In-vitro activity of cationic peptides alone and in combination with clinically used antimicrobial agents against Pseudomonas aeruginosa

A. Giacometti*, O. Cirioni, F. Barchiesi, M. Fortuna and G. Scalise

Institute of Infectious Diseases and Public Health, University of Ancona, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vitro activity of cecropin P1, indolicidin, magainin II, nisin and ranalexin alone and in combination with nine clinically used antimicrobial agents was investigated against a control strain, Pseudomonas aeruginosa ATCC 27853 and 40 clinical isolates of P. aeruginosa. Antimicrobial activities were measured by MIC, MBC and viable count. In the combination study, the clinically used antibiotics were used at concentrations close to their mean serum level in humans in order to establish the clinical relevance of the results. To select peptide-resistant mutants, P. aeruginosa ATCC 27853 was treated with consecutive cycles of exposure to each peptide at 1 x MIC. The peptides had a varied range of inhibitory values: all isolates were more susceptible to cecropin P1, while ranalexin showed the lowest activity. Nevertheless, synergy was observed when the peptides were combined with polymyxin E and clarithromycin. Consecutive exposures to each peptide at 1 x MIC resulted in the selection of stable resistant mutants. Cationic peptides might be valuable as new antimicrobial agents. Our findings show that they are effective against P. aeruginosa, and that their activity is enhanced when they are combined with clinically used antimicrobial agents, particularly with polymyxin E and clarithromycin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is a clinically important pathogen because of its natural resistance to many antimicrobial agents. It continues to pose a therapeutic problem because it produces a high rate of morbidity and mortality and because of the possibility of drug resistance developing during therapy. The main reason for this bacterial resistance is thought to be the organism's low outer membrane permeability to antimicrobial agents.1,2,3 One way to overcome these problems is to use new antimicrobial compounds and/or combination therapy. Such combination therapy is generally used to increase the in-vivo activity, to prevent the emergence of drug resistance and to broaden the antimicrobial spectrum.4,5,6,7

In recent years many positively charged polypeptides have been isolated from a wide range of animal, plant and bacterial species.8,9 ,10 ,11 ,12,13 In mammals, including man, they are the predominant protein species in neutrophils and they are also found on the surface of tongue, trachea, lungs and upper intestine, and are thought to be a major antibacterial defence on mucosal surfaces.14,15 It has been suggested that the mode of action of these compounds on the membranes of bacteria, fungi and protozoa involves the formation of ion-channel pores spanning the membranes without requiring a specific target receptor. The lethal events which occur at the biological membranes are not fully understood. 8,11,16

In this study we investigated the in-vitro activity of cecropin P1, indolicidin, magainin II, nisin and ranalexin alone and in combination with nine clinically used antimicrobial agents against the control strain P. aeruginosa ATCC 27853 and 40 clinical isolates of P. aeruginosa.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organisms

The control strain P. aeruginosa ATCC 27853 and 40 clinical isolates of P. aeruginosa were tested. The strains were identified according to the following criteria: Gram-negative bacillary non-spore-forming organisms, characteristic colonial morphology, production of diffusible pigments, positive oxidase test and substrate utilization (API-20 NE gallery, bioMérieux, Marcy l'Etoile, France).

Antimicrobial agents

Cecropin P1, indolicidin, magainin II, nisin and ranalexin were obtained from Sigma–Aldrich (Milan, Italy). The peptides were solubilized in phosphate-buffered saline (pH 7.2) yielding 1 mg/mL stock solution. Solutions of drugs were made fresh on the day of assay or stored at –80°C in the dark for short periods. The in-vitro activity of the following antibiotics was determined: doxycycline, netilmicin, ofloxacin and polymyxin E (all from Sigma– Aldrich), clarithromycin from Abbott (Rome, Italy), ceftazidime from Glaxo–Wellcome (Verona, Italy), meropenem from Zeneca (Rome, Italy), piperacillin and piperacillin + tazobactam from Wyeth–Lederle (Aprilia, Italy). Laboratory standard powders were diluted in accordance with manufacturers' recommendations yielding 1 mg/mL stock solution. Stock solutions of these antimicrobial drugs were stored at –80°C until they were used. The concentration range assayed for each antibiotic was 0.125–128 mg/L.

