Test results: characterising the antimicrobial activity of daptomycin B. Wiedemann

REVIEW Test results: characterising the antimicrobial activity of daptomycin B. Wiedemann University of Bonn, Bonn, Germany ABSTRACT Daptomycin is the first in a new class of antibiotics, the cyclic lipopeptides. This review discusses the extensive in vitro tests performed to characterise the antimicrobial activity of daptomycin. These tests have established daptomycin s activity against most Gram-positive bacteria, including methicillinsensitive and -resistant Staphylococcus aureus, vancomycin-resistant enterococci and multidrug-resistant coagulase-negative staphylococci. Daptomycin possesses rapid, concentration-dependent bactericidal activity against Gram-positive organisms. Interestingly, this in vitro bactericidal activity is maintained at high inoculum densities and against bacteria in the stationary phase of growth. In addition, in vitro tests have demonstrated a prolonged, concentration-dependent post-antibiotic effect against Gram-positive bacteria. The level of protein binding of daptomycin is high. However, daptomycin binds only weakly to albumin, and is two- to four-fold more active than would be predicted from its free drug concentration. In vitro studies have shown low spontaneous resistance rates of <10 )10 for S. aureus and <10 )9 for Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium. In summary, in vitro testing clearly demonstrates that daptomycin is an exciting new option for the treatment of Gram-positive infections. Keywords Daptomycin, Gram-positive bacteria, lipopeptides, Staphylococcus aureus Clin Microbiol Infect 2006; 12 (Suppl 8): 9 14 INTRODUCTION Daptomycin was originally isolated as a fermentation product of the soil-dwelling bacterium Streptomyces roseosporus. This cyclic lipopeptide antibiotic has recently been licensed in Europe for the treatment of complicated skin and soft tissue infections (csstis) in adults. During the drug development process and beyond, daptomycin has undergone rigorous tests to establish and characterise its antimicrobial activity. The objective of this article is to summarise and discuss the results of these tests, with particular emphasis on the most recent data describing the in vitro activity of daptomycin, its bactericidal activity, protein binding, activity in biofilms, post-antibiotic effect (PAE), synergistic interactions with other antibiotics, and potential for resistance development. STRUCTURE AND MODE OF ACTION Daptomycin is a 13-amino-acid lipopeptide comprising a hydrophilic core and a lipophilic tail. Corresponding author and reprint requests: B. Wiedemann, Böstens Hoi 15, 24882 Schaalby, Germany E-mail: pharmic@t-online.de This unique structure confers upon daptomycin a completely novel mode of action involving insertion into the cytoplasmic membrane of Grampositive bacteria and the formation of daptomycin pores. This Ca 2+ -dependent process leads to efflux of cell components, particularly potassium ions, and subsequent membrane depolarisation and cell death. Daptomycin kills bacteria with negligible cell lysis, so the theoretical risks associated with bacterial cell lysis including the release of bacterial exotoxins into the circulation are potentially minimised [1,2]. The mode of action of daptomycin has been described in some detail elsewhere [2,3]. IN-VITRO ACTIVITY Daptomycin is active in vitro against most Grampositive bacteria, including clinically important drug-resistant pathogens such as methicillinresistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and multidrug-resistant coagulase-negative staphylococci [4 6]. This broad-ranging activity was confirmed in a recent surveillance study involving the collection of Gram-positive isolates from 24 medical centres in 12 European countries Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

