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 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&#160;sterile PBS solution and amoxicillin in sterile DMSO solvent. Thereafter, store the antibiotics solution at 2-8 &#176;C as suggested.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Incubate bacteria at 37 &#176;C in a shaker at 200 revolutions per minute (rpm) until it reaches the logarithmic phase.</li>
</ol>
2. D2O incorporation treatment in the presence of antibiotics (Figure 2a)
<ol>
	<li>Check the bacterial concentration by measuring the optical density (OD) with a photometer at a wavelength of 600 nm.</li>
	<li>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.</li>
	<li>Prepare 300 &#181;L aliquots of the bacterial solution in seven 1.5 mL micro tubes, and 600 &#181;L aliquots of the bacterial solution in one 1.5 mL micro tubes.</li>
	<li>Add 4.8 &#181;L of antibiotic (gentamicin or amoxicillin) stock solution (1 mg/mL) into the micro tube containing 600 &#181;L of the bacterial solution, to make the final antibiotic concentration to 8 &#181;g/mL.</li>
	<li>Take 300 &#181;L of solution out of the 8 &#181;g/mL of antibiotic-containing bacteria solution, and add to another 300 &#181;L of bacterial solution, to make two-fold diluted antibiotic- (4 &#181;g/mL) containing bacteria solution.</li>
	<li>Repeat the two-fold serial dilution of the test antibiotics, gentamicin, or amoxicillin, until the micro tube with the lowest concentration (0.25 &#181;g/mL) is reached, and discard 300 &#181;L from the tube. For both gentamicin and amoxicillin, the serial concentrations range from 0.25 &#181;g/mL - 8 &#181;g/mL.
	<ol>
		<li>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.</li>
		<li>Leave one tube with no antibiotics and no&#160;D2O for the negative control.</li>
	</ol>
	</li>
	<li>Incubate the bacterial aliquot with the certain antibiotic (gentamicin or amoxicillin) containing MHB medium for 1 h.</li>
	<li>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 &#181;g/mL - 8 &#181;g/mL.</li>
	<li>After 1 h antibiotic treatment, add 700 &#181;L of serially diluted antibiotic and 100% D2O-containing MHB medium to the 300 &#181;L of antibiotic-pretreated bacteria in the same antibiotic concentration (prepared in step&#160;2.6), respectively.
	<ol>
		<li>For example, add 700 &#181;L of 100% D2O-containing MHB medium (containing 8 &#181;g/mL of antibiotic) to the 300 &#181;L of 8 &#181;g/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.</li>
		<li>Add 700 &#181;L of antibiotic-free 100% D2O-containing MHB medium to 300 &#181;L of antibiotic-free bacteria (prepared in step 2.6.1) as a blank control.</li>
		<li>Incubate at 37 &#176;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%.</li>
	</ol>
	</li>
	<li>First centrifuge the 1 mL of antibiotic and D2O-treated bacterial sample at 6200 x g for 5 min at 4 &#176;C, and then wash twice with purified water. Finally, fix samples in 10% formalin solution and store them at 4 &#176;C.</li>
</ol>
3. Preparation of bacteria in urine environment (Figure 2b)
<ol>
	<li>To prepare E. coli BW 25113 at the logarithmic phase, follow the steps at 1.4 and 1.5.</li>
	<li>Check the bacterial concentration by measuring the OD&#160;with a photometer at a wavelength of 600 nm.</li>
	<li>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.</li>
	<li>Filter the E. coli spiked urine using a 5 &#181;m filter, and then divide the bacterial solution in 300 &#956;L aliquots into seven 1.5 mL micro tubes, and 600 &#181;L aliquots of the bacterial solution in one 1.5 mL micro tubes.</li>
	<li>Perform D2O incorporation treatment in the presence of antibiotics and sample collection&#160;as described in the previous steps from 2.4 to 2.10.</li>
</ol>
4. Preparation of bacteria in blood environment (Figure 2c)
<ol>
	<li>To prepare Pseudomonas aeruginosa ATCC 47085 at the logarithmic phase, follow the steps at 1.4 and 1.5.</li>
	<li>To mimic the clinical bloodstream infections samples42,43, spike P. aeruginosa in 1 mL of deidentified human blood to reach a&#160;concentration of 107 CFU/mL.</li>
	<li>Add 9 mL of sterile purified water to lyse the blood.</li>
	<li>Filter the P. aeruginosa spiked blood using a 5 &#181;m filter. Then harvest bacteria to 1 mL volume by centrifugation at 6200 x g for 5 min at 4 &#176;C.&#160;Add 9 mL of pre-warmed normal MHB into the bacteria solution and gently vortex. The final concentration of bacteria is 106 CFU/mL</li>