MIC and MBC determinations

The MIC was determined using a microbroth dilution method with Mueller–Hinton (MH) broth (Becton Dickinson Italia, Milan, Italy) and an initial inoculum of 5 x 105 cfu/mL, according to the procedures outlined by the NCCLS.17 Polystyrene 96-well plates (Becton Dickinson and Co., Franklin Lakes, NJ, USA) were incubated for 18 h at 37°C in air. When peptides were tested, since they have a tendency to precipitate, plates were shaken throughout the study. The MIC was taken as the lowest drug concentration at which observable growth was inhibited. The MBC was taken as the lowest concentration of each drug that resulted in more than 99.9% reduction of the initial inoculum. Experiments were performed in triplicate.

Bacterial killing assay

The control strain ATCC 27853 was grown at 37°C in MH broth. Samples of exponentially growing bacteria were resuspended in fresh MH broth at approximately 107 cells/mL and exposed to each peptide (final concentration 32 mg/L) for 0, 5, 10, 15, 20, 30, 40, 50 and 60 min at 37°C. After these times samples were diluted serially and plated on to MH agar plates to obtain viable colonies.

Synergy studies

In-vitro interactions with doxycycline, netilmicin, ofloxacin, polymyxin E, clarithromycin, ceftazidime, meropenem, piperacillin and piperacillin + tazobactam were investigated. P. aeruginosa ATCC 27853 was used. The above-mentioned drugs, applied at a concentration close to their mean serum levels in order to establish the clinical relevance of the results, were combined with each peptide at 0.5 x MIC and 1 x MIC. In all tubes a 5 x 105 cfu/mL log-phase inoculum was added along with MH broth to give a final volume of 10 mL. All tubes were incubated overnight at 37°C and the bacterial growth in each tube was determined by performing consecutive 1:10 (v/v) dilution of a 0.1 mL sample from each tube in MH broth and by plating a 0.1 mL volume of each dilution on to MacConkey agar. Experiments were performed in triplicate. If a combination of the peptide with other drugs caused a decrease in viable cell count of >=2 log10 compared with the most active single agents, the effects of the combination were considered to be synergic. If the decrease in viable cell count was 1–2 log10 the effects of the combination were considered to be additive.18

Selection of peptide-resistant mutants

To select peptide-resistant mutants, P. aeruginosa ATCC 27853 was incubated at 37°C in 10 mL of MH broth until turbid and the turbidity of the cultures was adjusted to match an optical density of 1.25 at 550 nm, corresponding to approximately 109 cfu/mL. Aliquots (0.1 mL) were treated with seven consecutive cycles of overnight exposure to each peptide at 1 x MIC. Successively, cultures were treated with seven consecutive cycles of 1:100 (v/v) dilution in fresh, drug-free MH broth and overnight incubation at 37°C. Finally, each strain was retested for susceptibility to each peptide by the microbroth dilution method.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MIC and MBC determinations

The peptides showed a different range of inhibitory values: the 40 P. aeruginosa clinical isolates were more susceptible to cecropin P1 (MIC range, 4–64 mg/L) and less susceptible to ranalexin (MIC range, 64 to >128 mg/L). The results are summarized in the Table.


View this table:
[in this window]
[in a new window]
 
Table. Susceptibility of P. aeruginosa to cationic peptides
 
Bacterial killing assay

Viable counts of P. aeruginosaATCC 27853 treated with various agents are displayed graphically in the Figure. Killing by cecropin P1 was shown to be the most rapid: its activity was complete after a 20 min exposure period. Killing by magainin II and indolicidin was complete after a 30 min exposure period, while killing by nisin and ranalexin was complete after a 40 and 60 min exposure period, respectively.