10 Clinical Microbiology and Infection, Volume 12 Supplement 8, 2006 (H. Sader et al. ECCMID 2005). In this study, daptomycin was shown to be active against S. aureus and coagulase-negative staphylococci with MIC 90 values of 0.5 mg/l. In-vitro activity against these pathogens was maintained irrespective of the presence or absence of methicillin resistance. MIC distribution data While MIC 50 and MIC 90 data are undeniably useful, the in-vitro activity of antibiotics such as daptomycin can be described in more detail by MIC distribution data. The concept of antimicrobial wild-type susceptibility distributions has been developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [7]. Through its website (http://www.srga.org/ eucastwt/wt_eucast.htm), EUCAST displays aggregate MIC distributions for numerous antimicrobial microorganism combinations. Each aggregate is formed from individual MIC distributions obtained from a variety of worldwide sources, including peer-reviewed publications, national breakpoint committees, pharmaceutical companies and antimicrobial resistance surveillance projects. Only wild-type distribution data (i.e., data for bacteria without acquired resistance to the antibiotic agent of interest) are available from EUCAST. Aggregate MIC distributions for daptomycin against a range of Gram-positive bacteria are shown in Table 1. For each bacterial species, peaks of activity for daptomycin can be clearly seen. Pathogens with particular sensitivity to daptomycin identified from the EUCAST data include Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, S. aureus, S. epidermidis and the coagulase-negative staphylococci. Activity against glycopeptide-intermediate and -resistant pathogens The emergence of glycopeptide resistance in S. aureus is a well-established and worrying phenomenon. Although fully vancomycin-resistant S. aureus (VRSA) organisms are rare, vancomycinintermediate strains are more common [8]. Daptomycin has been shown to be active against VRSA, vancomycin-intermediate S. aureus (VISA) Table 1. MIC distributions for daptomycin [7] MIC (mg/l) 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 Streptococcus pyogenes (n ¼ 825) 1 16 64 17 2 0 0 0 0 0 0 Streptococcus bovis (n ¼ 91) 2 5 62 29 2 0 0 0 0 0 0 Streptococcus group G (n ¼ 269) 0 11 61 25 3 0 0 0 0 0 0 Streptococcus agalactiae (n ¼ 1686) 0 0 21 48 26 4 0 0 0 0 0 Corynebacterium jeikeium (n ¼ 10) 0 0 30 60 10 0 0 0 0 0 0 Streptococcus pneumoniae (n ¼ 7661) 0 0 3 63 30 4 0 0 0 0 0 Clostridium species (n ¼ 68) 0 3 26 35 24 4 3 0 0 3 1 Staphylococcus hominis (n ¼ 192) 0 0 1 16 64 18 1 0 0 0 0 Streptococcus mitis (n ¼ 142) 0 1 3 8 37 35 17 0 0 0 0 Streptococcus viridans (n ¼ 659) 0 1 9 18 30 29 13 0 0 0 0 Staphylococcus aureus (n ¼ 35 965) 0 0 2 22 51 23 2 0 0 0 0 Staphylococcus epidermidis (n ¼ 1979) 0 0 2 9 47 40 3 0 0 0 0 Staphylococcus, coagulase-negative (n ¼ 8966) 0 0 7 27 42 21 2 0 0 0 0 Staphylococcus haemolyticus (n ¼ 258) 0 0 0 12 48 37 3 0 0 0 0 Staphylococcus capitis (n ¼ 30) 0 0 0 0 10 53 37 0 0 0 0 Streptococcus anginosus (n ¼ 22) 0 0 5 0 41 55 0 0 0 0 0 Streptococcus oralis (n ¼ 50) 0 0 0 8 20 44 28 0 0 0 0 Enterococcus faecalis (n ¼ 9435) 0 0 0 1 5 31 42 18 3 0 0 Enterococcus faecium (n ¼ 12 308) 0 0 0 0 0 1 6 84 8 0 0 Listeria monocytogenes (n ¼ 78) 0 0 0 0 1 1 33 54 10 0 0 For each MIC value, data represent the percentage of tested isolates (n) with an equivalent MIC value. Values in bold represent the most common MIC for each species. For example, 64% of Streptococcus pyogenes isolates were associated with a daptomycin MIC of 0.064 mg/l. Data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) website (2005).

