INTRODUCTION

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Vascular prosthetic graft infection is one of the most feared complications that the vascular surgeon treats, frequently resulting in prolonged hospitalization, organ failure, amputation, and death. The patient can develop late-appearing signs of infection as commonly as early postoperative infection (1, 3). Coagulase-negative staphylococci are among the most common pathogens that cause biomaterial infections. In particular, Staphylococcus epidermidis, a commensal organism of the skin, is the most frequent cause of late-appearing vascular graft infection in humans (1-3, 21). Effective strategies for the prevention of prosthetic infection vary from device to device. The centerpiece of therapy is prophylactic systemic antibiotics (3, 7). In addition, in the case of vascular grafts, antimicrobials, such as rifampin, bound at high concentrations to prosthetic grafts have been proposed as adjunctive prophylaxis (6, 9, 15, 19, 20, 22, 24, 26).

In recent years several polycationic peptides, compounds that comprise a diverse class of molecules, have been isolated from a wide range of bacteria, plants, insects, fish, amphibians, birds, mammals, and humans (4, 5, 11). In mammals, including humans, they are the predominant protein species in the neutrophil, and they are also found on the surfaces of the tongue, trachea, lungs, and upper intestine and are thought to be a major antibacterial defense on mucosal surfaces (11, 12). Recent reports have demonstrated that the site for the antibacterial action of the peptides is the cytoplasmic membrane, where they cause the formation of ion-channel pores that span the membrane without requiring a specific target receptor. Therefore, these compounds must initially be able to cross or disintegrate the outer membranes of gram-negative bacteria and the peptidoglycan (10, 11, 12, 25). The essential property of the polycationic peptides is their net positive charge at neutral pH (usually +4, +5, or +6) by virtue of their possession of the amino acids arginine and lysine (11). In addition, these compounds are amphipathic molecules: they have both a hydrophobic face, comprising nonpolar amino acid side chains, and a hydrophilic face of polar and positively charged residues (11, 12). The selective antibiotic activity of the cationic peptides is determined by their mode of interaction with the bacterial surface: typically, their positively charged residues interact with the negatively charged lipids of the bacterial membranes. On the other hand, the low anionic lipid content of the eukaryotic cells leads to the selectivity of the activity of the peptides for bacteria (10, 11, 12). The surfaces of several synthetic materials used by microbiologists and surgeons, such as polystyrene, polyethylene terephthalate (Dacron), and polytetrafluoroethylene, bind cationic molecules, and this property has been evaluated and used during in vitro and in vivo studies (13, 17; R. E. W. Hancock, Laboratory methods, 1998 [http://www.interchg.ubc.ca/bobh/MIC.htm]). By virtue of this binding, the retention of the biologically active molecules is not due to passive entrapment in the plastic tissue but reflects an ionic interaction between the anionic ligands and the cationic compounds. In experimental models, if the antimicrobial agents have an insufficient number of positively charged residues, they are usually bound to the prosthetic graft via a binding compound such as collagen, albumin, fibrin, and tridodecylmethylammonium chloride (6, 9, 13, 15, 19, 20, 24, 26). In this study we investigated the in vivo efficacies of two polycationic peptides, ranalexin and buforin II, spontaneously bound to a Dacron graft in preventing S. epidermidis infection of the graft in a rat model.

	MATERIALS AND METHODS

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Organisms. A commercially available methicillin-susceptible (MS) quality control strain of S. epidermidis, strain ATCC 12228, and one clinical isolate of methicillin-resistant (MR) S. epidermidis (Se56-99) were used.

Drugs. Buforin II, ranalexin, rifampin, cefazolin, and oxacillin were obtained from Sigma-Aldrich S.r.l. (Milan, Italy). Buforin II and ranalexin were dissolved in distilled H₂O at 20 times the required maximal concentration. For in vitro studies, serial dilutions of the peptides were prepared in 0.01% acetic acid containing 0.2% bovine serum albumin in polypropylene tubes (Hancock, Laboratory methods). Rifampin was dissolved in 50% methanol-50% acetone at a concentration of 1 mg/ml. Cefazolin and oxacillin were dissolved in sterile distilled water at a concentration of 1 mg/ml. Solutions were made fresh on the day of assay or were stored at 80°C in the dark for short periods. The concentration range assayed for each antibiotic was 0.25 to 256 µg/ml.

