Use of Vital Dyes and Flow Cytometry to Establish Non-Antibacterial Activity of Chemically Modified Tetracyclines (CMTs)

Stephanie Sincock, J.Paul Robinson, and John G. Thomas

Purdue University Cytometry Laboratories, West Lafayette, Indiana, USA
West Virginia University, Pathology Department, Morgantown, West Virginia. USA

Tetracyclines are highly effective matrix metalloproteinase inhibitors and immunomodulators that can be chemically modified. Growing potential use of these chemically modified tetracyclines (CMTs) necessitated conformation of their reported non-antibacterial activity. The purpose of this study was to assess at sub-antimicrobial levels, antimicrobial levels and levels above serum Cmax whether CMTs had antibacterial activity comparable to doxycycline (DOX) using vital probes. Tetracycline susceptible Escherichia coli (ATCC 25922) was selected to evaluate the antibacterial activity of CMTs using EPICS XL flow cytometer. Membrane perturbation was assessed using the membrane integrity dye propidium iodide and the membrane potential-sensitive dye DiBAC4(3). Eleven concentrations of COL-3 (tetracycline analogue), COL-8 (doxycycline analogue) and DOX were added to early exponential phase E.coli cells and incubation was allowed to continue for 5 hours. Untreated and gentamicin (10.0 microgram/ml) treated E.coli cells were used as controls. At 8 timed intervals, aliquots of treated and untreated cells were removed, stained and analyzed. Flow cytometry combined with fluorescent probes allowed the measurement of CMT activity within minutes and provided information on the heterogeneity of the E.coli population response in presence of drug. Neither analogue had any antibacterial activity at sub-antimicrobial levels (0.03, 0.3, 0.5, 1.0 microgram/ml) or those equal to therapeutic levels (3.0, 4.0, 6.0, 10.0 microgram/ml) routinely achieved with doxycycline. Limited drug-induced membrane potential damage and loss of membrane integrity was noted at concentrations above physiologic levels (20.0, 30.0, 300.0 microgram/ml). Doxycycline demonstrated 100 to 1000X greater activity than COL-3 or COL-8. Hence, the use of flow cytometry further established the non-antibacterial activity of CMTs at sub-antimicrobial and therapeutic levels.

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