Rapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of D2O Metabolic Incorporation in a Single Bacterium

Abstract Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic‐resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D2O) metabolism is reported. Metabolic incorporation of D2O into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% D2O medium, the fastest among current technologies. Single‐cell metabolism inactivation concentration (SC‐MIC) is obtained in less than 2.5 h from colony to results. The SC‐MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of D2O metabolic incorporation and SC‐MIC determination after 1 h antibiotic treatment and 30 min mixture of D2O and antibiotics incubation of bacteria in urine or whole blood is demonstrated.


Introduction
Antimicrobial resistance has become a growing public threat, causing nearly 1 million related mortality each year globally. [1] To combat this crisis, rapid antimicrobial susceptibility testing (AST) is essential to slow down the emergence of antimicrobial resistance and consequently reduce the deaths caused by drugresistant infections. [3]The gold standard for AST is conducted by disc diffusion or broth dilution methods and used to determine whether the bacteria are susceptible, intermediate or resistant to antimicrobial agents tested. [4]After 16 to 20-h growth, an minimal inhibitory concentration (MIC) value is read as complete growth inhibition through visual inspection.Current culturebased phenotypic method for AST is too slow to guide immediate decision for infectious disease treatment. [5]For clinical samples, it usually takes at least 24 h for bacterial preincubation and at least additional 16 h for AST. [6]Genotypic methods [7] do not rely on culturing and provide faster results, but they only target specific known genetic sequences with resistance and thus, are not generally applicable to different bacterial species or mechanisms of resistance, nor providing MIC results. [8] overcome these limitations, novel phenotypic methods for rapid AST are under development, [9] including microfluidic devices that increase the detection sensitivity by confining the sample in a small area, [10] monitoring bacterial growth or morphological changes at single cell level, [10e, 11] phenotypic AST quantifying the nucleic acids copy number, [8a, 12] and Raman spectroscopy that probes the chemical content inside a bacterium. [13]While these methods reduce the time for AST, most of them only work for bacterial isolates.
Inside a cell, NADPH is ubiquitously used for biomolecular synthesis. [14]Based on rapid enzyme-catalyzed exchange between the redox-active H atom in NADPH and the D atom in deuterium oxide (D2O), so called heavy water, cellular metabolic activity can be probed via monitoring the intracellular conversion of D2O into C-D bonds of the biomolecules.
Spontaneous Raman spectroscopy of metabolic incorporation of D2O has been used to determine antimicrobial susceptibility. [15]However, the small spontaneous Raman scattering cross section does not allow high-throughput AST.By spontaneous Raman measurement, it usually takes ca. 10 minutes (30 second per spectrum) to acquire Raman spectra of 20 individual bacteria.Thus, to determine MIC via 10 concentrations of one antibiotics, the total Raman measurment time per strain would be 100 minutes.Thus, it would need at least 17 h to determine MIC of 10 antibiotics for one strain.In contrast, by focusing the excitation energy on the C-D vibration band, coherent Raman microscopy based on either coherent anti-Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS) provides orders-of-magnitude signal enhancement, thereby enabling high-speed and high-throughput chemical imaging of single cells. [16]For broad Raman bands such as CH and CD stretch vibrations, femtosecond pulse SRS further boosts the signal level. [17]re, we report a rapid phenotypic platform that can determine the susceptibility of bacteria in urine and whole blood by femtosecond SRS imaging of D2O metabolism in a single bacterium.Harnessing the high sensitivity of femtosecond SRS imaging, D2O metabolic incorption inside a signle bacterium to antibiotics is probed in as fast as 10 minutes.Unlike spontaneous Raman spectroscopy, a C-D SRS image covering tens of bacteria is recorded in ~ 1 second.In the presence of antibiotics, a single-cell metabolism inactivation concentration (SC-MIC) is determined in less than 2.5 h from colony to results.Compairsion of SC-MIC results with conventional MIC results among 37 sets of samples, including 8 major bacterial species and 14 different antibiotics often encountered in clinic, yields a category agreement of 94.6% and 5.4% minor error.Moreover, our method is able to determine the metabolic activity and susceptibility of bacteria in either urine or whole blood, which opens the opportunity for rapid single-cell phenotypic AST in clinic.

