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JAC Advance Access published online on September 16, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn393
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Protective effects of the combination of {alpha}-helical antimicrobial peptides and rifampicin in three rat models of Pseudomonas aeruginosa infection

Oscar Cirioni1,*, Carmela Silvestri1, Roberto Ghiselli2, Fiorenza Orlando3, Alessandra Riva1, Federico Mocchegiani2, Leonardo Chiodi2, Sefora Castelletti1, Eleonora Gabrielli1, Vittorio Saba2, Giorgio Scalise1 and Andrea Giacometti1

1 Institute of Infectious Diseases and Public Health, Università Politecnica delle Marche, Ancona, Italy 2 Department of General Surgery, I.N.R.C.A. I.R.R.C.S., Università Politecnica delle Marche, Ancona, Italy 3 Experimental Animal Models for Aging Units, Research Department, I.N.R.C.A. I.R.R.C.S., Ancona, Italy

* Corresponding author. Tel: +39-071-5963715; Fax: +39-071-5963468; E-mail: o.cirioni{at}univpm.it or anconacmi{at}interfree.it

Received 16 May 2008; returned 2 July 2008; revised 19 August 2008; accepted 25 August 2008


   Abstract
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Introduction: An experimental study has been performed to compare the in vitro activity and the in vivo efficacy of magainin II and cecropin A with or without rifampicin against control and multidrug-resistant Pseudomonas aeruginosa strains.

Methods: In vitro experiments included MIC determinations and synergy studies. For in vivo studies, animals were given an intraperitoneal injection of P. aeruginosa lipopolysaccharide, P. aeruginosa ATCC 27853 and one clinical multiresistant P. aeruginosa strain. Groups of animals received intravenously isotonic sodium chloride solution, 10 mg/kg rifampicin, 1 mg/kg magainin II or 1 mg/kg cecropin A. Two groups of animals received a combined treatment with magainin II + rifampicin or cecropin A + rifampicin at the same dosages as the singly treated groups. In addition, a further group was treated with tazobactam/piperacillin (120 mg/kg). Lethality, bacterial growth in blood and peritoneum, and endotoxin and TNF-{alpha} concentrations in plasma were evaluated.

Results: Combinations of {alpha}-helical antimicrobial peptides showed in vitro synergistic interaction. Magainin II and cecropin A exerted strong antimicrobial activity and achieved a significant reduction in plasma endotoxin and TNF-{alpha} concentrations when compared with control and rifampicin-treated groups. Rifampicin exhibited no anti-P. aeruginosa activity and good substantial impact on endotoxin and TNF-{alpha} plasma concentrations. Combined treatment groups had significant reductions in bacterial count, positive blood cultures and mortality rates when compared with singly treated and control groups.

Conclusions: Our results highlight the potential usefulness of these combinations that provide future therapeutic alternatives in P. aeruginosa infections.

Key Words: sepsis , multiresistant organisms , P. aeruginosa , synergy , animal model


   Introduction
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Gram-negative bacteria pose a particular difficulty for the healthcare community due to the emergence of multidrug-resistant strains.1–3 Among these resistant bacteria, Pseudomonas aeruginosa is of great concern because antimicrobial therapy for infections due to these multiresistant organisms remains a clinical dilemma in hospitalized patients.4–6 This growing problem has created a new antibiotic therapeutic void, in part because of the paucity of novel antibiotic development for Gram-negative infections as well as the sudden loss of previously effective agents.

After the discovery of polymyxins, a number of cationic peptides have been isolated from a wide range of bacterial species, plants and animals.7,8 They are able to kill both Gram-negative and Gram-positive bacteria, fungi, eukaryotic parasites and even enveloped viruses.9,10 They are known to have an amphipathic structure with clusters of hydrophobic and positively charged regions. This structural property appears to be closely related to their antimicrobial activity.11,12

Cecropins are positively charged peptides that were originally isolated from the blood lymph of the giant silk moth (cecropins A and B) and, successively, from the small intestine of the pig (cecropin P1). Previous studies showed that they exhibited an {alpha}-helical pattern in 15% hexafluoroisopropyl alcohol. The results suggested a highly amphipathic helix with hydrophobic and cationic-charged surfaces, a motif observed in many other cationic peptides.13

Magainins are positively charged peptides that were originally isolated from the skin of the African clawed frog Xenopus laevis. They were demonstrated to be active against numerous Gram-negative and Gram-positive bacteria, fungi and protozoa.14

