a Department of Infectious Diseases, Infection Control and Sexual Health, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane, Queensland 4102; b Department of Microbiology and Infectious Diseases, Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia
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Abstract |
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Introduction |
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Materials and methods |
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Test organisms consisted of four strains each of S. aureus, S. epidermidis and S. pneumoniae, three strains of E. faecalis and one strain of E. faecium. The test organisms included the reference strains S. aureus ATCC 25923, S. epidermidis ATCC 12228, S. pneumoniae ATCC 49619 and E. faecalis ATCC 29212. The other strains were recent clinical isolates from Princess Alexandra Hospital. Half of the staphylococci were methicillin susceptible and half were methicillin resistant. E. faecalis ATCC 29212 is vancomycin susceptible whereas the three clinical isolates of enterococci were vancomycin resistant. One strain of pneumococcus (strain 4H02517) was relatively resistant to penicillin with an MIC of 1.5 mg/L.
Antibiotic and determination of MICs
Linezolid was a gift from Pharmacia and Upjohn Company (Kalamazoo, MI, USA) and was stored and prepared according to the manufacturer's guidelines. Linezolid MICs were determined by Etest (AB Biodisk, Solna, Sweden) using a suspension of 0.5 McFarland turbidity plated on to MuellerHinton agar (staphylococci and enterococci) or MuellerHinton agar supplemented with 5% sheep blood (pneumococci). MICs were determined after 18 h of incubation at 35°C in ambient air (staphylococci and enterococci) or ambient air supplemented with 5% CO2 (pneumococci). MICs for all bacteria tested were <2 4 mg/L, the tentative breakpoints that have previously been proposed for linezolid against Gram-positive cocci.6
Method for determining the PAE
In vitro PAEs were determined by the viable plate count method2 using MuellerHinton broth (staphylococci and enterococci) (BBL, Becton Dickinson, Cockeysville, MD, USA) or brainheart infusion broth (pneumococci) (BBL, Becton Dickinson). 106 cfu/mL of logarithmic phase organisms were exposed for 1 h at 37°C to eight concentrations of linezolid (0.5 x, 1 x, 2 x, 4 x, 8 x, 16 x, 32 x and 64 x MIC). After 1 h, drug was removed by centrifuging the solution for 10 min at 2000g, decanting the supernatant and resuspending the organisms in fresh broth prewarmed to 37°C. This washing procedure was performed twice. Washing was selected as the preferred method of drug removal to avoid carry-over of drug from the high concentrations of drug used in the experiments. After drug removal, viable counts were plated hourly until visible regrowth had occurred. The following controls were included for each experiment: (i) a growth control, prepared and treated identically to the test solution but without exposure to antibiotic; and (ii) a residual antibiotic control, to which 1/1000 of the test antimicrobial concentration was added after centrifugation and washing. This last tube was included to ensure that, after centrifugation and washing, residual drug in the tubes containing the treated organism did not affect the rate of growth.
Quantification of the PAE
The PAE was calculated with the standard formula of Craig & Gudmundsson:2
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Mathematical modelling of the post-antibiotic effect
The results were analysed using the Hill (sigmoid Emax) doseresponse equation, a mathematical model that has been used to describe the relationship between PAE and AUC of drug exposure:35
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Results |
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Discussion |
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An in vivo study in mice recorded PAEs of 3.63.8 h for MSSA and 3.73.9 h for penicillin-susceptible pneumococci after iv doses of 2080 mg/kg of linezolid.7 Because enterococci were not tested, it is impossible to determine from this study whether there are in vivo interspecies differences in duration of PAE, as postulated for the in vitro studies with enterococci.
There are limited human data on the pharmacokinetics of linezolid. The average peak serum levels in healthy volunteers after an oral dose of 500 mg bd for 14 days was 15.3 mg/L, with an average trough of 5.04 mg/L.8 The bioavailability of the oral formulation is close to 100%, hence similar levels were obtained after the same dosage was given iv, with serum levels of >4 mg/L for 910 h of the 12 hourly dosing interval.9 The MIC90 of linezolid is 4 mg/L for the most resistant target pathogens, such as MRSA, VRE and penicillin-resistant pneumococci.6 A study in mice showed that time above MIC was the major pharmacodynamic parameter determining efficacy of linezolid.7 The percentage of time above MIC required for a bacteriostatic effect with linezolid varied from 33 to 49% (mean 40%) for pneumococci and from 33 to 59% (mean 41%) for staphylococci. Hence, based on a pharmacokinetic goal of time above MIC of at least 40% of the dosing interval, a dosage regimen of 500 mg orally or iv 12 hourly would be likely to achieve success against bacteria with MICs as high as 4 mg/L.7
Peak levels of 1648 mg/L linezolid (3264 x MIC) were used in our experiments, and the PAE was of maximum duration at these concentrations. Humans can readily achieve serum concentrations of up to 20 mg/L, and the drug is primarily renally excreted, with halving of peak serum levels as long as 68 h after the dose.8,9 Thus, the concentrations we used in vitro are achievable and sustainable in humans, and the results obtained are likely to be clinically relevant. However, an in vitro model could be used to study the dynamics of the PAE during one dosing interval of the drug.