SRS imaging of D2O metabolic incorporation in a single bacterium.
In cells, flavin enzymes catalyze the H-D exchange between water and NADPH's redox active hydrogen in D2O containing media.The deuterium labeled NADPH mediates fatty acid synthesis reaction with D2O incorporation, resulting in the deuterated fatty acids production (Figure 1a).Biosynthetic pathway of deuterated protein is through introducing deuterium atoms from D2O into reactions of amino acids (AAs). [14,18] he schematic of our SRS microscope is shown in Figure 1b.In brief, spatially and temporally overlapped pump and Stokes pulses are tuned to match the vibrational frequency of Raman-active modes.The SRS signal appears as an intensity gain in the Stokes beam and an intensity loss in the pump beam, which is extracted through a lock-in amplifier.Stimulated Raman loss is measured, in which most excitation power is in the 1040-nm Stokes beam having a high cellular damage threshold.
The carbon-deuterium (C-D) vibrational band, which is spectrally differentiated from endogenous Raman bands, is selectively detected with SRS using either chirped or non-chirped femtosecond laser pulses.Previously, [19] we used chirped femtosecond pulses for hyperspectral SRS imaging of C-D bonds in bacteria.To enhance the detection sensitivity and speed up the imaging process, we applied non-chirping femtosecond pulses and increased the signal to noise ratio by ~5 folds (Figure S4).With femtosecond SRS, C-D signals from all bacteria in the field of view could be obtained at a speed of ~1.2 s per image of 200 ×200 pixels, at a pixel dwell time of 30 µs.Therefore, femtosecond SRS imaging enables high-speed, high-throughput study of D2O incorporation at single bacterium level.
We then examined the toxicity of D2O to bacterial cells.Unlike mammalian cells, bacteria are much more resistant to D2O toxicity.Our experiments showed that 70% D2O concentration did not cause severe growth inhibition (Figure S1).Thus, we chose 70% D2O containing medium to culture bacteria in the following studies.By tuning the Raman shift to C-D vibration at ~2162 cm -1 , strong signals were observed at individual bacteria after culture in D2O containing medium for 1 h (Figure 1c and Figure S2).As a control, no C-D signal was observed for bacteria cultured in normal medium (Figure 1c and Figure S2).These results were further confirmed by SRS spectra (Figure 1d) obtained through temporal tuning of chirped pump and Stokes pulses, and spontaneous Raman spectra (Figure 1e), both showing a broad peak (from 2070 to 2250 cm -1 ) at C-D vibration only for bacteria cultured in D2O containing medium.
Therefore, SRS imaging at C-D vibrational region provides a good means to monitor D2O incorporation in a single bacterium.
To verify metabolic D2O incorporation in bacteria, we measured the cellular metabolic activity kinetics under different incubation conditions (Figure S3).As depicted in the SRS images, the live P. aeruginosa cells, cultured in D2O containing medium at 37 °C, had high metabolic activities and exibited increasingly stronger C-D intensities with increased incubation time.In contrast, neither live P. aeruginosa incubated at 4 °C, nor formalin-fixed P.
aeruginosa, incubated at 37 °C, showed observable C-D signals because of the metabolic activity inhibition.Our findings confirm that C-H bonds are unlikely to undergo abiotic H-D exchange.Instead, cellular metabolic activity directly relates to D2O incorporation, which is reflected by biochemical transformation of forming C-D bonds in newly synthesized biomolecules. [14,20] t, we investigated whether SRS imaging could resolve the fast D2O incorporation in biomolecule synthesis spatially and temporally.Time-lapse SRS images (Figure 1f) and statistical analysis (Figure 1g) showed that the average intensity of C-D signals in P. aeruginosa increases with time and saturates at ~2 h.With the enhanced detection sensitivity, C-D signals in individual P. aeruginosa can be observed after culture in as short as 10 min, which is shorter than the generation time of P. aeruginosa (24 to 27 min). [21]These results showed that the D2O incorporation of bacteria can be detected by SRS within one cell cycle.
With sub-micron spatial resolution, we further observed the differential distribution of C-D signals in 10-min, 30-min and longer culture time (Figure 1f).After 10-min culture, a stronger signal was observed at cell periphery than that at the center of bacteria (Figure 1f and Figure 1h).In contrast, with 30-min and longer culture times, the signal intensity was stronger at the cell center than that at the cell periphery (Figure 1f and Figure 1i).These results suggest that D2O is initially incorporated to synthesize lipids in plasma membrane and then used to synthesize proteins and nucleic acids inside the cell.Collectively, our studies demonstrated that D2O incorporation can be spatially and temporally monitored by SRS imaging at single bacterium level.

