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

Abstract

Source: Stone, M. R. L., et al. Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes. 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.

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). Streak glycerol stocks of bacterial strains appropriate for the antibiotic scaffold on…

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). <td c…

Materials

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