lowest concentration (0.25 µg/mL) is reached, and discard 300 µL from the tube. For both gentamicin and amoxicillin, the serial concentrations range from 0.25 µg/mL - 8 µg/mL.
    1. Leave one tube with no antibiotics for blank control. This will be the positive control to inspect the bacterial metabolic activity without antibiotics treatment but with D2O treatment.
    2. Leave one tube with no antibiotics and no D2O for the negative control.
  7. Incubate the bacterial aliquot with the certain antibiotic (gentamicin or amoxicillin) containing MHB medium for 1 h.
  8. During incubation, prepare a serial dilution of antibiotics with 100% D2O containing medium with the same concentration gradient of antibiotics prepared in step 2.6. For both gentamicin and amoxicillin, the serial concentrations range from 0.25 µg/mL - 8 µg/mL.
  9. After 1 h antibiotic treatment, add 700 µL of serially diluted antibiotic and 100% D2O-containing MHB medium to the 300 µL of antibiotic-pretreated bacteria in the same antibiotic concentration (prepared in step 2.6), respectively.
    1. For example, add 700 µL of 100% D2O-containing MHB medium (containing 8 µg/mL of antibiotic) to the 300 µL of 8 µg/mL antibiotic-pretreated bacteria. In the same manner, transfer to the corresponding tubes of the next concentration, and homogenize by pipetting up and down several times.
    2. Add 700 µL of antibiotic-free 100% D2O-containing MHB medium to 300 µL of antibiotic-free bacteria (prepared in step 2.6.1) as a blank control.
    3. Incubate at 37 °C in an incubation shaker at 200 rpm for an additional 30 min.
      NOTE: In this step, the final concentration of D2O in the medium for the test is 70%.
  10. First centrifuge the 1 mL of antibiotic and D2O-treated bacterial sample at 6200 x g for 5 min at 4 °C, and then wash twice with purified water. Finally, fix samples in 10% formalin solution and store them at 4 °C.

3. Preparation of bacteria in urine environment (Figure 2b)

  1. To prepare E. coli BW 25113 at the logarithmic phase, follow the steps at 1.4 and 1.5.
  2. Check the bacterial concentration by measuring the OD with a photometer at a wavelength of 600 nm.
  3. To mimic the clinical UTI samples14,18,41, spike the E. coli solution into 10 mL of deidentified urine to reach a final cell concentration of 106 CFU/mL.
  4. Filter the E. coli spiked urine using a 5 µm filter, and then divide the bacterial solution in 300 μL aliquots into seven 1.5 mL micro tubes, and 600 µL aliquots of the bacterial solution in one 1.5 mL micro tubes.
  5. Perform D2O incorporation treatment in the presence of antibiotics and sample collection as described in the previous steps from 2.4 to 2.10.

4. Preparation of bacteria in blood environment (Figure 2c)

  1. To prepare Pseudomonas aeruginosa ATCC 47085 at the logarithmic phase, follow the steps at 1.4 and 1.5.
  2. To mimic the clinical bloodstream infections samples42,43, spike P. aeruginosa in 1 mL of deidentified human blood to reach a concentration of 107 CFU/mL.
  3. Add 9 mL of sterile purified water to lyse the blood.
  4. Filter the P. aeruginosa spiked blood using a 5 µm filter. Then harvest bacteria to 1 mL volume by centrifugation at 6200 x g for 5 min at 4 °C. Add 9 mL of pre-warmed normal MHB into the bacteria solution and gently vortex. The final concentration of bacteria is 106 CFU/mL
  5. Divide the P. aeruginosa spiked blood solution in 300 μL aliquots into seven 1.5 mL micro tubes, and 600 µL aliquots of the bacterial solution in one 1.5 mL micro tubes.
  6. Perform D2O incorporation treatment in the presence of antibiotics and sample collection as described in the previous steps from 2.4 to 2.10.

5. SRS imaging of D2O metabolic incorporation in a single bacterium

  1. Wash 1 mL of fixed bacteria solution with purified water and then centrifuge at 6200 x g for 5 min at 4 °C. Remove the supernatant. Enrich the bacterial solution to about 20 µL.
  2. Deposit the bacterial solution on a poly-L-lysine coated coverglass. Sandwich and seal the sample for SRS imaging.
  3. Image bacteria at the C-D vibrational frequency at 2168 cm-1 using an SRS microscope.
    1. Input and tune the pump wavelength to 852 nm using the control software on a computer.
    2. Measure the laser power using a power meter. Set the power of pump laser at the sample to ~8 mW and the power of Stokes laser at the sample to ~40 mW by adjusting the half-wave plate in front of the laser output.
      NOTE: In the SRS microscope, a tunable femtosecond laser with an 80-MHz repetition rate provides the pump (680 to 1300 nm) and Stokes (1045 nm) excitation lasers.
  4. 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.
    1. Use a 60x water immersion objective to focus the pump and Stokes lasers on the sample.
    2. Use an oil condenser to collect the signals from the sample in the forward direction.
    3. Use a bandpass filter to filter out the Stokes laser before directing it into a photodiode.
    4. Extract the stimulated Raman signal by a lock-in amplifier and detect the signals by the photodiode.
  5. Set each SRS image to contain 200 x 200 pixels and the pixel dwell time for 30 µs in the software's control panel. The total acquisition time for one image is ~1.2 s. Set the Step size to 150 nm, so the image size is about 30 x 30 µm2. Image at least three field of views for each sample.