D2O incorporation in the presence of antibiotics
To examine how antibiotics affect the metabolic activity of D2O incorporation in bacteria, and to demonstrate that this effect can be used for rapid AST through SRS imaging, a cefotaximeresistant (MIC = 32 µg/ml) and gentamicin-susceptible (MIC = 4 µg/ml) P. aeruginosa strains were selected as a model system.P. aeruginosa were cultured for different time in D2O containing medium, with 20 µg/ml gentamicin or cefotaxime.SRS imaging (Figure 2a) and statistical analysis (Figure 2c We also, observed that P. aeruginosa tends to form filaments after culture with cefotaxime (Figure 2b).10e] Yet, it does not affect our metabolic activity measurements.
Next, we examined whether the rapid D2O incorporation inside bacteria can be used to differentiate the antimicrobial susceptibility.We used the relative C-D SRS intensity, the ratio between the antibiotic-treated group and the antibiotic-untreated group (Figure 1f), as a biomarker of antimicrobial susceptibility.To determine whether the SRS intensity ratio can be used to distinguish susceptible and resistant groups, the histogram of signal intensities for bacteria after 10-min culture was plotted over the intensity ratio (Figure 2e).The plots for susceptible and resistant groups were fitted with normal distribution.A cut-off at 0.62 was determined based on a 10-min culture of bacteria.The large area under curve (AUC = 0.985) in the corresponding receiver operating characteristic (ROC) curve plot clearly demonstrates the ability of this cut-off to separate the two groups (Figure 2f).These rusults indicate that our method is capable of determining susceptibility after 10-min D2O incubation time.The signal intensity ratio between the gentamicin-susceptible and cefotaxime-resistant groups showed more significant difference at longer culture time (Figure S5).A cut-off at 0.60 was obtained for the 30-min culture results (Figure 2g and Figure 2h).In the following studies, we use 30 min of D2O incubation time to ensure sufficient signal to noise ratio and apply 0.60 cut-off to separate the metabolism active and metabolism inhibited conditions for bacteria cultured at different concentrations of antibiotics.In particular, we use such cut-off to define a single cell metabolism inhibition concentration (SC-MIC) for a certain antibiotic: at or above SC-MIC, the bacteria is suscceptible and thus metabolically inactive; below SC-MIC, the bacteria is resistant and thus metabolically active.

Quantitation of susceptibility via SC-MIC
To explore whether SRS imaging of D2O metabolic incorporation can quantify the response of bacteria to antibiotics and generate a SC-MIC value comparable with the MIC, we tested P. aeruginosa with serially diluted gentamicin.Overnight cultured bacteria were diluted in cationadjusted Mueller-Hinton Broth (MHB) medium to a final concentration of 8×10 5 CFU/ml.The bacteria were first treated with selected antibiotic containing medium for 1 h, then a medium containing D2O and the same antibiotics was added to bacteria for an additional 30 min (Figure 3a).SRS imaging (Figure 3b) and statistical analysis (Figure 3c) showed that C-D signals at 2 µg/ml or higher gentamicin concentration were significantly lower than that in the control group (0 µg/ml).With the previous determined threshold, D2O incorporation in P. aeruginosa was inhibited at 2 µg/ml and above concentrations.Therefore, the SC-MIC was determined to be 2 µg/ml.This value is within the one-fold difference range with the MIC (4 µg/ml) determined by the broth microdilution method.

