Division of Microbiology and Infectious Diseases, Clinical Sciences Building, University of Nottingham, Nottingham City Hospital, Nottingham NG5 1PB, UK
Received 13 May 2005; accepted 20 May 2005
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Abstract |
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Methods: Using the Sorbarod model, biofilms were established. The strains tested included methicillin-susceptible and -resistant strains of Staphylococcus aureus and coagulase-negative staphylococci, as well as glycopeptide-intermediate S. aureus (GISA). The biofilms were exposed to exponentially decreasing concentrations of telavancin and four comparator antibiotics, vancomycin, teicoplanin, linezolid and moxifloxacin and the bactericidal activity of the antibiotics was assessed. The concentrations of the antibiotics used in these experiments corresponded to peak serum levels achievable in humans and the rates at which drug concentrations were decreased corresponded to their elimination half-lives.
Results: All of the drugs tested produced a reduction in the number of bacteria eluted from the biofilms. Telavancin was more effective than the commercially available glycopeptides, vancomycin and teicoplanin, and of the three, was the most active agent against both the non-GISA and GISA strains. Of all the antibiotics tested, moxifloxacin produced the greatest reduction in biofilm cells, but only against the non-GISA strains.
Conclusions: Telavancin exhibited substantial antimicrobial activity against staphylococcal biofilms, including GISA strains. This study supports the case for the evaluation of telavancin in the treatment of staphylococcal biofilm-associated infections.
Keywords: pharmacokinetic models , glycopeptides , GISA , indwelling medical devices
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Introduction |
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Coagulase-negative staphylococci (CoNS), although an uncommon cause of community-acquired infections, are a well established cause of nosocomial bacteraemia and indwelling medical device (IMD) associated infection. Such infections are frequently associated with adherent biofilms and are difficult to manage. Bacteria found in biofilms are often poorly controlled by antibiotics, which in particular may reflect a low growth rate and in some instances, a failure of the agent to penetrate the biofilm.7
A particular problem of IMD-associated infections is that conventional in vitro assessment of growth inhibition in liquid medium often fails to predict performance in vivo. In this study, we have investigated the effects of antibiotics on bacteria grown as biofilms, in a manner that more closely reflects the in vivo situation.
Furthermore, to address the problem of multidrug-resistant staphylococcal infections, a novel lipoglycopeptide, telavancin (TD-6424), which possesses greater bactericidal activity against staphylococci,8 has been investigated in a pharmacodynamic manner to evaluate its efficiency in controlling staphylococcal biofilms and has been compared with a selection of conventional antibiotics in a biofilm model. This permits the quantification of the effect of antibiotics on bacterial biofilms using exponentially decreasing concentrations of a drug.
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Materials and methods |
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The eight staphylococci studied included: a fully susceptible reference strain of Staphylococcus aureus (ATCC 29213); a methicillin-resistant S. aureus (MRSA, ATCC 33591); a methicillin-susceptible S. aureus (MSSA, MS 01); a methicillin-resistant Staphylococcus epidermidis (MRSE, RP 62A); a methicillin-susceptible S. epidermidis (MSSE, MS 501) and three glycopeptide intermediate S. aureus (GISA) strains [Mu 50 and Mu 3 isolated in Japan6 and HIP-5836 supplied by Theravance, Inc. (South San Francisco, CA, USA)]. Strains MS 01 and MS 501 were clinical isolates from blood cultures collected at the City Hospital, Nottingham, UK.
The following antibiotics were obtained as reference powders: telavancin (Theravance, South San Francisco, CA, USA), linezolid (Pharmacia, Kalamazoo, MI, USA), vancomycin (Sigma, Dorset, UK), teicoplanin (Aventis Pharma, Strasbourg, France) and moxifloxacin (Bayer, Wuppertal, Germany). Stock solutions were prepared and stored according to the recommendations of the British Society for Antimicrobial Chemotherapy (BSAC).9
MIC and MBC determinations
MICs and MBCs were determined in a 96-well microtitre plate using the BSAC standard microdilution method9 except MuellerHinton broth (MHB; Oxoid, Basingstoke, UK) was used with an inoculum of 105 cfu/mL.