View larger version (17K):
[in this window]
[in a new window]
 
Figure. Effect of cationic peptides on the viability of P. aeruginosa ATCC 27853. Peptides were tested at a concentration of 32 mg/L. Key: -{blacklozenge}-, cecropin P1; -{blacksquare}-, indolicidin; -{blacktriangleup}-, magainin II; -{square}-, nisin; -{circ}-, ranalexin; -•-, control.

 
Synergy studies

The clinically used antibiotics were tested at the following concentrations: doxycycline, ofloxacin and clarithromycin, 2 mg/L; polymyxin E and meropenem, 4 mg/L; netilmicin and ceftazidime, 8 mg/L; piperacillin, 32 mg/L; piperacillin + tazobactam 32 mg/L and 4 mg/L, respectively. Overall, an increase in killing at 24 h greater than 100-fold was observed when the peptides were combined with clarithromycin and polymyxin E, while additive effects were observed with the remaining combinations (data not shown). Particularly, a 4 log10 decrease in viable cell count was found when cecropin P1 at a concentration of 8 mg/L and magainin II at a concentration of 8 mg/L were combined with polymyxin E at a concentration of 4 mg/L. A 3 log10 decrease in viable cell count was found when cecropin P1 or magainin II at a concentration of 8 mg/L was combined with clarithromycin at a concentration of 2 mg/L.

Selection of peptide-resistant mutants

The consecutive exposures of P. aeruginosa ATCC 27853 to each peptide resulted in the selection of mutants with decreased susceptibility in which the MIC of cecropin P1, magainin II, indolicidin, nisin and ranalexin increased to 64, 128, 128, >128 and >128 mg/L, respectively. This decreased susceptibility was retained during seven passages in peptide-free growth medium, suggesting the selection of stable peptide-resistant mutants.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This report emphasizes the importance of the search for alternative antibacterial agents. Our data demonstrate that cationic peptides had a powerful bactericidal effect on clinical isolates of P. aeruginosa. These peptides are known to have variable antibacterial, antifungal and antiprotozoan in-vitro activity. Previous studies on the mode of action of the cationic peptides have demonstrated that these compounds cross the outer membrane of Gram-negative bacteria via the self-promoted uptake pathway.14,15 The initial step in this process is the binding of the peptide to the surface lipopolysaccharide (LPS) with a high affinity, causing the displacement of divalent cations that stabilize adjacent LPS molecules. The displacement of divalent cations destabilizes the Gram-negative outer membrane and leads to self-promoted uptake of the destabilizing compound across the outer membrane and subsequent channel formation in the cytoplasmic membrane, resulting in cell death. 14,19,20The lethal event which occurs at the cytoplasmic membrane is not fully understood: the association of several molecules would form a water-filled pore that would serve as an ion-conducting, anion-selective channel. Recent reports have shown that the peptides may act by inserting into the cytoplasmic membrane and triggering the activity of bacterial murein hydrolases, resulting in damage or degradation of the peptidoglycan and lysis of the cell.10,11,12

Our data demonstrate that all peptides were highly active against P. aeruginosa and showed a rapid bactericidal effect. There are few data on the concentration- or time-dependent killing kinetics of bacteria by polycationic peptides, nevertheless our observations are in agreement with recent reports that showed that killing by peptides was very rapid and resulted in log orders of cell death within minutes of peptide addition.11,13,21

Combination studies showed that each peptide acted synergically with polymyxin E and clarithromycin. The mechanism of the synergy between cationic peptides and polymyxins or macrolides appears to be complex. The polymyxins are a group of cyclic cationic polypeptides originally derived from Bacillus polymyxa; they share remarkable structural similarity with ranalexin. Like ranalexin, the polymyxins are amphipathic compounds, with a hydrophobic region at their amino terminus. Polymyxins and polymyxin-like peptides have both direct antibacterial activity and membrane-permeabilizing activity and present properties of synergy with lipophilic and amphiphilic agents such as rifampin, macrolides, fusidic acid and novobiocin. Recent reports demonstrate that polymyxin-like peptides allow maximal entry of several hydrophobic substrates into the cell.15,16,22The combination of cationic peptides with polymyxin E may be synergic since they have similar mechanisms of action, interacting with the phospholipids of the bacterial cell membrane, thereby increasing cell permeability and disrupting osmotic integrity.

