Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells

Abstract

Most antibiotics with bactericidal activity require that the bacteria be actively dividing to produce rapid killing. However, in many infections, such as endocarditis, prosthetic joint infections, and infected embedded catheters, the bacteria divide slowly or not at all. Daptomycin is a lipopeptide antibiotic with a distinct mechanism of action that targets the cytoplasmic membrane of gram-positive organisms, including Staphylococcus aureus. Daptomycin is rapidly bactericidal against exponentially growing bacteria (a 3-log reduction in 60 min). The objectives of this study were to determine if daptomycin is bactericidal against nondividing S. aureus and to quantify the extent of the bactericidal activity. In high-inoculum methicillin-sensitive S. aureus cultures in stationary phase (10(10) CFU/ml), daptomycin displayed concentration-dependent bactericidal activity, requiring 32 micro/ml to achieve a 3-log reduction. In a study comparing several antibiotics at 100 microg/ml, daptomycin demonstrated faster bactericidal activity than nafcillin, ciprofloxacin, gentamicin, and vancomycin. In experiments where bacterial cell growth was halted by the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or erythromycin, daptomycin (10 microg/ml) achieved the bactericidal end point (a 3-log reduction) within 2 h. In contrast, ciprofloxacin (10 microg/ml) did not produce bactericidal activity. Daptomycin (2 microg/ml) remained bactericidal against cold-arrested S. aureus, which was protected from the actions of ciprofloxacin and nafcillin. The data presented here suggest that, in contrast to that of other classes of antibiotics, the bactericidal activity of daptomycin does not require cell division or active metabolism, most likely as a consequence of its direct action on the bacterial membrane.

FIG. 1.

Daptomycin (2 μg/ml) exhibits rapid bactericidal activity against exponential-phase cultures of methicillin-sensitive S. aureus. DAP, daptomycin.

FIG. 2.

Daptomycin displays concentration-dependent bactericidal activity against high inoculum density stationary-phase cultures of S. aureus. Prior to exposure to daptomycin, cultures were incubated for >17 h to generate high cell density stationary-phase samples.

FIG. 3.

Effect of exposing stationary-phase S. aureus cultures to either daptomycin or a comparator antibiotic at 100 μg/ml. DAP, daptomycin; NAF, nafcillin; CIP, ciprofloxacin; GEN, gentamicin; VAN, vancomycin.

FIG. 4.

(A) Effect of cell physiology on the dose response and bactericidal activity of daptomycin against concentrated exponential-phase (Expo.) and stationary-phase (Stat.) S. aureus. (B) Log reductions of dilute and concentrated stationary-phase cultures. Cells were suspended in depMHBc and treated with either a high (100-μg/ml) or a low (10-μg/ml) concentration of daptomycin (DAP).

FIG. 5.

Effect of daptomycin on chemically arrested S. aureus. Exponentially growing cells were chemically arrested by a 1-h pretreatment with CCCP (a proton ionophore) or erythromycin (a protein synthesis inhibitor). Cells were treated with either daptomycin (A) or ciprofloxacin (B) at 10 μg/ml. CIP, ciprofloxacin; DAP, daptomycin; ERY, erythromycin.

FIG. 6.

Bactericidal activities of daptomycin, ciprofloxacin, and nafcillin (each at 2 μg/ml) against S. aureus that has been growth arrested by ice bath submersion. After 24 h, bacteria were warmed to 37°C. DAP, daptomycin; CIP, ciprofloxacin; NAF, nafcillin.