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...
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 ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.Many antibiotics derive bactericidal activity from their ability to lyse cells, which may cause liberation of potent proinflammatory bacterial components, resulting in the generation of a robust innate immune response that can potentially cause harm to the host (8). The lipopeptide antibiotic daptomycin is active against a wide range of gram-positive bacteria (Cubicin prescription information, 2005; Cubist Pharmaceuticals, Lexington, MA) (4) and is believed to possess a novel mechanism of action that does not involve cell lysis. Instead, the lipophilic acyl tail of daptomycin is inserted into the cytoplasmic membrane of the bacterium, leading to potassium efflux; destruction of the ion-concentration gradient; membrane depolarization; inhibition of protein, DNA, and RNA synthesis; and finally cell death (Cubicin prescription information, 2005; Cubist Pharmaceuticals, Lexington, MA) (5, 11, 13). Daptomycin is rapidly bactericidal in vitro against Staphylococcus aureus at low multiples of the MIC (12). Here, we demonstrate the bactericidal activity of daptomycin against S. aureus in the absence of cell lysis. (Portions of this work were presented previously at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy [10].)Cell lysis was initially monitored by measuring optical density during log-phase time kills. Late-exponential-phase cultures (approximately 10 8 CFU/ml) were used to allow samples to be obtained for transmission electron microscopy (TEM). Staphylococcus aureus ATCC 29213 was grown overnight in calcium-supplemented Mueller-Hinton broth (MHBc; 50 mg/ liter Ca 2ϩ ) and subcultured 1:1,000 into fresh MHBc. Cultures were grown at 37°C with shaking (200 rpm) to an optical density at 600 nm (OD 600 ) of 0.3 to ensure sufficient biomass for fixation and processing. Daptomycin was added at multiples (1ϫ to 8ϫ) of the MIC (0.5 g/ml). At the indicated time points, samples were removed, OD 600 and number of CFU/ml were measured as previously described (7), and cells were pelleted and resuspended in 1 ml MHBc plus 2.5% (vol/vol) glutaraldehyde. Glutaraldehyde-fixed samples were postfixed in 2.0% (wt/vol) osmium tetroxide, followed by en bloc staining with 2.0% (wt/vol) uranyl acetate. The cells were then dehydrated through an ethanol series and embedded in LR White resin. Samples were thin sectioned and stained by uranyl acetate; lead citrate TEM was performed using a LEO 912AB microscope under standard operating conditions at 100 kV, with a liquid nitrogen anticontaminator in place.As shown in Fig. 1, at 4 g/ml, daptomycin was rapidly bactericidal, producing a Ͼ1,000-fold decrease in viability in less than 120 min, with no concomitant drop in OD 6...
Background Violet laser diodes have recently become commercially available. These devices emit 5–25 mW in the range of 395–415 nm, and are available in systems that incorporate the diodes with collimating optics and regulated power supplies in housings incorporating thermoelectric coolers, which are necessary to maintain stable output. Such systems now cost several thousand dollars, but are expected to drop substantially in price. Materials and Methods A 4‐mW, 397‐nm violet diode system was used in a laboratory‐built flow cytometer to excite fluorescence of DAPI and Hoechst dyes in permeabilized and intact cells. Forward and orthogonal light scattering were also measured. Results DNA content histograms with good precision (G0/G1 coefficient of variation 1.7%) were obtained with DAPI staining; precision was lower using Hoechst 33342. Hoechst 34580, with an excitation maximum nearer 400 nm, yielded the highest fluorescence intensity, but appeared to decompose after a short time in solution. Scatter signals exhibited relatively broad distributions. Conclusions Violet laser diodes are relatively inexpensive, compact, efficient, and quiet light sources for DNA fluorescence measurement using DAPI and Hoechst dyes; they can also excite several other fluorescent probes. Cytometry 44:133–136, 2001. © 2001 Wiley‐Liss, Inc.
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