An investigation of the antimicrobial effects of linezolid on bacterial biofilms utilizing an in vitro pharmacokinetic model

Sarah Gander, Katy Hayward and Roger Finch,*

Division of Microbiology and Infectious Diseases, Clinical Sciences Building, University of Nottingham, Nottingham City Hospital, Nottingham NG5 1PB, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
Biofilms of methicillin-susceptible and -resistant Staphylococcus aureus, a strain of coagulase-negative staphylococcus and glycopeptide-intermediate strains of S. aureus (GISA) were exposed to the oxazolidinone linezolid, and four comparator antibiotics (quinupristin/ dalfopristin, vancomycin, teicoplanin and ciprofloxacin) using a Sorbarod model. The effects of these antibiotics were assessed by monitoring the reduction in the number of cells eluted from the biofilms. The biofilms were exposed to the antibiotics by two methods. The first was an exponentially decreasing drug concentration method, where the rate of dilution was matched to the half-lives of the antibiotics and the initial concentration matched peak serum levels. The second was a constant drug concentration method, in which biofilms were exposed to antibiotics for 2 h, with the concentration of the antibiotic equalling the total amount of drug used in the exponentially decreasing method. The results indicate that linezolid produces a greater reduction in the number of cells eluted with the exponentially decreasing method compared with the constant concentration exposure against all strains tested except for one of the GISA strains, Mu 50. Overall, ciprofloxacin produced the greatest effects in the exponentially decreasing concentration experiments, but only against non-resistant strains. In the constant concentration exposure no one drug was responsible for the largest reductions in cell numbers observed. Linezolid and quinupristin/dalfopristin produced a reduction in the number of cells eluted from the biofilms of all of the strains tested in both methods of exposure and should be considered for further clinical studies of the treatment of staphylococcal biofilm-associated infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
For several decades there has been a steady increase in the incidence of bacterial infections caused by Gram-positive bacteria. Gram-positive infections have become a serious problem, especially in the nosocomial setting, and the treatment of these infections is complicated by the emergence of multidrug-resistant pathogens.1 Staphylococci are among the most frequent causes of both community- and hospital-acquired infection,2 and the prevalence of these infections appears to be increasing. Consequently, the isolation of clinical strains of Staphyloccus aureus exhibiting reduced susceptibility to the glycopeptides3–5 is extremely worrying. The rise in antimicrobial resistance among Gram-positive species has highlighted concerns regarding the use of antibiotics and the need for novel agents.6

One such novel antibiotic is linezolid. Linezolid is the first of a new class of antibiotic, the oxazolidinones, with a unique mechanism of action. Bacterial protein synthesis is inhibited by blocking the formation of ribosomal initiation complexes.7 This antibiotic has been shown to have a broad spectrum of activity against Gram-positive organisms, including methicillin-resistant S. aureus (MRSA).8 The effects of linezolid on bacterial biofilms, a common complication of medical device-associated staphylococcal infections, have not as yet been investigated. Biofilm-associated infections are becoming more common, this is largely due to the increase seen in the use of indwelling medical devices. Bacteria found in biofilms typically exhibit increased MICs resulting in reduced susceptibility to antibiotics. It is thought that a number of factors contribute to this increase in resistance, such as a low growth rate and failure of the agent to penetrate the biofilm.9 The majority of in vitro assessments of antibiotics from which conclusions are applied in vivo are still performed using bacterial cells cultured in liquid medium. In this study we have investigated the effects of antibiotics on bacteria grown as biofilms, which more closely represents the in vivo situation of infection complicating indwelling medical devices than liquid-cultured bacteria.

This study was designed to assess the effectiveness of linezolid and four comparator antibiotics against staphylococcal biofilms. A range of strains was selected, including MRSA, a coagulase-negative staphylococcus (CoNS) and three strains of S. aureus with reduced susceptibility to the glycopeptide antibiotics [glycopeptide-intermediate S. aureus (GISA)]. In vivo, intermittent administration of antibiotics usually results in the organisms being subjected to decreasing concentrations of the drug. Therefore, experiments in vitro that expose bacteria to constant levels of antibiotics do not reflect the true in vivo situation.10 To compare in vitro activity and simulate the situation found in vivo, we have studied biofilms exposed to exponentially decreasing and constant concentrations of the drugs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
Organisms

The strains chosen for investigation were: a reference strain S. aureus ATCC 29213; a MRSA (MR 038); a methicillin-susceptible S. aureus (MSSA) (MS 020); a coagulasenegative Staphylococcus epidermidis (CoNS 01); and three GISA stains, Mu 3 and Mu 50 isolated in Japan3,11 and SMH 24 isolated in Bristol12 (kindly supplied by Dr Alasdair MacGowan, Bristol, UK). All strains except ATCC 29213 and the GISA strains were clinical strains from Nottingham City Hospital. Clinical strains from Nottingham were blood culture isolates. Association with invasive devices was not determined.

