In vitro activity of a new antibacterial rhodanine derivative against Staphylococcus epidermidis biofilms

Journal of Antimicrobial Chemotherapy (2006) 58, 778 783 doi:10.1093/jac/dkl314 Advance Access publication 30 July 2006 In vitro activity of a new antibacterial rhodanine derivative against Staphylococcus epidermidis biofilms Maxime Gualtieri 1, Lionel Bastide 2, Philippe Villain-Guillot 1, Sylvie Michaux-Charachon 3,4, Jaqueline Latouche 1 and Jean-Paul Leonetti 1 * 1 CNRS UMR 5160, Centre de Pharmacologie et Biotechnologie pour la Santé, Faculté de Pharmacie, Montpellier, France; 2 Selectbiotics, Nîmes, France; 3 INSERM U-431, Faculté de Médecine, Nîmes, France; 4 Laboratoire de Bactériologie, CHU de Nîmes, Nîmes, France Introduction Received 24 April 2006; returned 8 June 2006; revised and accepted 7 July 2006 bjectives: Staphylococcus epidermidis biofilms form at the surface of implants and prostheses and are responsible for the failure of many antibiotic therapies. nly a few antibiotics are relatively active against biofilms, and rifampicin, a transcription inhibitor, is among the most effective molecules for treating biofilm-related infections. Having recently selected a new potential transcription inhibitor, we attempted to evaluate its efficacy against S. epidermidis biofilms. Methods: Biofilm-forming S. epidermidis strains were grown planktonically or as biofilms and their susceptibility to this transcription inhibitor was compared with reference antibiotics with different mechanisms of action. Conclusions: ur results demonstrate that this new molecule is active; its effects are fast and kinetically related to those of rifampicin, but unlike rifampicin it does not select for resistant bacteria. Keywords: RNA polymerase, inhibitors, antibiotics Biofilm-related infections are very common nosocomial infections 1 4 and account for significant morbidity and mortality. Biofilms are formed by the colonization of solid supports (bones, implants and catheters) by adherent bacteria. Several mechanisms have been proposed to explain why only very few molecules are active against biofilms: biofilm-embedded bacteria enter a nongrowing (stationary) state, in which they are less susceptible to growth-dependent antimicrobial killing, 5 physicochemical interaction of certain antibiotics with slime 6 and lower diffusion, 7,8 or changes in the bacterial envelope following adhesion. However, the presence in the biofilms at a high frequency of persisters, bacteria that do not grow but do not die in the presence of the antibiotic, might be the cause of these recalcitrant infections. 9 Vancomycin, for example, is often used to treat biofilm-related infections because of the frequent occurrence of methicillinresistant coagulase-negative staphylococci, but its efficacy is low within a biofilm. Consequently, vancomycin must be used in association with other molecules. n the other hand, despite its tendency to trigger the appearance of resistance, the efficacy of rifampicin in treating bacteria adhered to biomaterials has been broadly demonstrated in vitro 10 and in clinical trials. 11 Rifampicin derivatives with high antistaphylococcal activity are among the most lipophilic antibiotics. Their association with other lipophilic bacteriostatic agents such as erythromycin and fusidic acid results in antimicrobial activities far more effective than the individual agents against non-growing bacteria. 12 We have recently selected relatively hydrophobic new bactericidal agents that inhibit transcription in enzymatic studies and affect bacterial RNA synthesis. 