a Departments of Medical Microbiology,
b Biochemistry and
c Chemistry, University and University Hospital of Troms, Norway
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Abstract |
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Introduction |
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Some antimicrobial peptides have also been reported to have intracellular targets. Buforin II inhibits cellular functions by binding to DNA and RNA, while indolicidin and the cecropin PR-39 have been demonstrated to inhibit DNA and/or protein synthesis.68 Since several of these peptides also exhibit effects on the outer and inner membranes of bacteria, controversy has arisen about their bactericidal mode of action. Whether or not the killing event is a result of the effects on the membrane or disturbance of cellular metabolism, or a combination thereof, is under debate.9
In addition to the benefits of improved effects of antibacterial therapy, evaluation of the synergic actions of antimicrobial agents may be used to explore possible modes of action of new antibiotics. There are four known mechanisms by which antibacterial synergy can arise: serial inhibition of a common biochemical pathway; inhibition of protective bacterial enzymes; combinations of cell wall active agents; and enhancing the uptake of other antibacterial agents.10 Several synergy studies performed on antimicrobial peptides have been reported.1118 Most of these experiments were performed on naturally occurring peptides. The antimicrobial magainin peptide PGLa has been demonstrated to act synergically with magainin 2, two naturally occurring peptides from the same host.13,19 These studies point out the importance of peptide-enhanced uptake of antibiotics owing to the peptide-induced permeabilization of the bacterial membranes.18 Even though synthesis of antimicrobial peptides with enhanced activity has been reported,2022 few reports of synergy testing have been published.
Permeabilizing ability is predicted to be influenced, amongst other factors, by the length of the peptide. In order to elucidate whether different sized peptides too short to span the membrane as monomers, exerted the same antibacterial effects, five peptides ranging from 6 to 18 residues with equal antibacterial efficacy, were designed. The peptides were tested in combination with clinically used antibiotics as an approach to achieve a better understanding of the possible modes of action of these peptides. To elucidate the peptidepeptide interactions and to compare the effect of these peptides with magainin, we also investigated the combined action of our peptides and PGLa.
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Materials and methods |
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Bacterial strains used were Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. The strains were stored at 70°C, and further grown in 2% bacto peptone water (BPW) (Difco 1807-17-4; Difco, Detroit, MI, USA) at pH 6.80 and 37°C, before the bacterial suspension was adjusted in 2% BPW to give a final density of 1 x 106 cfu/mL. All tests were performed with bacteria in exponential growth phase.
Determination of the MICs of the peptides was performed using a broth microdilution method with a final concentration of 1% BPW, consistent with recommended methods and as described previously by Vorland et al.23 All assays were performed in parallel, and repeated at least twice.
The effects of two-agent combinations were analysed as described previously.24 Serial dilutions of two antimicrobial agents were mixed in chequerboard fashion in a microtitre plate so that each row (and column) contained a fixed amount of one agent and increasing amounts of the second agent. The concentration ranges used were based upon the MICs of each antimicrobial agent and bacteria. The dilutions covered 4 x MIC (antagonistic action) and 0.25 x MIC (synergistic action). Aliquots of 75 µL bacteria (c. 1 x 106 cfu/mL) and 75 µL of each agent were added to the microtitre tray. As controls, the MIC of the agents acting alone was determined in every tray. The trays were incubated overnight at 37°C and inspected visually for bacterial growth, and confirmed by reading optical density at 540 nm (Reader 510; Organon Teknika, Boxtel, The Netherlands).
The fractional inhibitory concentration (FIC) index was calculated for all of the combination experiments using the following formula: FIC = (MIC drug A in combination)/ (MIC drug A alone) + (MIC drug B in combination)/ (MIC drug B alone).
Each chequerboard test generated many different combinations, and by convention, the FIC indices of the most efficient combination are used in calculating the FIC index.12 Synergic action was defined as FIC < 0.5, indifference as FIC 14, and antagonism as FIC > 4.24
For combinations that showed tendencies towards synergy or antagonism, isobolograms were drawn by plotting changes in the MICantibiotics as a function of the changes in MICpeptides when tested in combination.25 A concave isobol illustrates synergy, and a convex isobol antagonism.
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Results |
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The results from the synergy testing are given as FIC indices in Table III. A combination of the synthetic peptides and antibiotics with different mode of action resulted in synergy between all peptides and erythromycin (FIC < 0.5) when acting on E. coli (illustrated for P18 in Figure 1
). The FIC index also reveals synergy between the RNA polymerase inhibitor rifampicin and P15 and P12. Antagonistic effect was observed with combinations of the two shortest peptides, P6 and P9, and the peptide PGLa (Figure 2
). Neither synergy nor antagonism was revealed when combining the peptides with the cell wall acting antibiotics, ampicillin or vancomycin.
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The antagonism observed between PGLa and the peptides acting on E. coli showed a correlation with chain length with the two shortest peptides giving the highest FIC indices. For the synergy observed between the peptides and erythromycin and rifampicin against E. coli, none of the combinations seemed to be dependent upon the number of residues in the synthetic peptides. In the case of S. aureus, ampicillin and peptides P18 through P9 (the four longest peptides) showed positive correlation between chain length and FIC index. Furthermore, rifampicin in combination with the peptides revealed a negative correlation between chain length and FIC index when tested against S. aureus.