Rifampicin was introduced for clinical use in 1968 as an effective anti-tuberculous drug and has primary activity against Gram-positive bacteria. It exerts its bactericidal activity by forming a stable complex with bacterial DNA-dependent RNA polymerase, preventing the chain initiation process of DNA transcription.15 Recent studies showed that, against multidrug-resistant P. aeruginosa, increased activity in vitro was achieved by the combination of rifampicin and polymyxins.16 Previous studies have also reported the positive interaction between antimicrobial peptides and clinically used antibiotics, even though the mechanism remains unknown.17

In order to broaden our knowledge, we evaluated the activity of the combination of two {alpha}-helical peptides (magainin II and cecropin A) and rifampicin in vitro and in vivo using two P. aeruginosa strains with different susceptibilities to antibiotics.


   Materials and methods
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Drugs

Magainin II (Gly-Ile-Gly-Lys-Phe-Leu-His-Ala-Ala-Lys-Lys-Phe-Ala-Lys-Ala-Phe-Val-Ala-Glu-Ile-Met-Asn-Ser-NH2), cecropin A (Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly-Gln-Asn-Ile-Arg-Asp-Gly-Ile-Ile-Lys-Ala-Gly-Pro-Ala-Val-Ala-Val-Val-Gly-Gln-Ala-Thr-Gln-Ile-Ala-Lys-NH2), rifampicin (Sigma-Aldrich, Milan, Italy) and tazobactam/piperacillin (Wyeth-Lederle, S.p.A., Rome) powders were diluted in accordance with manufacturers' recommendations. Solutions of drugs were made fresh on the day of assay or stored at –80°C in the dark for 20 days.

Organisms and reagents

The commercially available quality control strain of P. aeruginosa ATCC 27853 was used, and a multidrug-resistant P. aeruginosa strain was isolated from a clinical specimen submitted for routine bacteriological investigation to the Institute of Infectious Diseases and Public Health, Polytechnic University of Marche, Ancona, Italy. This strain was resistant to all clinically used antibiotics, with the exception of colistin and tazobactam/piperacillin. Endotoxin [lipopolysaccharide (LPS) from P. aeruginosa 10; Sigma-Aldrich S.r.l.] was prepared in sterile saline, aliquotted and stored at –80°C for short periods.

Susceptibility testing

Susceptibility testing was performed by a microbroth dilution method, according to the procedures outlined by the CLSI (formerly the NCCLS).18 The MIC was taken as the lowest antibiotic concentration at which observable growth was inhibited. Experiments were performed in triplicate.

Synergy studies

In interaction studies, P. aeruginosa ATCC 27853 was used to test the antibiotic combinations by a chequerboard titration method using 96-well polypropylene microtitre plates. The ranges of drug dilutions used were: 0.125–64 mg/L for magainin II and cecropin A and 0.25–256 mg/L for rifampicin and tazobactam/piperacillin. The FIC index for combinations of two antimicrobials was calculated according to the equation: FIC index = FICA + FICB = A/MICA + B/MICB, where A and B are the MICs of drug A and drug B in the combination, MICA and MICB the MICs of drug A and drug B alone, and FICA and FICB the FICs of drug A and drug B, respectively. The FIC indexes were interpreted as follows: ≤0.5, synergy; >0.5–4.0, no interaction and >4.0, antagonism.

In addition, time–kill synergy studies were performed at the recommended subinhibitory concentrations (one-fourth and one-half the MIC).19 Synergy or antagonism was defined as a 100-fold increase or decrease, and indifference was defined as a <10-fold increase or decrease in killing after incubation with the combination compared with that of the most active single agent.

Animals

Adult male Wistar rats weighing 200–300 g were used. All animals were housed in individual cages under constant temperature (22°C) and humidity with 12 h light/dark cycles and had access to food and water as much as desired throughout the study. The study was approved by the animal research Ethics Committee of the I.N.R.C.A. I.R.R.C.S., Ancona, Italy.

Experimental design

Sepsis was induced by three different experimental methods involving a total of 21 groups of animals: (i) intraperitoneal administration of LPS; (ii) intraperitoneal injection of 2 x 1010 cfu of P. aeruginosa ATCC 27853; and (iii) intraperitoneal injection of 2 x 1010 cfu of the multiresistant P. aeruginosa strain.