13 These molecules are active in the micromolar range against planktonic bacteria. Unlike rifampicin, which triggers the appearance of resistance due to point mutation at the surface of the polymerase, these molecules are far less likely to select resistance. We describe here our investigation of the efficacy of one of them, called SB13 (Figure 1), against biofilms of Staphylococcus epidermidis. Materials and methods Bacterial strains and antimicrobial agents S. epidermidis reference strains ATCC 35984 (RP62A) and DSM 3269 and two clinical isolates (strains 40004 and 48155) obtained from patients with staphylococcal infection at the University Hospital of Nîmes were used. All four isolates were chosen for their ability to... *Corresponding author. Tel: +33-467-548-607; Fax: +33-467-548-610; E-mail: jp.leonetti@cpbs.univ-montp1.fr... 778 Ó The Author 2006. Published by xford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RNA polymerase inhibitors active on biofilms H Table 1. MICs and MBCs of SB13 and reference antibiotics determined by the broth microdilution method for planktonic S. epidermidis (ATCC 35984) Antibiotic MIC (mg/l) MBC (mg/l) Figure 1. Structure of SB13. form a biofilm. The following antimicrobial agents belonging to different antibiotic classes were selected, most of them for their common use in human medicine: rifampicin (Sigma-Aldrich), vancomycin (Sigma-Aldrich), minocycline (Sigma-Aldrich), fusidic acid (Sigma-Aldrich), novobiocin (Sigma-Aldrich), fosfomycin (Sigma-Aldrich), a mixture of amoxicillin (84%)/clavulanic acid (16%) (AugmentinÒ, GlaxoSmithKline), a mixture of imipenem (50%)/cilastatin (50%) (TienamÒ, Merck Sharp and Dohme Chibret) and SB13 (Chembridge Corp., San Diego, CA, USA). Susceptibility testing methods MICs were determined as recommended by the CLSI. 14 Antibiotics were tested at final concentrations (prepared from serial 2-fold dilutions) ranging from 20 to 1.5 10 5 mg/l. The MIC was defined as the lowest antibiotic concentration that yielded no visible growth. The test medium was Mueller Hinton broth (MHB) and the inoculum was 5 10 5 cfu/ml. The inoculated microplates were incubated at 37 C for 18 h before reading. MBCs were established by extending the MIC procedure to the evaluation of bactericidal activity. After 24 h, 10 ml was drawn from the wells, serially diluted and then spotted onto suitable agar plates. The plates were incubated at 37 C overnight. The MBC read 18 h later was defined as the lowest concentration of antibiotic that resulted in 0.1% survival in the subculture. All the experiments were performed in triplicate. The results are summarized in Table 1. Growth of biofilms in 96-well polystyrene microtitre plates The wells of 96-well polystyrene microtitre plates (Falcon Microtest TM ) were filled with 0.1 ml aliquots of S. epidermidis inoculum (10 7 cfu/ml), and the plates were incubated for 24 h at 37 C. The wells were rinsed twice with 0.2 ml of sterile water to discard non-adherent bacteria. Subsequently, 0.1 ml of MHB containing the desired antibiotic concentration was added to the wells and the plates were incubated at 37 C for 1 24 h without shaking. After the challenge, the plates were washed twice with sterile water and then 0.1 ml of MHB was added. Adherent bacteria were sonicated with a Branson 450 Sonifier with a microtip (4 2 s, 10% of the maximal amplitude) and bacteria were quantified by a serial dilution method. Aliquots were spotted onto MHB-agar plates. The plates were incubated at 37 C for 24 h before counting. All the experiments were performed in triplicate. Antibiotic combination study on biofilms S Twenty-four hours after biofilm formation, the biofilms were washed twice with sterile water. Subsequently 0.1 ml of MHB containing S N SB13 2.5 2.5 Rifampicin 0.001 0.002 Fosfomycin 20 40 Vancomycin 1.25 1.25 Minocycline 0.02 2.5 Fusidic acid 0.04 1.25 Novobiocin 0.08 0.16 Amoxicillin/clavulanic acid 2.5 5 Imipenem/cilastatin 1.25 2.5 antibiotics 1 and 2 at 0.5, 1, 2 and 4 MIC was added to the wells. The plates were incubated at 37 C for 24 h without shaking. They were then processed as described above. All the experiments were performed in triplicate. Results The bacterial strain ATCC 35984 used in the study is known for its ability to colonize solid supports such as plastic culture dishes and catheters and has been already tested by numerous laboratories. 