JAC Advance Access originally published online on February 12, 2004
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Journal of Antimicrobial Chemotherapy (2004) 53, 530-532
© 2004 The British Society for Antimicrobial Chemotherapy
Daptomycin synergy with rifampicin and ampicillin against vancomycin-resistant enterococci
Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA
Received 28 August 2003; returned 29 October 2003; revised 5 December 2003; accepted 8 December 2003
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Abstract |
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We used a novel screening method to look for synergy between daptomycin and 18 other antibiotics against 19 strains of high-level vancomycin-resistant enterococci (VRE) (vancomycin MIC
256 mg/L). In this approach, daptomycin was incorporated into Ca2+-supplemented Mueller–Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Etest MICs on agar with and without daptomycin. A striking reduction in the rifampicin MIC was seen in 11/15 (73.3%) VRE that were resistant to rifampicin, from 12 mg/L to a mean ± S.D. of 0.22 ± 0.21 mg/L at daptomycin 0.25 x MIC and 0.85 ± 0.90 mg/L at daptomycin 0.125 x MIC. Synergy was also observed for 13/19 (68%) isolates with ampicillin (MIC 128 mg/L). There was no significant synergy between daptomycin and any other antibiotic by this screening method. If confirmed by further studies, daptomycin with either rifampicin or ampicillin may be useful in the management of infections caused by VRE. Keywords: antibiotic synergy, Enterococcus, VRE
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Introduction |
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Daptomycin is a novel lipopeptide antibiotic with bactericidal activity against a wide range of clinically important Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA).1,2 The mechanism of action appears to involve Ca2+-dependent binding to the Gram-positive cell membrane, leading to channel formation, K+ leakage and ultimately inhibition of protein and nucleic acid synthesis.1–3 This unusual mechanism of action raised the possibility that unexpected synergic interactions with other antibiotics might be found. We used a novel screening method to look for synergy between daptomycin and 18 other antibiotics against 19 strains of high-level VRE (vancomycin MIC
256 mg/L). In this approach, daptomycin was incorporated into Ca2+-supplemented Mueller–Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Etest MICs on agar with and without daptomycin. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Strains
Nineteen isolates of vancomycin-resistant Enterococcus faecium (Etest MIC 256 mg/L) were obtained from the clinical microbiology laboratory at Shands Hospital at the University of Florida, Gainesville, FL, USA. All were identified by MicroScan (BBL, Cockeysville, MD, USA). All isolates also had ampicillin MICs 128 mg/L by Etest and gentamicin MICs 6 mg/L (MIC50 256) by Etest. Enterococcus faecalis ATCC 29212 QC strain was included for quality control testing of daptomycin, and was always within the acceptable NCCLS range (1–8 mg/L).
Antimicrobial agents
Daptomycin (lot no. 710403A) was obtained from Cubist Pharmaceuticals, Lexington, MA, USA. Rifampicin (lot no. 1854) was kindly supplied by VersaPharm, Marietta, GA, USA. Ampicillin sodium was obtained from Sigma Scientific, St Louis, MO, USA. Etest strips were obtained from AB Biodisk, Solna, Sweden. All testing was carried out in Mueller–Hinton agar (BBL) or Mueller–Hinton broth (Becton Dickinson, Sparks, MD, USA), both supplemented to 50 mg/L Ca2+ as recommended.4
Synergy screen
For synergy screening, daptomycin was tested with the following antibiotics by Etest: ampicillin, oxacillin, piperacillin, ceftriaxone, cefepime, imipenem, gentamicin, amikacin, azithromycin, tetracycline, chloramphenicol, clindamycin, linezolid, synercid, rifampicin, trimethoprim–sulfamethoxazole, vancomycin and levofloxacin. Daptomycin was incorporated into Mueller–Hinton agar supplemented to 50 mg/L Ca2+ at 0.125, 0.25, 0.5, 1 and 2x the agar MIC for each strain to be tested. Etest strips were placed on the agar containing 0, 0.125 and 0.25x the daptomycin MIC. For the 0, 0.125 and 0.25 x MIC plates, six different Etest strips were placed on each 150 mm plate after inoculation with a suspension equivalent to a 0.5 McFarland standard prepared by the direct colony suspension method.5 The 0.5, 1 and 2x daptomycin MIC containing agar plates did not have Etest strips placed on them, and were included so that an accurate daptomycin MIC would be obtained for each test strain. In some experiments, rifampicin discs were included for comparison with the Etest (Figure 1). Etest MICs were read after 20–24 h of incubation at 35°C in air. The Etest MIC on the 0.125 and 0.25x daptomycin MIC plates was compared with that on the plate without daptomycin. The decrease in MIC of the Etest at 0.25 or 0.125x the daptomycin agar MIC was used to calculate the fractional inhibitory concentration (FIC) for the combinations. Synergy was defined as an FIC 
0.5, as conventionally used in chequerboard synergy studies.6
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Results |
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For those isolates of rifampicin-resistant VRE showing synergy between daptomycin and rifampicin, the effect was dramatic. Figure 1 shows a rifampicin Etest on agar containing 0, 0.125 and 0.25x daptomycin MIC for a representative synergic strain of high-level VRE. In Figure 1(a), with no daptomycin in the agar, growth was observed all the way to the edge of the rifampicin Etest strip and disc. At 0.125x daptomycin MIC in the agar, the rifampicin Etest MIC was 0.25 mg/L (Figure 1b), and at 0.25x daptomycin MIC in the agar, it was 0.047 mg/L (Figure 1c). The corresponding rifampicin disc zones were 21 and 28 mm, respectively.
