The in vitro activity of daptomycin against 514 Gram-positive aerobic clinical isolates

Anna King*, and Ian Phillips

Department of Infection, KCL, St Thomas' Hospital Campus, London SE1 7EH, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activity of daptomycin was assessed in comparison with that of vancomycin and penicillin against a wide range of Gram-positive aerobic clinical isolates. MICs were determined by an agar dilution method on Mueller–Hinton agar (NCCLS/EUCAST) and on Isotonic agar adjusted to contain 50 mg/L free calcium (BSAC). Both media were enriched with 5% horse blood for fastidious organisms. Daptomycin MICs for all 172 staphylococci, including methicillin-susceptible and methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus, were 0.03–0.5 mg/L. For 99 of the 100 enterococci (Enterococcus faecalis, n = 50; Enterococcus faecium, n = 50), including 37 vancomycin-resistant isolates, they were 0.25–2 mg/L. For all 108 ß-haemolytic streptococci, including Streptococcus pyogenes and Streptococcus agalactiae, daptomycin MICs were 0.016– 0.25 mg/L; for 101 {alpha}-haemolytic streptococci, including Streptococcus pneumoniae and ‘viridans’ streptococci, they were 0.016–2 mg/L. For miscellaneous vancomycin-resistant isolates including Lactobacillus spp., Lactococcus spp., Leuconostoc spp., Pediococcus spp. and isolates of Enterococcus casseliflavus and Enterococcus gallinarum, daptomycin MICs were 0.03–2 mg/L; MICs for the seven isolates of Listeria monocytogenes were 0.25–4 mg/L. There was little difference between the results on Mueller–Hinton agar and on supplemented Isotonic agar The discrepant results occasionally obtained tended to be one dilution higher on supplemented Isotonic agar. Daptomycin was active (MICs <= 2 mg/L) against all the isolates tested with the exception of one isolate each of E. faecium and L. monocytogenes (MICs = 4 mg/L). Our results indicate that daptomycin MICs are independent of methicillin and vancomycin MICs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The recent increase in infections caused by resistant Gram-positive aerobic pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and penicillin-resistant Streptococcus pneumoniae, and the reports of S. aureus with intermediate resistance to glycopeptides are causing concern and have created interest in new antimicrobials with activity against these organisms. Daptomycin, a cyclic lipopeptide antibiotic1 with in vitro bactericidal activity against Gram-positive bacteria, including isolates resistant to other antimicrobial agents,2 is one such antibiotic.

Daptomycin was discovered in the early 1980s and was shown to be effective in patients with skin and soft tissue infections; however, because of mild skeletal muscle toxicity at high doses and treatment failures in patients with S. aureus endocarditis,1,3 clinical trials were suspended. Since this time the marked increase in isolation of resistant Gram-positive organisms prompted Cubist Pharmaceuticals to conduct further clinical trials with dosing regimens unlikely to cause toxicity. The precise mechanism of action of daptomycin is not completely understood, but it is know that it kills bacteria by disrupting membrane function; possible mechanisms include inhibition of peptidoglycan synthesis, inhibition of lipotechoic acid synthesis and alterations of cytoplasmic membrane potential.46 The bactericidal activity of daptomycin is concentration-dependent and is influenced by pH and ionized calcium concentration.7

In this study we compared the in vitro activity of daptomycin with that of penicillin and vancomycin against a wide variety of Gram-positive aerobic bacteria, including those known to be resistant to other antibiotics. We also compared results obtained by NCCLS/EUCAST810 and BSAC11,12 methods.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial agents

The following antimicrobial agents were tested as powders of known potency: daptomycin (Cubist Pharmaceuticals, Cambridge, MA, USA), penicillin (SmithKline Beecham, Harlow, UK) and vancomycin (Sigma–Aldrich Co. Ltd, Poole, UK). Breakpoints for penicillin and vancomycin were as stated either in the NCCLS8,9 or BSAC11,12 methods. Tentative MIC breakpoints for daptomycin are: susceptible <=2 mg/L, intermediate 4 mg/L and resistant >=8 mg/L; tentative NCCLS zone diameter breakpoints are: <=12 mm for resistant and >=16 mm for susceptible.13

Organisms

The organisms (n = 514) included in the study were all clinical isolates from St Thomas' Hospital and were selected to represent the different Gram-positive aerobic species isolated from infections. The isolates were further selected to include, where possible, those known to have specific resistance mechanisms including vancomycin, methicillin and erythromycin resistance. For commonly isolated species the isolates were recent but for less common species the isolates were from collections made over the past 10 years.