15,16 Preliminary control experiments demonstrated unambiguously that this adherent strain generated reproductively a bacterial count about 100-fold greater than non-adherent S. epidermidis strains (data not shown). The dose-dependent effects of control antibiotics and SB13 incubated on 24 h biofilms were evaluated first. As shown in Table 2, only rifampicin and SB13 decreased the bacterial count by more than 3 log 10 U. At 20 mg/l, amoxicillin/clavulanic acid or imipenem/cilastatin had little or no activity, whereas fosfomycin, fusidic acid, vancomycin, novobiocin and minocycline had intermediate activity. It is commonly accepted that biofilms become more resistant to antibiotics with ageing. 17 Six hour biofilms generally respond well in vitro to the treatment, but resistance increases at 24 and 48 h. We did not attempt to evaluate the effects of SB13 on 6 h biofilms because they behave more like planktonic bacteria than true biofilms. However the comparison between 24 and 48 h biofilms correlates with data from the literature. When the age of the biofilm increased, the biofilms became more resistant and the effects of all the antibiotics tested became negligible (data not shown). In order to investigate the time dependence of the activity of rifampicin and SB13, the biofilms were incubated for an increased period of time with these molecules (Tables 3 and 4). The kinetics of action of SB13 were slower than those of rifampicin. After a 3 h challenge with 8 MIC, reductions of 1.79 and 3.7 log 10 Uof the bacterial counts were observed for SB13 and rifampicin, respectively. However, at 8 MIC and treatment for 24 h, SB13 and rifampicin decreased the bacterial viability respectively by more than 3.9 and 3.14 log 10 U (Figure 2). Despite the fact that SB13 exhibited the highest MIC of the antibiotics tested, it was the most efficient molecule on biofilm after rifampicin in terms of concentration and the best molecule in terms of the reduction of the bacterial count. 779

Gualtieri et al. Table 2. Effects of SB13 and references antibiotics incubated for 24 h with an S. epidermidis (ATCC 35984) biofilm Imipenem/ cilastatin Amoxicillin/ clavulanic acid Antibiotic concentration (mg/l) SB13 Rifampicin Fosfomycin Vancomycin Minocycline Fusidic acid Novobiocin 20.00 3.90 0.083 a 3.48 0.087 1.59 0.103 2.40 0.087 2.12 0.065 2.90 0.079 2.23 0.085 0.77 0.102 0.58 0.139 10.00 2.74 0.088 3.65 0.247 1.03 0.066 2.27 0.118 1.97 0.117 2.84 0.078 2.25 0.103 0.37 0.063 0.10 0.018 5.00 1.06 0.026 3.62 0.123 0.98 0.087 0.23 0.155 2.30 0.069 2.65 0.079 2.06 0.057 0.06 0.121 0.18 0.073 2.5 0.33 0.105 3.59 0.090 1.09 0.120 0.11 0.090 2.20 0.099 2.32 0.051 2.20 0.059 0.03 0.042 0.00 0.047 1.25 0.31 0.120 3.37 0.130 0.85 0.064 0.03 0.155 2.22 0.086 2.40 0.110 2.28 0.139 0.56 0.150 0.12 0.025 0.63 0.08 0.113 3.32 0.087 0.73 0.183 0.02 0.039 2.10 0.139 2.02 0.087 1.79 0.081 0.08 0.021 0.18 0.091 0.31 0.03 0.083 3.39 0.175 0.57 0.076 0.24 0.039 2.40 0.107 1.84 0.097 1.76 0.068 0.03 0.110 0.03 0.116 0.16 0.42 0.056 2.97 0.055 0.52 0.046 0.18 0.087 2.22 0.089 0.84 0.087 1.49 0.107 0.09 0.130 0.04 0.100 0.08 0.06 0.059 3.11 0.032 0.52 0.032 0.14 0.110 1.96 0.073 0.75 0.090 1.42 0.087 0.13 0.202 0.00 0.052 0.04 0.47 0.064 3.10 0.139 0.11 0.068 0.20 0.092 1.52 0.055 0.89 0.158 0.79 0.177 0.07 0.116 0.08 0.102 0.02 0.21 0.064 3.18 0.081 0.27 0.104 0.02 0.186 1.10 0.157 0.08 0.066 0.41 0.108 0.09 0.147 0.03 0.090 0.01 0.25 0.019 3.14 0.102 0.08 0.017 0.08 0.064 0.39 0.061 0.08 0.072 0.47 0.124 0.02 0.032 0.11 0.029 0.005 0.19 0.051 1.97 0.041 0.21 0.057 0.27 0.102 0.38 0.084 0.01 0.079 0.38 0.093 0.01 0.051 0.09 0.032 2.4 10 3 0.00 0.079 0.67 0.036 0.04 0.056 0.09 0.158 0.03 0.068 0.05 0.159 0.06 0.122 0.00 0.007 0.02 0.160 1.2 10 3 0.27 0.165 0.17 0.202 0.16 0.085 0.03 0.110 0.12 0.040 0.11 0.128 0.04 0.064 0.09 0.069 0.01 0.117 a Mean value standard error of the difference in log10 cfu/ml between untreated and treated samples observed in 24 h S. epidermidis (ATCC 35 984) biofilms. Antibiotic combinations are often necessary in the treatment of S. epidermidis infection. 