Wiedemann Antimicrobial activity of daptomycin 11 and other vancomycin-intermediate staphylococci [9,10]. Recent reports have proposed a correlation between reduced daptomycin susceptibility and vancomycin resistance in MRSA isolates from patients exposed to vancomycin, VISA and S. aureus passaged in vancomycin-containing medium [11,12]. However, neither of these studies looked at minimum bactericidal concentrations. Tolerance of S. aureus to vancomycin is common, while tolerance to daptomycin has not yet been observed [13,14]. Therefore, it remains to be clarified whether reports of reduced susceptibility to daptomycin following exposure to vancomycin represent true cross-resistance. In-vitro testing methods Consistent with the Ca 2+ -dependent mode of action of daptomycin, in-vitro testing methods require physiological concentrations of free calcium ions (50 mg/l) [15]. Broth microdilution is the gold standard susceptibility testing method for daptomycin, and E-test methods are also reliable. Agar dilution methods are not recommended Ca 2+ levels in agar vary and daptomycin does not diffuse easily through agar. Daptomycin has a high molecular mass (1620 Da); all antibiotics with a molecular mass over approximately 700 Da have poor diffusion characteristics from paper disks into the surface agar (e.g., teicoplanin, 1993 Da) [15]. Disk diffusion methods are not available for susceptibility testing with daptomycin. Daptomycin panels and cards are available for use with several automated and semi-automated susceptibility testing systems [15]. The following daptomycin MIC breakpoints have been established by EUCAST for both staphylococci and streptococci (excluding Streptococcus pneumoniae): 1 mg/l (susceptible) and >1 mg/l (resistant) [16]. BACTERICIDAL ACTIVITY Bactericidal antibiotic agents offer potential advantages over bacteriostatic agents, including the potential suppression of resistance selection and rapid bactericidal effects that are independent of the host immune response [17,18]. In-vitro time-kill studies have shown that daptomycin possesses rapid, concentration-dependent bactericidal activity against Gram-positive organisms [4,19]. For example, daptomycin has been shown to reduce S. aureus viable cell counts (including MRSA) by more than three orders of magnitude in <2 h at concentrations two to four times the MIC [19]. The bactericidal activity of daptomycin is reduced only slightly by an increase in inoculum density. In an in-vitro study utilising simulated bacterial vegetations, daptomycin retained its bactericidal activity against both moderate- and high-density MRSA and methicillin-susceptible S. aureus (MSSA) inocula [20]. In contrast, no reduction in cell counts was seen at either inoculum density with the bacteriostatic agent linezolid, while the bactericidal activities of nafcillin and vancomycin were greatly diminished at high inoculum densities. The bactericidal activity of daptomycin is also maintained in the stationary phase of bacterial growth [21]. In a recent study, the in-vivo bactericidal activity of daptomycin, unlike those of ciprofloxacin and nafcillin, was maintained against cold-arrested, non-growing S. aureus (C. Mascio et al. ICAAC 2005). PROTEIN BINDING Pharmacokinetic studies have shown that daptomycin binds reversibly to human plasma proteins, primarily albumin, in a concentration-dependent manner [22]. Although the level of protein binding is high (90% at 4 g/100 ml), daptomycin binds only weakly to albumin and daptomycin is two- to four-fold more active than would be predicted from its free drug concentration (W. Craig et al. IDSA 2004). Further evidence for weak and/or highly reversible protein binding was provided in a recent in-vitro study demonstrating only minimal effects of human serum on the bactericidal activity of daptomycin (I. Morissey et al. ECCMID 2006). In this study, exponentially growing MSSA, MRSA, vancomycin-susceptible Enterococcus faecium and VRE were exposed to daptomycin, vancomycin, teicoplanin, piperacillin tazobactam or linezolid in the presence and absence of 50% human serum. To compare the bactericidal activities of each antibiotic agent, bactericidal indices were calculated by plotting the log reduction in viable cell counts over time and measuring the resulting area-under-the-curve. With or without the addition of human serum, daptomycin was

