JAC Advance Access originally published online on March 16, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):1017-1020; doi:10.1093/jac/dkm045
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Daptomycin-reversible rifampicin resistance in vancomycin-resistant Enterococcus faecium
1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA 2 Cubist Pharmaceuticals, Lexington, MA 02421, USA
* Corresponding author. Tel: +1-352-392-5621; Fax: +1-352-392-4693; E-mail: rand{at}pathology.ufl.edu
Received 20 October 2006; returned 20 November 2006; revised 12 January 2007; accepted 22 January 2007
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Abstract |
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Objectives: In a previous study, we observed marked synergy between daptomycin and rifampicin against 73% of rifampicin-resistant, vancomycin-resistant Enterococcus faecium (VRE), with approximately 100-fold reductions in rifampicin MICs observed at one-eighth to one-fourth daptomycin MIC. The purpose of this study was to determine whether the synergy between daptomycin and rifampicin could be explained by enhanced entry of rifampicin into the cell or was related to amino acid substitutions in the rifampicin-binding site in the ß subunit (rpoß) of the RNA polymerase.
Methods: We developed a bioassay for rifampicin to measure cell-bound rifampicin levels as well as metabolic inactivation of rifampicin. In addition, we sequenced the rifampicin-binding site in the rpoß of VRE strains with and without synergy between daptomycin and rifampicin.
Results: Cell-bound rifampicin levels were the same in rifampicin-susceptible VRE as in rifampicin-resistant VRE showing daptomycin synergy and were not affected by the presence of daptomycin. In contrast, rifampicin-resistant VRE without daptomycin synergy had undetectable cell-bound rifampicin. Sequencing the rpoß rifampicin-binding site revealed that the synergistic strains had the same sequence as rifampicin-susceptible wild-type E. faecium. The daptomycin synergy-resistant strains all had mutations in known rifampicin-binding sites.
Conclusions: Daptomycin is able to reverse rifampicin resistance in some strains of VRE, but the mechanism could not be explained by an effect of daptomycin on entry of rifampicin into or transport out of the cell, by inactivation of rifampicin or by mutation involving the rifampicin-binding site.
Keywords: rpoß , rifampicin-binding site , E. faecium , VRE
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Introduction |
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Daptomycin is a cyclic lipopeptide active against a wide range of Gram-positive bacteria, including problematic organisms such as vancomycin-resistant Enterococcus faecium (VRE). Daptomycin is believed to act at the cell membrane by insertion of its lipophilic ‘tail’, ultimately leading to the leakage of ions and cell death.1 In view of the relatively limited therapeutic options for VRE, a better understanding of the mechanism of action of effective antibiotics is important. We observed synergy between daptomycin and rifampicin for 73% of VRE with about 100-fold reductions in rifampicin MIC at one-fourth MIC daptomycin.2 In the current study, we extended the previous work to investigate the possible mechanisms of the synergy.
Rifampicin acts by entering the cell cytoplasm, binding to RNA polymerase (rpoß) and inhibiting transcription. Resistance to rifampicin is typically due to amino acid substitutions in the three known resistance clusters in the rifampicin-binding site of rpoß,3 but can also be mediated by efflux mechanisms or by enzymatic inactivation of the drug.4–7 Synergy between daptomycin and rifampicin against rifampicin-resistant VRE implies that daptomycin can overcome or reverse the mechanism of resistance in these isolates. Cell-bound rifampicin has been measured using [14C]rifampicin and shows rapid accumulation and intracellular binding in both Escherichia coli and Staphylococcus aureus.8 We used the procedure described by Williams and Piddock8 to measure cell-bound rifampicin, except that we developed a bioassay because radiolabelled rifampicin was not available. If binding site mutations are present, we expected to find little or no cell-bound rifampicin. The purpose of this study was to determine whether daptomycin affected levels of cell-bound rifampicin, which together with the binding site mutation data, would explain the synergy between daptomycin and rifampicin.