SC-MIC measurement in 37 sets of samples
To validate the broad applicability of our method, we tested 8 major bacterial species and 14 different antibiotics often encountered in clinic (Table 1).The antibiotics cover major bacterial inhibition mechanisms of action: inhibition of cell wall synthesis, protein synthesis, DNA synthesis, and/or cell membrane disruption.Typical SRS imaging (Figure S6) and statistical analysis (Figure 3d-3f) showed that antibiotics with all the mechanisms of action affect D2O incorporation in bacteria: the β-lactam amoxicillin, the aminoglycoside gentamicin, the fluoroquinolone ciprofloxacin, and the cell membrane targeting daptomycin.We performed 37 sets of the experiments (Table 1), where SC-MIC was obtained after 1.0-h incubation with antibiotics and additional 0.5-h incubation with D2O and antibiotics.For each set, the SC-MIC determination by quantifying the SRS signal intensities versus the concentration of antibiotics is presented as a heatmap.SC-MIC, MIC and the defined susceptibility category interpreted as "susceptible," "resistant," or "intermediate," according to Clinical and Laboratory Standards Institute criteria are presented for each tested bacterial strain.As compared with MIC determined by conventional broth microdilution assay, the SC-MIC (highlighted in black boxes in Table 1) achieved a category agreement of 94.6% (35 out of 37), with 5.4% minor error (2 out of 37), no major error, and no very major error.These results satisfy US Food and Drug Administration (FDA) requirements for AST systems.Most of the SC-MIC results were obtained after 1-h culture in antibiotic containing medium followed by 0.5-h culture in D2O and antibiotics-containing medium.We observed that methicillin-resistant S. aureus (MRSA) grew slower than susceptible S. aureus.Therefore, MRSA strains were cultured in D2O medium for 1 h to achieve comparable C-D signals.With automated imaging and data analysis (Figure S7), the whole procedure from colony to results took less than 2.5 h for most of the bacterial strains tested, and 3 h for MRSA strains.Collectively, these results validate SRS imaging of D2O metabolic incorporation as a rapid and accurate AST method.
We further analyzed the SC-MIC results in the 37 sets of samples based on bacterial species.The 2 minor errors were both from Gram-negative bacteria, resulting in a category agreement of 100% (11 of 11) for Gram-positive samples (9 S. aureus samples and 2 E. faecalis samples), and a category agreement of 92.3% (24 of 26) with 7.7% minor error (2 of 26) for Gram-negative samples.Though the category agreement in Gram-negative bacterial strains was lower than that in Gram-positive strains, these results still meet the FDA requirements (category agreement ≥ 90%, minor error ≤ 10.0%, major error ≤ 3.0%, very major error ≤ 1.5%).
As shown in Table 1, 32 SC-MICs are identical or have one-fold difference with MIC results, resulting in an essential agreement of 86.5% (32 of 37).Four SC-MIC results have three-fold difference, and one result has more than three-fold difference.To better understand the good aggrement and the residual discrepancy between SC-MICs and MICs in these specimens, we obtained MICs of the 37 sets of samples by conventional broth microdilution assay in a blinded manner, using 70% D2O MHB as the culture medium.The results are listed in Table S1.Most of the MICs determined in 70% D2O MHB are identical or show only onefold difference with the MICs in normal MHB.Interestingly, for the five results that had the most differences between MIC and SC-MIC, the MICs determined in 70% D2O MHB agreed more with SC-MICs than MICs determined in normal MHB.Specifically, when P. aeruginosa was treated with colistin, a polypeptide that targets bacterial cell membrane, the SC-MIC values were much lower than the MICs in normal MHB, but had much smaller difference from the MICs in 70% D2O MHB.This comparison indicates that 70% D2O might increase the venerability of some bacteria to certain antibiotics.The discrepancy between the SC-MICs in 70% D2O MHB and the MICs in normal MHB can be resolved by using a smaller cut-off value.

SC-MIC for bacteria in urine environment
To investigate the potential of rapid AST by SRS imaging of D2O metabolic incorporation for clinical applications, we first tested bacteria in urine.For bacteria in urine, we tested E. coli, which is the most common pathogen in urinary tract infection (UTI). [22]To mimic the clinical UTI samples, we used spiked samples by adding E. coli to the urine at a final concentration of 10 6 CFU/ml.After filtration with 5-µm filter and centrifugation, the purified bacteria were used for SC-MIC measurements (Figure 4a).This sample preparation procedure took about 15 min.The clean background in SRS images showed that this convenient sample preparation procedure was favorable for rapid AST (Figure 4b).SC-MIC for E. coli in urine with amoxicillin was determined to be 4 µg/ml (Figure 4b-4c), which has the same essential and category agreement with the SC-MIC or MIC for pure E. coli (Figure 4d).These results showed that rapid AST by SRS imaging of D2O metabolic incorporation is suitable for clinical application to bacteria in the urine.