The mechanisms of the synergy between peptides and clarithromycin remains largely unknown. The antibacterial activity of macrolides is known to result from their ability to inhibit protein synthesis by binding to the transpeptidation site of the larger ribosomal subunit. Today there is increasing evidence of a role for macrolides as anti-pseudomonal agents. Macrolides might act in vivo by two main mechanisms: via effects on the immune system to modify the inflammatory response to infection, or via direct effects on P. aeruginosa to decrease its virulence.23 These mechanisms, however, do not explain how macrolides would show in-vitro anti-pseudomonal activity. Nevertheless, a recent report suggests that macrolides may inhibit P. aeruginosa cell growth in vitro: although clarithromycin and erythromycin at 2 mg/L had no effect on cell growth at 24 h, they were bactericidal for P. aeruginosa when incubation was continued for 48 h.24 The permeabilization of the outer membrane to other molecules might explain the synergy of peptides with macrolides. Large hydrophobic antibiotic molecules are usually ineffective against Gram-negative bacteria, since they cannot diffuse across the outer membrane: one can speculate that the peptides increased the activity of clarithromycin by increasing permeability of the outer membrane and promoting the above-mentioned bactericidal effect.11,24 Finally, regarding the other antimicrobial agents studied, additive effects were observed when they were combined with peptides. Even in this case, the peptides might have increased the activity of the antibiotics by increasing their access across the outer membrane of P. aeruginosa.

In spite of this speculated mode of peptide interaction, proofs of clinical benefits are lacking. However, the intrinsic antibacterial activity and the synergic interactions demonstrated by several combinations make the cationic peptides potentially valuable as an adjuvant for antimicrobial chemotherapy. Future research towards this objective based on animal models are needed.


    Notes
 
* Correspondence address. Institute of Infectious Diseases and Public Health, c/o Azienda Ospedaliera Umberto I, Piazza Cappelli 1, Ancona, I-60121, Italy. Tel: +39-071-5963467; Fax: +39-071-5963468; E-mail: cmalinf{at}popcsi.unian.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Mouton, J. W., den Hollander J. G. & Horrevorts A. M. (1993). Emergence of antibiotic resistance amongst Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Journal of Antimicrobial Chemotherapy 31, 919–26.[Abstract]

2 . Masuda N. & Ohya S. (1992). Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 36, 1847–51.[Abstract]

3 . Margaret, B. S., Drusano, G. L. & Standiford H. C. (1989). Emergence of resistance to carbapenem antibiotics in Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 161–7. [ISI][Medline]

4 . den Hollander, J. G, Horrevorts, A. M., van Goor, M. L. P. J., Verbrugh, H. A. & Mouton, J. W. (1997). Synergism between tobramycin and ceftazidime against a resistant Pseudomonas aeruginosa strain, tested in an in vitro pharmacokinetic model. Antimicrobial Agents and Chemotherapy 41, 95–100.[Abstract]

5 . Giacometti, A., Cirioni, O., Greganti, G., Quarta, M. & Scalise, G. (1998). In vitro activities of membrane-active peptides against gram-positive and gram-negative aerobic bacteria. Antimicrobial Agents and Chemotherapy 42, 3320–4.[Abstract/Free Full Text]

6 . Livermore, D. M. & Chen, H. Y. (1997). Potentiation of ß-lactams against Pseudomonas aeruginosa strains by Ro 48-1256, a bridged monobactam inhibitor of AmpC ß-lactamases. Journal of Antimicrobial Chemotherapy 40, 335–43.[Abstract]