Model i. Seven groups, each containing 15 animals, were anaesthetized by intramuscular injection of ketamine and xylazine (30 and 8 mg/kg of body weight, respectively) and injected intraperitoneally with 1 mg of P. aeruginosa 10 LPS in a total volume of 500 µL of sterile saline. Immediately after injection, animal groups received intravenously a single dose of isotonic sodium chloride solution (control group C0), 1 mg/kg magainin II, 1 mg/kg cecropin A or 10 mg/kg rifampicin. Two groups of animals received a combined treatment with magainin II + rifampicin or cecropin A + rifampicin at the same dosages as the singly treated groups. Tazobactam/piperacillin (120 mg/kg), one of the most commonly used anti-Pseudomonas agents, was used as a control agent.

Models ii and iii. P. aeruginosa ATCC 27853 or the clinical isolate was grown in brain heart infusion broth. When bacteria were in the log phase of growth, the suspensions were centrifuged at 1000 g for 15 min, the supernatants were discarded and the bacteria were resuspended and diluted into sterile saline. All animals (14 groups, each containing 15 animals) were anaesthetized, as described earlier. The abdomen of each animal was shaved and prepared with iodine. The rats received, intraperitoneally, 1 mL of saline containing 2 x 1010 cfu of P. aeruginosa ATCC 27853 (model ii) or the multiresistant strain (model iii). Immediately after bacterial challenge, animal groups received intravenously a single dose of isotonic sodium chloride solution (control groups C1 for model ii and C3 for model iii), 1 mg/kg magainin II, 1 mg/kg cecropin A or 10 mg/kg rifampicin. Two groups of animals received a combined treatment with magainin II + rifampicin or cecropin A + rifampicin at the same dosages as the singly treated groups. Tazobactam/piperacillin (120 mg/kg) was used as a control agent. To better investigate the clinical situation in which there is an interval between the onset of sepsis and the initiation of therapy, the same experiments were performed with the administration of the drugs 360 min after the bacterial challenge. In this set of experiments, the control group C2 received P. aeruginosa ATCC 27853, whereas the control group C4 received the multiresistant clinical isolate.

For each animal model, toxicity was evaluated on the basis of the presence of any drug-related adverse effect, i.e. local signs of inflammation, anorexia, weight loss, diarrhoea, fever and behavioural alterations. In particular, to evaluate the physiological effects of the two peptides, leucocyte count, serum creatinine, rectal temperature, pulse, blood pressure, breathing rate and oxygenation were monitored daily in a supplementary peptide-treated group without infection or LPS.

Evaluation of treatment

On the basis of the type of experiment, at the end of the study, the rate of blood culture positivity, the quantities of bacteria in the intra-abdominal fluid, the rate of lethality, and plasma endotoxin and TNF-{alpha} levels were evaluated. The animals were monitored for the subsequent 72 h.

In all models, the presence of systemic symptoms was defined in analogy to the criteria applied for humans. Each animal was considered to be endotoxic if it satisfied at least two of the following criteria: (i) increased pulse rate; (ii) rectal temperature above 38°C or below 36°C; (iii) increased breathing rate; and (iv) more than 12 000 or <4000 white blood cells/µL. The surviving animals were sacrificed with 4% isofluorane, and blood samples for culture were obtained by aseptic percutaneous transthoracic cardiac puncture. In addition, to perform quantitative evaluations of the bacteria in the intra-abdominal fluid, 10 mL of sterile saline was injected intraperitoneally, samples of the peritoneal lavage fluid were serially diluted and a 0.1 mL volume of each dilution was spread onto blood agar plates. The limit of detection was ≤1 log10 cfu/mL. The plates were incubated both in air and under anaerobic conditions at 35°C for 48 h. P. aeruginosa was identified by biochemical assay.

For blood cultures (models ii and iii) and determination of endotoxin and TNF-{alpha} in plasma (all models), 0.2 mL of blood samples were collected from a tail vein 0, 2, 6, 12 and 36 h after injection of LPS or bacteria into a sterile syringe and transferred to tubes containing ethylenediaminetetraacetic acid tripotassium salt (EDTA-K3).

Biochemical assays

Endotoxin concentrations were measured by the commercially available Limulus amebocyte lysate test (E-TOXATE®; Sigma-Aldrich). Plasma samples were serially 2-fold diluted with sterile endotoxin-free water and were heat-treated for 5 min in a water bath at 75°C to destroy inhibitors that can interfere with the activation. The endotoxin content was determined as described by the manufacturer. Endotoxin solutions from the manufacturer (0, 0.015, 0.03, 0.06, 0.125, 0.25 and 0.5 EU/mL) were used in each run, in order to obtain a standard curve, and the concentration of endotoxin in the animal samples was calculated by comparison with this standard curve. TNF-{alpha} levels were measured using a solid-phase sandwich enzyme-linked immunosorbent assay. The intensity of the colour was measured in an MR 700 Microplate Reader (Dynatech Laboratories, Guernsey, UK) by reading the absorbance at 450 nm. The results for the samples were compared with the standard curve to determine the amount of TNF-{alpha} present. All samples were run in duplicate. The lower limit of sensitivity for TNF-{alpha} by this assay was 0.05 ng/mL.