12 Clinical Microbiology and Infection, Volume 12 Supplement 8, 2006 the most bactericidal agent; however, the serum concentration used was far greater than physiological levels. ACTIVITY IN BIOFILMS Biofilms are extracellular matrices that allow bacteria to accumulate at high densities. By acting as a physical barrier to antibiotics, and through other mechanisms, biofilms may prevent effective treatment and/or promote the development of antibiotic resistance. Raad et al. evaluated the activity of daptomycin, linezolid, minocycline and quinupristin dalfopristin against bacteria in biofilms generated from vancomycin-resistant E. faecium isolates from patients with catheter-related bacteraemia [23]. While the colonisation densities of linezolidtreated and control water-treated biofilms were comparable, daptomycin produced an approximately 97% reduction in the number of CFUs (Fig. 1). Daptomycin was the most effective antibiotic tested, producing a greater reduction in the number of E. faecium CFUs than minocycline, quinupristin dalfopristin and linezolid. At a simulated dose of 6 mg/kg, daptomycin also demonstrated potent bactericidal activity in vivo against implanted endocardial MRSA vegetations in rats (L. Mortin et al. American Society for Microbiology 2003). The activity in biofilms of daptomycin and other antibiotic agents is an important area for future research. POST-ANTIBIOTIC EFFECT AND SYNERGY The maintained suppression of bacterial growth following exposure to an antibiotic is referred to as the post-antibiotic effect (PAE). Although their clinical significance has not been fully determined, prolonged PAEs measured in vitro may indicate the suppression of bacterial growth in vivo during periods of low drug concentration [24]. In-vitro experiments with daptomycin have revealed a prolonged, concentration-dependent PAE against Gram-positive bacteria [25,26]. Hanberger et al. measured the PAE of daptomycin using viable cell counts and a bioluminescence assay of bacterial ATP [25]. At clinically achievable concentrations, and in the presence of physiological levels of free Ca 2+, daptomycin produced a PAE of over 6 h against Enterococcus faecalis and S. aureus. Synergistic interactions between daptomycin and some other antibiotics have been demonstrated in vitro. For example, both rifampicin and ampicillin show synergistic activity with daptomycin against VRE [27,28]. Importantly, antagonistic activity following combination of daptomycin with other antibiotics has not been reported. RESISTANCE The development of antibiotic resistance is a complex process and first requires the presence of a gene complex mediating resistance to an antibiotic agent (Fig. 2). Either by mutation or by uptake of resistance plasmids, this gene complex must enter the genome of a suitable pathogen. Selection pressure must then favour the drugresistant pathogen. Providing that it is able to survive in its specific ecosystem, the drug-resistant pathogen may then be able to spread to the wider environment. Fig. 1. Activity of daptomycin and other antibiotics in biofilms. The activity of daptomycin (DAP), minocycline (MIN), quinupristin dalfopristin (Q-D) and linezolid (LIN) was tested against bacteria in biofilms generated from vancomycin-resistant Enterococcus faecium isolates obtained from patients with catheter-related bacteraemia. Data represent the reduction in number of CFUs measured following the addition of each antibiotic. Adapted from Raad et al. 2005 [23]. In-vitro resistance Studies have shown that daptomycin has low potential for the development of resistance in vitro. Silverman et al. found no spontaneous resistant mutants among organisms exposed to daptomycin at eight times the MIC; resulting spontaneous mutation rates were <10 )10 for S. aureus and <10 )9 for S. epidermidis, E. faecalis, E. faecium and