Biofilm studies
The biofilm model used in this study was a modification of the Sorbarod model10 (Figure 1) described previously.11 Sorbarods (Ilacon Ltd, Kent, UK) consist of a compacted concertina of cellulose fibres encased within a cylindrical paper sleeve. The Sorbarod filter is contained within a length of PVC tubing and has bacteria loaded on to it from a syringe. A biofilm is established by inoculating the Sorbarod and incubating overnight at 37°C. Cells attach to the cellulose fibre plug and are perfused with medium (MuellerHinton broth) from one side; cells shed from the opposite side are collected and counted by plating out. After the initial loss of loosely attached cells into the eluted medium, a steady state is established in which the adherent biomass and the rate of cell release from the fibres becomes constant. The number of cells eluted from the biofilm reflects the number of actively dividing cells within the biofilm. The model can then be used to quantify the effects of antibiotics on the biofilm cells.
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The bactericidal index of each drug/bacteria combination was calculated using FigP software. This calculates the AUC for each curve when log10 reduction in viable count is plotted against log10 time (h).12
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Results |
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Telavancin gave the most consistent and extensive bactericidal effects of the three glycopeptides tested. Furthermore, telavancin showed the greatest reductions at the end of the experiments, although some re-growth was observed. Telavancin was again the most effective glycopeptide against the GISA strains in reducing the number of bacteria eluted from the biofilms. Following the first dose of telavancin, the number of eluted bacteria of the GISA strains HIP-5836, Mu 50 and Mu 3 fell by 3, 3 and 2 log10, respectively. This contrasts with the lesser effects produced by vancomycin (1.0, 0.4 and 0.5 log10) and teicoplanin (0, 0.8 and 0.5 log10). The reductions observed after the second dose (24 h) of telavancin were smaller than those following the first dose. The second (12 h) and third (24 h) doses of vancomycin and teicoplanin produced no effect on any of the strains with the exception of HIP-5836 following exposure to vancomycin where a reduction of 0.8 log10 was observed. At the end of the experiments, all three GISA strains exposed to vancomycin and teicoplanin had recovered to the extent that the biofilms were eluting the same number of bacteria as at the beginning of the experiments. Mu 3 also recovered after exposure to telavancin. However, the other two GISA strains, HIP-5836 and Mu 50, showed reductions of 1.5 and 1.0 log10.
Similar results were obtained with the non-GISA strains after exposure to the glycopeptide drugs to those seen with the GISA strains. Telavancin again demonstrated the most consistent and extensive reductions in the number of bacteria eluted, especially following the first dose. Of the three glycopeptide drugs, telavancin was the only one to inhibit the growth of all non-GISA strains at the end of the experiments.
Of all the antibiotics tested, moxifloxacin produced the greatest reduction in biofilm bacteria, but only against the non-GISA strains (range of 06.0 log10) and following the first dose (GISA range 01.0 log10). All GISA strains had recovered fully by the end of the experiments. Exposure to moxifloxacin however, resulted in a reduction of the number of bacteria eluted from the biofilms of all of the non-GISA strains except MSSE strain MS 501; at the end of the experiments, these reductions were greater than seen with any of the other antibiotics tested. The reductions obtained with linezolid, following the first dose, were more uniform with a range of 1.12.5 log10 and was equally effective against both the GISA and non-GISA strains. All GISA strains had recovered fully by the end of the experiments.
Table 3 gives the sample times at which the maximum log10 reductions in the number of bacteria eluted from the biofilms were observed. Following the initial doses, the range of times was smaller for the three glycopeptides (1.54.5 h) than the other two drugs (linezolid 1.511 h, moxifloxacin 1.57.5 h). The longest time for the maximum reduction to be seen occurred with linezolid and strain ATCC 33591 and was 11 h. Only three reductions were seen following the 12 h dose and these were all observed at the 12.5 h sample. Following the final doses, the range of times for the maximum reduction was 25.531.5 h. Furthermore, there were 5 and 4 strains, respectively, which failed to show any reduction after the final doses of vancomycin and teicoplanin.
Table 4 shows the bactericidal indices (BI) of the antibiotics against the bacterial strains used. The greater the BI, the greater the bactericidal activity of the antibiotic. Generally, moxifloxacin gave the highest BI values, but only against susceptible strains; the resistant strains resulted in some of the lowest values. Telavancin gave the next highest range of BI values followed by linezolid and then vancomycin and teicoplanin. Overall, the resistant strains resulted in the lower BIs.