Statistical analysis

Mortality rates between groups were compared by use of Fisher's exact test. Qualitative results for blood cultures were analysed by the {chi}2 test (eventually corrected according to Yates method) or Fisher's exact test, depending on the sample size. Quantitative evaluations of the bacteria in the intra-abdominal fluid cultures were presented as means ± SD; statistical comparisons between groups were made by analysis of variance. Post hoc comparisons were performed by Bonferroni's test. Plasma endotoxin and TNF-{alpha} mean values were compared between groups by non-parametric analysis of variance (Kruskal–Wallis test, followed by the standard procedure for multiple comparisons), due to the presence of censored data. Each comparison group contained 15 animals. The significance level was fixed at 0.05.


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In vitro studies

According to the microbroth dilution method, P. aeruginosa ATCC 27853 and the multiresistant strain showed different susceptibilities to magainin II (MICs of 4 mg/L for both the control strain and the clinical isolate), cecropin A (8 mg/L for both the control strain and the clinical isolate) and tazobactam/piperacillin (4 mg/L for the control strain and 32 mg/L for the clinical isolate), whereas the MICs of rifampicin, as expected, were higher than 256 mg/L for both strains. In the interaction study, a strong synergy (FIC index of 0.312) was observed by testing magainin II and cecropin combined with rifampicin. Time–kill synergy studies showed no effect when the compounds were tested at one-fourth the MIC. In contrast, synergism was clearly observed at one-half the MIC: actually, the combination of the drugs produced at 24 h a decrease in the colony count of 3 log (5.34 ± 1.09 x 103 and 6.20 ± 1.12 x 103), compared with magainin II or cecropin A, the most active single agents, that produced at 24 h colony counts of 5.34 ± 1.28 x 106 and 5.28 ± 1.17 x 106, respectively.

In vivo studies

Model i: intraperitoneal administration of LPS. Plasma peak levels of endotoxin and TNF-{alpha} were observed 6 h after the intraperitoneal administration of 1.0 mg of P. aeruginosa 10 LPS. Nevertheless, intravenous (iv) magainin II and cecropin treatment with or without rifampicin resulted in a marked decrease (P < 0.05) in the plasmatic TNF-{alpha} and endotoxin levels compared with those found in the control (C0), tazobactam/piperacillin-treated and rifampicin-treated groups (Table 1). Interestingly, there was no significant difference in the effect on TNF-{alpha} between magainin II and cecropin A with or without rifampicin. In contrast, there was no significance to endotoxin levels of control, tazobactam/piperacillin-treated and rifampicin-treated groups.


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  		Table 1. Endotoxin and TNF-{alpha} plasma levels in a mouse model 6 h after the intraperitoneal administration of 1.0 mg of P. aeruginosa 10 LPS

 
Model ii: intraperitoneal injection of 2 x 1010 cfu of P. aeruginosa ATCC 27853. All animals were monitored for 72 h. The rate of lethality in control groups C1 and C2 (antibiotics administered immediately or 360 min after bacterial challenge) was 100% within 48 h. All antibiotic treatments, with the exception of rifampicin, led to decreased mortality (P < 0.05). In particular, in the singly treated group, tazobactam/piperacillin showed the lowest lethality rates (40%). Lethality rates of 46.6% and 53.3% were observed for the groups treated with magainin II and cecropin A immediately after the bacterial challenge, whereas a rate of 26.6% was observed in the peptide + rifampicin-treated groups (Table 2). In both control groups, bacteriological evaluation showed 100% positive blood cultures, and 8.2 x 108 ± 2.4 x 108 cfu/mL were counted in the intra-abdominal fluid. Both peptides showed a good antimicrobial activity, comparable to that of tazobactam/piperacillin. When they were combined with rifampicin, the positive interaction produced low bacterial counts (4.0 x 102 ± 1.0 x 102 cfu/mL for magainin II and 4.5 x 102 ± 0.8 x 102 cfu/mL for cecropin A) that were statistically significant versus all other groups (P < 0.05). Similar effects on lethality and bacterial counts were observed when the drugs were administered 360 min after the intervention (Table 2). Magainin II or cecropin A and rifampicin had a different effect on plasma endotoxin and TNF-{alpha} levels. Both peptides produced a strong reduction in the plasmatic level of endotoxin and TNF-{alpha} compared with that found in the control, tazobactam/piperacillin-treated and rifampicin-treated animals, either when the drugs were administered immediately or 360 min after the bacterial challenge. Significant differences were also observed between the rifampicin-treated group and the tazobactam/piperacillin-treated group and the control, whereas the combination treatment groups exhibited the best results with regard to these parameters. The results are summarized in Figures S1 and S2 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].


