Antibiotic susceptibility testing by flow cytometry.

Flow cytometry can provide a rapid indication of antibiotic susceptibility. This unit describes a number of fluorescent dyes that allow the cytometer to distinguish the heterogeneic nature of populations exposed to antibiotics. No single method can be used for multiple organisms, since it is the interference with the organisms growth and structural integrity that signals the impact of the antibiotic so rapidly. This unit discusses membrane structural changes as well as alterations in nucleic acid and the probes that can be successfully used with flow cytometry.

Flow cytometric monitoring of antibiotic-induced injury in Escherichia coli using cell-impermeant fluorescent probes.

Three fluorescent nucleic acid binding dyes-propidium iodide, TO-PRO-1, and SYTOX green-were evaluated, and their abilities to distinguish between bacterial cells with and without an intact cytoplasmic membrane were compared. Each dye was readily able to discriminate between healthy and permeabilized cells of Escherichia coli, although SYTOX green showed a greater enhancement in fluorescence intensity on staining-compromised, as opposed to healthy, cells in log-phase growth, than either PI or TO-PRO-1. Flow cytometric analysis of E. coli stained with these dyes after exposing them to several antimicrobial agents showed that all three dyes were able to detect antimicrobial action. Notably, however, the intensity of the cell-associated fluorescence was related to the mechanism of action of the antimicrobial agent. Large changes in fluorescence intensity were observed for all the dyes subsequent to beta-lactam antibiotic action, but smaller changes (or no change) were seen subsequent to exposure to antimicrobials acting directly or indirectly on nucleic acid synthesis. Furthermore, cell-associated fluorescence did not relate to loss of viability as determined by plate counts. Despite offering much insight into antimicrobial mechanisms of action, these fundamental problems become relevant to the development of rapid antimicrobial susceptibility tests if colony formation is used as the standard.

A fluorescent Gram stain for flow cytometry and epifluorescence microscopy.

The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.