No abstract

The objective of this study was to further elucidate the role of membrane potential in the mechanism of action of daptomycin, a novel lipopeptide antibiotic. Membrane depolarization was measured by both fluorimetric and flow cytometric assays. Adding daptomycin (5 g/ml) to Staphylococcus aureus gradually dissipated membrane potential. In both assays, cell viability was reduced by >99% and membrane potential was reduced by >90% within 30 min of adding daptomycin. Cell viability decreased in parallel with changes in membrane potential, demonstrating a temporal correlation between bactericidal activity and membrane depolarization. Decreases in viability and potential also showed a dose-dependent correlation. Depolarization is indicative of ion movement across the cytoplasmic membrane. Fluorescent probes were used to demonstrate Ca 2؉ -dependent, daptomycin-triggered potassium release from S. aureus. Potassium release was also correlated with bactericidal activity. This study demonstrates a clear correlation between dissipation of membrane potential and the bactericidal activity of daptomycin. A multistep model for daptomycin's mechanism of action is proposed.Daptomycin is a novel lipopeptide antibiotic in late-stage clinical development for the treatment of serious gram-positive infections. Daptomycin exhibits rapid in vitro bactericidal activity against clinically significant strains of gram-positive pathogens including hemolytic streptococci, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci (4,10,12,14,19,22,23). Daptomycin acts at the cytoplasmic membrane of susceptible bacteria (8), as demonstrated by binding and fractionation studies. Additionally, the activity of daptomycin is dependent on the presence of physiologic levels of free calcium ions (50 mg/liter).Debate over daptomycin's mechanism of action has continued for more than a decade. One hypothesis suggests that daptomycin bactericidal activity is mediated by inhibition of lipoteichoic acid (LTA) biosynthesis (7,8). However, a recent investigation has failed to find evidence of a role for LTA in the mechanism of action of daptomycin in S. aureus or Enterococcus faecalis, suggesting that the in vitro bactericidal activity of daptomycin is independent of LTA biosynthesis (V. Laganas, J. Alder, and J. A. Silverman, submitted for publication). A second proposed mechanism of action for daptomycin is that the antibiotic causes dissipation of bacterial membrane potential, resulting in disruption of multiple aspects of cellular function (1, 2). Bactericidal activity via disruption of membrane potential is the proposed mechanism of action for a variety of antimicrobial peptides, including the pore-forming antibiotic nisin (18,20). We wished to further investigate the role of bacterial membrane potential in the mechanism of action of daptomycin. In this study, we demonstrate a significant correlation between membrane depolarization and bactericidal activity. Furthermore, we demonstrate that one possible mechanism of membrane depol...

Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably – apoptosis. The cyanine dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.

Background: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size‐ and MP‐sensitive measurement, and an MP‐independent, size‐sensitive measurement, should provide a better estimate of MP. Methods: Flow cytometry and spectrofluorometry were used to detect red (488 → >600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 → 530 nm) fluorescent. Results: In bacteria incubated with 30 μM dye, aggregate formation increases with the magnitude of the interior‐negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from −50 mV through −120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4°C and washing in glucose‐free medium. Conclusions: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms. Cytometry 35:55–63, 1999. © 1999 Wiley‐Liss, Inc.

Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts.Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC 2 (3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC 2 (3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 g/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 g/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose-and time-dependent decreases in MP. Tetracycline concentrations of <1 g/ml did not change permeability, while a tetracycline concentration of 4 g/ml permeabilized 50% of the bacteria; 4 g of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (H0342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring H0342 fluorescence at 430-470 nm and PY fluorescence at 590-650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY The addition of RNA content estimation to the flow cytometric determination of DNA content commonly used for studies of cell kinetics has been shown to provide valuable information concerning cells' transitions between quiescent and proliferative states.Darzynkiewicz et al.

Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work.

No abstract