1 Faculty of Applied Science, University of the West of England, Bristol BS16 1QY; 2 Bristol Centre for Antimicrobial Research and Evaluation, Department of Medical Microbiology, Southmead Hospital, Bristol BS10 5NB, UK
Received 27 February 2003; returned 6 April 2003; revised 19 May 2003; accepted 19 May 2003
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Abstract |
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Keywords: bioluminescence, Streptococcus pneumoniae, oxazolidinones
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Introduction |
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Linezolid is the first of a new class of synthetic antimicrobial agents, oxazolidinones, to be approved for clinical use against many resurgent Gram-positive pathogens.2 It has a potent range of activity against multiresistant staphylococci, enterococci and streptococci. Oxazolidinones disrupt bacterial growth by inhibiting the initiation process of protein synthesis. Specifically, they block the formation of the 70S initiation complex by binding to the 50S ribosomal subunit.3 This site of inhibition is different from those of other protein synthesis inhibitors that interfere with the translation elongation process; hence, cross-resistance with other protein synthesis inhibitors has not been reported.4 Linezolid, which has little activity against Gram-negative bacteria, is known to be bacteriostatic against staphylococci and enterococci, and has a modest bactericidal effect on most streptococci.5
Microorganisms expressing the lux operon are able to emit light, as a result of the activity of bacterial luciferase. This activity involves the oxidation, by molecular oxygen, of reduced flavin mononucleotide (FMNH2) and a long-chain aldehyde to produce FMN, acid and bluegreen light. Since FMNH2 production depends upon functional electron transport, only metabolically active cells can produce light. Bioluminescence is an extremely sensitive, non-destructive, real-time reporter of cell metabolism that has been used successfully to monitor the effect of antimicrobials.6,7 In this study, we used bioluminescence to investigate the pharmacodynamics of linezolid on a clinical isolate of S. pneumoniae, modified to express the lux operon. We compared the method with that of traditional colony counting. The transformed strain retained its ability to form capsules and demonstrated growth and antimicrobial susceptibility identical to the parent strain.7
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Materials and methods |
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S. pneumoniae SMH 11662/pAL2, a clinical isolate from Southmead Hospital (Bristol, UK), was transformed with pAL2 plasmid containing lux ABCDE operon from Photorhabdus luminescens.7 This isolate emits light constitutively and stably at 37°C and thus is an accurate reporter of cellular metabolic activity. The strain was maintained on blood agar supplemented with erythromycin (150 mg/L). Studies on S. pneumoniae were performed in brainheart infusion (BHI) broth (Oxoid, Basingstoke, UK). Linezolid (a gift from Pharmacia Corporation, Peapack, NJ, USA) was stored and prepared according to the manufacturers guidelines. The MIC of linezolid for S. pneumoniae SMH 11622/pAL2 was determined by a broth macrodilution method.8
Timekill measurements
The activity of linezolid against S. pneumoniae/pAL2 expressing the lux operon (MIC, 1.0 mg/L) was determined by monitoring bioluminescence and viable colony counts in static cultures at 37°C. Linezolid was added at concentrations of 6 mg/L (minimum concentration in serum, Cmin), 13 mg/L (intermediate concentration, Cint) and 20 mg/L (maximum concentration in serum, Cmax). At intervals, bioluminescence was measured in an automated bioluminometerphotometer (Lucy1, Anthos, Salzburg, Austria), and viable counts were determined by plating cells onto blood agar plates with a spiral plater (Autoplate model 3000, Spiral Biotech, Maryland, USA).
Measurement of bacterial regrowth
Log-phase cultures of S. pneumoniae SMH 11622/pAL2 were incubated with linezolid (6, 13 and 20 mg/L) for 1 h at 37°C. Light output and viable counts were determined before and after the addition of antibiotic. After incubation for 1 h, the cells were immediately spun down, washed and diluted (1:10) in fresh, pre-warmed BHI broth and allowed to recover at 37°C. Samples were taken to measure bioluminescence and viable counts over a 24 h period. A control culture was prepared and treated identically to the test cultures, but without exposure to linezolid. The post-antibiotic effect (PAE) was obtained from the regrowth curves by calculating the difference in time taken by experimental and control cultures to increase 1 log10 above the count observed immediately after drug removal. The control effective regrowth time (CERT) was calculated similarly, as the difference in time required for the bacteria to resume logarithmic growth and return to the pre-exposure inoculum.
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Results |
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Figure 1 depicts the killing rates of three different concentrations of linezolid against S. pneumoniae SMH 11622/pAL2, as determined by monitoring viable plate counts (cfu/mL) and bioluminescence (relative light units, RLU). The kill curves show that linezolid produced only minimal bactericidal activity (1 log10 decrease over 6.5 h) when measured by either method. In addition, independent of concentration, linezolid inhibited bioluminescence and viable counts.
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The regrowth of S. pneumoniae SMH 11622/pAL2 culture after treatment for 1 h with linezolid was monitored by viable counts and bioluminescence (Figure 2). Bacterial recovery was similar following exposure to the various drug concentrations. However, the recovery rate was modified by the method used, and commenced 1 h after drug exposure when monitored by bioluminescence and 4 h after drug exposure when measured by viable counts. Moreover, the PAE of linezolid at 6, 13, and 20 mg/L, when measured by colony counts, was considerably longer (4.3, 4.6 and 4.7 h) than the PAE determined by bioluminescence (1.7, 1.9 and 2.3 h) of the same culture. The calculated CERT values of 5.0, 5.4 and 5.5 h based on viable counts were also slightly longer than those for bioluminescence ( 4.4, 5.1 and 5.3 h)
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Discussion |
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It is interesting that CERT, which has previously been found to be method-independent,11 is also slightly longer when measured by viable counts than by bioluminescence monitoring. This supports the view that following exposure to linezolid, metabolic activity may recover more rapidly than cell replication.
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Acknowledgements |
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Footnotes |
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References |
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