Susceptibility testing

MICs were determined by an agar dilution method on Mueller–Hinton agar (Oxoid Ltd, Basingstoke, UK) and Isotonic agar adjusted to contain 50 mg/L Ca2+ supplemented Isotonic agar (Mast Laboratories, Bootle, UK). Both media were enriched with 5% horse blood (E & O Laboratories, Bonnybridge, UK) for fastidious organisms. Organisms were grown overnight in brain–heart infusion broth (Oxoid) and diluted in sterile distilled water, or suspended in water directly from a fresh culture to match a 0.5 McFarland turbidity standard. These suspensions were further diluted 1/10 in IsoSensitest broth (Oxoid) and inoculated on the agar with a multipoint inoculator (Denley, Burgess Hill, UK) to give a final inoculum size of c. 104 cfu/spot. The plates were incubated for 20–24 h at 37°C in air (with 5% added CO2 for S. pneumoniae).

Disc diffusion susceptibility was determined on either Mueller–Hinton agar (NCCLS) or supplemented Isotonic agar (BSAC). Both media were enriched with 5% horse blood for fastidious organisms. The inoculum for the NCCLS method was prepared by making a suspension in sterile distilled water to match a 0.5 McFarland turbidity standard. For the BSAC method the inoculum was prepared by diluting the suspension equivalent to 0.5 McFarland standard either 1/10 or 1/100, depending on the species, to produce semi-confluent growth. The disc contents tested were 30 µg daptomycin (BBL, Becton Dickinson, Cockysville, MD, USA) for both methods, 10 µg (NCCLS) or 1 µg (BSAC) penicillin (Oxoid), and 30 µg (NCCLS) or 5 µg (BSAC) vancomycin (Oxoid). Plates were incubated for 20–24 h at 37°C in air (with 5% added CO2 for S. pneumoniae). Methicillin susceptibility was determined for all staphylococci by disc diffusion11 with 5 µg discs (Oxoid). The presence of mec(A) in all S. aureus and Staphylococcus epidermidis shown to be resistant by disc testing was confirmed by PCR.14


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The range of MICs, MIC50 and MIC90 for each species for each method are listed in the TableGo. Daptomycin was active against all isolates of staphylococci (MICs 0.03–0.5 mg/L on Mueller–Hinton agar by the NCCLS/EUCAST method, and 0.03–1 mg/L on supplemented Isotonic agar by the BSAC method). Daptomycin MICs for enterococci were in the range 0.25–2 mg/L, with the exception of one vancomycin-resistant E. faecium, which had a daptomycin MIC of 4 mg/L on both media. Daptomycin was highly active against ß-haemolytic streptococci Groups A, C and G (MICs 0.016–0.06 mg/L) and was only slightly less active against Streptococcus agalactiae and S. pneumoniae (MICs 0.06–0.25 mg/L on Mueller–Hinton agar, and 0.125–1 mg/L on supplemented Isotonic agar). Daptomycin was also active against the ‘viridans’ streptococci (MICs 0.016– 2 mg/L on both agars) and against the miscellaneous Gram-positive isolates, mostly vancomycin resistant, included in the study. Among isolates of Listeria monocytogenes, one isolate had a daptomycin MIC of 4 mg/L on both agars, and a further two isolates had daptomycin MICs of 1 mg/L on Mueller–Hinton agar but 4 mg/L on supplemented Isotonic agar; most were inhibited by 2 mg/L or less. There was no evidence that daptomycin MICs were increased for isolates known to be resistant to other antimicrobials, particularly penicillin and vancomycin.


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Table. In vitro activity of daptomycin, penicillin and vancomycin (MICs, mg/L)
 
There was little difference in results between the methods, although daptomycin MICs tended to be one dilution higher on supplemented Isotonic agar than on Mueller– Hinton agar, and penicillin MICs for methicillin-susceptible but penicillin-resistant S. aureus were two- to 32-fold lower on supplemented Isotonic agar than on Mueller– Hinton agar. Differences were greater for organisms with higher penicillin MICs on Mueller–Hinton agar. Correlation between NCCLS/EUCAST and BSAC methods for daptomycin was good (r = 0.892) and, for the majority of isolates, the daptomycin MICs were either the same on both media or one dilution higher on supplemented Isotonic agar. However, for 43 (7.6%) of the isolates (nine of 68 S. epidermidis, 27 of 50 S. agalactiae, three of seven L. monocytogenes and one each of Streptococcus pyogenes, S. pneumoniae, Streptococcus bovis and Pediococcus pentosaceus) results were two dilutions higher on supplemented Isotonic agar than on Mueller–Hinton agar. The daptomycin MIC for one isolate of Enterococcus faecium was two dilutions lower on supplemented Isotonic agar and there was one isolate of S. pyogenes for which the MIC was repeatedly three dilutions higher on supplemented Isotonic agar than on Mueller–Hinton agar.

