In Vitro Bactericidal Activities of Daptomycin against Staphylococcus aureus and Enterococcus faecalis Are Not Mediated by Inhibition of Lipoteichoic Acid Biosynthesis

Abstract

Previous studies have suggested that lipoteichoic acid biosynthesis inhibition is the mechanism of action of daptomycin. In this investigation, daptomycin inhibited all macromolecular synthesis in Staphylococcus aureus, Enterococcus faecalis, and Enterococcus hirae without kinetic or dose specificity for lipoteichoic acid. Daptomycin remained bactericidal in the absence of ongoing lipoteichoic acid synthesis. Inhibition of lipoteichoic acid synthesis is apparently not the mechanism of action of daptomycin in these pathogens.

Daptomycin is a novel lipopeptide antibiotic in late-stage clinical development for the treatment of serious gram-positive infections, including complicated skin and soft tissue infections. This antibiotic exhibits rapid in vitro bactericidal activity against a number of clinically significant gram-positive pathogens (3, 10, 13, 14, 16, 18, 19). Daptomycin acts at the cytoplasmic membrane of susceptible bacteria, based on the results of binding and fractionation studies (7). Additionally, the activity of daptomycin is dependent on the presence of physiologic levels of free calcium ions (50 mg/liter).

Previously, other authors have suggested two mechanisms of action for daptomycin: dissipation of the bacterial membrane potential (1, 2) or inhibition of lipoteichoic acid (LTA) biosynthesis (5, 7). LTA is a cell surface polymer anchored in the cytoplasmic membrane of gram-positive bacteria and extends out into the peptidoglycan layer and external environment. The membrane-anchored core is highly conserved, whereas the extended polymer is highly divergent in daptomycin-susceptible organisms (8, 9). Although LTA is essential for cell viability, its function in bacteria is not fully understood.

Previous studies have reported that in Enterococcus hirae daptomycin exhibited kinetic specificity for LTA, inhibiting its biosynthesis before that of other macromolecules (e.g., DNA and protein) (5, 7). Daptomycin also exhibited dose specificity, inhibiting only LTA synthesis at doses at or very near the MIC, yet inhibiting multiple pathways at higher doses. Kinetic and dose specificity are characteristic of most antibiotics that target macromolecular synthesis and suggest that LTA is the primary target of daptomycin action. However, this specificity was not observed for Staphylococcus aureus (7). We have reexamined the effect of daptomycin on macromolecular synthesis in S. aureus, Enterococcus faecalis, and E. hirae (V. Laganas and J. A. Silverman, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1802, 2001).

Three bacterial strains, S. aureus ATCC 29213, E. faecalis ATCC 49452, and E. hirae ATCC 9760, were utilized in this study. Bacterial growth, MIC determination, and bactericidal activity were determined as previously described by Silverman et al. (17). Ciprofloxacin, rifampin, vancomycin, and purified S. aureus LTA were purchased from Sigma (St. Louis, Mo.). Daptomycin and N_α,N_ɛ-diacetyl-Lys-d-Ala-d-Ala were provided by Cubist Pharmaceuticals, Inc. (Lexington, Mass.). Radiolabeled chemicals were purchased from Perkin-Elmer Life Sciences (Boston, Mass.).

Macromolecular synthesis was monitored via incorporation of radiolabeled precursors during mid-exponential-phase (optical density at 600 nm, 0.3) growth in calcium-supplemented Mueller-Hinton broth (17). S. aureus cells were labeled by a 5-min pulse exposure to radioactive precursors. Labeling of E. faecalis and E. hirae was initiated at the beginning of each assay and maintained continuously throughout the time course. Synthesis of RNA was monitored by measuring the incorporation of [5′-³H]uridine (5 μCi/ml) into trichloroacetic acid-precipitable material. Labeled samples were quenched into cold 10% trichloroacetic acid and transferred to a 96-well filter plate (96-well Packard Unifilter GF/B; Perkin-Elmer) by using a Filtermate cell harvester (Perkin-Elmer). Radioactivity was measured with a TopCount NXT microplate scintillation and luminescence counter (Perkin-Elmer). Lipid and LTA biosynthesis were monitored separately by incorporation of [³H]glycerol (5 μCi/ml) following the procedures described by Canepari et al. (7). Specific radioactive counts for LTA were determined by hot phenol extraction (12), and lipid fractions were obtained by methanol-chloroform extraction (4). The radioactivity in all samples was measured by liquid scintillation (1600TR liquid scintillation analyzer; Packard) by using standard methods and materials.

