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

Overview

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.

Protocol

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).

1. Streak glycerol stocks of bacterial strains appropriate for the antibiotic scaffold onto lysogeny broth agar (LB, prepared per manufacturer's instructions), and grow overnight at 37 °C.
   NOTE: The choice of bacteria to test antibacterial activity must be made based on the antibiotic scaffold being used. A representative range of 5–10 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.
2. Pick a single colony from the plate, and culture overnight in 5 mL of cation adjusted Mueller-Hinton Broth (CAMHB, prepared per manufacturer’s instructions) at 37 °C.
3. Dilute the overnight cultures ~40-fold in CAMHB and grow to a mid-log phase, optical density at 600 nm (OD₆₀₀) = 0.4–0.8, volume 5 mL).
4. Prepare stock solutions of each fluorescent antibiotic at 1.28 mg/mL in sterile water, and pipette 10 μL of antibiotic to the first column of a 96 well plate.
5. Add 90 μL of CAMHB to the first column and 50 μL to all other wells. Then, perform serial 2-fold dilution across the plate.
6. Thoroughly mix, then dilute the mid-log phase cultures to ~10⁶ colony forming units (CFU)/mL and add 50 μL 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
7. Cover the plates with lids and incubate at 37 ˚C for 18–24 h without shaking.
8. 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.

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.

1. Streak glycerol stocks of the bacterial strains onto LB agar and grow overnight at 37 °C.
2. Pick a single colony from the plate and culture overnight in LB at 37 °C.
3. Dilute overnight cultures ~50-fold in media and grow to mid-log phase (OD₆₀₀ = 0.4–0.8).
4. Centrifuge the cultures at 1,470 x g for 25 min and decant the media.
5. Resuspend the bacteria in 1 mL of phosphate-buffered saline (PBS), then centrifuge at 1,470 x g for 15 min.
6. Decant the media and resuspend the washed pellets in PBS to a final OD₆₀₀ = 2.
7. If desired, add 10.1 μL of carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10 mM in PBS) to 1 mL of bacteria (final concentration 100 μM) and incubate at 37 ˚C for 10 min.
   NOTE: CCCP is an efflux pump inhibitor. Addition of CCCP will allow the examination of the impact of efflux.
8. Centrifuge the cultures at 18,000 x g for 4 min at 20 °C and decant the media.
9. Add 1 mL of fluorescent antibiotic solution (10–100 μM in PBS) to the pellet, and incubate at 37 °C for 30 min.
10. Centrifuge the cultures at 18,000 x g for 7 min at 4 °C and decant the media.
11. Resuspend the bacteria in 1 mL of cold PBS, and repeat step 2.9.
12. Repeat step 2.10 a total of 4x.
13. If desired, lyse bacteria by adding 180 μL of lysis buffer (20 mM Tris-HCl, pH 8.0, and 2 mM sodium EDTA) then 70 μL of lysozyme (72 mg/mL in H₂O).
14. Incubate at 37°C for 30 min, then freeze-thaw 3x (−78 °C for 5 min, then 34 °C for 15 min).
15. Sonicate the samples for 20 min, then heat to 65 °C for 30 min.
16. Centrifuge the lysed samples (18,000 x g, 8 min), then filter through a 10 kDa filter membrane.
17. Wash the filter the 4x with 100 μL of water.
18. 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]: λ_ex = 400 nm, λ_em = 490 nm; nitrobenzoxadiazole [NBD]: λ_ex = 475 nm, λ_em = 545 nm).
    NOTE: See Figure 1 for examples of spectrophotometric analyses of bacteria using the fluorescent NBD-labeled ciprofloxacin antibiotic.

Representative Results

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).

		MIC (µg/mL)
Species	Strain	Cipro	Cipro-NBD	Cipro-DMACA	TMP	TMP-NBD	TMP-DMACA	Linezolid (Lz)	Lz-NBD	Lz-DMACA
Staphylococcus aureus	ATCC 25923	0.125 - 0.5	32 - ≥64	16	1	16	>64
	ATCC 43300							1	16	>64
Streptococcus pneumoniae	ATCC 700677							1	4	64
Enterococcus faecium	ATCC 35667	1 - 8	32	32 - ≥64
	ATCC 51559							2	16	32
Klebsiella pneumoniae	ATCC 13883	0.015 - 0.06	8 - 16	8 - 32
Pseudomonas aeruginosa	ATCC 27853	0.25 - 1	32 - ≥64	32 - ≥64
Escherichia coli	ATCC 25922	≤0.004	8	2	0.5	>64	>64
	Mutant ΔtolC				0.125	0.25	2

Figure 1: Plate reader measurement of antibiotic-derived probe accumulation. Fluorescence spectroscopic measurement of cellular accumulation of TMP-NBD (50 μM) in wild type (1, ATCC 25922) and ΔtolC (2, ATCC 25922) E. coli incubated (A) with and (B) without addition of CCCP (100 μM). Statistical significance (**p ≤ 0.01; ***p ≤ 0.001) is shown between the absence or presence of CCCP and between wild type and ΔtolCE. coli. Data reported are the mean ± SD for three experiments. This figure is adapted from our previous publication, and illustrates the use of spectroscopy to elucidate the role of efflux on intracellular accumulation.

Materials

Name	Company	Catalog Number	Comments
Amicon Ultra-0.5 centrifugal filter unit with Ultracel- 10 membrane	Merck	UFC501096
Bruker Avance 600 MHz spectrometer	Bruker
CCCP	Sigma-Aldrich	C2759
Celite 545	Sigma-Aldrich	22140-5KG-F
Cygel	ABCAM	Ab109204
FM4-64FX, fixable analog of FM™ 4-64 membrane stain	Life Technologies Australia Pt	F34653
Gamma 2-16 LSCplus lyophilise	CHRIST
Hettich Zentrifugen Rotofix 32	Hettich
LB	AMRESCO	J106
Lysozyme from chicken egg white lyophilized powder	Sigma-Aldrich	L6876
Mueller Hinton II Broth Cation adjusted	Becton Dickinson	212322
Sigma 1-15 Microcentrifuge	Sigma-Aldrich
TECAN Infinite M1000 PRO	TECAN