Flow cytometric assessment of susceptibilities of Streptococcus pyogenes to erythromycin and rokitamycin

Abstract

The effects of erythromycin (a 14-membered ring macrolide) and rokitamycin (a 16-membered ring macrolide) on the viability of the Streptococcus pyogenes M phenotype were studied by means of flow cytometry and fluorescence microscopy by using a combination of two fluorochromes (syto 9 and propidium iodide) that stains live bacteria green and dead bacteria red. In order to apply the flow cytometry, a bacterial sonication procedure was expressly set up to separate single cells from the long, intralaced S. pyogenes chains of up to 30 to 40 cells that have previously prevented the application of flow cytometry to this type of bacteria. The association of flow cytometry using an appropriate sonication procedure, together with a combination of fluorescent probes, offered the possibility of very quickly investigating the different microbiological effects of rokitamycin at 2 microg/ml, which was active on the S. pyogenes M phenotype, and of erythromycin at doses of up to 32 microg/ml, which was not.

FIG. 1.

Examples of interlaced chains of untreated S. pyogenes before sonication (A) and the individual cells obtained after sonication (B). Examples of the corresponding density dot plots (FL-I, syto 9; FL-III, propidium iodide) of the chains (C) and individual cells after sonication (D).

FIG. 2.

Growth curves of S. pyogenes incubated with no drug (control), erythromycin (ERY) (32 μg/ml), and rokitamycin (RKM) (2 μg/ml).

FIG. 3.

Examples of the random distribution of red cells inside chains of S. pyogenes incubated with half the rokitamycin MIC (A to C). Density dot plots (FL-I, syto 9; FL-III, propidium iodide) of S. pyogenes incubated with half the rokitamycin MIC before (D) and after sonication (E).