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Discussion |
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The experiments demonstrated considerable variation in the effects of the various antibiotics on the maximum log10 reductions in the number of bacteria eluted from the biofilms. The glycopeptide antibiotics generally elicited a more rapid effect than either linezolid or moxifloxacin following the initial dose. This may be related to the fact that vancomycin and teicoplanin have been reported to be bactericidal against staphylococci;13 however, moxifloxacin is also normally recognized to be rapidly bactericidal against susceptible strains.14 Linezolid, on the other hand is considered bacteriostatic against staphylococci.15 The same patterns of response were seen with the GISA and non-GISA strains in the times to maximum reductions and were not related to the MICs.
Some of the observations made will reflect the fact that the drugs were being challenged by bacteria grown as biofilms. It is known that biofilm cells do not behave in a similar fashion to planktonically grown bacteria. Routine susceptibility tests, such as the determination of the MIC, often fail to predict therapeutic success where biofilm-associated infections are involved. For example König et al.16 found that although clinical isolates of coagulase-negative staphylococci were highly susceptible to vancomycin when tested in vitro as planktonic cells, the same organisms were resistant or tolerant to the antibiotic when grown as biofilms. The recalcitrant nature of biofilm infections is well recognized clinically.17
It is important to note that none of the drugs came close to eliminating the bacteria, even though the dosage regimen, which was selected to reflect that used in humans, meant that for much of the experimental period, the concentrations were above the MICs of the bacteria tested. The exceptions were GISA strain Mu 50 and linezolid where the drug was above the MIC (4.0 mg/L) for 9 h of the 48 h experiment and moxifloxacin which failed to exceed the MIC for all GISA strains and MSSE strain MS 501. These findings underscore the challenge of eradicating bacteria growing as biofilms.
The pharmacodynamic properties of the antibiotics tested may provide some explanation of the observed results. Antibiotics frequently demonstrate bactericidal activity that is either concentration- or time-dependent.18,19 In the case of concentration-dependent antibiotics, the parameters that best correlate with bactericidal action are the ratio of area under the concentration curve at 24 h (AUC24) to MIC (AUC24/MIC) and the ratio of maximum serum concentration (Cmax) to MIC (Cmax/MIC). In contrast, the pharmacodynamic parameters which best predict bactericidal effect of time-dependent agents varies20 between those antibiotics which exhibit only mild or moderate persistent effects where the important parameter is the time (T) above the MIC (T > MIC), compared with those exhibiting a prolonged persistent effect where the important parameter is AUC24/MIC.18 The three parameters, Cmax/MIC, T > MIC and AUC24/MIC, are really simplifications of complex relationships.21
Among the glycopeptide antibiotics, vancomycin is generally considered to exhibit time-dependent inhibition; however, as it exhibits prolonged persistence effects, AUC24/MIC is the parameter which best correlates with efficacy.20,2224 In our studies, AUC24/MIC ranged from 0.5 (strain HIP-5836, MIC 16 mg/L) to 7.9 (strains with an MIC of 1 mg/L). The results, i.e. the log10 reduction in bacteria eluted from the biofilms, do not correlate with the AUC24/MIC values.
In the case of teicoplanin, the majority of studies have reported time-dependent bactericidal activity22,2527 although, one recent study suggested a concentration-dependent bactericidal effect.28 In our experiment, the antibiotic concentration was above the MIC for all strains and there was no apparent correlation between the MICs and the maximum reductions observed emphasizing the clear differences between biofilm and planktonically growing cells.
Unlike other glycopeptide antibiotics, telavancin exhibits concentration-dependent activity and the pharmacodynamic parameter associated with efficacy is AUC24/MIC.29 The results obtained demonstrated no distinction between GISA and non-GISA strains and were again unrelated to MIC, although the number of cells eluted from the biofilms was reduced for all strains following both doses of the drug, with the exception of GISA strain HIP-5836 where there was no reduction following the 24 h dose. Additionally, only one strain, GISA strain Mu 3, had recovered fully by the end of the experiment.
Linezolid is reported as showing time-dependent killing.20,30 In our experiments, the antibiotic was above the MIC for all strains (2.0 mg/L) with the exception of GISA strain Mu 50 (MIC of 4.0 mg/L), against which the first dose of antibiotic produced the smallest reduction (1.1 log10) of all the strains tested. At the end of the experiment, three of the eight strains tested failed to recover fully (S. aureus strains MS 01, ATCC 29213 and ATCC 33591). This lack of concordance with published data for planktonically grown cells suggests that linezolid is unlikely to prove effective against biofilm-associated infections.