SC-MIC for bacteria in blood environment
As compared with urine, blood includes a lot of blood cells and presents a much bigger challenge for in situ analysis of bacterial activity.To investigate the potential of rapid AST by SRS imaging of D2O metabolic incorporation for clinical bloodstream infections (BSI) samples, we tested P. aeruginosa spiked in human blood.Bacteria were first added to blood at a final concentration of ~10 6 CFU/ml (Figure 5a).Then, water was added to the mixture to lyse the blood cells.After filtration and centrifugation, the purified bacterial samples were used for SC-MIC measurements.The whole procedure for sample preparation took about 15 min.After culture in D2O medium, SRS images at the C-H vibration showed a lot of debris or blood cells still left in the purified bacterial samples (Figure 5b).While, at the same area, the SRS image of C-D vibration was dominatated by bacterial signal.The reason is that deris or red blood cells do not have metabolic activity to incorporate D2O unlike live bacteria.The weak background mostly comes from the cross-phase modulation or photothermal signal of interferent species, which does not affect the quantification of SC-MICs.The off-resonance SRS images further confirmed that the signals in bacteria largely came from the C-D vibration (Figure 5b).The SC-MIC value for P. aeruginosa in blood after 1-h culture was determined to be 2 µg/ml (Figure 5c-d), which agreed with the SC-MIC or MIC for P. aeruginosa in growth medium (Figure 5e).These results showed that SRS imaging of D2O metabolic incorporation can rapidly determine SC-MIC for bacteria in blood environment.We note that bacterial concentration in the spiked urine and blood samples was 10 6 CFU/ml in our tests (Figure 4a and Figure 5a).8b] The bacterial concentrations in positive blood cultures range from 10 6 to 10 9 CFU/ml. [23]Therefore, our SC-MIC measurement can be directly used for UTI or positive blood culture samples.

Discussion
The current work demonstrates a rapid, high-throughput platform that can determine the susceptibility of bacteria in MHB medium, urine and blood by SRS imaging of D2O incorporation at a single bacterium level.Metabolic incorporation of D2O, which is used for biomolecule synthesis, was monitored in a single bacterium by SRS imaging of C-D bonds.
Metabolic response was probed in as short as 10 min after culture in D2O medium.SC-MIC was obtained in less than 2.5 h from colony to results.The SC-MIC results of 37 sets of samples, which included 8 major bacterial species and 14 different antibiotics, were systematically studied and validated by MIC determined by the broth microdilution method, with a category agreement of 94.6% and 5.4% minor error.Furthermore, we investigated the feasibility of our method to study samples in complex biological environments.The SC-MIC can be determined after 1-h culture of bacteria in urine and blood, which is considered a tremendous reduction in analysis time as compared with the conventional broth microdilution method.
Previously, we monitored the metabolic incorporation of glucose-d7 in isolated bacteria or fungi using SRS microscopy. [19,24] hough glucose is the preferred carbon source for most bacterial growth, [25] glucose-d7 itself contains C-D bonds which cause a large background in the SRS image.20a, 26] In the present study, we monitored the D2O metabolic incorporation by tracking the speed and amount of C-D bond formation.Significantly, the C-D vibration is spectrally separated from the O-D vibration in D2O, allowing for background-free SRS measurement of bacterial metabolic activity in a complex environment such as whole blood.
Another innovation of this study is the use of femtosecond pulses, which significantly increased the signal to noise ratio and the imaging speed accordingly.
It is known that stationary-phase and non-dividing bacteria are common in many persistent infections (e.g., endocarditis and osteomyelitis) and in biofilm-associated infectious diseases (e.g., periodontitis and cystic fibrosis). [27]To evaluate the potential of our SRS metabolic imaging method for non-dividing bacteria, we investigated the metabolic dynamics of D2O incorporation in E. coli starting from different phases, lag, log, and stationary phase (Figure S8).Interestingly, we observed similar metabolic dynamics during the same period of time, which is consistent with the growth curves with optical density measurements.Hence, our SRS metabolic imaging measurement can be potentially applied to determine the susceptibility of non-dividing bacteria, which is beyond the reach of conventional culture method.
A few groups reported coherent Raman imaging of D2O activity inside mammalians.
Potma et al. used CARS microscopy to minor D2O entry into a cell in real time. [28]20a] Compared to mammalian cells, imaging D2O metabolic activity in a micron-sized bacterium is challenging.Here, we deployed a few strategies to achive good signal to noise ratio in a single scan.First, stimulated Raman loss is measured, where most excitation power in on the Stokes beam to minimize photodamage to the specimen.Second, femtosecond pulses are used for excitation of the broad C-D vibrational bands, which improved the signal to noise ratio by 5 times compared to picosecond pulses.Third, the cross phase modulation background is minimized by placing the bacteria on a poly-L-lysine-coated glass substrate and covered with phosphate-buffered saline solution.
Because NADPH is ubiquitously used in cell metabolism, our SRS metabolic imaging method has the potential of being broadly used for rapid AST in various strains and can be extended to determine the susceptibility in fungal infections.Another exciting application of this method is for slowly growing bacteria, like Mycobacterium tuberculosis which doubles roughly once per day and has a remarkably slow growth rate. [29]r clinical specimens, each sample requires hours of pre-incubation to obtain bacterial isolates.Methods based on nanoliter array, [8b] digital nucleic acid quantification, [8a] and Raman spectroscopy [15c] have been developed to perform AST for clinical urine samples.Compared with urinary tract infections, bloodstream infections or sepsis are more life-threatening cases, [8a, 30] where rapid AST is urgently needed.Methods based on microscopic imaging, [31] commercial automatic systems, [32] and mass spectrometry [33] allow for direct AST from positive blood cultures.Yet, these methods rely on bacterial growth and take at least 6 h of incubation.In this work, we demonstrate in situ SRS imaging of D2O metabolic incorporation in single bacteria at a clinically relevant concentration (10 5 ~10 6 CFU/ml) in either urine or whole blood.This capacity paves the way toward clinical translation of our technology.While we need to know the antimicrobial susceptibility of bacteria, MIC determination is even more significant in clinics to avoid excess dosage of antibiotics to patients to cause potential side effects. [34]We would emphasize that our SC-MIC method is capable of detecting MICs and susceptibility classification for each strain/antibiotic.Compared with the spontaneous Raman microscopy, our method requires tremendously reduced data acquisitiom time (ca.600 times less) to obtain MIC results due to orders-of-magnitude signal enhancement.Based on our method, the MICs are determined after 1-h antibiotics treatment and 30-min mixture of D2O and antibiotics incubation into bacteria in urine and blood.Each SRS image, containing at least 10 bacteria, was acquired within ca. 1 second in one single shot, while it takes about 10 min by spontaneous Raman measurement.We estimate the total MIC assay time to study 10 antibiotics per strain/antibiotic set is less than 2.5 h from sample to MIC results, which is much more efficient and competitive in determining MICs.
33b, 35], Integration of these in situ analysis tools and translation into clinic could potentially eliminate the "culture to colony" paradigm, thus allowing for on-time identification of correct antimicrobial agents for precise treatment.

