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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, June 2007, p. 2005-2010, Vol. 51, No. 6
0066-4804/07/$08.00+0 doi:10.1128/AAC.01576-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
ukasiak,3
Giorgio Scalise,1
Vittorio Saba,2 and
Andrea Giacometti1
Institute of Infectious Diseases and Public Health, Università Politecnica delle Marche, Ancona, Italy,1 Department of General Surgery, I.N.R.C.A. I.R.R.C.S., Università Politecnica delle Marche, Ancona, Italy,2 Faculty of Pharmacy, Medical University of Gdansk, Gdansk, Poland,3 Experimental Animal Models for Aging Units, Research Department, I.N.R.C.A. I.R.R.C.S., Ancona, Italy4
Received 19 December 2006/ Returned for modification 12 January 2007/ Accepted 23 March 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antimicrobial therapy for infections due to these multiresistant organisms remains a clinical dilemma with hospitalized patients. Strategies for prevention and therapy include both infection control and modifications in antibiotic use, such as antibiotic cycling or rotation, combination therapies, and the use of "old" and new drugs for the management of infections with gram-negative bacilli resistant to all other alternatives (4, 16).
Since the discovery of polymyxins, a number of cationic peptides have been isolated from a wide range of bacterial, plant, and animal species (2, 12, 14). They are a recently emerged class of antibiotics with therapeutic potential. These molecules are important components of the innate immune response in most multicellular organisms, used by animals to effectively deal with pathogenic microorganisms in their environments (12, 14, 18). In mammals, these peptides are found in circulating phagocytes, where they contribute to the killing of engulfed microorganisms, and in epithelial surfaces, where they act as a local defense mechanism that protects anatomical compartments from microbial invasion.
Tachyplesins are a group of antimicrobial peptides isolated from horseshoe crabs. Tachyplesin III (KWCFRVCYRGICYRKCR-NH2), isolated from the lymph hemolysate of Southeast Asian horseshoe crabs Tachypleus gigas and Carcinoscorpius rotundicauda and consisting of 17 amino acids with two disulfide bridges, is a representative antimicrobial peptide with a cyclic ß-sheet. It is similar in structure and activity profile to the protegrins, a family of peptides found in pig intestines. Because of its potency and relatively small size, this peptide is an attractive target for structure/activity studies that may lead to therapeutics to treat infections (13, 19). Tachyplesin III exhibits broad-spectrum activity against gram-negative and -positive bacteria, fungi, and even enveloped viruses at low concentrations. Furthermore, it has also been shown to play a role in the proinflammatory response because it forms complexes with bacterial lipopolysaccharides (LPS) that neutralize the factor C-activating activity of LPS in a manner similar to that of anti-LPS factor (19). It was reported, even though the precise action mechanism of these cationic peptides remains to be determined, that they rapidly perturbed the membrane function of pathogenic microorganisms.
Recent reports have shown that a synergistic effect was observed in several clinically isolated bacterial strains when some antimicrobial peptides were combined with several clinically used antibiotics. Therefore, the presence of this synergistic effect makes the cationic peptides potentially valuable as an adjuvant for antimicrobial chemotherapy against antibiotic-resistant bacterial strains (9, 25).
In order to broaden our knowledge of this role, we evaluated the activities of the combination of tachyplesin III or colistin and imipenem in vitro and in vivo using two P. aeruginosa strains.
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MATERIALS AND METHODS |
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Synthetic peptide. Tachyplesin III (molecular mass, 2,495.5 Da) was synthesized by 9-fluorenylmethoxycarbonyl solid-phase chemistry (5, 8). The protected peptidyl resin was treated with 92% trifluoroacetic acid, 2% ethanedithiol, 2% water, and 2% triisopropylosilane for 2 h. After cleavage, the solid support was removed by filtration, and the filtrate was concentrated under reduced pressure. The cleaved peptide was precipitated with diethyl ether, dissolved in 20% acetic acid, and oxidized with 0.1 M iodine in methanol. The tachyplesin III was purified and analyzed by high-performance liquid chromatography. The resulting fractions with purity greater than 94 to 95% were tested by high-performance liquid chromatography. The peptide was analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The peptide was solubilized in phosphate-buffered saline (PBS) (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.