6. Image processing and data analysis ( Figure 3)

  1. To obtain the average C-D signal intensity, open and process SRS images with ImageJ software.
  2. First, convert SRS images into 8-bit type images with inverted color by clicking Image | Type | 8-bit, and then Edit | Invert buttons in the ImageJ software.
  3. Then, filter the images with Gaussian blur by clicking Process | Filters | Gaussian blur buttons and set the Sigma (Radius) to 1.
  4. Use image threshold adjustment to select the bacterial area. Click Image | Adjust | Threshold to ensure the selected bacterial sizes match those in the original SRS images. Eliminate small particles by adjusting the size threshold to determine the particles. Click Apply.
  5. Apply Analyze | Particles Analysis buttons to label and determine the area of bacteria.
  6. By clicking the Show All button in the ROI manager to the original unprocessed SRS image, label the same area of bacteria, determine the average intensity of each data point by clicking the Measure button in the ROI manager.
  7. Circle the background area in the original SRS image and measure the average intensity of the background. The average C-D intensities of each bacterium is obtained by deducting the background signal intensity.

7. Quantitation of antimicrobial susceptibility via SC-MIC

NOTE: The cut-off value at 0.60 to determine the SC-MIC is established according to the statistical analysis of the SRS C-D intensities of the metabolism-active and metabolism-inhibited conditions for bacteria upon various concentrations of drug exposure40. The C-D intensities for the antibiotic-susceptible and antibiotic-resistant groups were fitted with normal distribution.

  1. Plot the receiver operating characteristic (ROC) curve and evaluate the cut-off threshold at 0.60. Based on this cut-off value, the SC-MIC as an indicator of the efficacy of antibiotics can be defined to determine the metabolically inactive and metabolically active group.
  2. To quantitatively analyze the SRS imaging data, plot the histograms of C-D signal intensities for each bacteria group treated with the serially diluted antibiotic concentration. The colored data points stand for different individual bacterium.
  3. Normalize the C-D intensities of antibiotic-treated group to the mean intensity of the control group without antibiotic treatment. Determine the SC-MIC results of different bacteria and antibiotic combinations by quantifying the SRS signal intensities at C-D region versus various concentration of antibiotics using the cut-off value at 0.60.
  4. Validate and compare the SC-MIC readout with the MIC determined using conventional broth microdilution assay.
  5. According to the Clinical and Laboratory Standards Institute (CLSI), the susceptibility category based on the SRS metabolic imaging results for each tested bacterial strain is interpreted as "susceptible", "resistant", or "intermediate".

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 microbia...

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...

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

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

Materials

NameCompanyCatalog NumberComments
Acousto-optic modulationGooch&HousegoR15180-1.06-LTDModulating stokes laser beam
AmoxicillinSigma AldrichA8523-5G
Bandpass filterChromaHQ825/150mBlock the stokes laser beam before the photodiode
Calcium chlorideSigma AldrichC1016-100GCation adjustment
Cation-adjusted Mueller-Hinton BrothFisher ScientificB12322Antimicrobial susceptibility testing of microorganisms by broth dilution methods
CentrifugeThermo Scientific75002542
Cover GlassesVWR16004-318
Culture tube with snap capFisher brand149569B
DaptomycinAcrosA0386346
Deuterium oxide151882Organic solvent to dissolve antibiotics
Deuterium oxide-d6Sigma Aldrich156914Organic solvent as a standard to calibrate SRS imaging system
Escherichia coli BW 25113The Coli Genetic Stock Center7636
Eppendorf polypropylene microcentrifuge tubes 1.5 mLFisher brand05-408-129
Gentamicin sulfateSigma AldrichG4918
Hydrophilic Polyvinylidene Fluoride filtersMillipore-SigmaSLSV025NBpore size 5 µm
ImageJ softwareNIHVersion: 2.0.0-rc-69/1.52tImage processing and analysis
Incubating orbital shaker set at 37 °CVWR97009-890
Inoculation loopSigmaBR452201-1000EA
InSight DeepSee femtosecond pulsed laserSpectra-PhysicsModel: insight X3Tunable laser source and fixed laser source at 1045 nm for SRS imaging
Lock-in amplifierZurich InstrumentHF2LIDemodulate the SRS signals
Oil condenserOlympusU-AACNA 1.4
Pseudomonas aeruginosa ATCC 47085 (PAO1)American Type Culture CollectionATCC 47085
PhotodiodeHamamatsuS3994-01Detector
Polypropylene conical tube 15 mLFalcon14-959-53A
Polypropylene filtersThermo Scientific726-2520pore size 0.2 µm
Sterile petri dishesCorning07-202-031
Syringe 10 mLFisher brand14955459
UV/Vis SpectrophotometerBeckman CoulterModel: DU 530Measuring optical density at wavelength of 600 nm
Vortex mixerVWR97043-562
Water objectiveOlympusUPLANAPO/IR60×, NA 1.2

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