SRS microscope.
A femtosecond (fs) pulsed laser (InSight DeepSee, Spectra-Physics) with an 80-MHz repetition rate and dual outputs was employed for excitation.One 120 fs laser with tunable 680-1100 nm wavelength served as the pump beam.The other 220 fs laser, centered at 1040 nm wavelength and served as the Stokes beam, was modulated by an acousto-optical modulator (AOM, 1205-C, Isomet) at ~2.4 MHz.The two beams were collinearly combined through a dichroic mirror.When spectral focusing is needed for hyperspectral SRS, both beams were chirped with two 15 cm long SF57 glass rods.After chirping, the pulse durations of the pump and Stokes laser were 1.9 ps and 1.3 ps, respectively.For implementation of SRS imaging with femtosecond pulses, the glass rods were removed from the path.The pump and Stokes beams were directed into a lab-built laser scanning microscope with a 2D galvo mirror for laser scanning.A 60× water objective (NA=1.2,UPlanApo/IR, Olympus) was used to focus the lasers to the sample, and an oil condenser (NA=1.4,U-AAC, Olympus) was used to collect the signal from the sample.Two filters (HQ825/150m, Chroma) were used to filter out the Stokes beam, the pump beam was detected by a photodiode (S3994-01, Hamamatsu) and the stimulated Raman signal was extracted by a lock-in amplifier (HF2LI, Zurich Instrument).
To image bacteria at the C-D vibrational frequency, the pump wavelength was tuned to 852 nm, and the power at the sample was ~8 mW; the Stokes power at the sample was ~40 mW.
Each image contains 200 × 200 pixels and the pixel dwell time is 30 µs, resulting in an image acquisition time of ~1.2 seconds.
Bacterial strains.Bacterial strains used in this study (Table S2) were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) and the American Type Culture Collection (ATCC).