The following antibiotics were supplied as reference powders by their respective manufacturers: linezolid (Pharmacia and Upjohn, Kalamazoo, MI, USA), quinupristin/ dalfopristin (Aventis Pharma, West Malling, UK), vancomycin (Eli Lilly, Basingstoke, UK), teicoplanin (Aventis Pharma) and ciprofloxacin (Bayer, Wuppertal, Germany). Stock solutions were prepared and stored according to the recommendations of the BSAC.13

MIC and MBC determinations

MICs and MBCs were determined using the standard broth dilution method13 and Mueller–Hinton broth (MHB; Oxoid, Basingstoke, UK).

Biofilm studies

The biofilm model used in this study was a modification of the Sorbarod model,14 in which quarter Sorbarods were used. A Sorbarod (Ilacon Ltd, Kent, UK) consists of a cylindrical paper sleeve encasing a compacted concertina of cellulose fibres. Bacteria attach to the cellulose fibre plug and are subsequently perfused with MHB (Oxoid) from one side and bacterial cells are shed from the opposite side. After initial loss of loosely attached bacteria into the eluted medium, a steady state is achieved in which the adherent biomass and the rate of bacterial cell release from the membrane remain constant. The number of bacteria eluted from the biofilm reflects the number of actively dividing bacteria in the biofilm, and so it can be used to quantify the effects of antibiotics on the biofilm. The system is maintained at 37°C.

Exposure to exponentially decreasing drug concentrations

Perfusing biofilms with media via a dilution vessel into which drugs are added allows the biofilm cells to be exposed to exponentially decreasing concentrations of antibiotic. A modified version of a previously described method15 was used. Briefly, test and control biofilms are established as above, except that the tubing carrying the medium from the medium reservoir to the test biofilm goes via a dilution vessel. Once the biofilms have reached a steady state, i.e. a constant number of bacteria are being eluted (determined by performing viable counts on the eluate), the antibiotic is added directly to the dilution vessel. Owing to the design and tubing layout, a vacuum is created in the dilution vessel that draws medium out from the reservoir, thus diluting the drug in the dilution vessel at the same rate as the medium is delivered to the biofilm. This results in the test biofilms being exposed to an exponentially decreasing concentration of the antibiotic. The concentration of antibiotic to which the biofilms were exposed was calculated to match those reported for in vivo Cmax values (Table 1Go). This concentration was added to the dilution vessel three times at 12 hourly intervals. Using the equation tH = 0.6931 V/r, based on first-order decay kinetics,15 to determine the volume of media required in the dilution vessel, the dilution rate of the drugs was matched to the half-lives of the antibiotics (Table 1Go). Viable counts were performed on the cells eluted from the biofilms before and during perfusion with the antibiotics. The control biofilms were perfused with MHB without exposure to the antibiotics. The viable counts were determined by spiral plating (Spiral Systems, Cincinnatti, OH, USA) of serially diluted samples of eluate subcultured on to nutrient agar (Oxoid). Using this method the sample is deposited on to the agar plate in a spiral, with the rate of sample deposition decreasing exponentially, resulting in the greatest amount of sample being near the centre, with ever decreasing amounts towards the edge of the plate. This virtually removes the need for serial dilutions. Determination of bacterial density is made using a specially designed grid placed on the plate after incuba-tion.


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Table 1. Human pharmacokinetic details of the antibiotics studieda
 
Exposure to constant drug concentrations

Test and control biofilms were set up and perfused with MHB. When the biofilms had reached a steady state, the test biofilms were perfused for 2 h with MHB containing the appropriate concentration of the antibiotic. Following this exposure perfusion continued in the absence of the drug. The concentrations of antibiotic to which the biofilms were exposed were equal to the total amount of drug to which the biofilms were exposed in the exponentially decreasing concentration model (see Table 1Go). This was calculated using the equation given in the Appendix. Viable counts, again determined by spiral plating, were performed on the cells eluted from the biofilms before, during and after exposure to the antibiotics.

The flow rate remained at 15 mL/h throughout all experiments, which were performed in triplicate.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
The MICs and MBCs for the bacterial strains tested are given in Table 2Go. The results from the exponentially decreasing drug concentration experiments are given in Table 3Go. This shows the maximum log10 reduction in cell numbers eluted from the biofilms after each of the three doses of antibiotics given and whether this reduction resulted in no viable cells being eluted (no growth), and the reduction in the number of cells, if any, seen at the end of the experiment (30 h).