As demonstrated in Fig. 1A, daptomycin concentrations at two times the MIC inhibited the biosynthesis of LTA, lipids, and RNA with similar kinetics in S. aureus. In contrast (and as expected), at two times the MIC, the RNA polymerase inhibitor rifampin inhibited the biosynthesis of RNA earlier than that of LTA or lipids (Fig. 1B). Against E. faecalis and E. hirae, daptomycin at two times the MIC also inhibited RNA and LTA synthesis with essentially identical kinetics (Fig. 2). Note that in this continuous labeling assay, background levels of LTA and RNA synthesis were approximately 5 and 30% of those of the control, respectively. As observed with S. aureus, rifampin also demonstrated kinetic specificity for inhibition of RNA biosynthesis compared with LTA biosynthesis in both Enterococcus spp. (data not shown). Overall, the kinetics for daptomycin inhibition of RNA and LTA biosynthesis were similar in S. aureus, E. faecalis, and E. hirae. The lack of kinetic specificity at these low doses for any synthetic pathway suggested that LTA biosynthesis was not the primary target of action in these species.

FIG. 1.

Effect of daptomycin on the biosynthesis of LTA, lipids, and RNA in S. aureus. S. aureus ATCC 29213 was incubated with two times the MIC of daptomycin (A) or rifampin (B) at time zero. Data are plotted as the means ± standard deviations of triplicate experiments.

FIG. 2.

Effect of daptomycin biosynthesis of LTA and RNA in E. faecalis and E. hirae. Daptomycin at two times the MIC was added to E. faecalis (A) or E. hirae (B) cultures at time zero, following 10 min of labeling in the absence of drug. Data are plotted as the means ± standard deviations of triplicate experiments.

The possibility that the bactericidal activity of daptomycin might require ongoing LTA biosynthesis, even if it was not the primary target, was also investigated. This study took advantage of the observation that treatment of S. aureus and E. faecalis for 1 h with rifampin results in a complete cessation of macromolecular synthesis, including that of LTA (Fig. 1B and data not shown), with no significant loss in bacterial viability (Fig. 3). If active (or concurrent) LTA biosynthesis is required for the action of daptomycin, then pretreatment with rifampin should protect bacteria from the activity of daptomycin. The in vitro bactericidal activity of daptomycin was measured against both exponentially growing S. aureus cultures and cultures that were growth arrested by a 60-min pretreatment with rifampin. For comparison, we tested ciprofloxacin, which requires active cell division, as well as RNA and protein synthesis, to manifest its bactericidal activity (15). A 1-h exposure to daptomycin at eight times the MIC effectively killed (≥3 log reduction in cell viability) both exponentially growing and rifampin-growth-arrested S. aureus. In contrast, the growth-arrested S. aureus was protected against the bactericidal activity of ciprofloxacin due to the lack of active macromolecular synthesis (Fig. 3A). Similar results were observed for E. faecalis (Fig. 3B) for both antibiotics. These data suggest that active LTA biosynthesis is not required for the bactericidal activity of daptomycin against these species.

FIG. 3.

Growth arrest does not inhibit daptomycin bactericidal activity. Exponentially growing or rifampin-growth-arrested S. aureus (A) or E. faecalis (B) was incubated for 1 h with daptomycin (Dap), ciprofloxacin (Cipro), or rifampin (Rif) at eight times the MIC. Representative data are shown.

We have also investigated the possibility that LTA functions as a daptomycin-binding molecule or membrane receptor. If LTA is the daptomycin receptor, then exogenous LTA should protect cells from the effects of the drug, as has been previously demonstrated with the peptidoglycan pentapeptide target of vancomycin (N_α,N_ɛ-diacetyl-Lys-d-Ala-d-Ala) (6). MICs of daptomycin and vancomycin were measured in the presence or absence of purified S. aureus LTA or N_α,N_ɛ-diacetyl-Lys-d-Ala-d-Ala. The addition of purified LTA at concentrations up to 500 μg/ml (above the critical micelle concentration) did not significantly affect MICs of daptomycin for S. aureus or E. faecalis, whereas the addition of 100-μg/ml N_α,N_ɛ-diacetyl-Lys-d-Ala-d-Ala inhibited vancomycin (Table 1). The data for daptomycin were consistent with previous observations that LTA polymers exhibit significant structural diversity among gram-positive bacteria, including daptomycin-susceptible strains (11), making it unlikely that LTA would be functioning as a global daptomycin-specific binding molecule or membrane receptor.

TABLE 1.

Effect of LTA on daptomycin and vancomycin MICs

Antibiotic	Concn of compound (μg/ml)		MIC (μg/ml)
	LTA	Nα,Nɛ- diacetyl-Lys- d-Ala-d-Ala*	S. aureus	E. faecalis
Daptomycin			0.39	1.56
	5		0.78	1.56
	50		0.39	1.56
	500		0.39	1.56
		100	0.78	1.56
Vancomycin (control)			0.78	1.56
		100	25	>25

In summary, there was no evidence of a role for LTA in the mechanism of action of daptomycin in S. aureus, E. faecalis, or E. hirae, lending support to the hypothesis that daptomycin acts via the dissipation of bacterial membrane potential in these clinically relevant pathogens (1, 2, 17a). Further investigation would be necessary to rule out a role for daptomycin-mediated inhibition of LTA biosynthesis in other pathogens.