Multiparameter flow cytometry of bacteria: Implications for diagnostics and therapeutics

Abstract

Background: Flow cytometric studies of antibiotic susceptibilities of bacteria have typically measured a single fluorescence parameter, such as membrane potential (indicating viability), or permeability to nucleic acid stains such as propidium (indicating nonviability). Cytometry of bacteria stained simultaneously with a membrane potential dye and a permeability indicator reveals unanticipated complexity. Methods: Aliquots of cultures of three bacterial species were stained with the potential-sensitive dye hexamethylindocarbocyanine [DiICI(3)] and the permeability indicator TO-PRO-3™ in the presence and absence of a proton ionophore which collapses the potential gradient. They were analyzed using a dual-laser flow cytometer. Results: Cultures grown under suboptimal conditions appear to contain cells that take up TO-PRO-3 while maintaining membrane potential, although some events showing both high DiICI(3) fluorescence and high TO-PRO-3 fluorescence may represent clumps. Conclusions: Variations in metabolic patterns between species and within organisms under suboptimal culture conditions or following antibiotic exposure may make it difficult to develop flow cytometric clinical assays for antibiotic susceptibility. However, transient permeabilization of otherwise resistant organisms by sublethal doses of antibiotics may make it possible to treat infections by such organisms with suitably derivatized, otherwise toxic molecules; multiparameter cytometry should be useful in pursuing this approach to therapy. © 2001 Wiley-Liss, Inc.