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Materials and methods |
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Twelve strains of E. faecium (vancomycin MIC
256 mg/L, by Etest, AB Biodisk, Solna, Sweden) were obtained from the clinical microbiology laboratory at the Shands at the University of Florida Hospital, as previously described.2 Four strains were rifampicin-susceptible, whereas eight were rifampicin-resistant (MIC 32 mg/L by Etest). Of the eight rifampicin-resistant isolates, four displayed synergy with daptomycin (synergy-positive) and four did not (synergy-negative). Daptomycin MICs by broth dilution in Ca2 +-supplemented (50 mg/L) Mueller–Hinton broth were 4 mg/L for all strains, except strain 11 (2 mg/L). Strain typing by PFGE at the Mayo Medical Laboratories (Rochester, MN, USA) showed that all strains were distinguishable from one another, except strains 10 and 11. A bioassay for rifampicin was modified from that described by Dabbs4 using a highly susceptible indicator strain of coagulase-negative Staphylococcus. For a standard curve, Ca2 +-supplemented (50 mg/L) Mueller–Hinton agar was inoculated with the indicator strain, and a series of 4 mm holes punched in the agar. The holes were then filled with rifampicin standards, 25 µL/well containing 30, 7.5, 1.875 and 0.45 ng of rifampicin. Zone diameters were recorded after 18–24 h at 34°C in air. Assay sensitivity was increased by using 15 mL of agar per 100 mm plate, instead of 30 mL.
The bioassay was used to measure the levels of cell-bound rifampicin with and without daptomycin. Volumes (10 mL) of Ca2 +-supplemented (50 mg/L) Mueller–Hinton broth were inoculated with 0.5 mL of log phase bacteria (OD = 0.5) and incubated for 3–4 h at 37°C. A 50 µL sample was removed for titration and rifampicin was added to a final concentration of 2 mg/L to each of eight tubes. Duplicate tubes received daptomycin at 1, 0.5, 0.25 or 0 mg/L final concentrations and were incubated for 1 h at 37°C. Cells were pelleted at 10 000 g for 15 min at 4°C, washed with 25 mL of cold PBS and pelleted for 20 min at 10 000 g. Pellets were resuspended in 1 mL of cold PBS and pelleted in an Eppendorf centrifuge for 2 min at 14 000 g. Final pellets were resuspended in 25 µL of 5% DMSO in PBS, incubated at 56°C for 30 min and assayed as described for the rifampicin standard in the paragraph above. Zone sizes surrounding the cell pellets were converted into nanogram equivalents from the standard curve included in each experiment and normalized per 109 cells. Input viable counts ranged from 8 x 108 to 2.5 x 109 cfu. Control experiments indicated that daptomycin carryover did not contribute to bioassay results (data not shown).
Sequencing was performed at the University of Florida DNA Sequencing Core Laboratory by standard methods. Primers were as described previously,9 except that the forward primer CTATTGCATGCGATCTTTG corresponding to E. faecium positions +2728 to +2746 was used.
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Results |
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The rifampicin bioassay was linear between 2 and 30 ng (data not shown). When cell-bound rifampicin was measured for synergy-positive strains (Table 1), an average of
5 ng of rifampicin/109 cfu was detected. This value did not change in the presence of daptomycin up to 1 mg/L. A range of rifampicin values (0.9–13 ng/109 cfu) were observed for the different isolates tested, but in all cases, addition of daptomycin had no significant effect on rifampicin levels. In contrast, no cell-bound rifampicin (<2 ng/109 cfu) was observed for synergy-negative isolates. This observation suggests fundamental differences in the mechanism of rifampicin resistance in synergy-positive and synergy-negative strains. Furthermore, since rifampicin-susceptible strains had cell-bound rifampicin in the same range as that of the synergy-positive strains (data not shown), rifampicin efflux is unlikely to be responsible for resistance in synergy-positive strains.
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Bacteria resistant to rifampicin frequently have mutations involving the rifampicin-binding site on the ß subunit of RNA polymerase.3 Therefore, the rpoß subunit of RNA polymerase was sequenced. Figure 1 shows that synergy-negative strains 1, 7 and 8 had an R529S substitution at a position known to interact with rifampicin in resistance Cluster I3 and an additional amino acid substitution S574R in resistance Cluster II. Synergy-negative strain 11 had an L533F substitution at a position known to interact with rifampicin in Cluster I, as well as a G522D substitution in Cluster I. Strain 10 also had the L533F substitution, despite rifampicin susceptibility (MIC = 0.094 mg/L). No other significant differences were found. In contrast, synergy-positive strains had no amino acid differences from the reference E. faecium strain 343-3 (GenBank AY167138 [GenBank] ),9 strongly suggesting differences in the mechanism of resistance.