MIC determination and bacterial killing assay. Tachyplesin III, colistin (Sigma-Aldrich, Milan, Italy), and imipenem (Merck, Sharp & Dohme, Milan, Italy) powders were diluted in accordance with the manufacturers’ recommendations. Solutions of drugs were made fresh on the day of assay or stored at –80°C in the dark until 20 days. The MICs were determined using a broth microdilution method with Mueller-Hinton (MH) broth (Becton Dickinson Italia, Milan, Italy) and initial inocula of 5 x 105 CFU/ml, according to the procedures outlined by CLSI (formerly NCCLS) (20). Polypropylene 96-well plates (Becton Dickinson and Co., Franklin Lakes, NJ) were incubated for 18 h at 37°C in air. When the peptide tachyplesin III was tested, the plates were shaken throughout the study, since this compound has a tendency to precipitate. The MIC was taken as the lowest drug concentration at which observable growth was inhibited. Experiments were performed in triplicate. The minimum bactericidal concentration was taken as the lowest concentration of each drug that resulted in a more than 99.9% reduction of the initial inoculum. Experiments were performed in triplicate.
Both strains were grown at 37°C in MH broth. Aliquots of exponentially growing bacteria were resuspended in fresh MH broth at approximately 107 cells/ml and separately exposed to each peptide at 2x MIC for 0, 2, 5, 10, 20, 30, 40, 50, and 60 min at 37°C. After these times, 0.1-ml samples were serially diluted and plated onto MH agar plates to obtain viable colonies. The limit of detection for this method was approximately 10 CFU/ml. In preliminary experiments, antibiotic carryover was ruled out by plating samples of bacterial suspensions. Killing was also investigated in combination studies, to evaluate the activity of imipenem at a concentration of 4 mg/liter when combined with each peptide at 0.5x MIC and 1x 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 35°C, and the bacterial growth in each tube was determined by performing consecutive 1:10 (vol/vol) dilutions of a 0.1-ml aliquot from each tube in MH broth and by plating a 0.1-ml volume of each dilution onto MH agar. Experiments were performed in triplicate. If a combination caused a decrease in viable cell count of 
log102 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 log101 to log102, the effects of the combination were considered to be additive.
Synergy studies. In interaction studies, the strains were tested against the antibiotic combinations by using a checkerboard titration method using 96-well polypropylene microtiter plates. The ranges of drug dilutions used were 0.125 to 64 mg/liter for tachyplesin III and 0.25 to 256 mg/liter for colistin and imipenem. The fractionary inhibitory concentration (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, respectively, in the combination, MICA and MICB are the MICs of drug A and drug B, respectively, alone, and FICA and FICB are the FICs of drug A and drug B, respectively. The FIC indexes were interpreted as follows: <0.5, synergy; 0.5 to 4.0, indifference; and >4.0, antagonism (7). In addition, time-kill synergy studies were performed at recommended subinhibitory concentrations (one-fourth and one-half the MIC). 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 to killing with the most active single agent.
Hemolysis of human red blood cells and cytotoxicity assay. Fresh human red blood cells (hRBC) with EDTA were rinsed three times with PBS (35 mM phosphate buffer-0.15 M NaCl [pH 7.3]) by centrifugation for 10 min at 800 x g at 35°C and resuspended in PBS. Tachyplesin dissolved in PBS was then added to 50 µl of a solution of the stock hRBC in PBS to reach a final volume of 100 µl (final erythrocyte concentration, 4% vol/vol). The resulting suspension was incubated with agitation for 60 min at 37°C. The samples were then centrifuged at 800 x g for 10 min. The release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm. Controls for 0% hemolysis (blank) and 100% hemolysis consisted of hRBC suspended in PBS and 1% Triton X-100, respectively.
For the cytotoxicity assay, A-549 cells from human lung carcinoma (BioWhittaker Inc., Walkersville, MD) were cultured in 25-cm2 tissue culture flasks. The medium consisted of Dulbecco's modified Eagle's medium with 10% fetal calf serum (Bio-Whittaker). The cytotoxicities of tachyplesin III A at 1x minimum bactericidal concentration were determined by using a CellTiter 96 AQ cell proliferation assay (Promega Corp., Lyon, France).
Animals. BALB/c male mice weighting 25 to 30 g were used for all the experiments. Each mouse was housed in an individual cage under constant temperature (22°C) and humidity with a 12-h light/dark cycle and had access to chow and water ad libitum 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., Polytechnic University of Marche, Ancona, Italy.
Preparation and implantation of the inoculum and antibiotic therapy. P. aeruginosa ATCC 27853 and the clinical isolate strain were grown overnight at 37°C in brain heart infusion broth. When bacteria were in the log phase of growth, the suspension was centrifuged at 1,000 x g for 15 min, the supernatant was discarded, and the bacteria were resuspended in sterile saline to achieve a concentration of approximately 1 x 108 CFU/ml. All animals were anesthetized by an intramuscular injection of ketamine (30 mg/kg of body weight). The mice were injected intravenously via the tail vein with 0.2 ml of one of the above-mentioned bacterial suspensions (2.0 x 107 CFU) on day 0 and monitored for 72 h.
Immediately after bacterial challenge, the mice were randomly chosen to receive, intravenously, isotonic sodium chloride solution (control group), 20 mg/kg imipenem, 1 mg/kg tachyplesin III, 1 mg/kg colistin, or 20 mg/kg imipenem combined with 1 mg/kg tachyplesin III or 1 mg/kg colistin. Each group included 20 mice. The animals were returned to individual cages and monitored for the subsequent 72 h. The end points of the study were lethality rates, quantitative blood cultures, and endotoxin, tumor necrosis factor alpha (TNF-
), and interleukin-6 (IL-6) plasma levels. Toxicity was evaluated on the basis of the presence of drug-related adverse effects (local signs of inflammation, weight loss, vomiting, diarrhea, and fever) in the supplementary tachyplesin III-treated group without challenge.
Evaluation of treatment. Blood samples for culture were obtained from the tail vein by aseptic percutaneous puncture 24 h after bacterial challenge. The animals that died before this time were not tested. To perform quantitative bacterial cultures, blood samples were serially diluted and a 0.1-ml volume of each dilution was spread on blood agar plates and cultured at 35°C for 48 h and the CFU were counted. The limit of detection was 10 CFU/ml.
For determination of endotoxin, TNF-, and IL-6 levels in plasma, blood samples (0.2 ml for each animal) were collected from the tail vein at 0, 6, 12, 24, and 48 h postinjection. Endotoxin concentrations were measured by using the commercially available Limulus amebocyte lysate test (E-TOXATE; Sigma-Aldrich). Endotoxin standards (0, 0.015, 0.03, 0.06, 0.125, 0.25, and 0.5 EU/ml) were tested in each run, and the concentration of endotoxin in the tested samples was calculated by comparison to the standard curve.
IL-6 and TNF- levels were measured by enzyme-linked immunosorbent assay according to the manufacturer's specifications (Walter Occhiena srl, Turin, Italy). The lower limits of sensitivity for IL-6 and TNF- were 12 pg/ml and 0.05 ng/ml, respectively. The assays were performed in duplicate.
Statistical analysis.
Lethality rates were compared between groups by use of Fisher's exact test. Data from quantitative blood cultures are presented as means ± standard deviations of the means; statistical comparisons between groups were made by analysis of variance. Post hoc comparisons were performed by Bonferroni's test. Plasma endotoxin, IL-6, and TNF- mean values were compared between groups by analysis of variance. Significance was accepted when the P value was 
0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cytotoxic effect was practically absent at the concentrations tested. Our data revealed that tachyplesin showed low hemolytic activity in spite of its high activity against the organism. In fact, its hemolytic activity was observed at concentrations higher than the MICs (4.5%, 12.6%, and 26.7% at concentrations of 8, 16, and 32 mg/liter, respectively).
In vivo studies. As shown in Table 1, the control groups showed 100% bacterial lethality rates within 72 h. In contrast, for both the bacterial strains, immediate treatment with drugs demonstrated efficacies significantly higher than that of the control treatment (P < 0.05). For P. aeruginosa ATCC 27853, lethality rates of 30%, 30%, and 40% were observed for groups treated with imipenem, tachyplesin III, and colistin A, respectively. For the clinical strain, imipenem showed a higher lethality rate (80%), while the same lethality rates were observed for peptide-treated groups (30%). The combination of tachyplesin III or colistin and imipenem showed the lowest lethality rates, 5% and 10% for the control strain and 10% and 15% for the clinical isolate, respectively. Quantitative blood culture showed high bacterial numbers both in the ATCC 27853 control group (5.8 x 107 ± 0.8 x 107 CFU/ml) and in the clinical isolate control group (7.9 x 107 ± 2.2 x 107 CFU/ml), as shown in Table 1. Tachyplesin III demonstrated a good antibacterial activity against the ATCC strain, reducing the bacterial growth to 3.6 x 103 ± 0.6 x 103 CFU/ml, comparable to the activities of colistin and imipenem (6.0 x 104 ± 1.7 x 104 CFU/ml and 2.8 x 103 ± 0.4 x 103 CFU/ml, respectively). When imipenem was combined with tachyplesin III and colistin, the positive interactions produced the lowest bacterial counts (1.1 x 101 ± 0.1 x 101 CFU/ml for tachyplesin III plus imipenem and 4.6 x 101 ± 0.5 x 101 for colistin plus imipenem). Against the panresistant strain, tachyplesin III and colistin showed antibacterial activities comparable to those against the ATCC strain, while imipenem showed weak activity. Similar to the results of the previous experiment, when imipenem was combined with tachyplesin III or colistin, the positive interaction produced the lowest bacterial counts. Overall, any combination-treated group had significantly lower bacterial counts than the groups treated with a single agent (P < 0.05).