Correlation between NCCLS and BSAC methods for daptomycin disc diffusion was also good (r = 0.815) but daptomycin zone diameters were 2–3 mm larger on supplemented Isotonic agar than on Mueller–Hinton agar because of the smaller inoculum. However, the correlation between MIC and zone diameter was poor for both methods (r = 0.472 NCCLS and 0.437 BSAC), as shown in the FigureGo. A tentative NCCLS breakpoint of 16 mm is appropriate for the susceptible isolates. However, the two isolates with decreased susceptibility (daptomycin MICs 4 mg/L) had zone diameters >16 mm. Similar discrimination by the BSAC method would be a zone breakpoint of 18 mm, but again the four isolates with decreased susceptibility would not be detected. Correlations between the two methods of MIC determination were very good for penicillin (r = 0.975) and vancomycin (r = 0.988), as were the correlations between the methods for zone diameters of penicillin (r = 0.921) and vancomycin (r = 0.937). Correlations between MICs and zone diameters for both penicillin (r = 0.933 for NCCLS and 0.903 for BSAC) and vancomycin (r = 0.888 for NCCLS and 0.881 for BSAC) were also very good. Species-specific breakpoints for penicillin discriminated well in both methods and there were no errors of interpretation. The breakpoints for vancomycin were likewise species-specific for the NCCLS method but discriminated well, with the exception of one enterococcus, with a vancomycin MIC of 8 mg/L and a zone diameter of 17 mm. The lower disc content of vancomycin for the BSAC method and smaller zone breakpoint discriminated well between susceptible and resistant isolates.



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Figure. Correlation of daptomycin MICs and zone diameters for all isolates on (a) Mueller–Hinton agar and (b) supplemented Isotonic agar. The dashed line represents susceptibility breakpoints.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Daptomycin was active against all the isolates tested with the exception of two isolates, an E. faecium and a L. monocytogenes, both of intermediate susceptibility. There was no apparent increase in daptomycin MICs for isolates known to be resistant to other antimicrobials.

The potency of daptomycin is known to be dependent on free calcium content.13 Preliminary results on both Oxoid IsoSensitest agar and Mast Isotonic agar showed that the Ca2+ content was insufficient, resulting in high MICs and small zone diameters. The recommended BSAC media for susceptibility testing were therefore initially considered unsuitable for testing daptomycin. However, Mast Laboratories supplemented Isotonic agar with 50 mg/L Ca2+ and this agar was used for this study. MIC results on supplemented Isotonic agar were similar to those on Mueller– Hinton agar and for the majority of isolates were either the same or differed by only one doubling dilution. Where there were differences, MICs were more often higher on supplemented Isotonic agar (mean MIC 0.306 mg/L) than on Mueller–Hinton (mean MIC 0.201 mg/L). This appears to reflect either the difference in Ca2+ content, another chemical difference in the media or the fact that most isolates grew slightly better on supplemented Isotonic agar than on Mueller–Hinton agar. However, since results for penicillin and vancomycin did not differ in this way, the discrepancy is probably not associated with the quality of growth. This small difference in MICs had no implications for designating isolates as resistant. However, two isolates were susceptible on Mueller–Hinton but intermediate on supplemented Isotonic agar. No isolate had a daptomycin MIC in the resistant range on either agar. Penicillin MICs for methicillin-susceptible, penicillin-resistant S. aureus were lower on supplemented Isotonic agar than on Mueller–Hinton agar, possibly because of sodium chloride content, but this had no implications in designating the isolates as resistant. MIC determinations were repeated for some of the isolates and the results were reproducible. This phenomenon clearly needs to be investigated further.

