The application of flow cytometry to the study of bacterial responses to antibiotics

Abstract

Summary. Experiments were performed to determine whether a modern flow cytometer could be used to study bacterial populations in suspension, with particular reference to their morphological characteristics and their responses to antibiotics. The FACScan, a commercial benchtop flow cytometer fitted with an air-cooled laser, designed primarily for the study of eukaryotic peripheral blood mononuclear cells, yielded reproducible data relating to bacterial shape and internal architecture. It was sensitive enough to detect changes in bacterial morphology on entry into the growth cycle and after exposure to antibiotics. Antibioticinduced morphological changes affecting subpopulations of bacteria were sufficiently specific to allow differentiation between antibiotics with different cell-wall enzyme targets. Simultaneously, the effect of such antibiotics on the integrity of the outer cell membrane of Escherichia coli was assessed by measurement of the association of the nucleic acid-binding dye propidium iodide with the bacteria. These experiments demonstrated complex patterns of probable cell-wall leakage, related to the modes of action of the antibiotics. The FACScan is a useful and sensitive tool for the study of the morphology and physiology of bacterial populations in suspension, and is especially applicable to the study of antibiotic action.

Behavior and changes in cell morphology ofEscherichia coliO157:H7 in liquid medium and skim milk in the presence of caffeine

“…We observed changes in the integrity of the bacterial cell after treatment with different concentrations of caffeine. Our findings are consistent with the morphological changes of ampicillin-treated E. coli detected by flow cytometry previously reported (Gant, Warnes, Phillips, & Savidge, 1993). We speculate that the mode of action of caffeine involves the inhibition of cell wall synthesis leading to bacterial filamentation, which could be an indication of the initial process of cell lysis.…”

Rapid Differentiation of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus by Flow Cytometry after Brief Antibiotic Exposure

“…An important question from a clinical perspective is whether these changes can be effectively measured to accurately differentiate susceptible from resistant bacteria before they can be differentiated on the basis of cell viability. Antibiotics have previously been shown to induce membrane potential and membrane permeability changes in bacteria (6,8). Thus, live bacteria adapting to stress are probably changing in measurable ways.…”

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

“…Flow cytometry was performed using a BD LSR-Fortessa X-20 (BD Biosciences, San Jose, CA) equipped with a 488-nm excitation wavelength from a blue air laser at 50 mW and two light scatter detectors measuring forward and side scatter lights. Forward-scattered light (FSC) is proportional to the volume of the cells and was used to evaluate cell filamentation (50,52,53). Treated cultures and controls (200 l) were diluted with 1 ml of 0.85% NaCl for mitomycin C-, heat-, acid-, or pressure-treated samples, or with 1 ml of phosphate-buffered saline (PBS) containing 0.2% (wt/vol) sodium thiosulfate (Sigma-Aldrich) for H 2 O 2 -treated samples.…”