Sample preparation.
To make D2O containing Lauria-Bertani broth (LB) (Sigma Aldrich) or cation-adjusted cation-adjusted Mueller-Hinton Broth (MHB) (Thermo Fisher Scientific) media, D2O was first mixed with purified water, then LB or MHB powder was added to the solution.The solution was sterilized by filtering.To prepare bacterial samples for SRS imaging, bacterial strains were cultured in normal medium to reach the logarithmic phase, then bacteria were diluted to ~10 6 CFU/ml in the D2O-containing medium.After a controlled culture time, 500 µl sample was centrifuged, washed twice with purified water, fixed by 10% (w/v) formalin solution (Thermo Fisher Scientific) and deposited to an agarose gel pad or poly-L-lysine coated coverglasses.To prepare samples for spontaneous Raman spectroscopy, bacteria were cultured in normal or 70% D2O-containing medium for 2 h, and then centrifuged and washed twice with purified water to remove the culture medium.For the time lapse experiment, P. aeruginosa ATCC 47085 were cultured in 70% D2O containing medium for up to 3 h.At different time points, 500 µl bacteria were centrifuged, washed twice with purified water, and deposited on a poly-L-lysine coated slide for imaging.D2O toxicity measurement.D2O toxicity measurement was done in 96-well plates.P. aeruginosa ATCC 47085, E. coli BW25113, or S. aureus ATCC6538were cultivated into D2O-containing LB medium with D2O concentrations ranging from 0 to 100%, and incubated at 37 °C.At each time point, optical densities (OD) of the samples were measured at 600 nm wavelength.
Data analysis.To get the average C-D signal intensity, the SRS images were opened in ImageJ software (ImageJ, NIH), single bacterial cells were selected, and the average intensity of the selected area was calculated.The same procedure was applied for other bacteria in the same image.For bacteria that have filamentary formation, a section of one filament was selected for calculation of SRS intensity.Spontaneous Raman spectroscopy.Bacteria in solution were deposited on a coverglass for spontaneous Raman measurement.Spontaneous Raman spectra of bacteria were acquired with an inverted Raman spectrometer (LabRAM HR evolution, Horiba scientific) with 532 nm laser source.The laser power at the sample was ~12 mW after a 40× air objective, and the acqusition time was 10 s.The grating was 600 l/mm.Broth microdilution method.Bacteria were cultured in D2O-containing MHB medium in 96well plates.Antibiotics, using triplicate samples, were added to the plate and two-fold serially diluted.Plates were then, incubated aerobically at 37° C for ~20 h.MIC was categorized as the concentration at which no visible growth of bacteria was observed.
Additional experimental results and data analysis are available in the supplementary information.

Figure 1 .
Figure 1.SRS imaging of D2O metabolic incorporation in a single bacterium.(a) Scheme for D2O labeling of lipid and protein.(b) Schematic illustration of SRS setup.AOM: acoustooptic modulation.PD: photodiode.Lock-in: lock-in amplifier.DAQ: data acquisition board.(c) SRS and corresponding transmission images of P. aeruginosa after culture in normal and D2Ocontaining medium for 3 h.Scale bar: 20 µm.(d, e) SRS spectra (d) and spontaneous Raman spectra (e) of P. aeruginosa after culture in normal and D2O containing medium for 3 h.(f) Time lapse of P. aeruginosa after culture in D2O containing medium.(g) Average C-D intensity ) showed that C-D signals of bacteria were significantly reduced after culture with gentamicin, indicating inhibition of D2O incorporation in P. aeruginosa by gentamicin.On the contrary, Figure 2b and d shows that the C-D signals of P. aeruginosa cultured with cefotaxime increased with time, indicating active D2O incorporation in bacteria.

Figure 2 .
Figure 2. SRS-based AST of P. aeruginosa as a function of culture time.(a, c) Time lapse SRS at C-D and transmission images of P. aeruginosa after culture in D2O-containing medium with the addition of 20 µg/ml gentamicin (a) or cefotaxime (b).(c, d) Average C-D intensity plot over time for P. aeruginosa after culture in D2O-containing medium with gentamicin (c) or cefotaxime (d) treatment.Number of cells per group ≥ 10.Error bars represent SD.Scale bar: 20 µm.(e, g) Histogram plot of the count of bacteria as a function of C-D intensity ratio of antibiotic-treated group over the control group after 10 min (e) and 30 min (g) treatment.(f, h) ROC curves of 10 min (f) and 30 min (h) treatment illustrating the ability of the C-D intensity ratio to distinguish susceptible and resistant groups.AUC: area under the curve.