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  		Table 2. Efficacy of administration of iv magainin II, cecropin A, tazobactam/piperacillin and rifampicin in a rat model after intraperitoneal injection of 2 x 1010 cfu of P. aeruginosa ATCC 27853 at 0 and 360 min

 
Model iii: intraperitoneal injection of 2 x 1010 cfu of multiresistant clinical strain. The rate of lethality in the control group C3 (antibiotics administered immediately after the bacterial challenge) was 100% within 48 h. Magainin II or cecropin A treatment (alone or combined with rifampicin) led to decreased mortality (P < 0.05), even though this rate was less than that in model ii. Specifically, at 72 h, lethality rates of 53.3% and 26.6% were observed for groups treated with magainin II alone or in combination with rifampicin, respectively (Table 3). The tazobactam/piperacillin-treated group showed lethality rates of 46.6% and 66.6% for the group treated immediately and the group treated after 360 min, respectively. Finally, lethality rates of 60.0% and 33.3% were observed for groups treated with cecropin A alone or in combination with rifampicin, respectively (Table 3). Bacteraemia was detected in all animals of the control group C3, and 8.8 x 108 ± 2.6 x 108 cfu/mL were counted in their intra-abdominal fluids. Magainin II combined with rifampicin showed the highest antimicrobial activities. Similar effects on lethality and bacterial counts were observed when the drugs were administered 360 min after the intervention (Table 3). As shown in model ii, magainin II and cecropin A had a strong effect on plasma endotoxin and TNF-{alpha} concentrations both when they were administered immediately and when they were administered 360 min after the bacterial challenge.


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  		Table 3. Efficacy of administration of iv magainin II, cecropin A, tazobactam/piperacillin and rifampicin in a mouse model after intraperitoneal injection of 2 x 1010 cfu of multiresistant P. aeruginosa at 0 and 360 min

 
Finally, treatment with peptides did not result in any clinical evidence of drug-related adverse effects, and no changes in physiological parameters were observed in the supplementary 1 mg/kg peptide-treated rats without infection.


   Discussion
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Outer membranes in Gram-negative bacteria perform diverse functional roles in Gram-negative bacteria and act as an effective protective barrier to antibiotics that might otherwise be active. This protective barrier, formed by a divalent cation-cross-linked matrix of LPS molecules on the outer leaflet of the outer membrane, can be breached via displacement of the LPS-bound metals by polycations of diverse structural classes.5,20–22 Polymyxins are considered a prototype membrane-perturbing agent, whose antibacterial action is manifested via its binding to the lipid A moiety of LPS.23

Similar to polymyxins, the dominating targets of other antimicrobial cationic peptides are the bacterial membranes. The common feature shared among these compounds is their tendency to form amphipathic structures that cluster basic and hydrophobic amino acids into distinctive regions.24,25 In virtue of their positive charges, polycationic peptides bind to the negatively charged residues of LPS by electrostatic and hydrophobic interactions and so have anti-endotoxin activity.26,27

In the present study, we evaluated the efficacy of the combination between two {alpha}-helical peptides (magainin II and cecropin A) and rifampicin using two P. aeruginosa strains with different patterns of susceptibilities to antibiotics first in vitro and then in vivo. Their efficacies were also compared with that of tazobactam/piperacillin. In in vitro studies, both magainin II and cecropin A exhibited good antimicrobial activities against both the control strain and the multiresistant clinical isolate. Actually, this fact confirms that the peptides work on a different target compared with the traditional antibiotics. Furthermore, time–killing curves and a chequerboard titration method showed a synergistic effect between the peptides and rifampicin. In in vivo studies, we selected as main outcome measures lethality, quantitative blood and peritoneal fluid cultures, and detection of endotoxin and TNF-{alpha} plasma levels to have different parameters and better define the efficacies of the treatment. Magainin II and cecropin A exerted strong antimicrobial activity, had good survival rates and, finally, achieved a significant reduction in plasma endotoxin and, consequently, TNF-{alpha} concentration. In fact, it is known that LPS associated with cell membranes of Gram-negative bacteria activates the host effector cells through stimulation of receptors on their surface: these target cells secrete large quantities of inflammatory cytokines, such as TNF.28 Interestingly, both antimicrobial activity and survival rates were comparable to those obtained with tazobactam/piperacillin treatment, whereas the peptides showed a greater activity on endotoxin and TNF-{alpha} than β-lactam. Our data demonstrated that the administration of drugs at 0 or 360 min after the bacterial challenge had comparable impact on all outcomes evaluated. Interestingly, a strong reduction in the LPS and TNF-{alpha} levels was also obtained in the groups treated with 6 h of delay, in which a dramatic build-up of cytokine levels was observed.

