an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells

An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells

For probe accumulation analysis, streak glycerol stocks of the bacterial strains onto LB agar plates for an overnight incubation at 37 degrees Celsius. The next morning, pick a single colony from the plate for overnight culture in lysogeny broth at 37 degrees Celsius. The next morning, dilute the overnight culture approximately 50-fold in fresh medium.
When the culture reaches mid-log phase, pellet the bacteria by centrifugation, and decant the medium. Resuspend the bacteria in 1 milliliter of PBS, and centrifuge the bacteria again. Decant the supernatant, and resuspend the washed pellet in PBS to an optical density at 600 nanometers of two.
Add 10.1 microliters of 10 millimolar CCCP in PBS to 1 milliliter of bacteria, and incubate the bacteria at 37 degrees Celsius for 10 minutes. At the end of the incubation, collect the bacteria by centrifugation, and resuspend the pellet in 1 milliliter of 10-to-100 micromolar fluorescent antibiotic solution in PBS.
After a 30-minute incubation at 37 degrees Celsius, wash the cells by centrifugation four times in 1 milliliter of cold PBS per wash. After the wash, lyse the bacteria with 180 microliters of lysis buffer, and 70 microliters of lysozyme.
After 30 minutes at 37 degrees Celsius, freeze-thaw the bacteria three times at minus 78 degrees Celsius for 5 minutes and 34 degrees Celsius for 15 minutes respectively. After the last round of freeze-thawing, sonicate the sample for 20 minutes, followed by a 30-minute incubation at 65 degrees Celsius.
At the end of the incubation, collect the lysed sample by centrifugation, and strain the tube contents through a 10-kilodalton filter membrane. Wash the filter four times with 100 microliters of water per wash, and aliquot each wash into individual wells of a black flat-bottom 96-well plate. Then measure the fluorescence intensity on a plate reader with excitation and emission wavelengths appropriate to the fluorophore.

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JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