	<li>Divide the P. aeruginosa spiked blood solution in 300 &#956;L aliquots into seven 1.5 mL micro tubes, and 600 &#181;L aliquots of the bacterial solution in one 1.5 mL micro tubes.</li>
	<li>Perform D2O incorporation treatment in the presence of antibiotics and sample collection as described in the previous steps from 2.4 to 2.10.</li>
</ol>
5. SRS imaging of D2O metabolic incorporation in a single bacterium
<ol>
	<li>Wash 1 mL of fixed bacteria solution with purified water and then centrifuge at 6200 x g for 5 min at 4 &#176;C. Remove the supernatant. Enrich the bacterial solution to about 20 &#181;L.</li>
	<li>Deposit the bacterial solution on a poly-L-lysine coated coverglass. Sandwich and seal the sample for SRS imaging.</li>
	<li>Image bacteria at the C-D vibrational frequency at 2168 cm-1 using an SRS microscope.
	<ol>
		<li>Input and tune the pump wavelength to 852 nm using the control software on a computer.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Use a 60x water immersion objective to focus the pump and Stokes lasers on the sample.</li>
		<li>Use an oil condenser to collect the signals from the sample in the forward direction.</li>
		<li>Use a bandpass filter to filter out the Stokes laser before directing it into a photodiode.</li>
		<li>Extract the stimulated Raman signal by a lock-in amplifier and detect the signals by the photodiode.</li>
	</ol>
	</li>
	<li>Set each SRS image to contain 200 x 200 pixels and the pixel dwell time for 30 &#181;s in the software&#39;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 &#181;m2. Image at least three field of views for each sample.</li>
</ol>
6. Image processing and data analysis (&#160;Figure 3)
<ol>
	<li>To obtain the average C-D signal intensity, open and process SRS images with ImageJ software.</li>
	<li>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.</li>
	<li>Then, filter the images with Gaussian blur by clicking Process | Filters | Gaussian blur buttons and set the Sigma (Radius) to 1.</li>
	<li>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.</li>
	<li>Apply Analyze | Particles Analysis buttons to label and determine the area of bacteria.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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&#160;different individual bacterium.</li>
	<li>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.</li>
	<li>Validate and compare the SC-MIC readout with the MIC determined using conventional broth microdilution assay.</li>
	<li>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 &#34;susceptible&#34;, &#34;resistant&#34;, or &#34;intermediate&#34;.</li>
</ol>

The authors have no conflicts of interest to disclose.

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><tbody><tr><td>Acousto-optic modulation</td><td>Gooch&amp;amp;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 &amp;micro;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 &amp;deg;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 &amp;micro;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&amp;times;, 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 ...

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Research

JoVE Journal

Biology

2.6K Views.  Boston University. Snelle antimicrobiële gevoeligheidstests kunnen binnen 2,5 uur in urine en bloed worden verkregen, wat wordt beschouwd als een enorme vermindering van de analysetijd in vergelijking met de conventionele bouillonmicroverdunningsmethode. Het kan bacteriële metabole activiteit in een complexe omgeving, zoals volbloed, volgen. Controleer om te beginnen de bacteriële concentratie van de monsters door de optische dichtheid te meten met een fotometer op een golflengte van 600 nanometer.Om ...

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

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Immunology and Infection

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Bioengineering

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...

Chemistry

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

Biochemistry

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

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

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