1 Department of Pathology, Case Western Reserve University and University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106; 2 Department of Pathology, Hershey Medical Center, Hershey, PA, USA
Received 24 February 2003; returned 29 June 2003; revised 1 July 2003; accepted 5 August 2003
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Abstract |
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Keywords: post-antibiotic effects, PAEs, ketolides, streptococci, staphylococci
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Introduction |
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Telithromycin is a recently developed member of the ketolide family, a new class of semi-synthetic 14-membered ring macrolides derived from erythromycin A, with human pharmacokinetic properties similar to erythromycin A.612 Previous studies have documented in vitro activity of ketolides against Streptococcus pneumoniae regardless of macrolide or lincosamide susceptibility.1,7,9,10 Ketolides are active against macrolide-susceptible staphylococci and strains with inducible macrolide resistance and do not induce resistance in the latter group.1 However, these agents are inactive against staphylococci and Streptococcus pyogenes with constitutive macrolide resistance.10
Post-antibiotic effect (PAE) is the term used to describe suppression of bacterial growth that persists after short exposure of organisms to antibacterials.1315 This effect is the result of the prior antibacterial exposure rather than to persisting subinhibitory concentrations of the compound. PAE is one of the factors that has a clinical impact on antibacterial dosing regimens, allowing less frequent dosing than with agents with no PAE.13 The post-antibiotic subinhibitory effect (PA-SME), on the other hand, is used to determine the additional effect of subinhibitory concentrations of the agent, which will again increase the effect of the agent and further decrease the dosing frequency.13,1619
This study examined the PAE and PA-SME of the new ketolide, telithromycin, against selected strains of Gram-positive cocci, including strains with no macrolide resistance, as well as macrolide-resistant strains with constitutively and inducibly expressed erm(A) or erm(B) determinants, and streptococci with macrolide efflux mediated resistance associated with mef(A) or mef(E) determinants.
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Materials and methods |
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Genotypes of macrolide-resistant strains were determined by PCR using primers for the presence of erm(A), erm(A) subclass erm(TR), erm(B), erm(C) and mef(A) as shown in Table 1.3,5,6
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PAE was determined by the viable plate count method.13,17 For experiments with staphylococci, MuellerHinton broth (Difco Laboratories, Detroit, MI, USA) (MHB) was used. MHB supplemented with 5% lysed horse blood was used for testing streptococci. Inocula were prepared by suspending growth from an overnight trypticase soy blood agar plate in broth. The broth was incubated at 35°C for 24 h in a shaking water bath until turbidity matched a 2 McFarland standard (approximately 109 cfu/mL). Glass tubes containing 5 mL of broth with antibiotic at 10 x MIC were inoculated with 50 µL of the above suspension to produce inocula of 107108 cfu/mL. Tubes were mixed well by vortexing and viability counts determined. Growth controls with inoculum but no antibiotic were included with each experiment. Inoculated tubes were then placed in a shaking water bath at 35°C for an exposure period of 1 h. At the end of the exposure period, cultures were diluted 1:1000 in prewarmed broth to remove the antibiotic. An additional control culture was diluted in broth containing the antibiotic at a concentration of 0.01 x MIC to confirm that after dilution the activity of the antibiotic had been effectively eliminated.
Viability counts were determined initially, before and immediately after dilution (0 h), and then every 2 h until the turbidity of the tube reached that of a 1 McFarland standard. Recovery plates were inoculated for at least 72 h and colony counts carried out on plates yielding 30300 colonies.
The PAE was defined, according to Craig & Gudmundsson,13 as PAE = T C, where T = time required for viability counts of an antibiotic-exposed culture to increase by 1 log10 above the counts observed immediately after dilution, and C = corresponding time for the antibiotic-free growth control. Viability counts, expressed as log10 cfu/mL, were plotted against time.
For PA-SME determination, the PAE was induced as described above.13,1619 Following the 1:1000 dilution in broth to remove the antibiotic, three additional tubes were prepared containing subinhibitory concentrations of the compound at 0.12x, 0.25x and 0.5x the MIC of the compound for that strain. Tubes were then handled as for PAE determination above. The PA-SME was defined according to Odenholt-Tornqvist et al.1719 as PA-SME = Tpa C, where Tpa = time for cultures previously exposed to antibiotic and then re-exposed to different subinhibitory concentrations to increase by 1 log10 above the counts observed immediately after dilution, and C = corresponding time for the antibiotic-free control.
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Results |
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Discussion |
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The PAE was short (0.30.9 h) for three of the strains, and longer (1.53.8 h) for the remaining strains, with the four S. pneumoniae strains all belonging to the latter group. PA-SME values were most consistent for the S. aureus strains, but were either the same as or slightly higher than PAE values for two of the strains. PA-SME values for S. pyogenes were generally one- to almost five-fold longer than PAE values. For S. pneumoniae, PA-SME values were similar to the PAE values for macrolide-resistant strains, and were prolonged by up to two-fold only for the macrolide-susceptible strain.
These results indicate potential benefits of these PAE and PA-SME effects against all three species. For S. pneumoniae, the benefit was demonstrated in this study from both effects for macrolide-susceptible strains, and from the PAE for macrolide-resistant strains. Both effects were significant for S. pyogenes and S. aureus strains with all three common macrolide-resistance determinants, although they were more modest for the latter group. The concentrations used to document PAE and PAE-SME effects were within achievable concentrations of telithromycin for all experiments except for those using the constitutively macrolide-resistant strain of S. pyogenes, which had a telithromycin MIC of 4 mg/L.
These results indicate that the dosage regimen of telithromycin might be affected by the combination of its serum half-life and the length of the PAE and/or PA-SME, as these represent the total period of time during which regrowth does not occur. Additional pharmacokinetic/pharmacodynamic and clinical studies will be necessary to validate this hypothesis.
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Acknowledgements |
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Footnotes |
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References |
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