17 These combinations are used in treatments involving rifampicin to avoid the appearance of rifampicin resistance. The combinations can also enhance the effects of individual antimicrobial agents by synergic action. When we used 96-well plates to challenge the biofilm with rifampicin and SB13 for several days, no resistant bacteria were selected (data not shown). However, when 6-well plates were used, the decrease of the bacterial count was only transient with rifampicin, whereas it was stable with SB13. This result can account for the fact that the number of bacteria present in the 6-well plates was greater then in the 96-well plates and that spontaneous rifampicin-resistant bacteria were selected. Under the same conditions, SB13 did not select any resistant bacteria (Table 5). When we tested different antibiotic combinations (Table 6), the strongest synergic effect was observed with SB13 and vancomycin, the difference being attributed to synergy that reached 1.21 log 10 U followed by SB13 and imipenem/cilastatin (difference = 0.68 log 10 U) and amoxicillin/clavulanic acid (difference = 0.58 log 10 U). It was striking to notice that most of the antibiotics synergic or antagonistic with rifampicin behaved similarly when combined with SB13. A comparison of the effects of SB13 on three other S. epidermidis strains, another reference strain and two strains isolated from catheters, demonstrated that these observations were not restricted to the model strain (Table 7). SB13 decreased the amount of viable bacteria by 3 4 log 10 U after a 24 h treatment. Discussion SB13 is a member of a family of synthetic molecules with strong bactericidal properties and low toxicity. 13 It inhibits transcription in enzymatic assays and selectively inhibits bacterial RNA synthesis. 13 Due to the absence of spontaneous resistant mutants, 13 we cannot exclude that this relatively hydrophobic molecule targets additional proteins. However, the fast and selective transcription inhibition by SB13 observed in vivo and the similarities between the bactericidal kinetics of rifampicin and SB13 on planktonic bacteria are encouraging facts. 13 We attempted to compare the activity of rifampicin with that of SB13 and reference antibiotics on S. epidermidis biofilms. It has been suggested that the protein target could be the major determinant of antibiotic efficacy against biofilms 18 and that molecules affecting cell wall synthesis are among the least efficient. It is also clear that rifampicin is one of the best molecules for eradicating S. epidermidis. 10,18,19 Due to its very low MIC, rifampicin is more active in the present study than SB13 in terms of concentration. However at a concentration close to its MIC and comparable to the antibiotic concentration often used in the literature on biofilms 10,18 SB13 is as efficient as rifampicin. This confirms that antibiotics targeting transcription do not differ in their efficacy against biofilms. To our knowledge this is the first comparison of two transcription inhibitors with different structures and more detailed studies with other inhibitors are ongoing. Incidentally, we also observed that molecules targeting different proteins involved in cell synthesis can differ strongly in their efficacy to eradicate the biofilm. Vancomycin and fusidic acid decreased the bacterial counts by 2 3 log 10 U, while mixtures 780

