Download Pharmacodynamics of linezolid in a clinical isolate of Streptococcus

Journal of Antimicrobial Chemotherapy (2003) 52, 511–513
DOI: 10.1093/jac/dkg334
Advance Access publication 29 July 2003
Pharmacodynamics of linezolid in a clinical isolate of
Streptococcus pneumoniae genetically modified to express lux genes
Habib M. Alloush1*, Vyvyan Salisbury1, Roger J. Lewis1 and Alasdair P. MacGowan2
1Faculty
of Applied Science, University of the West of England, Bristol BS16 1QY; 2Bristol 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
A bioluminescent clinical isolate of Streptococcus pneumoniae was used to test the real-time effects of the
oxazolidinone antibiotic, linezolid, on metabolism compared with effects on cell replication. Viable counts
and bioluminescence measurements showed that linezolid has little bactericidal effect, which was similar
at minimum (6 mg/L), intermediate (13 mg/L) and maximum (20 mg/L) serum concentrations. The postantibiotic effect, however, was shorter when measured by light output than by viable counts. The results
demonstrate that bioluminescence provides a rapid and sensitive means of measuring the effect of
antimicrobials on bacterial metabolism, and that the latter recovers earlier than commencement of cell
replication after linezolid exposure.
Keywords: bioluminescence, Streptococcus pneumoniae, oxazolidinones
Introduction
Streptococcus pneumoniae is the most frequent bacterial cause of
pneumonia, acute meningitis and otitis media in children and adults.
Management of pneumococcal infections is complicated by the emergence worldwide of penicillin and multi-antibiotic resistance among
S. pneumoniae isolates, which is mainly caused by the unnecessary
use of antibiotics.1 As a result, there is an urgent need for new agents
with good activity against S. pneumoniae and other drug-resistant
bacterial pathogens.
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 Gramnegative 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 mono-
nucleotide (FMNH2) and a long-chain aldehyde to produce FMN,
acid and blue–green light. Since FMNH2 production depends upon
functional electron transport, only metabolically active cells can
produce light. Bioluminescence is an extremely sensitive, nondestructive, 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
Materials and methods
Test organism, antibiotics and media
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 brain–heart infusion (BHI) broth (Oxoid, Basingstoke, UK). Linezolid (a gift from Pharmacia Corporation, Peapack, NJ,
USA) was stored and prepared according to the manufacturer’s guidelines. The MIC of linezolid for S. pneumoniae SMH 11622/pAL2 was
determined by a broth macrodilution method.8
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*Corresponding author. Tel: +44-0117-3442473; Fax: +44-0117-3442904; E-mail: [email protected]
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© The British Society for Antimicrobial Chemotherapy 2003; all rights reserved.
H. M. Alloush et al.
Time–kill 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 bioluminometer–photometer (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.
Results
Concentration-independent killing
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.
Bacterial recovery after exposure to linezolid
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)
Discussion
In this work, we have demonstrated that light output from a selfbioluminescent Gram-positive pathogen can be used for rapid and
real-time evaluation of antimicrobial pharmacodynamics. Kill curve
Figure 1. Killing rates of linezolid against S. pneumoniae SMH 11622/pAL2
measured by colony plate counts (a) and bioluminescence (b). The antibiotic
concentrations used were 0 mg/L (diamonds), Cmin (triangles), Cint (circles) and
Cmax (squares).
data showed that during 20 h in static broth culture, the effect of
linezolid on both bioluminescence and colony counts of S. pneumoniae was similar and concentration-independent. Cultures exhibited
a slow exponential decrease, indicating that the bactericidal effect
of linezolid was minimal. Similar results have been reported by
Zurenko et al.5 However, the PAE of linezolid was considerably
shorter when determined by light output than when determined by the
traditional viable count method. This is in contrast to previous work
where direct methods have given either longer,6,9 or very similar
PAEs7 compared to viable counts. The shorter PAEs of viable counts
are thought to be the result of long chain or spheroplast formation,
which have been shown elsewhere to give falsely low post-exposure
viable counts.9 Here the viable counts and bioluminescence show a
very similar post-exposure drop (Figure 2), possibly because of the
limited bactericidal action of linezolid. The observed reduced PAE of
linezolid, when measured by direct methods, indicates that recovery
of cellular metabolism (as measured by bioluminescence) is more
rapid than that of cell replication (as determined by colony plate
count). There is an absence of data from other direct methods monitoring metabolic activity, but our findings are in accordance with
those from animal models, which show little or no PAEs of linezolid
in S. pneumoniae.10
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
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Bioluminescent S. pneumoniae to study linezolid effects
References
Figure 2. Regrowth kinetics of S. pneumoniae SMH 11622/pAL2 after 1 h treatment with linezolid at 0 mg/L (diamonds), Cmin (triangles), Cint (circles) and Cmax
(squares). Regrowth was monitored by viable counts (a) and bioluminescence (b).
view that following exposure to linezolid, metabolic activity may
recover more rapidly than cell replication.
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Acknowledgements
We would like to thank Drs Robin Howe and Karen Bowker for
useful suggestions and guidance, and the Bristol Centre for Antimicrobial Research and Evaluation, Southmead Hospital, Bristol.
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