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Discussion |
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Antagonism can arise as a result of different mechanisms: competition between binding sites; alteration of the binding site by the other drug; or inactivation of the drug(s) caused by the other drug.26 The peptides showed either indifferent effect or partial antagonism when combined with PGLa against E. coli. A similar, but less marked effect was observed in S. aureus. The antagonism was dependent on the chain length, with only the two shortest peptides showing antagonistic interactions with PGLa. PGLa and magainin have been reported to act synergistically owing to a combined action on the same target, i.e. the cytoplasmic membrane.19 Magainin 2 consists of 23 amino acids with a hydrophobic tail at the C terminus.27 None of our peptides has such a tail, and it seems likely that this feature combined with a certain length is necessary to give the reported apparent synergy between PGLa and magainin 2. The partial antagonism may be owing to either aggregation of our synthetic peptides and PGLa with subsequent inactivation, or competition of initial binding sites. Magainin 2 has been shown to act synergically with ß-lactam antibiotics, a feature that none of our peptides exhibited.17 Thus it seems that these shorter amphipatic peptides have a different mode of action than magainin.
In a study performed on the interaction of bovine lactoferricin and a series of antibiotics, no synergy was detected against S. aureus.14 This is also valid for the five peptides tested here, although FIC indices close to 0.5 were calculated. However, the low MIC of S. aureus makes the detection of synergy technically difficult, and this should be taken into consideration when evaluating the results. The low FIC indices indicating possible synergy should be validated using additional techniques.
Synergic interactions between erythromycin and the peptides were observed against E. coli. In addition, we observed synergy between rifampicin and peptides designated P15 and P12. In combination with our peptides, the in vitro antibacterial effect of erythromycin increased up to 24-fold. Macrolides are large hydrophobic antibiotic molecules usually ineffective against Gram-negative bacteria owing to the outer membrane barrier or efflux of the antibiotic.28,29 Several cationic antimicrobial peptides are known to interact with the outer membrane of Gramnegative bacteria, making this outer protective shield more permeable.30 It is possible that our peptides may cause an increase in this permeability by interacting with the LPS. Hence, erythromycin will have easier access to its cytoplasmic target. Another possible mode of action may be a blocking of macrolide efflux pumps by the peptides. Giacometti et al.18 report on synergy between a macrolide (clarithromycin) and cationic peptides, concluding that this is a complex mechanism probably involving a peptide induced entrance of large lipophilic, amphiphilic molecules into the cell. A synergic interaction between peptides and rifampicin was reported previously by Mangoni et al.31 They suggest that synergy between rifampicin and temporin H, a 10-mer isolated from frog secretions, is caused by the ability of temporin H to facilitate the entrance of rifampicin into the bacterial cell.
No synergy was detected when combining our peptides with vancomycin or ampicillin, antibiotics usually excluded by the LPS and therefore inactive against E. coli.32 Hence, a general permeability increasing effect on the LPS by the peptides is not likely to be the main reason for synergy. Increased permeability may not be adequate to allow a synergic interaction to be revealed. It is possible that the synergic interaction is a result of a combined effect of increased access to the intracellular target for erythromycin and secondary effects of the peptides themselves.
Erythromycin acts on the ribosomal 50S subunit, inhibiting translation by blocking either the peptidyltransferase reaction or the translocation step.33 Whilst not entirely excluding the hypothesis of increased uptake as a result of increased permeability of the LPS, it is possible that antibacterial peptides and erythromycin may inhibit sequential steps in the protein biosynthesis. The data from the vancomycin and ampicillin trials (no synergy caused by increased permeability) and the tetracycline trials (indifferent effect when combined with 30S binding antibiotics) argue in favour of this possibility. A hypothesis including increased uptake and accessibility to the target, combined with drugs acting on a common pathway, can explain the observed synergy for erythromycin.
In conclusion, we have shown synergy between erythromycin and rifampicin and antibacterial peptides when tested against E. coli. The synergy observed in this study could be a result of both enhanced uptake owing to increased permeability of the cytoplasmic membrane and/or LPS layer, or the sequential inhibition of the same biosynthetic pathway (i.e. protein synthesis). The synergic effects observed cannot be explained by increased permeability of the outer membrane alone, since no synergy is observed with vancomycin. In the experiments performed here, a blocking effect of possible efflux pumps by the peptides cannot be ruled out.
We further show that there is no clear-cut correlation between the ability to interact synergically or antagonistically and the length of the peptides. The synergy observed with erythromycin is independent of chain length, while the antagonistic interaction with PGLa appears to be chain length dependent. Given the two-step hypothesis of increased permeabilization and sequential inhibition of a common pathway, this suggests that the peptides are capable of exerting similar effects independent of chain length. Our results indicate a mode of action of these antibacterial peptides which differs from the antibacterial peptide magainin.
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Acknowledgments |
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Notes |
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References |
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Received 29 January 2001; returned 28 March 2001; revised 19 April 2001; accepted 8 May 2001