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1. Evaluation of Antimicrobial Activity
NOTE: All work involving bacteria should be carried out under sterile conditions to avoid contamination of either the assay or the laboratory. All media should be autoclaved before use, and plasticware and equipment such as pipettes must be kept sterile. It is recommended that work be done in a biocontainment hood (type 2).
<ol>
	<li>Streak glycerol stocks of bacterial strains appropriate for the antibiotic scaffold onto lysogeny broth agar (LB, prepared per manufacturer&#39;s instructions), and grow overnight at 37 &#176;C. 
	NOTE: The choice of bacteria to test antibacterial activity must be made based on the antibiotic scaffold being used. A representative range of 5&#8211;10 bacteria should be chosen from the species that are known to be susceptible to the antibiotic, with consideration given to the logistical capabilities of the lab. If possible, resistant bacteria should also be tested. The protocol given below will work on most bacteria, but check if special conditions are required (e.g., CO2, special media) and make alterations as necessary. Bacteria successfully assayed using these conditions include Staphylococci, Streptococci, Bacilli, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterococcus faecium.</li>
	<li>Pick a single colony from the plate, and culture overnight in 5 mL of cation adjusted Mueller-Hinton Broth (CAMHB, prepared per manufacturer&#8217;s instructions) at 37 &#176;C.</li>
	<li>Dilute the overnight cultures ~40-fold in CAMHB and grow to a mid-log phase, optical density at 600 nm (OD600) = 0.4&#8211;0.8, volume 5 mL).</li>
	<li>Prepare stock solutions of each fluorescent antibiotic at 1.28 mg/mL in sterile water, and pipette 10 &#956;L of antibiotic to the first column of a 96 well plate.</li>
	<li>Add 90 &#956;L of CAMHB to the first column and 50 &#956;L to all other wells. Then, perform serial 2-fold dilution across the plate.</li>
	<li>Thoroughly mix, then dilute the mid-log phase cultures to ~106 colony forming units (CFU)/mL and add 50 &#956;L to all wells, to provide a final concentration of ~5 x 105 CFU/mL. 
	volume of culture (mL) = (media volume in mL)/(OD600 x 1,000) 
	e.g., for an OD600 = 0.5 culture in a desired media volume of 12 mL, add (12/(0.5 x 1,000) = 0.024 mL of culture to 12 mL of media</li>
	<li>Cover the plates with lids and incubate at 37 &#730;C for 18&#8211;24 h without shaking.</li>
	<li>Visually inspect the plates, with the MIC being the lowest concentration well with no visible growth. 
	NOTE: See Table 1 for some examples of active and inactive fluorescent antibiotics.</li>
</ol>
2. Analysis of Probe Accumulation by Spectrophotometry
NOTE: These centrifugation times have been optimized for E. coli, so slight alterations may be needed for other species. Representative data for probe accumulation is reported for the NBD-labeled ciprofloxacin probe.
<ol>
	<li>Streak glycerol stocks of the bacterial strains onto LB agar and grow overnight at 37 &#176;C.</li>
	<li>Pick a single colony from the plate and culture overnight in LB at 37 &#176;C.</li>
	<li>Dilute overnight cultures ~50-fold in media and grow to mid-log phase (OD600 = 0.4&#8211;0.8).</li>
	<li>Centrifuge the cultures at 1,470 x g for 25 min and decant the media.</li>
	<li>Resuspend the bacteria in 1 mL of phosphate-buffered saline (PBS), then centrifuge at 1,470 x g for 15 min.</li>
	<li>Decant the media and resuspend the washed pellets in PBS to a final OD600 = 2.</li>
	<li>If desired, add 10.1 &#956;L of carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10 mM in PBS) to 1 mL of bacteria (final concentration 100 &#956;M) and incubate at 37 &#730;C for 10 min. 
	NOTE: CCCP is an efflux pump inhibitor. Addition of CCCP will allow the examination of the impact of efflux.</li>
	<li>Centrifuge the cultures at 18,000 x g for 4 min at 20 &#176;C and decant the media.</li>
	<li>Add 1 mL of fluorescent antibiotic solution (10&#8211;100 &#956;M in PBS) to the pellet, and incubate at 37 &#176;C for 30 min.</li>
	<li>Centrifuge the cultures at 18,000 x g for 7 min at 4 &#176;C and decant the media.</li>
	<li>Resuspend the bacteria in 1 mL of cold PBS, and repeat step 2.9.</li>
	<li>Repeat step 2.10 a total of 4x.</li>
	<li>If desired, lyse bacteria by adding 180 &#956;L of lysis buffer (20 mM Tris-HCl, pH 8.0, and 2 mM sodium EDTA) then 70 &#956;L of lysozyme (72 mg/mL in H2O).</li>
	<li>Incubate at 37&#176;C for 30 min, then freeze-thaw 3x (&#8722;78 &#176;C for 5 min, then 34 &#176;C for 15 min).</li>
	<li>Sonicate the samples for 20 min, then heat to 65 &#176;C for 30 min.</li>
	<li>Centrifuge the lysed samples (18,000 x g, 8 min), then filter through a 10 kDa filter membrane.</li>
	<li>Wash the filter the 4x with 100 &#956;L of water.</li>
	<li>Transfer the lysate to a flat-bottom black 96 well plate and measure the fluorescence intensity on a plate reader with excitation and emission wavelengths appropriate for the fluorophore (i.e., 7-(dimethylamino)-2-oxo-2H-chromen-4-yl [DMACA]: &#955;ex = 400 nm, &#955;em = 490 nm; nitrobenzoxadiazole [NBD]: &#955;ex = 475 nm, &#955;em = 545 nm). 
	NOTE: See Figure 1 for examples of spectrophotometric analyses of bacteria using the fluorescent NBD-labeled ciprofloxacin antibiotic.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amicon Ultra-0.5 centrifugal filter unit with Ultracel- 10 membrane</td>
			<td>Merck</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Avance 600 MHz spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2759</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite 545</td>
			<td>Sigma-Aldrich</td>
			<td>22140-5KG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cygel</td>
			<td>ABCAM</td>
			<td>Ab109204</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64FX, fixable analog of FM&#8482; 4-64 membrane stain</td>
			<td>Life Technologies Australia Pt</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma 2-16 LSCplus lyophilise</td>
			<td>CHRIST</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hettich Zentrifugen Rotofix 32</td>
			<td>Hettich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB</td>
			<td>AMRESCO</td>
			<td>J106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme from chicken egg white 
			lyophilized powder</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Mueller Hinton II Broth Cation adjusted</td>
			<td>Becton Dickinson</td>
			<td>212322</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma 1-15 Microcentrifuge</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TECAN Infinite M1000 PRO</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Stone, M. R. L., et al. <a href="https://app.jove.com/v/60743">Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.

Table 1. Antibiotic activities of fluorescent antibiotic probes based on ciprofloxacin, trimethoprim, and linezolid against appropriate clinically relevant bacterial strains, as measured by broth microdilution MIC assays. In most cases, the probes lost some activity compared to the parent drug, but retained some measurable antibiotic potency (sufficient to be useful in further studies).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td c...

1. Evaluation of Antimicrobial Activity
NOTE: All work involving bacteria should be carried out under sterile conditions to avoid contamination of either ...

Take assay tubes containing bacterial samples.
In the test sample, add inhibitor molecules to block the antibiotic efflux pump of the bacteria.
Centrifuge and discard the supernatant.
Add fluorophore-conjugated antibiotic probes that enter the bacterial cells.
In the control sample, the efflux pump extrudes the probes into the extracellular medium.
In the inhibitor-treated sample, the blocked efflux pump cannot expel the probes, causing their accumulation.
Centrifuge the samples. Discard the supernatant containing extracellular probes.
Add lysis buffer followed by lysozyme to weaken the bacterial cell wall.
Perform freeze-thaw cycles, sonication, and high-temperature exposure to lyse the cells.
Centrifuge. Transfer the supernatant to a filter membrane.
Wash and collect the filtrate containing the probes.
Measure the fluorescence intensity of the filtrate.
A higher fluorescence intensity in inhibitor-treated bacterial samples suggests loss of efflux function, leading to intracellular probe accumulation.

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An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells

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An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells