AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Laganas, V.
Right arrow Articles by Silverman, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Laganas, V.
Right arrow Articles by Silverman, J. A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, August 2003, p. 2682-2684, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2682-2684.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Valerie Laganas, Jeffrey Alder, and Jared A. Silverman*

Cubist Pharmaceuticals, Inc., Lexington, Massachusetts

Received 23 December 2002/ Returned for modification 17 March 2003/ Accepted 7 May 2003


   ABSTRACT
Top
 Abstract
Text
 References
 
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.


   TEXT
Top
 Abstract
 Text
References
 
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{alpha},N{varepsilon}-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.



View larger version (30K):
[in this window]
[in a new window]
  		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.

 


View larger version (29K):
[in this window]
[in a new window]
  		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.



View larger version (23K):
[in this window]
[in a new window]
  		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{alpha},N{varepsilon}-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{alpha},N{varepsilon}-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{alpha},N{varepsilon}-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.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effect of LTA on daptomycin and vancomycin MICs

 
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.


   ACKNOWLEDGMENTS
 
We thank Nicole Oliver for valued assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Cubist Pharmaceuticals, Inc., 65 Hayden Ave., Lexington, MA 02421. Phone: (781) 860-8405. Fax: (781) 861-1164. E-mail: jared.silverman{at}cubist.com. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Alborn, W. E., Jr., N. E. Allen, and D. A. Preston. 1991. Daptomycin disrupts membrane potential in growing Staphylococcus aureus. Antimicrob. Agents Chemother. 35:2282-2287.[Abstract/Free Full Text]
  2. Allen, N. E., W. E. Alborn, Jr., and J. N. Hobbs, Jr. 1991. Inhibition of membrane potential-dependent amino acid transport by daptomycin. Antimicrob. Agents Chemother. 35:2639-2642.[Abstract/Free Full Text]
  3. Barry, A. L., P. C. Fuchs, and S. D. Brown. 2001. In vitro activities of daptomycin against 2,789 clinical isolates from 11 North American medical centers. Antimicrob. Agents Chemother. 45:1919-1922.[Abstract/Free Full Text]
  4. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.
  5. Boaretti, M., P. Canepari, M. D. M. Lleo, and G. Satta. 1993. The activity of daptomycin on Enterococcus faecium protoplasts: indirect evidence supporting a novel mode of action on lipoteichoic acid synthesis. J. Antimicrob. Chemother. 31:227-235.[Abstract/Free Full Text]
  6. Breukink, E., I. Wiedemann, C. van Kraaij, O. P. Kuipers, H. Sahl, and B. de Kruijff. 1999. Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 286:2361-2364.[Abstract/Free Full Text]
  7. Canepari, P., M. Boaretti, M. del Mar Lleo, and G. Satta. 1990. Lipoteichoic acid as a new target for activity of antibiotics: mode of action of daptomycin (LY146032). Antimicrob. Agents Chemother. 34:1220-1226.[Abstract/Free Full Text]
  8. Fischer, W. 1988. Physiology of lipoteichoic acids in bacteria. Adv. Microb. Physiol. 29:233-302.[Medline]
  9. Fischer, W. 1994. Lipoteichoic acid and lipids in the membrane of Staphylococcus aureus. Med. Microbiol. Immunol. (Berlin) 183:61-76.[CrossRef][Medline]
  10. Fuchs, P. C., A. L. Barry, and S. D. Brown. 2002. In vitro bactericidal activity of daptomycin against staphylococci. J. Antimicrob. Chemother. 49:467-470.[Abstract/Free Full Text]
  11. Greenberg, J. W., W. Fischer, and K. A. Joiner. 1996. Influence of lipoteichoic acid structure on recognition by the macrophage scavenger receptor. Infect. Immun. 64:3318-3325.[Abstract]
  12. Kessler, R. E., and G. D. Shockman. 1979. Precursor-product relationship of intracellular and extracellular lipoteichoic acids of Streptococcus faecium. J. Bacteriol. 137:869-877.[Abstract/Free Full Text]
  13. King, A., and I. Phillips. 2001. The in vitro activity of daptomycin against 514 Gram-positive aerobic clinical isolates. J. Antimicrob. Chemother. 48:219-223.[Abstract/Free Full Text]
  14. Lamp, K. C., M. J. Rybak, E. M. Bailey, and G. W. Kaatz. 1992. In vitro pharmacodynamic effects of concentration, pH, and growth phase on serum bactericidal activities of daptomycin and vancomycin. Antimicrob. Agents Chemother. 36:2709-2714.[Abstract/Free Full Text]
  15. Ramon, M. S., E. Canton, J. Peman, A. Pastor, and J. P. Martinez. 1999. Mechanisms of action of quinolines against staphylococci and relationship with their in vitro bactericidal activity. Chemotherapy 45:175-182.[CrossRef][Medline]
  16. Rybak, M. J., E. Hershberger, T. Moldovan, and R. G. Grucz. 2000. In vitro activities of daptomycin, vancomycin, linezolid, and quinupristin-dalfopristin against staphylococci and enterococci, including vancomycin-intermediate and -resistant strains. Antimicrob. Agents Chemother. 44:1062-1066.[Abstract/Free Full Text]
  17. Silverman, J. A., N. Oliver, T. Andrew, and T. Li. 2001. Resistance studies with daptomycin. Antimicrob. Agents Chemother. 45:1799-1802.[Abstract/Free Full Text]
  18. Silverman, J. A., N. G. Perlmutter, and H. M. Shapiro. 2003. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob. Agents Chemother. 47:2538-2544.[Abstract/Free Full Text]
  19. Snydman, D. R., N. V. Jacobus, L. A. McDermott, J. R. Lonks, and J. M. Boyce. 2000. Comparative in vitro activities of daptomycin and vancomycin against resistant gram-positive pathogens. Antimicrob. Agents Chemother. 44:3447-3450.[Abstract/Free Full Text]
  20. Wise, R., J. M. Andrews, and J. P. Ashby. 2001. Activity of daptomycin against Gram-positive pathogens: a comparison with other agents and the determination of a tentative breakpoint. J. Antimicrob. Chemother. 48:563-567.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, August 2003, p. 2682-2684, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2682-2684.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Laganas, V.
Right arrow Articles by Silverman, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Laganas, V.
Right arrow Articles by Silverman, J. A.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS