Institute of Infectious Diseases and Public Health, University of Ancona, Italy
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 SigmaAldrich (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 GlaxoWellcome (Verona, Italy), meropenem from Zeneca (Rome, Italy), piperacillin and piperacillin + tazobactam from WyethLederle (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 0.125128 mg/L.
MIC and MBC determinations
The MIC was determined using a microbroth dilution method with MuellerHinton (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 12
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The peptides showed a different range of inhibitory values: the 40 P. aeruginosa clinical isolates were more susceptible to cecropin P1 (MIC range, 464 mg/L) and less susceptible to ranalexin (MIC range, 64 to >128 mg/L). The results are summarized in the Table.
|
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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Masuda N. & Ohya S. (1992). Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 36, 184751.[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, 1617. [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, 95100.[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, 33204.
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, 33543.[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, 163744.[Abstract]
8 . Bevins, C. L. & Zasloff, M. (1990). Peptides from frog skin. Annual Review of Biochemistry 59,395414.[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, 1084955.
10 . Jack, R. W., Tagg, J. R. & Ray, B. (1995). Bacteriocins of gram-positive bacteria. Microbiological Review 59, 171200.[ISI]
11 . Moore, A. J., Beazley, W. D., Bibby, M. C. & Devine, D. A. (1996). Antimicrobial activity of cecropins. Journal of Antimicrobial Chemotherapy 37, 107789.[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, 3417.[Abstract]
14 . Falla, T. J. & Hancock, R. E. W. (1997). Improved activity of a synthetic indolicidin analog. Antimicrobial Agents and Chemotherapy 41, 7715.[Abstract]
15 . Hancock, R. E. (1997). Antibacterial peptides and the outer membranes of gram-negative bacilli. Journal of Medical Microbiology 46, 13.[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, 18015.[Abstract]
17 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow AerobicallyThird 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, 1624. 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, 19298303.
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, 9518.[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, 544953.[ISI]
22 . Viljanen, P., Matsunaga, H., Kimura, Y. & Vaara, M. (1991). The outer membrane permeability-increasing action of deacylpolymyxins. Journal of Antibiotics 44, 51723.[ISI][Medline]
23
.
Howe, R. A. & Spencer, R. C. (1997).
Macrolides for the treatment of Pseudomonas aeruginosa infections? Journal
of
Antimicrobial Chemotherapy 40, 1535.
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