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Table 2. MICs and MBCs (mg/L) for the bacterial strains used
 

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Table 3. Maximum (± S.D.) log10 reductions in bacteria eluted from the biofilms following exposure to exponentially decreasing concentrations of antibiotics administered on three occasions and the log10 reduction observed at the end of the experiment (30 h)
 
The most consistent and extensive bactericidal effects seen with the non-GISA strains in the exponentially decreasing concentration experiments were produced by linezolid, quinupristin/dalfopristin and ciprofloxacin. CoNS 01, however, was not affected at all by ciprofloxacin and only slightly by quinupristin/dalfopristin.

Ciprofloxacin produced the greatest effect with S. aureus ATCC 29213 and the MRSA MR 038, with no detectable growth following the second dose of the drug. Linezolid was the only antibiotic to produce a reduction in the number of viable bacteria eluted from the biofilms throughout the experiment. For the GISA strains, the antibiotics producing the greatest bactericidal effects were linezolid and quinupristin/dalfopristin, with the greatest reductions in cell numbers being seen with SMH 24 exposed to linezolid. Mu 50 was affected after all three doses by quinupristin/dalfopristin, but only following the initial dose of linezolid. A reduction in the number of cells eluted from the Mu 3 biofilms was only observed following the first dose of all of the antibiotics tested. Ciprofloxacin did not produce any detectable effect against any of the GISA strains. Vancomycin and teicoplanin caused a reduction in the number of cells eluted following the first dose for all strains except Mu 3, where no reduction was observed throughout the experiments.

The results obtained with the constant antibiotic concentration experiments are summarized in Table 4Go, which shows the maximum log10 reduction in the number of cells eluted from the biofilms and the final reduction in the number of cells eluted at the end of the experiment.


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Table 4. Maximum (± S.D.) log10 reductions in bacteria eluted from the biofilms following constant concentration exposure to the antibiotics over 2 h and the log10 reduction seen at the end of the experiment (30 h)
 
Less variation was observed with exposure to constant concentrations than with exposure to exponentially decreasing concentrations, with all antibiotics producing a reduction in the number of cells eluted from the biofilms for all strains tested. Quinupristin/dalfopristin produced the greatest reduction with vancomycin-susceptible strains, resulting in no detectable growth with MSSA strain MS 020 after exposure to the drug for 1.5 h. Surprisingly vancomycin appeared to produce the greatest reductions with the GISA strain SMH 24. The reductions in eluted cells for all the antibiotics against the GISA strains ranged from 0.6 to 2.5 log10. Linezolid produced a consistent effect, reducing the number of cells eluted by at least 1.1 log10. By the end of the experiment the majority of strains had shown recovery, with the biofilms eluting the same number of cells as at the beginning of the experiment.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
Overall the results of this study indicate that for linezolid, exposure to three doses of exponentially decreasing concentrations of this antibiotic appeared to be more effective at reducing the number of cells eluted from staphylococcal biofilms than exposure to the same amount as a constant exposure for 2 h. For the two glycopeptides and quinupristin/dalfopristin, constant exposure for 2 h was more effective than the administration of three doses of exponentially decreasing concentrations. For ciprofloxacin, constant exposure appears to be more effective against the GISA strains and the CoNS 01 strain, whereas with the other strains the exponentially decreasing method of exposure appears to be more effective at reducing the number of cells eluted from the biofilms. It is important to be aware that the observed reductions in the numbers of cells eluted from the biofilms could be due to either an inhibition of cell growth or the death of the cells.

It was noted that with the exponentially decreasing method of exposure for four of the five drugs tested and all strains of bacteria, the maximum log10 reduction in the number of cells eluted from the biofilms occurred after the first dose. The exception was quinupristin/dalfopristin, where the maximum reduction with strains MSSA 020 and SMH 24 occurred following the second dose.

The experiments performed where the biofilms were exposed to exponentially decreasing concentrations of antibiotics could be considered to be simulating the in vivo administration of antibiotics at 12 h intervals, which is comparable to the recommended regimen for linezolid. Exposure of the biofilms to constant antibiotic concentrations could be compared to the in vivo administration of drug by infusion over 2 h. With the exponentially decreasing method of drug exposure the biofilm cells were exposed to a decreasing concentration of the antibiotics with the rate of this decrease depending on their half-lives.