Wiedemann Antimicrobial activity of daptomycin 13 endocarditis and/or concurrent immunosuppression. There have been no post-marketing reports of daptomycin-resistant isolates in patients treated for csstis. The safety and efficacy of daptomycin for the treatment of S. aureus endocarditis and/or bacteraemia was assessed in a recent phase III clinical study [31]. In this study, daptomycin MICs crept above the CLSI breakpoint (1 mg/l) in seven patients. All of these patients had deepseated infections that required adjunctive therapy (e.g., valve surgery, drainage of infection foci, removal of foreign material). Six patients none of whom received the required adjunctive therapy had persisting or relapsing infection. The seventh patient received adjunctive therapy and was considered a clinical success. SUMMARY Fig. 2. Stages in the development of antibiotic resistance. Streptococcus pneumoniae [29]. In the same study, daptomycin-resistant isolates were difficult to select by serial passage requiring over 20 passages and were infrequently selected by chemical mutagenesis. Consistent with the novel mode of action of daptomycin, none of the daptomycinresistant isolates were cross-resistant to vancomycin or ampicillin. In-vivo resistance The low potential for resistance in vitro is reflected by the low incidence of daptomycin resistance in the clinical setting. More than 1000 patients were treated with daptomycin during phase II and III studies, and resistance emerged in just two of these patients (<0.2%) [16,30]. Since it was launched in the USA in September 2003, more than 120 000 patients have been treated with daptomycin; resistance to daptomycin has been reported in 13 patients (S. aureus, n ¼ 8; E. faecium, n ¼ 4; E. faecalis, n ¼ 1). In all 13 cases, patients had bacteraemia with osteomyelitis, Extensive testing has shown that daptomycin possesses rapid bactericidal activity against a broad range of Gram-positive bacteria. This activity is maintained against both high inocula and non-growing bacterial cells. Daptomycin has a prolonged PAE and shows synergy with certain other antibiotic agents. The potential to induce daptomycin-resistant mutants in vitro is low, and resistant isolates have only rarely been recovered from daptomycin-treated patients. Interesting areas for future testing include further characterisation of daptomycin s mode of action, its protein binding and its activity in biofilms. It is already clear, however, that daptomycin represents an exciting new option for the treatment of Grampositive infections. REFERENCES 1. Ginsburg I. The role of bacteriolysis in the pathophysiology of inflammation, infection and post-infectious sequelae. APMIS 2002; 110: 753 770. 2. Tedesco KL, Rybak MJ. Daptomycin. Pharmacotherapy 2004; 24: 41 57. 3. Silverman JA, Perlmutter NG, Shapiro HM. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob Agents Chemother 2003; 47: 2538 2544. 4. Rybak MJ, Hershberger E, Moldovan T, Grucz RG. In vitro activities of daptomycin, vancomycin, linezolid, and quinupristin dalfopristin against staphylococci and enterococci, including vancomycin-intermediate and - resistant strains. Antimicrob Agents Chemother 2000; 44: 1062 1066.

14 Clinical Microbiology and Infection, Volume 12 Supplement 8, 2006 5. Sader HS, Streit JM, Fritsche TR, Jones RN. Antimicrobial activity of daptomycin against multidrug-resistant Gram-positive strains collected worldwide. Diagn Microbiol Infect Dis 2004; 50: 201 204. 6. Streit JM, Jones RN, Sader HS. Daptomycin activity and spectrum: a worldwide sample of 6737 clinical Grampositive organisms. J Antimicrob Chemother 2004; 53: 669 674. 7. EUCAST. Antimicrobial wild type MIC distributions of microorganisms. 2005. http://www.srga.org/eucastwt/ WT_EUCAST.htm (accessed 11 October 2006). 8. Ruef C. Epidemiology and clinical impact of glycopeptide resistance in Staphylococcus aureus. Infection 2004; 32: 315 327. 