Figure 3 .
Figure 3. SC-MIC determination via SRS imaging of D2O metabolic incorporation in single bacteria.(a) Workflow of rapid AST with SC-MIC determination by SRS imaging of D2O metabolic incorporation.(b) SRS at C-D and corresponding transmission images of P. aeruginosa after culture in D2O containing medium with the addition of serially diluted gentamicin.(c) Statistical analysis of C-D intensity in P. aeruginosa in (b).(d-f) SC-MIC determination for antibiotics with different mechanisms of action.Error bars represent the standard error of the mean (SEM).Scale bar: 20 µm.

Figure 4 .
Figure 4. SC-MIC determination after 1-h culture of E. coli in urine.(a) Bacterial purification protocol for bacteria in urine for rapid AST by SRS imaging of D2O metabolic incorporation.(b) SRS and corresponding transmission images of E. coli in urine after 1-h culture in D2Ocontaining medium with the addition of serially diluted amoxicillin.(c) Statistical analysis of C-D intensity in bacteria in (b).Number of cells per group ≥ 10.(d) Comparison of SC-MIC and susceptibility category for E. coli isolate and E. coli in urine.Error bars represent SEM.Scale bar: 10 µm.

Figure 5 .
Figure 5. SC-MIC determination after 1-h culture of P. aeruginosa in blood.(a) Bacterial purification protocol for bacteria in blood for rapid AST by SRS imaging of D2O metabolic incorporation.(b) SRS images at C-D, off-resonance (2407 cm -1 ), and C-H of bacteria in blood after 1-h culture in D2O containing medium.(c) SRS and corresponding transmission images of P. aeruginosa in blood after 1-h culture in D2O-containing medium with the addition of serially diluted gentamicin.(d) Statistical analysis of C-D intensity in bacteria in (c).(e) Comparison of SC-MIC and susceptibility category for P. aeruginosa isolate and P. aeruginosa in blood.Error bars represent SEM.Scale bar: 10 µm.

Figure S1 .
Figure S1.Testing D2O toxicity to bacterial growth.Growth curve of E. coli.(a), S. aureus (b) and P. aeruginosa (c) cultured in LB medium with different D2O concentrations.Error bars indicate standard deviation values (number of measurements = 5).

Figure S2 .
Figure S2.SRS and corresponding transmission images of E. coli and S. aureus after being cultured in LB and D2O containing LB medium for 3 h.Scale bar: 20 µm.

Figure S4 .
Figure S4.Femtosecond SRS improves signal to noise ratio (SNR) at C-D vibrational region over the chirped SRS.(a) SRS image at ~2162 cm -1 of P. aeruginosa 47085 cultured in 70% D2O containing LB medium for 30 min with picosecond pulses generated by chirping with two SF57 glass rods.(b) Intensity plot of the orange line over bacteria in (a).(c) SRS image at ~2162 cm -1 of P. aeruginosa cultured in 70% D2O containing LB medium for 30 min with femtosecond pulses.(d)Intensity plot of the orange line over bacteria in(c).Scale bar: 20 µm.

Figure S5 .
Figure S5.Statistical analysis of C-D intensity in P aeruginosa without treatment and with gentamicin or cefotaxime treatment for different time.Black bars indicate the threshold that discriminates susceptible (gentamicin) and resistant (cefotaxime) groups.

Figure S6 .
Figure S6.SC-MIC determination for S. aureus and P. aeruginosa treated with different mechanism of action.(a-c) SRS and corresponding images of bacteria cultivated in antibiotics for 30 min and D2O containing medium for another 30 min.Scale bar: (a, c) 10 µm; (b) 20 µm.

Figure S8 .
Figure S8.SRS imaging of D2O metabolic activity in E. coli BW 25113 of (a) lag phase, (b) log phase and (c) stationary phase cultured in 70% D2O MHB medium with different incubation time.Scale bar: 20 µm.

Table S1 .
List of MICs obtained in normal MHB medium and in 70% D2O-containing MHB medium.