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, and IL-6 were observed 6 and 12 h after bacterial challenge. Tachyplesin III and colistin treatments (alone or the combinations) resulted in marked decreases (P < 0.05) of endotoxin, TNF-, and IL-6 plasma levels compared to those of control and vancomycin-treated groups. (Fig. 1 to 3). The strongest reduction in endotoxin and cytokine plasma levels was observed in the groups treated with the combination of tachyplesin III and imipenem.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we evaluated the efficacy of the combination of tachyplesin or colistin and imipenem against two strains of P. aeruginosa with different patterns of susceptibility, first in vitro and then in vivo. We used one susceptible and one multiresistant strain that can be associated with clinical failure following antibiotic therapy. In time-killing curves and with the checkerboard titration method, a synergistic effect between the peptides and imipenem was observed. This synergistic pattern was also clearly observed in the in vivo setting. Previous studies have reported contradictory results for interactions among peptides and antibiotics (7, 9, 25, 26). The peptide acts by inserting into the cytoplasmic membrane and triggering the activity of bacterial murein hydrolases, resulting in damage to or degradation of bacterial membranes and lysis of the cell. In addition, antimicrobial peptides can induce, by the loss of effective outer membrane porin channels, a membrane permeability that facilitates the penetration of antibiotics and the consequent enhancement of their activity (6, 17, 25, 27).
As has been described previously, a sepsis mouse model can be used to compare the efficacies of different antibiotic therapies and also the efficacy of a therapy for strains with different susceptibilities. We selected as the main outcome measures lethality, quantitative blood cultures, and detection of endotoxin, TNF-, and IL-6 plasma levels, to have different parameters and better define the efficacies of the treatments. We used a short period of treatment of 24 h in order to increase the relevance of the model, since this is the critical period of such an acute infection (15). In our model, tachyplesin III and colistin showed antimicrobial activities superior (clinical isolate) or similar (ATCC control strain) and lethality rates comparable to those of imipenem. They demonstrated a greater ability than imipenem to inhibit the effects of endotoxin and, consequently, the release of cytokines.
Finally, the best results for mortality rates and bacteremia were obtained when tachyplesin III was combined with imipenem. This combination was also most effective in decreasing the levels of cytokines, confirming the capacity of the peptides to neutralize membrane components that are the inducers of cytokine activation. Several studies have shown that exposure of gram-negative organisms to bactericidal agents such as carbapenems can initially result in endotoxin release, even though the subsequent decrease in endotoxin release due to the inhibitory effect on the bacterial growth appears more relevant (24). Therefore, new drugs able to neutralize molecules released by gram-negative organisms and block their interaction with specific receptors on immune cells at the beginning of antimicrobial treatment can be an attractive concept. Today, the clinically used antimicrobial peptides are the microbial cyclic molecules such as colistin and polymyxin B. Our results emphasized that tachyplesin III, a linear invertebrate peptide, has an in vitro activity and a systemic in vivo efficacy equivalent to those of colistin.
Few treatment options remain for serious infections caused by multidrug-resistant bacteria. Carbapenems are active against some isolates of P. aeruginosa, and although the polymyxins remain the most consistently effective agents in vitro against this organism, a growing number of reports have documented resistance to these antibacterials (10). In this situation, novel compounds and antibiotic combinations will be necessary to treat infections caused by these multidrug-resistant organisms and to reduce the increasing selection pressure by antibiotics on gram-negative pathogens. One way to overcome the problems of emergence of resistance is the correct use of new antimicrobial compounds and/or combination therapies. In fact, infections with isolates that are susceptible to only one or a few agents may be treated with a single drug, but the risk of progressive resistance must be considered. Specific combination therapies can be used to increase the in vivo activity, to prevent the emergence of drug resistance, and to broaden the antimicrobial spectrum. In particular, sublethal alterations of the gram-negative bacterial outer membrane in combination with the use of antibiotics that, because of resistance, are now ineffective alone may further extend therapeutic opportunities. The good antimicrobial activity toward gram-negative bacteria with high levels of antibiotic resistance and the synergistic interaction with imipenem illustrate additional important attributes that may support tachyplesin III as a promising candidate for an adjuvant in antimicrobial therapy against multiresistant organisms. Further studies should be performed to address the development of tachyplesins for this use.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Italian Ministry of Education, University, and Research (PRIN 2005).
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FOOTNOTES |
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Published ahead of print on 2 April 2007.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ukasiak, and G. Scalise. 2005. In vitro activity of the histatin derivative P-113 against multidrug-resistant pathogens responsible for pneumonia in immunocompromised patients. Antimicrob. Agents Chemother. 49:1249-1252.This article has been cited by other articles:
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