Moxifloxacin was the only antibiotic studied which is characterized by concentration-dependent killing.20 The pharmacodynamic parameters AUC24/MIC and Cmax/MIC ranged from 0.125 to 16.67 and 0.625 to 83.34, respectively. The findings did support a relationship to the MIC values. Strains with MICs of 4.0 mg/L (the three GISA strains and the MSSE strain MS 501) resulted in the smallest reductions in the number of bacteria eluted from the biofilms and they were also the only strains to show full recovery at the end of the experiment. In contrast, exposure to moxifloxacin demonstrated extensive reductions for all non-GISA strains and showed strong bactericidal activity even after 48 h.
In all the experiments regardless of the agent tested and bacterial strain exposed, the maximum log10 reduction in the number of cells eluted from the biofilms occurred with the first dose. The most likely explanation for this is that the more susceptible cells are killed or inhibited by the first dose in comparison with subsequent doses. Alternatively, it may be that the first dose damages a proportion of the cells which are unable to recover fully before the second dose is administered. To test this theory, sequential MICs would need to be performed on both the biofilm and eluted cells at various times throughout the experiments.
The BI is an analytical tool developed to assess the bactericidal activity of antibiotics,12 and was calculated for all the drug/bacteria combinations investigated in this study. Using this method of analysis, the antibiotic showing the greatest bactericidal activity against the susceptible strains is moxifloxacin. The two glycopeptides vancomycin and teicoplanin generally had the lowest BIs. Interestingly, it appears that the values of BIs calculated corresponded most closely to the log10 reduction in the number of bacteria eluted following the first dose of antibiotic (Table 3). Moxifloxacin resulted in the highest BI values and also showed the greatest reduction in numbers of bacteria eluted. Telavancin generally gave the next highest BI values and reduction in numbers of bacteria eluted, followed by linezolid and then vancomycin and teicoplanin, which both gave similar results. This suggests that calculation of the BI is a useful method for assessing the bactericidal activity of antibiotics against biofilm-grown bacteria.
In conclusion, this study has demonstrated that telavancin exhibits substantial antimicrobial activity against staphylococcal biofilms and compares favourably with the other antibiotics tested. Using exponentially decreasing drug concentrations, exposure to telavancin reduced the number of cells eluted from the biofilms of all strains tested. In addition, by the end of the experiments, all but one strain (Mu 3) had failed to recover fully, that is the biofilms were eluting fewer cells than at the beginning of the experiments, although in some cases, the observed reduction in numbers of bacteria eluted from the biofilms at the end of the experiments was only 1.0 log10. These studies indicate that telavancin appears to be a promising antibiotic against multidrug-resistant staphylococcal biofilms and supports the case for its evaluation in clinical studies of IMD biofilm-associated infections.
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Archer GL. Staphylococcus aureus: a well-armed pathogen. Clin Infect Dis 1998; 26: 117981.[ISI][Medline]
3. Plouffe JF. Emerging therapies for serious gram-positive bacterial infections: a focus on linezolid. Clin Infect Dis 2000; 31 Suppl 4: S1449.[CrossRef][ISI][Medline]
4. Public Health Laboratory Service. VISAvancomycin intermediate resistant Staphylococcus aureus discovered in Scotland. PHLS News Bull 1999; 9.
5. May J, Shannon K, King A et al. Glycopeptide tolerance in Staphylococcus aureus. J Antimicrob Chemother 1998; 42: 18997.[Abstract]
6.
Hiramatsu K, Hanaki H, Ino T et al. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother 1997; 40: 1356.
7. Gander S. Bacterial biofilms: resistance to antimicrobial agents. J Antimicrob Chemother 1996; 37: 104750.[ISI][Medline]
8. Pace JL, Krause K, Johnston D et al. In vitro activity of TD-6424 against Staphylococcus aureus. Antimicrob Agents Chemother 2003; 47: 36024.[CrossRef]
9.
Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother 2001; 48 Suppl 1: 516.
10. Hodgson AE, Nelson SM, Brown MRW et al. A simple in vitro model for growth control of bacterial biofilms. J Appl Bacteriol 1995; 79: 8793.[ISI][Medline]
11.