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It was possible that the rifampicin-resistant VRE without rpoß mutations were resistant to rifampicin because it was able to inactivate it. Several studies have demonstrated that species such as Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chelonae, Mycobacterium fortuitum, Nocardia, Gordona and Tsukumarella spp. can inactivate rifampicin primarily by ribosylation.4–7 We tested the ability of the four synergy-positive VRE strains to inactivate rifampicin using the bioassay. VRE (107 log phase) were incubated with 2 mg/L of rifampicin for 24 h at 37°C. After incubation, bacteria were removed from the turbid suspension by centrifugation and 25 µL of the supernatant was assayed for rifampicin as described. Rifampicin concentrations in the supernatant from the synergy-positive strains were identical to that of broth alone incubated under the same conditions, suggesting that no rifampicin inactivation was occurring.
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Discussion |
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Our findings indicate that the daptomycin synergy-positive, rifampicin-resistant VRE differ from the daptomycin synergy-negative strains in several important ways: first, they are capable of binding rifampicin, whereas the synergy-negative strains do not; secondly, the rifampicin-binding site is the same as that of susceptible VRE, whereas the synergy-resistant VRE have mutations in known resistance clusters and thirdly, daptomycin can reverse the rifampicin resistance in the synergy-positive strains, but not in the synergy-resistant strains.2 We were unable to demonstrate inactivation of rifampicin by any of our VRE, even after incubating strains for 24 h under conditions in which growth had become completely turbid. Since the daptomycin synergy-positive strains are phenotypically resistant to rifampicin (MIC >32 mg/L),2 there must be another mechanism of rifampicin resistance besides mutation in the rpoß gene and rifampicin inactivation.
We suggest that the mechanism of this novel rifampicin resistance in the daptomycin synergy-positive strains of VRE be termed daptomycin-reversible resistance, until the explanation for the reversal is worked out.
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J. A. S. is employed by Cubist Pharmaceuticals. K. H. R. and H. J. H. have nothing to declare.
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Acknowledgements |
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We gratefully acknowledge the support of the staff of the Shands at the University of Florida Hospital Clinical Microbiology Laboratory. This work is supported in part by a grant from Cubist Pharmaceuticals (Lexington, MA, USA) and by the Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.
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References |
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1 Steenbergen JN, Alder J, Thorne GM, et al. (2005) Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. J Antimicrob Chemother 55:283–8.
2
Rand KH and Houck H. (2004) Daptomycin synergy with rifampicin and ampicillin against vancomycin-resistant enterococci. J Antimicrob Chemother 53:530–2.
3 Campbell EA, Korzheva N, Mustaev A, et al. (2001) Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104:901–12.[CrossRef][Web of Science][Medline]
4 Dabbs ER. (1987) Rifampicin inactivation by Rhodococcus and Mycobacterium species. FEMS Microbiol Lett 44:395–9.[CrossRef][Web of Science]
5 Dabbs ER, Yazawa K, Tanaka Y, et al. (1995) Rifampicin inactivation by Bacillus species. J Antibiot (Tokyo) 48:815–9.[Medline]
6 Dabbs ER, Yazawa K, Mikami Y, et al. (1995) Ribosylation by mycobacterial strains as a new mechanism of rifampin inactivation. Antimicrob Agents Chemother 39:1007–9.[Abstract]
7 Quan S, Venter H, Dabbs ER. (1997) Ribosylative inactivation of rifampin by Mycobacterium smegmatis is a principal contributor to its low susceptibility to this antibiotic. Antimicrob Agents Chemother 41:2456–60.[Abstract]
8
Williams KJ and Piddock LJ. (1998) Accumulation of rifampicin by Escherichia coli and Staphylococcus aureus.. J Antimicrob Chemother 42:597–603.
9
Enne VI, Delsol AA, Roe JM, et al. (2004) Rifampicin resistance and its fitness cost in Enterococcus faecium.. J Antimicrob Chemother 53:203–7.
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