7 . Hall, L. M., Livermore, D. M., Gur, D., Akova, M. & Akalin H. E. (1993). OXA-11, an extended-spectrum variant of OXA-10 (PSE-2) ß-lactamase from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 37, 1637–44.[Abstract]

8 . Bevins, C. L. & Zasloff, M. (1990). Peptides from frog skin. Annual Review of Biochemistry 59,395–414.[ISI][Medline]

9 . Clark, D. P., Durell, S., Maloy, W. L. & Zasloff, M. (1994). Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catasbeiana) skin, structurally related to the bacterial antibiotic, polymyxin. Journal of Biological Chemistry 269, 10849–55.[Abstract/Free Full Text]

10 . Jack, R. W., Tagg, J. R. & Ray, B. (1995). Bacteriocins of gram-positive bacteria. Microbiological Review 59, 171–200.[ISI]

11 . Moore, A. J., Beazley, W. D., Bibby, M. C. & Devine, D. A. (1996). Antimicrobial activity of cecropins. Journal of Antimicrobial Chemotherapy 37, 1077–89.[Abstract]

12 . Sahl, H. G., Jack, R. W. & Bierbaum, G. (1995). Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. European Journal of Biochemistry 230,827 –53.[Abstract]

13 . Severina, E., Severin, A. & Tomasz, A. (1998). Antibacterial efficacy of nisin against multidrug-resistant Gram-positive pathogens. Journal of Antimicrobial Chemotherapy 41, 341–7.[Abstract]

14 . Falla, T. J. & Hancock, R. E. W. (1997). Improved activity of a synthetic indolicidin analog. Antimicrobial Agents and Chemotherapy 41, 771–5.[Abstract]

15 . Hancock, R. E. (1997). Antibacterial peptides and the outer membranes of gram-negative bacilli. Journal of Medical Microbiology 46, 1–3.[ISI][Medline]

16 . Vaara, M. & Porro, M. (1996). Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria. Antimicrobial Agents and Chemotherapy 40, 1801–5.[Abstract]

17 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Third Edition: Approved Standard M7-A3. NCCLS, Villanova, PA.

18 . Hindler, J. (1992). Tests to assess bactericidal activity. In Clinical Microbiology Procedures Handbook, (Eisenberg, H. D., Ed.), p. 5, 14, 16–24. American Society for Microbiology, Washington, DC.

19 . Falla, T. J., Karunaratne, D. N. & Hancock, R. E. W. (1996). Mode of action of the antimicrobial peptide indolicidin. Journal of Biological Chemistry 271, 19298–303.[Abstract/Free Full Text]

20 . Piers, K. L. & Hancock, R. E. W. (1994). The interaction of a recombinant cecropin/mellitin hybrid peptide with the outer membrane of Pseudomonas aeruginosa. Molecular Microbiology 12, 951–8.[ISI][Medline]

21 . Zasloff, M. (1987). Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proceedings of the National Academy of Science of the United States of America 84, 5449–53.[ISI]

22 . Viljanen, P., Matsunaga, H., Kimura, Y. & Vaara, M. (1991). The outer membrane permeability-increasing action of deacylpolymyxins. Journal of Antibiotics 44, 517–23.[ISI][Medline]

23 . Howe, R. A. & Spencer, R. C. (1997). Macrolides for the treatment of Pseudomonas aeruginosa infections? Journal of Antimicrobial Chemotherapy 40, 153–5.[Free Full Text]

24 . Tateda, K., Ishii, Y., Matsumoto, T., Furuya, N., Ohno, A., Miyazaki, S. et al. (1996). New evidence of anti-pseudomonal activity of macrolides. In Program and Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, 1996.Abstract C82, p. 49. American Society for Microbiology, Washington, DC.

Received 15 January 1999; returned 13 April 1999; revised 26 May 1999; accepted 10 July 1999