RNA polymerase inhibitors active on biofilms Table 3. Time dependence of the activity of SB13 against S. epidermidis (ATCC 35984) biofilm SB13 concentration (mg/l ) MIC 1 h 3 h 6 h 12 h 24 h 20.00 8 0.83 0.081 a 1.79 0.090 2.04 0.075 2.74 0.174 3.90 0.083 10.00 4 0.08 0.115 1.07 0.028 1.26 0.042 2.08 0.071 2.74 0.088 5.00 2 0.10 0.055 0.54 0.045 0.52 0.067 0.90 0.062 1.06 0.026 2.5 1 0.04 0.065 0.28 0.114 0.26 0.075 0.47 0.157 0.33 0.105 1.25 0.5 0.12 0.021 0.09 0.096 0.13 0.078 0.33 0.139 0.31 0.120 0.63 0.25 0.08 0.061 0.09 0.088 0.10 0.068 0.01 0.080 0.08 0.113 a Mean value standard error of the difference in log 10 cfu/ml between untreated and treated samples observed in 24 h S. epidermidis (ATCC 35984) biofilms. Table 4. Time dependence of the activity of rifampicin against S. epidermidis (ATCC 35984) biofilm Rifampicin concentration (mg/l) MIC 1 h 3 h 6 h 12 h 24 h 20.00 16 383 1.12 0.056 a 3.49 0.061 2.88 0.101 3.01 0.094 3.48 0.087 10.00 8192 0.38 0.082 2.73 0.107 2.68 0.069 2.73 0.130 3.65 0.247 5.00 4096 0.18 0.035 2.76 0.059 2.61 0.080 2.79 0.043 3.62 0.123 2.5 2048 0.15 0.069 2.79 0.108 2.60 0.063 2.99 0.071 3.59 0.090 1.25 1024 0.31 0.065 2.63 0.152 2.72 0.132 2.85 0.174 3.37 0.130 0.63 512 0.31 0.058 2.83 0.099 2.10 0.032 2.92 0.092 3.32 0.087 0.31 256 0.38 0.124 2.73 0.025 2.10 0.059 2.85 0.059 3.39 0.175 0.16 128 0.43 0.122 2.81 0.110 2.07 0.112 2.92 0.083 2.97 0.055 0.08 64 0.48 0.087 2.87 0.051 1.99 0.107 3.00 0.256 3.11 0.032 0.04 32 0.46 0.088 2.81 0.068 1.89 0.087 3.23 0.069 3.10 0.139 0.02 16 0.31 0.124 2.72 0.142 1.93 0.095 3.26 0.201 3.18 0.081 0.01 8 0.33 0.080 2.70 0.071 1.99 0.091 2.58 0.087 3.14 0.102 0.005 4 0.18 0.113 2.00 0.113 2.16 0.036 1.37 0.068 1.97 0.041 2.4 10 3 2 0.03 0.079 1.43 0.046 1.44 0.110 0.04 0.014 0.67 0.036 1.2 10 3 1 0.05 0.066 1.25 0.133 1.31 0.051 0.04 0.082 0.17 0.202 6.1 10 4 0.5 0.05 0.038 0.43 0.203 0.14 0.120 0.04 0.101 0.03 0.178 3.1 10 4 0.25 0.00 0.097 0.09 0.075 0.15 0.056 0.22 0.154 0.11 0.044 a Mean value standard error of the difference in log 10 cfu/ml between untreated and treated samples observed in 24 h S. epidermidis (ATCC 35984) biofilms. Table 5. Comparison of the propensity of SB13 and rifampicin to select spontaneous resistant mutants on an S. epidermidis (ATCC 35984) biofilm Antibiotic treatment duration Antibiotic (20 mg/l) 24 h 48 h 120 h 168 h 216 h Rifampicin 2.23 0.03 a 0.67 0.14 0.16 0.09 0.24 0.17 0.12 0.07 SB13 3.66 0.07 3.67 0.04 4.22 0.12 3.64 0.09 3.50 0.18 a Mean value standard error of the difference in log 10 cfu/ml between untreated and treated samples observed in 24 h S. epidermidis (ATCC 35984) biofilms. of amoxicillin/clavulanic acid or imipenem/cilastatin demonstrated poor efficacy. When used in combination with several other antibiotics, we also observed that most of the antibiotics that are synergic or antagonistic with rifampicin behave similarly with SB13. This suggests that a similar mechanism of action leads to similar synergism or antagonism between these two molecules. The synergic effect of a combination between rifampicin and vancomycin on a biofilm has already been documented 10,19 and we extend it to our new transcription inhibitor. A major advantage of this molecule, when compared with rifampicin, is the absence of selection of resistant mutants. We previously attempted to select spontaneous resistant mutants to SB13 at a concentration of 3 MIC without success, suggesting 781

Gualtieri et al. Difference between untreated and treated samples log 10 cfu/ml 5.00 4.00 3.00 2.00 1.00 0.00 1.00 1 2 3 4 5 6 7 8 Antibiotic concentration ( MIC) Figure 2. Effect of antibiotics at different concentrations on 24 h S. epidermidis (ATCC 35984) biofilms treated for 24 h. pen diamonds, vancomycin; filled squares, minocycline; filled triangles, fusidic acid; open circles, novobiocin; open squares, SB13; filled diamonds, rifampicin; filled circles, amoxicillin/clavulanic acid; open triangles, imipenem/cilastatin. Table 6. Comparison of the effects of SB13 and rifampicin in combination with reference