Interestingly, the synergistic effect between peptides and rifampicin described in the in vitro studies was also observed in the in vivo setting. The best results on mortality rates and bacteraemia were obtained when {alpha}-helical peptides were combined with rifampicin, suggesting that their mode of action might be complementary. This combination was also most effective in decreasing the levels of cytokines, confirming the capacity of the peptides to neutralize cell wall components that are the inducers of cytokine activation and also the immunomodulatory activity of rifampicin.29 Nevertheless, our results show that the effect of rifampicin on LPS and TNF-{alpha} levels was lower than that of magainin II and cecropin A.

Previous studies have reported the positive interaction between antimicrobial peptides and hydrophobic antibiotics.17,24,30 Data from our study could be explained by different mechanisms: (i) the peptides, which are bacterial membrane-permeabilizing compounds, allow rifampicin to gain access to its intra-cytoplasmic target; (ii) the peptides detoxify the cytokine associated with Gram-negative sepsis; and (iii) the peptides stimulate the host immune response that allows rifampicin to exert its antimicrobial activity, independent of the peptides.24,25,31

However, in spite of several positive facts associated with antimicrobial peptides, there are still problems such as fewer data available on the unknown in vitro and in vivo toxicities and cost of their production on a large scale. Antibiotic resistance has become a threat in hospital settings, and Gram-negative bacteria are the dominant killers among microorganisms in the intensive care unit; our interest in examining {alpha}-helical peptides and rifampicin lies not only in evaluating their intrinsic antimicrobial properties but rather in exploring the feasibility to utilize such compounds in combination therapy.

The intrinsic antibacterial and anti-endotoxin activities of magainin II and cecropin A, and the synergistic interactions demonstrated upon rifampicin highlight the potential usefulness of these combinations and provide future therapeutic alternatives in P. aeruginosa severe infections.


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 Funding
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 Supplementary data
 References
 
This work was supported by the Italian Ministry of Education, University and Research (PRIN 2005).


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None to declare.


   Supplementary data
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Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
We wish to express our thanks to Silvana Esposito for her technical assistance.


   References
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 Materials and methods
 Results
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 Transparency declarations
 Supplementary data
 References
 
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12 . Wade D, Boman A, Wåhlin B, et al. All-D amino acid-containing channel-forming antibiotic peptides. Proc Natl Acad Sci USA (1990) 87:4761–5.[Abstract/Free Full Text]

13 . Hultmark D, Steiner H, Rasmuson T, et al. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem (1980) 106:7–16.[Web of Science][Medline]

14 . Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA (1987) 84:5449–53.[Abstract/Free Full Text]

15 . Steffee CH, Morrell RM, Wasilauskas BL. Clinical use of rifampin during routine reporting of rifampin susceptibilities: a lesson in selective reporting of antimicrobial susceptibility data. J Antimicrob Chemother (1997) 40:595–8.[Abstract/Free Full Text]

16 . Cirioni O, Ghiselli R, Orlando F, et al. Efficacy of colistin/rifampin combination in experimental rat models of sepsis due to multiresistant Pseudomonas aeruginosa strain. Crit Care Med (2007) 35:1717–23.[CrossRef][Web of Science][Medline]

17 . Cirioni O, Giacometti A, Ghiselli R, et al. Citropin 1.1-treated central venous catheters improve the efficacy of hydrophobic antibiotics in the treatment of experimental staphylococcal catheter-related infection. Peptides (2006) 27:1210–6.[CrossRef][Web of Science][Medline]

18 . National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard M7-A6 (2003) Villanova, PA, USA: NCCLS.

19 . Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother (2003) 52:1.[Free Full Text]

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