Daptomycin Exerts Bactericidal Activity without Lysis of Staphylococcus aureus

Abstract

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...

The action mechanism of daptomycin

“…Studies using both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicate that daptomycin does not cause cell lysis of S. aureus and E. faecalis, although some morphological changes are observed. 76,77 With Staphylococcus aureus in liquid culture, daptomycin at 4 µg/ml produced a 10 3 -fold decrease in cell viability with no concomitant drop in optical density. 76 Electron microscopy showed that the cells remained intact, and the cell membranes free of discontinuities, but formation of aberrant division septa was common.…”

“…76,77 With Staphylococcus aureus in liquid culture, daptomycin at 4 µg/ml produced a 10 3 -fold decrease in cell viability with no concomitant drop in optical density. 76 Electron microscopy showed that the cells remained intact, and the cell membranes free of discontinuities, but formation of aberrant division septa was common. When the cells were loaded with calcein, an anionic fluorescent small molecule, the dye was retained after exposure to daptomycin even after the cells had lost viability.…”

“…Similarly, the nucleic acid stain TO-PRO3 remained excluded by cells even after treatment with daptomycin. 76 Another nucleic acid stain whose exclusion by cell membranes is often used as an indicator of cell viability is propidium iodide. Using this assay, a fraction of the cells in a liquid culture of Staphylococcus epidermidis were found to be permeabilized after daptomycin treatment.…”

“…Later, this same group, in collaboration with others, using essentially the same assay, reported only 7% calcein release from DOPC/PG liposomes at 32 µg/mL of daptomycin, 32 which seems more in line with the previously discussed results obtained with bacterial cells. 76 Using a coupled fluorescence assay that is based on the pH-sensitive indicator pyranine, 80 Zhang et al recently demonstrated that daptomycin can form pores in DMPC/DMPG (1:1) liposomes. 81 The pores are selective for cations, with permeabilities being highest for Na + , K + , and other alkali metal ions.…”

“…59 Bacillus subtilis cells grown with daptomycin at below MIC appeared bent and elongated. 72 While elongated cell forms suggest that division is inhibited, the observation of additional, aberrant septa in Staphylococcus aureus 76 could mean that cell division may be dysregulated rather than disrupted.…”