antibiotics on S. epidermidis (ATCC 35984) biofilm Antibiotic combination (antibiotic 1 + antibiotic 2) Antibiotic concentration Sum of D values of the individual antibiotics D value for the combination SB13 + vancomycin MIC 0.30 a 0.67 b 2 MIC 1.17 1.87 4 MIC 2.97 4.18 Rifampicin + vancomycin 0.5 MIC 0.03 0.40 MIC 0.2 0.10 2 MIC 0.78 1.00 4 MIC 2.20 3.08 SB13 + amoxicillin/ MIC 0.36 0.81 clavulanic acid 2 MIC 1.00 1.82 Rifampicin + amoxicillin/ clavulanic acid SB13 + imipenem/ cilastatin Rifampicin + imipenem/cilastatin 4 MIC 3.11 3.51 MIC 0.14 0.41 2 MIC 0.61 1.24 4 MIC 2.34 2.92 0.5 MIC 0.13 0.35 MIC 0.21 0.47 2 MIC 1.06 1.45 4 MIC 2.56 3.24 MIC 0.29 0.40 2 MIC 0.67 0.81 4 MIC 1.79 2.40 a Sum of D1 + D2. D1 = mean value standard error of the difference in log 10 cfu/ml between untreated and treated samples with antibiotic 1 (SB13 or rifampicin) observed in 24 h S. epidermidis (ATCC 35984) biofilms. D2 = mean value standard error of the difference in log 10 cfu/ml between untreated and treated samples with antibiotic 2 observed in 24 h S. epidermidis (ATCC 35984) biofilms. b Mean value standard error of the difference in log 10 cfu/ml between untreated and treated samples with antibiotic combination (antibiotic 1 + antibiotic 2) observed in 24 h S. epidermidis (ATCC 35984) biofilms. Table 7. Effects of SB13 on the reference S. epidermidis (ATCC 35984) biofilm and on three S. epidermidis clinical isolates SB13 concentration (mg/l) a mutation frequency lower than 10 9. 13 We also failed to isolate resistant bacteria by slow adaptation at subinhibitory concentrations (not shown). In a biofilm and under the same conditions, rifampicin selects resistant bacteria that rapidly re-colonize the surface of the plate. ur study suggests that antibiotics with the same target but having different molecular weights and hydrophobicity do not differ in their efficacy against biofilm cells, and we confirm that these new molecules present a strong interest. Data on the peak serum concentration of this molecule are still missing, but the relative hydrophobicity of SB13 and its binding to serum proteins precludes for now systemic uses and topical applications are under evaluation. Modifications are also ongoing to decrease this binding to serum proteins. Strikingly, very few transcription inhibitors have been tested on biofilms and other molecules such as lipiarmycin, a macrocyclic antibiotic currently under development under the name of PT-80 (ptimer Pharmaceuticals, Inc., San Diego, CA, USA), should be evaluated. Acknowledgements We thank Dr S. L. Salhi for the editorial revision of the manuscript. This work was supported by institutional funds from the Centre National de la Recherche Scientifique and the Grant Biosécurité 2004. Transparency declarations J.-P. L. receives research support from Selectbiotics, and Lionel bastide is researcher for Selectbiotics. References Strain ATCC 35984 DSM 3269 40004 48155 20.00 3.9 0.083 a 4 0.239 3.8 0.055 3.4 0.032 a Mean value standard error of the difference in log 10 cfu/ml between untreated and treated samples observed in different 24 h S. epidermidis biofilms. 1. Gara JP, Humphreys H. Staphylococcus epidermidis biofilms: importance and implications. J Med Microbiol 2001; 50: 582 87. 2. von Eiff C, Peters G, Heilmann C et al. Pathogenesis of infections due to coagulase negative staphylococci. Lancet Infect Dis 2002; 2: 677 85. 3. Costerton JW, Stewart PS, Greenberg EP et al. Bacterial biofilms: a common cause of persistent infections. Science 1999; 284: 1318 22. 4. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002; 15: 167 93. 5. Gilbert P, Collier PJ, Brown MRW et al. Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob Agents Chemother 1990; 34: 1865 8. 6. Gordon CA, Hodges NA, Marriott C et al. Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic 782

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