Although the presence or absence of in vivo PAE is usually predicted by in vitro studies, in vivo PAEs are usually longer than those observed in vitro.10 In vivo PAEs of linezolid against penicillin-susceptible pneumococci and MSSA were c. 2 h longer7 than the in vitro PAEs obtained by us or by Rybak et al.1 Other organisms such as enterococci, S. epidermidis, penicillin-resistant pneumococci and MRSA should therefore also be tested in animal models as host defence mechanisms, concentrations of drug within cells, tissue binding of drug and sub-MIC effects may be important.
In a previous study we demonstrated a significant relationship between E50 and imipenem MIC,4 although a subsequent study of the ketolides telithromycin (HMR 3647) and HMR 3004 did not show a significant correlation between E50 and MIC.5 Linear regression of the linezolid results shows no correlation between linezolid MIC and PAEmax or n (P > 0.1), and a weak correlation between linezolid MIC and E50 (P = 0.6). One explanation for this could be the much narrower range of linezolid and ketolide MICs compared with those of imipenem. This spread of MICs could be too narrow to detect any significant correlation.
In conclusion, linezolid exhibits moderate concentration-dependent in vitro PAE against S. aureus, S. epidermidis, E. faecalis, E. faecium and S. pneumoniae. Resistance to methicillin (for staphylococci), vancomycin (for enterococci) and penicillin (for pneumococci) had no effect on the duration of the PAE. Results of PAE testing support twice-daily dosing of linezolid in humans.
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Acknowledgments |
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Notes |
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References |
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2 . Craig, W. A. & Gudmundsson, S. (1996). The postantibiotic effect. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed), pp. 296329. Williams and Wilkins, Baltimore.
3 . Turnidge, J. D. (1990). Prediction of antibiotic dosing intervals from in vitro susceptibility, pharmacokinetics and post-antibiotic effect: theoretical considerations. Scandanavian Journal of Infectious Diseases, Suppl. 74, 13741.
4 . Munckhof, W. J., Olden, D. & Turnidge, J. D. (1997). The postantibiotic effect of imipenem: relationship with drug concentration, time of exposure and minimum inhibitory concentration. Antimicrobial Agents and Chemotherapy 41, 17357.[Abstract]
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Munckhof, W. J., Borlace, G. & Turnidge, J. D. (2000). Postantibiotic suppression of growth of erythromycin A-susceptible and -resistant Gram-positive bacteria by the ketolides telithromycin (HMR 3647) and HMR 3004. Antimicrobial Agents and Chemotherapy 44, 174953.
6 . Wise, R., Andrews, J. M., Boswell, F. J. & Ashby, J. P. (1998). The in-vitro activity of linezolid (U-100766) and tentative breakpoints. Journal of Antimicrobial Chemotherapy 42, 7218.[Abstract]
7 . Andes, D., Van Ogtrop, M. L. & Craig, W. A. (1998). Pharmacodynamic activity of a new oxazolidinone, linezolid, in an animal model. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract A-9, p. 3. American Society for Microbiology, Washington, DC.
8 . Stalker, D. J., Wajszczuk, C. P. & Batts, D. H. (1997). Linezolid safety, tolerance and pharmacokinetics following oral dosing twice daily for 14.5 days. In Program and Abstracts of the Thirty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract A-115, p. 23. American Society for Microbiology, Washington, DC.
9 . Stalker, D. J., Wajszczuk, C. P. & Batts, D. H. (1997). Linezolid safety, tolerance and pharmacokinetics after intravenous dosing twice daily for 7.5 days. In Program and Abstracts of the Thirtyseventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract A-116, p. 23. American Society for Microbiology, Washington, DC.
10 . Craig, W. A. (1993). Postantibiotic effects in experimental infection models: relationship to in vitro phenomena and to treatment of infections in man. Journal of Antimicrobial Chemotherapy 31, Suppl. D, 14958.[ISI][Medline]
Received 2 October 2000; returned 5 December 2000; revised 29 January 2001; accepted 20 February 2001