The antibacterial action of the glycopeptides vancomycin and teicoplanin, and the streptogramin quinupristin/ dalfopristin, is time dependent.16 This means that the important pharmacodynamic parameter predicting their antimicrobial activity is the total amount of drug or the area under the curve over 24 h (AUC24)/MIC ratio or the time above the MIC. When calculated for the two methods of exposure, it was found that the time above the MIC was greater in the exponentially decreasing concentration model for all of the bacterial strains. With two exceptions, S. aureus ATCC 29213 with quinupristin/dalfopristin and MSSA 020 with teicoplanin, the maximum log10 reductions in the number of bacteria eluted from the biofilms was seen with the constant concentration exposure experiments. The total log10 reduction, if any, seen at the end of the experiment was, however, generally greater with the exponentially decreasing concentration model.

Ciprofloxacin kills in a concentration-dependent manner. Here the pharmacodynamic parameters that best correlate with bactericidal action are the maximum serum concentration (Cmax)/MIC ratio and also AUC24/MIC. This drug would therefore be expected to have a greater effect in the constant concentration exposure experiments. This was true for those bacterial strains resistant to ciprofloxacin, the three GISA strains and CoNS 01 (see Table 2Go), but was not observed for the other strains tested, where ciprofloxacin had a much greater effect in the exponentially decreasing concentration experiments. Linezolid has been quoted as inhibiting in a time-dependent manner.16 This is supported by the lack of concentration-dependent killing against staphylococci.17 In this study the drug appeared to be more effective in the exponentially decreasing concentration experiments, which would be in accordance with a half-life of 4.5 h, Cmax of 16 mg/L and activity (MIC) against all of the strains tested of 2 mg/L, resulting in the time above MIC being greater in the exponentially decreasing concentration model compared with the constant concentration model.

The results of this study indicate that linezolid is an effective antibiotic against staphylococcal biofilms and compares favourably with the other four antibiotics tested. Exposure to linezolid resulted in the reduction of the number of cells eluted from the biofilms of all strains tested with both methods of exposure. It should be mentioned, however, that total eradication of the biofilm was not demonstrated, and with infections associated with indwelling medical devices the total eradication of the biofilm cells is important, as recolonization of the surface by any surviving bacteria is very likely to occur. To determine whether treatment with linezolid can result in total eradication of the bacteria, further experiments would have to be performed, with the biofilms being exposed to the antibiotic for longer periods of time or to further repeated dosages.

Many studies have shown linezolid to be effective against a broad range of Gram-positive bacteria, including multidrug-resistant organisms.18–22 Only one other study has looked at the effects of linezolid on biofilms.23 In this study, drug concentrations within the biofilm were measured and concentrations of vancomycin in the biofilm were found to be higher than those of linezolid after antibiotic infusion. A study of the activity of linezolid against GISA strains found the antibiotic to be active against the three strains tested.24 In the present study, the GISA strains tested were all found to be susceptible to linezolid. Although quinupristin/dalfopristin also performed well against these strains, issues of resistance and patient tolerance must be considered before recommending an antibiotic. Resistance to quinupristin/dalfopristin has already been documented,25 including in enterococcal strains that have never been exposed to the drug.26

Quinupristin/dalfopristin is difficult to administer because it is associated with venous irritation,27 the most frequently reported events being pain and/or inflammation.28 Linezolid, on the other hand, is reported to be well tolerated and is available in both an oral and intravenous form. In addition, studies have shown that resistance is not easily induced,29 and that owing to the oxazolidinones' mechanism of action the appearance of resistant mutants during treatment is unlikely.30 However, caution is necessary in view of the recent reports of resistance among enterococci31 and MRSA32 occurring during therapy. Based on the observations in the present study, linezolid appears to be a promising antibiotic with the potential for treating multidrug-resistant staphylococcal biofilm-associated infections. Clinical studies are awaited with interest.


    Appendix
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
Calculation used to determine the total amount of drug the biofilms were exposed to in the exponentially decreasing concentration model

When the first dose of drug is administered the concentration is C0.

This drug concentration then exponentially decreases according to the equation lnC/C0 = -r/vt.

From this equation it can be worked out what the concentration (C{alpha}) of drug is just before the next dose is administered (t{alpha}).

When the drug is added the concentration = C{alpha} + C0C0 is the contribution from the new dose and C{alpha} is the contribution from the residual drug from the first dose.

Using the decay equation with the initial concentration of C0 + C{alpha} the same process can be carried out to work out Cß at tß and so on.



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Figure A. Explanation of the calculation of the total amount of drug used.

 

    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
We wish to acknowledge receipt of an educational grant from Pharmacia to support these studies.


    Notes
 
* Corresponding author. Tel: +44-115-840-4741; Fax: +44-115-840-4742; E-mail: r.finch{at}nottingham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
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Received 18 June 2001; returned 29 August 2001; revised 21 September 2001; accepted 29 October 2001