Table 1 shows a summary of the agar Etest synergy studies for daptomycin with both rifampicin and ampicillin. Of the 15 rifampicin-resistant strains of VRE (rifampicin Etest MIC 12 mg/L), a striking reduction in the rifampicin MIC was seen in 11/15 (73.3%) when daptomycin was in the agar at 0.25 x MIC (see strains 1–11 in Table 1). The mean ± S.D. rifampicin Etest MIC of these 11 strains was 0.22 ± 0.21 mg/L at 0.25x daptomycin MIC and 0.85 ± 0.90 mg/L at 0.125x daptomycin MIC (data not shown).
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Table 1 also shows a summary of the daptomycin and ampicillin synergy testing. All VRE were resistant to ampicillin by Etest (
128 mg/L; 16/19, 256 mg/L). By the screening method, ampicillin synergy with daptomycin at 0.25 x MIC was found for 13/19 (68.4%) strains, 10 of which also showed synergy between daptomycin and rifampicin. None of the other 16 antibiotics showed significant synergy with daptomycin by the agar Etest screening method.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Striking synergy between daptomycin and rifampicin was demonstrated by an agar diffusion method, showing that at 0.25 and 0.125x daptomycin MIC, 11/15 (73.3%) rifampicin-resistant VRE strains (MIC
12 mg/L) became inhibited at 1 mg/L rifampicin. At 0.25x daptomycin MIC the mean ± S.D. rifampicin MIC was 0.22 ± 0.21 mg/L. This observation was confirmed by time–kill studies using daptomycin at 0.5 x MIC and a clinically achievable rifampicin concentration of 5 mg/L (data not shown). Despite the fact that the agar diffusion Etest measures inhibition of bacterial growth while the time–kill measures killing as well as inhibition, categorical agreement was found for 89.5% of strains, which is statistically significant.
The screening method used here also suggested that there was synergy between daptomycin and ampicillin, even though all VRE strains were resistant to 128 mg/L ampicillin. Surprisingly, all 19 strains showed synergy by time–kill at 0.5x daptomycin MIC with ampicillin at the clinically achievable concentration of 32 mg/L (data not shown). Synergy between ampicillin and daptomycin at 5 and 10 mg/L was previously reported by Bingen et al.7 for six out of six strains of VRE, although their strains were significantly more susceptible to ampicillin (MIC90 32 mg/L) than ours. In addition, they observed >1000x killing by the combination versus the inoculum for the 10 mg/L daptomycin concentration.
Daptomycin is believed to act by Ca2+-dependent insertion of its acyl tail into the Gram-positive cell membrane, which is followed by the development of potassium efflux channels, depolarization of the membrane and cell death.1,2 A recent study by Silverman et al.3 showed that membrane depolarization and potassium leakage correlate well with decreased viability at concentrations above the daptomycin MIC. Our results suggest that daptomycin may have significant effects on the cell below the MIC. A plausible explanation for the synergy between daptomycin and rifampicin would be that at subinhibitory concentrations, daptomycin binds and opens channels that alone are insufficient to produce killing but can allow specific entry of rifampicin. Other membrane-acting polypeptide antibiotics such as nisin and ranalexin have been reported to show synergy with rifampicin against S. aureus.8 Among Gram-negative bacteria, 100-fold decreases in rifampicin MICs have been observed with other membrane active polypeptide antibiotics such as the polymyxins and certain synthetic cationic peptides.9,10 The mechanism is believed to involve permeabilization of the outer membrane to hydrophobic antibiotics such as erythromycin, fusidic acid, rifampicin and novobiocin. In view of these studies, it is plausible that the action of daptomycin at the cell membrane could promote entry of a hydrophobic drug, such as rifampicin.
Since the penicillins do not act intracellularly, the synergy between daptomycin and ampicillin would be unlikely to involve pore formation and remains unexplained at this time.
In summary, we describe a novel screening method for determining antibiotic synergy that can rapidly test one antibiotic with relatively large numbers of combinations. Screening daptomycin by this method led to the finding of two potentially important antibiotic combinations. Further study of these daptomycin-containing regimens in vitro and in vivo are needed to improve our understanding of the mechanism of the synergy as well as to determine the pharmacokinetics and pharmacodynamics for potential clinical use. At this point, daptomycin with either rifampicin or ampicillin appears to have potential in the treatment of VRE.
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Acknowledgements |
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We gratefully acknowledge the support of the Department of Pathology, Immunology and Laboratory Medicine, University of Florida and the staff of the Clinical Microbiology Laboratory, Shands Hospital at the University of Florida, Gainesville, FL, USA. We appreciate the careful reviews and many helpful suggestions by Jared Silverman, PhD (Cubist Pharmaceuticals, Lexington, MA, USA). We thank Ms Claire Noegel for preparation of this manuscript. This study was supported in part by a grant from Cubist Pharmaceuticals.
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Footnotes |
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* Corresponding author. Tel: +1-352-392-5621; Fax: +1-352-392-4693; E-mail: Rand{at}pathology.ufl.edu
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References |
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1 . Tally, F. P. & DeBruin, M. F. (2000). Development of daptomycin for Gram-positive infections. Journal of Antimicrobial Chemotherapy 46, 523–6.
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6 . Eliopoulos, G. M. & Moellering, R. C., Jr (1996). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 330–96. Williams & Wilkins, Baltimore, MD, USA.
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Bingen, E., Lambert-Zechovsky, N., Leclercq, R. et al. (1990). Bactericidal activity of vancomycin, daptomycin, ampicillin and aminoglycosides against vancomycin-resistant Enterococcus faecium. Journal of Antimicrobial Chemotherapy 26, 619–26.
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