Post-antibiotic growth suppression of linezolid against Gram-positive bacteria

W. J. Munckhofa,*, C. Gilesa and J. D. Turnidgeb

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


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The in vitro post-antibiotic effects (PAEs) of eight different concentrations of linezolid against Gram-positive cocci were investigated and the results analysed using the sigmoid Emax model for mathematically modelling the PAE. Mean maximal linezolid PAEs against strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and Streptococcus pneumoniae were 2.2, 1.8, 2.8, 2.0 and 3.0 h, respectively. Resistance to methicillin (for the staphylococci), vancomycin (for the enterococci) and penicillin (for the pneumococci) had no effect on the duration of the PAE. Results of PAE testing support twice-daily dosing of linezolid in humans.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Linezolid (formerly PNU-100766) is a member of the new synthetic class of antibacterial agents known as oxazolidinones, which are unrelated to other currently available agents. Linezolid has excellent antibacterial activity against Gram-positive bacteria, including methicillin-resistant strains of Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), Enterococcus spp. including vancomycin-resistant strains (VRE) and Streptococcus spp. including penicillin-resistant strains.1 Dosing regimens of new antibiotics such as linezolid may be influenced by pharmacodynamic phenomena such as the post-antibiotic effect (PAE).2 We therefore investigated the in vitro PAEs of linezolid against S. aureus, S. epidermidis, Enterococcus faecalis, Enterococcus faecium and Streptococcus pneumoniae, and analysed the results using the sigmoid Emax mathematical model, which has been used to describe the relationship between PAE and the area under the concentration–time curve (AUC) of drug exposure.35


    Materials and methods
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 Materials and methods
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Test organisms

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 Mueller–Hinton agar (staphylococci and enterococci) or Mueller–Hinton 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 Mueller–Hinton broth (staphylococci and enterococci) (BBL, Becton Dickinson, Cockeysville, MD, USA) or brain–heart 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

where T is the time required for the count of cfu in the test culture to increase 1 log10 above the count observed immediately after drug removal, and C is the time required for the count of cfu in an untreated control culture to increase 1 log10 above the count observed immediately after completion of the same procedure used on the test culture for drug removal.

Mathematical modelling of the post-antibiotic effect

The results were analysed using the Hill (sigmoid Emax) dose–response equation, a mathematical model that has been used to describe the relationship between PAE and AUC of drug exposure:35

where PAEmax is the estimated maximum PAE, E50 is the estimated AUC at which 50% of the maximum PAE is reached, and n is a constant associated with the steepness of the exposure-response curve. The parameters PAEmax, E50 and n were estimated for each bacterial strain using the non-linear regression module of Systat, version 8.0 (SPSS Inc., Chicago, IL, USA). MICs were then compared with each of the three parameters by linear regression performed in Systat.


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 Materials and methods
 Results
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 References
 
The PAEs after 1 h exposure to linezolid are shown in the TableGo and FigureGo. Maximum PAEs for the staphylococci ranged from 1.2 to 2.2 h, and results for the enterococci were similar with maximal PAEs of 1.4–2.1 h. Maximal PAEs for the pneumococci were slightly longer and ranged from 2.3 to 2.5 h. The TableGo also shows the correlation between the PAEs determined experimentally and those estimated using the Hill (sigmoid Emax) equation, with corrected r2 (defined as the correlation squared between the observed values and the predicted values) ranging from 0.656 to 0.990. For the majority of strains, correlation between experimental results and estimates using the Hill equation was very good, with corrected r2 > 0.900.


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Table.  PAE parameters after 1 h exposure of bacterial strains to linezolid in vitro
 


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Figure.  Relationship between duration of PAE after 1 h exposure to linezolid, and linezolid concentration (expressed as multiples of the MIC). (a) Strains of S. aureus (symbols: {diamondsuit}, MSSA ATCC 25923; {blacksquare}, MSSA MK803549; {blacktriangleup}, MRSA ATCC 49476; x, MRSA MK810545); (b) strains of S. epidermidis (symbols: {diamondsuit}, MSSE ATCC 12228; {blacksquare}, MSSE M35732; {blacktriangleup}, MRSE MK506349; x, MRSE MK711617); (c) strains of enterococci (symbols: {diamondsuit}, E. faecalis ATCC 29212; {blacksquare}, VR-E. faecalis M106009; {blacktriangleup}, VR-E. faecium M31838; x, VR-E. faecalis M23304); (d) strains of S. pneumoniae (symbols: {diamondsuit}, S. pneumoniae ATCC 49619; {blacksquare}, S. pneumoniae 4H02517; {blacktriangleup}, S. pneumoniae M48191; x, S. pneumoniae 4G12043).

 

    Discussion
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 Materials and methods
 Results
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Two other studies have examined post-antibiotic growth suppression of linezolid.1,7 In one study, after 1 h exposure to linezolid at 1 x MIC, the in vitro PAEs were 0.1–0.8 h against S. aureus, S. epidermidis and vancomycin-resistant E. faecalis and E. faecium, with PAEs increasing to 0.1–1.4 h when linezolid concentrations of 4 x MIC were tested.1 However, this study tested only a single strain of each species and did not test concentrations of linezolid higher than 4 x MIC, which we predict would not have detected the maximal effect. This study also noted higher linezolid MICs and shorter PAEs for E. faecalis compared with E. faecium, although only one strain of each species was tested. However, our study did not show any significant differences in MIC or duration of PAE between E. faecalis and E. faecium, although admittedly few (four) strains of enterococci were tested.

An in vivo study in mice recorded PAEs of 3.6–3.8 h for MSSA and 3.7–3.9 h for penicillin-susceptible pneumococci after iv doses of 20–80 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 9–10 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 16–48 mg/L linezolid (32–64 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 6–8 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.


    Acknowledgments
 
We are grateful for the assistance of Jacqui Schooneveldt and Dr Graeme Nimmo from the Department of Microbiology, Princess Alexandra Hospital for the supply of bacterial isolates. This work was partly supported by a grant from Pharmacia and Upjohn, Sydney, Australia.


    Notes
 
* Corresponding author. Tel: +61-732-405918; Fax: +61-732-405540; E-mail: wendy_munckhof{at}health.qld.gov.au Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 .  Rybak, M. J., Cappelletty, D. M., Moldovan, T., Aeschlimann, J. R. & Kaatz, G. W. (1998). Comparative in vitro activities and post-antibiotic effects of the oxazolidinone compounds eperezolid (PNU-100592) and linezolid (PNU-100766) versus vancomycin against Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Antimicrobial Agents and Chemotherapy 42, 721–4.[Abstract/Free Full Text]

2 .  Craig, W. A. & Gudmundsson, S. (1996). The postantibiotic effect. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed), pp. 296–329. 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, 137–41.

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, 1735–7.[Abstract]

5 .  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, 1749–53.[Abstract/Free Full Text]

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, 721–8.[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, 149–58.[ISI][Medline]

Received 2 October 2000; returned 5 December 2000; revised 29 January 2001; accepted 20 February 2001