Gander S, Finch R. The effects of exposure at constant (1 h) or exponentially decreasing concentrations of quinupristin/dalfopristin on biofilms of Gram-positive bacteria. J Antimicrob Chemother 2000; 46: 617.
12. Morrissey I. Bactericidal index: a new way to assess quinolone bactericidal activity. J Antimicrob Chemother 1997; 39: 7137.[Abstract]
13. Greenwood D. Glycopeptides. In: Finch R, Greenwood D, Norrby SR, Whitley RJ, eds. Antibiotic and Chemotherapy, 8th edn. London: Churchill Livingstone, 2003; 3004.
14. Andriole VT. Quinolones. In: Finch R, Greenwood D, Norrby SR, Whitley RJ, eds. Antibiotic and Chemotherapy, 8th edn. London: Churchill Livingstone, 2003; 34973.
15. Ni Riain U, MacGowan AP. Oxazolidinones. In: Finch R, Greenwood D, Norrby SR, Whitley RJ, eds. Antibiotic and Chemotherapy, 8th edn. London: Churchill Livingstone, 2003; 3448.
16. König C, Schwank S, Blaser J. Factors compromising antibiotic activity against biofilms of Staphylococcus epidermidis. Eur J Clin Microbiol Infect Dis 2001; 20: 206.[CrossRef][ISI][Medline]
17.
Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother 2001; 45: 9991007.
18. Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998; 26: 110.[ISI][Medline]
19. Drusano GL. Antimicrobial pharmacodynamics: critical interactions of bug and drug. Nat Rev Microbiol 2004; 2: 289300.[CrossRef][ISI][Medline]
20. Zhanel GC. Influence of pharmacokinetic and pharmacodynamic principles on antibiotic selection. Curr Infect Dis Rep 2001; 3: 2934.[Medline]
21. MacGowan AP. Moxifloxacin (Bay 12-8039) a new methoxy quinolone antibacterial. Expert Opin Investig Drugs 1999; 8: 18199.[CrossRef][Medline]
22. MacGowan AP. Pharmacodynamics, pharmacokinetics, and therapeutic drug monitoring of glycopeptides. Ther Drug Monit 1998; 20: 4737.[CrossRef][ISI][Medline]
23. Peetermans WE, Hoogeterp JJ, Hazekamp-van Dokkum AM et al. Antistaphylococcal activities of teicoplanin and vancomycin in vitro and in an experimental infection. Antimicrob Agents Chemother 1990; 34: 186974.[ISI][Medline]
24. Cantoni L, Glauser MP, Bille J. Comparative efficacy of daptomycin, vancomycin, and cloxacillin for the treatment of Staphylococcus aureus endocarditis in rats and role of test conditions in this determination. Antimicrob Agents Chemother 1990; 34: 234853.[ISI][Medline]
25. Chambers HF, Kennedy S. Effect of dosage, peak and trough concentrations in serum, protein binding and bactericidal rate of efficacy of teicoplanin in a rabbit model with endocarditis. Antimicrob Agents Chemother 1990; 34: 5104.[ISI][Medline]
26. Contrepois A, Joly V, Abel L et al. The pharmacokinetics and extravascular diffusion of teicoplanin in rabbits and comparative efficacy with vancomycin in an experimental endocarditis model. J Antimicrob Chemother 1988; 21: 62131.[Abstract]
27.
Knudsen JD, Fuursted K, Raber S et al. Pharmacodynamics of glycopeptides in the mouse peritonitis model of Streptococcus pneumoniae or Staphylococcus aureus infection. Antimicrob Agents Chemother 2000; 44: 124754.
28. Odenholt I, Lowdin E, Cars O. In vitro studies of the pharmacodynamics of teicoplanin against Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium. Clin Microbiol Infect 2003; 9: 9307.
29.
Hegde SS, Reyes N, Wiens T et al. Pharmacodynamics of telavancin (TD-6424), a novel bactericidal agent, against Gram-positive bacteria. Antimicrob Agents Chemother 2004; 48: 304350.
30. Goldberg J, Owens RC. Optimizing antimicrobial dosing in the critically ill patient. Curr Opin Crit Care 2002; 8: 43540.[CrossRef][Medline]
31. Balfour JA, Wiseman LR. Moxifloxacin. Drugs 1999; 57: 36373.[ISI][Medline]
32. Clemett D, Markham A. Linezolid. Drugs 2000; 59: 81527.[ISI][Medline]
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