JAC Advance Access originally published online on July 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):778-783; doi:10.1093/jac/dkl314
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In vitro activity of a new antibacterial rhodanine derivative against Staphylococcus epidermidis biofilms
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
*Corresponding author. Tel: +33-467-548-607; Fax: +33-467-548-610; E-mail: jp.leonetti{at}cpbs.univ-montp1.fr
Received 24 April 2006; returned 8 June 2006; revised 7 July 2006; accepted 7 July 2006
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Abstract |
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Objectives: Staphylococcus epidermidis biofilms form at the surface of implants and prostheses and are responsible for the failure of many antibiotic therapies. Only 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: Our 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
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Introduction |
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Biofilm-related infections are very common nosocomial infections1–4 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 non-growing (stationary) state, in which they are less susceptible to growth-dependent antimicrobial killing,5 physicochemical interaction of certain antibiotics with slime6 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 methicillin-resistant coagulase-negative staphylococci, but its efficacy is low within a biofilm. Consequently, vancomycin must be used in association with other molecules. On 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 vitro10 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.
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Materials and methods |
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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 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%) (Augmentin®, GlaxoSmithKline), a mixture of imipenem (50%)/cilastatin (50%) (Tienam®, 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 x 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 x 105 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 µL 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.
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Growth of biofilms in 96-well polystyrene microtitre plates
The wells of 96-well polystyrene microtitre plates (Falcon MicrotestTM) were filled with 0.1 mL aliquots of S. epidermidis inoculum (107 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 x 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
Twenty-four hours after biofilm formation, the biofilms were washed twice with sterile water. Subsequently 0.1 mL of MHB containing antibiotics 1 and 2 at 0.5, 1, 2 and 4x 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.
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Results |
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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 log10 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).
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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 8x MIC, reductions of 1.79 and 3.7 log10 U of the bacterial counts were observed for SB13 and rifampicin, respectively. However, at 8x MIC and treatment for 24 h, SB13 and rifampicin decreased the bacterial viability respectively by more than 3.9 and 3.14 log10 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.
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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).
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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 log10 U followed by SB13 and imipenem/cilastatin (difference = 0.68 log10 U) and amoxicillin/clavulanic acid (difference = 0.58 log10 U). It was striking to notice that most of the antibiotics synergic or antagonistic with rifampicin behaved similarly when combined with SB13.
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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 log10 U after a 24 h treatment.
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Discussion |
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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 biofilms18 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 biofilms10,18 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 log10 U, while mixtures 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 documented10,19 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 3x MIC without success, suggesting 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.
Our 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 OPT-80 (Optimer Pharmaceuticals, Inc., San Diego, CA, USA), should be evaluated.
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Transparency declarations |
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J.-P. L. receives research support from Selectbiotics, and Lionel bastide is researcher for Selectbiotics.
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Acknowledgements |
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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.
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References |
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