Zone diameters determined by the NCCLS method were smaller than those determined by the BSAC method. This is not surprising since the inoculum size is different. The tentative breakpoints suggested by NCCLS seem relevant and a tentative breakpoint of >=18 mm for susceptible organisms would be suitable for the BSAC method. However, the two isolates of intermediate susceptibility included in this study both had zone diameters larger than the breakpoint and would not have been detected by either method. This is consistent with an earlier study13 in which 12 enterococci with daptomycin MICs of 4 mg/L all had zone diameters >18 mm with a 30 µg daptomycin disc. There were no resistant isolates to test the proposed resistance breakpoint. In view of the two isolates of intermediate susceptibility and the poor correlation between MIC and zone diameter when all isolates are analysed together (NCCLS r = 0.472, BSAC r = 0.437), it may be necessary to introduce species-specific breakpoints, as with penicillin and vancomycin. Furthermore, the tentative breakpoints suggested may well be altered when the results of Phase III clinical trials have been analysed.

The distribution of daptomycin MICs seen in this study, which agree with those of other reports,1,3 show daptomycin to be active in vitro against a wide range of Gram-positive aerobic species, including those resistant to ß-lactams and glycopeptides. Daptomycin is potentially a useful antimicrobial agent, particularly against isolates resistant to other classes of antibiotic, but its clinical role will also depend on pharmacokinetic and safety profiles.


    Acknowledgments
 
We thank N. Percival and D. Kinnear for technical assistance. This study was supported by a grant from Cubist Pharmaceuticals, Inc.


    Notes
 
* Corresponding author. Tel: +44-20-7928-9292 ext. 2456; Fax: +44-20-7928-0730; E-mail: anna.king{at}kcl.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Tally, F. P., Zeckel, M., Wasilewski, M. M., Carini, C., Berman, C. L., Drusano, G. L. et al. (1999). Daptomycin: a novel agent for Gram-positive infections. Expert Opinion on Investigational Drugs 8, 1223–38.

2 . Jacobus, N. V., McDermott, L., Lonks, J. R., Boyce, J. M. & Snydman, D. R. (1998). In-vitro activity of daptomycin against resistant gram-positive pathogens. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. Abstract F112, p. 260. American Society for Microbiology, Washington, DC.

3 . Rybak, M. J., Hershberger, E., Moldovan, T. & Grucz, R. G. (2000). In vitro activities of daptomycin, vancomycin, linezolid, and quinupristin–dalfopristin against staphylococci and enterococci, including vancomycin-intermediate and -resistant strains. Antimicrobial Agents and Chemotherapy 44, 1062–6.[Abstract/Free Full Text]

4 . Canepari, P., Boaretti, M., Del Mar Lleo, M. & Satta G. (1990). Lipoteichoic acid as a target for activity of antibiotics. Mode of action of daptomycin (LY146032). Antimicrobial Agents and Chemotherapy 34, 1220–6.[ISI][Medline]

5 . Allen, N. E., Hobbs, J. N. & Alborn, W. E. (1987). Inhibition of peptidoglycan biosynthesis in gram-positive bacteria by LY146032. Antimicrobial Agents and Chemotherapy 31, 1093–9.[ISI][Medline]

6 . Alborn, W. E., Jr, Allen, N. E. & Preston, D. A. (1991). Daptomycin disrupts membrane potential in growing Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 35, 2282–7.[ISI][Medline]

7 . Lamp, K. C. & Rybak, M. J. (1993). Teicoplanin and daptomycin bactericidal activities in the presence of albumin or serum under controlled conditions of pH and ionized calcium. Antimicrobial Agents and Chemotherapy 37, 605–9.[Abstract]

8 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

9 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Disk Susceptibility Tests—Sixth Edition: Approved Standard M2-A6. NCCLS, Wayne, PA.

10 . European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). (2000). Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Clinical Microbiology and Infection 6, 509–15.[ISI][Medline]

11 . Andrews, J. M. for the BSAC Working Party on Susceptibility Testing. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.[Abstract/Free Full Text]

12 . Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract/Free Full Text]

13 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (1999). Daptomycin susceptibility tests: Provisional criteria, Quality Control, and importance of Ca++ concentrations in test media. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA. Abstract 350, p. 199. American Society for Microbiology, Washington, DC.

14 . Kobayashi, N., Wu, H., Kojima, K., Taniguchi, K., Urasawa, S., Uehara, N. et al. (1994). Detection of mecA, femA and femB genes in clinical strains of staphylococci using polymerase chain reaction. Epidemiology and Infection 113, 259–66.[ISI][Medline]

Received 30 January 2001; returned 9 April 2001; revised 9 May 2001; accepted 22 May 2001