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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: antibiotic synergy, Enterococcus, VRE
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (18 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 MuellerHinton agar (BBL) or MuellerHinton 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, trimethoprimsulfamethoxazole, vancomycin and levofloxacin. Daptomycin was incorporated into MuellerHinton 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 2024 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
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 111 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).
|
None of the other 16 antibiotics showed significant synergy with daptomycin by the agar Etest screening method.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 timekill 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.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Thorne, G. M. & Alder, J. (2002). Daptomycin: a novel lipopeptide antibiotic. Clinical Microbiology Newsletter 24, 3340.[CrossRef]
3
.
Silverman, J. A., Perlmutter, N. G. & Shapiro, H. M. (2003). Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 47, 253844.
4 . Jones, R. N. & Barry, A. L. (1987). Antimicrobial activity and spectrum of LY146032, a lipopeptide antibiotic, including susceptibility testing recommendations. Antimicrobial Agents and Chemotherapy 31, 6259.[ISI][Medline]
5 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A6. NCCLS, Villanova, PA, USA.
6 . Eliopoulos, G. M. & Moellering, R. C., Jr (1996). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 33096. Williams & Wilkins, Baltimore, MD, USA.
7 . 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, 61926.[Abstract]
8 . Giacometti, A., Cirioni, O., Barchiesi, F. et al. (2000). In-vitro activity and killing effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and interactions with clinically used antibiotics. Diagnostic Microbiology and Infectious Disease 38, 1158.[CrossRef][ISI][Medline]
9 . Savage, P. B. (2001). Multidrug-resistant bacteria: overcoming antibiotic permeability barriers of gram-negative bacteria. Annals of Medicine 33, 16771.[ISI][Medline]
10 . Vaara, M. & Porro, M. (1996). Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria. Antimicrobial Agents and Chemotherapy 40, 18015.[Abstract]