9. Chang S, Sievert DM, Hageman JC et al. Infection with vancomycin-resistant Staphylococcus aureus containing the vana resistance gene. N Engl J Med 2003; 348: 1342 1347. 10. Tenover FC, Weigel LM, Appelbaum PC et al. Vancomycin-resistant Staphylococcus aureus isolate from a patient in Pennsylvania. Antimicrob Agents Chemother 2004; 48: 275 280. 11. Sakoulas G, Alder J, Thauvin-Eliopoulos C, Moellering RC Jr, Eliopoulos GM. Induction of daptomycin heterogeneous susceptibility in Staphylococcus aureus by exposure to vancomycin. Antimicrob Agents Chemother 2006; 50: 1581 1585. 12. Cui L, Tominaga E, Neoh HM, Hiramatsu K. Correlation between reduced daptomycin susceptibility and vancomycin resistance in vancomycin-intermediate Staphylococcus aureus. Antimicrob Agents Chemother 2006; 50: 1079 1082. 13. Sader HS, Fritsche TR, Streit JM, Jones RN. Daptomycin in vitro activity tested against Gram-positive strains collected from European and Latin American medical centers in 2003. J Chemother 2005; 17: 477 483. 14. Sader HS, Fritsche TR, Jones RN. Antimicrobial activity of daptomycin tested against clinical strains of indicated species isolated in North American medical centers (2003). Diagn Microbiol Infect Dis 2005; 53: 329 332. 15. Chiron Biopharmaceuticals. Antibiotic susceptibility testing with daptomycin. Chiron Biopharmaceuticals, 2006. 16. CUBICIN Summary of Product Characteristics. 2006. http:// www.emea.eu.int/humandocs/humans/epar/cubicin/ cubicin.htm (accessed 11 October 2006). 17. Finberg RW, Moellering RC, Tally FP et al. The importance of bactericidal drugs: future directions in infectious disease. Clin Infect Dis 2004; 39: 1314 1320. 18. Stratton CW. Dead bugs don t mutate: susceptibility issues in the emergence of bacterial resistance. Emerg Infect Dis 2003; 9: 10 16. 19. Thorne GM, Alder J. Daptomycin: a novel lipopeptide antibiotic. Clin Microbiol Newslett 2002; 24: 33 40. 20. LaPlante KL, Rybak MJ. Impact of high-inoculum Staphylococcus aureus on the activities of nafcillin, vancomycin, linezolid, and daptomycin, alone and in combination with gentamicin, in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 2004; 48: 4665 4672. 21. Lamp KC, Rybak MJ, Bailey EM, Kaatz GW. In vitro pharmacodynamic effects of concentration, ph, and growth phase on serum bactericidal activities of daptomycin and vancomycin. Antimicrob Agents Chemother 1992; 36: 2709 2714. 22. Lee BL, Sachdeva M, Chambers HF. Effect of protein binding of daptomycin on MIC and antibacterial activity. Antimicrob Agents Chemother 1991; 35: 2505 2508. 23. Raad II, Hanna HA, Boktour M et al. Vancomycin-resistant Enterococcus faecium: catheter colonization, esp gene, and decreased susceptibility to antibiotics in biofilm. Antimicrob Agents Chemother 2005; 49: 5046 5050. 24. Eisenstein BI. Lipopeptides, focusing on daptomycin, for the treatment of Gram-positive infections. Expert Opin Invest Drugs 2004; 13: 1159 1169. 25. Hanberger H, Nilsson LE, Maller R, Isaksson B. Pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca 2+ and albumin on these drugs. Antimicrob Agents Chemother 1991; 35: 1710 1716. 26. Bush LM, Boscia JA, Wendeler M, Pitsakis PG, Kaye D. In vitro postantibiotic effect of daptomycin (LY146032) against Enterococcus faecalis and methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains. Antimicrob Agents Chemother 1989; 33: 1198 1200. 27. Pankey G, Ashcraft D, Patel N. In vitro synergy of daptomycin plus rifampin against Enterococcus faecium resistant to both linezolid and vancomycin. Antimicrob Agents Chemother 2005; 49: 5166 5168. 28. Rand KH, Houck H. Daptomycin synergy with rifampicin and ampicillin against vancomycin-resistant enterococci. J Antimicrob Chemother 2004; 53: 530 532. 29. Silverman JA, Oliver N, Andrew T, Li T. Resistance studies with daptomycin. Antimicrob Agents Chemother 2001; 45: 1799 1802. 30. European Public Assessment Report for CUBICIN. 2006. http://www.emea.eu.int/humandocs/humans/epar/ cubicin/cubicin.htm (accessed 11 October 2006). 31. Flower VG Jr, Boucher HW, Corey GR et al. Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphyloccus aureus. N Engl J Med 2006; 355: 653 665.