Susceptibility testing. The antimicrobial susceptibilities of the strains were determined by using the broth microdilution method by the procedures outlined by the National Committee for Clinical Laboratory Standards (14). The MIC was taken as the lowest antibiotic concentration at which observable growth was inhibited. However, the MICs of buforin II and ranalexin were determined by the procedures recently proposed for the testing of antimicrobial peptides (Hancock, Laboratory methods). Particularly, since cationic peptides bind to polystyrene, 96-well polypropylene plates (Sigma-Aldrich) were substituted for polystyrene plates and the plates were incubated for 18 h at 37°C in air. The plates were shaken throughout the study. The MIC was considered the lowest peptide concentration that reduced growth by more than 50% of that in the control well. The viable count in each well was determined by preparing 10⁶ dilutions and plating 10 µl of each dilution onto Mueller-Hinton agar plates to obtain overnight cultures. Experiments were performed in triplicate.

Rat model. Adult male Wistar rats (weight range, 300 to 350 g) were studied. The study included a control group (no graft contamination) and two series composed of eight groups (groups MS1 to MS8 and MR1 to MR8) for each of the staphylococcal strains. Each of the series included one contaminated group (groups MS1 and MR1) that received intraperitoneally isotonic sodium chloride solution, one contaminated group (groups MS2 and MR2) to which perioperative intraperitoneal cefazolin prophylaxis (30 mg/kg of body weight), was administered, three contaminated groups (groups MS3 to MS5 and MR3 to MR5) that received a buforin II-, a ranalexin-, or a rifampin-soaked graft, respectively, and three contaminated groups (groups MS6 to MS8 and MR6 to MR8) that received a buforin II-, a ranalexin-, or a rifampin-soaked graft, respectively, and perioperative intraperitoneal cefazolin prophylaxis (30 mg/kg). Each group consisted of 15 animals. The rats were anesthetized with ether, the hair of the inbacks was shaved, and the skin was cleansed with 10% povidone-iodine solution. One subcutaneous pocket was made on each side of the median line with 1.5-cm incision. Aseptically, 1-cm² sterile collagen-sealed Dacron grafts (Albograft; Sorin Biomedica Cardio, S.p.A., Saluggi VC, Italy) were implanted into the pockets. Prior to implantation, the Dacron graft segments were impregnated with 10 µg of buforin II per ml (groups MS2, MS5, MR2, and MR5), 10 µg of ranalexin per ml (groups MS3, MS6, MR3, and MR6), and 5 mg of rifampin per ml (groups MS4, MS7, MR4, and MR7). Antibiotic soaking was done immediately before implantation by placing the grafts for 20 min in a sterile solution of the agents mentioned above. Groups MS1, MS8, MR1, and MR8 received nonantibiotic-impregnated Dacron graft segments. In addition, the effect of preoperative intraperitoneal cefazolin administered 30 min before implantation at the standard dose of 30 mg/kg was evaluated in groups MS5 to MS8 and MR5 to MR8. The pockets were closed by means of skin clips, and sterile saline solution (1 ml) containing S. epidermidis ATCC 12228 or the MR strain S. epidermidis Se56-99 at a concentration of 2 × 10⁷ CFU/ml was inoculated onto the graft surface by using a tuberculin syringe to create a subcutaneous fluid-filled pocket (2). The animals were returned to individual cages and were thoroughly examined daily. All grafts were explanted 7 days following implantation.

Assessment of infection. The explanted grafts were placed in sterile tubes, washed in sterile saline solution, placed in tubes containing 10 ml of phosphate-buffered saline solution, and sonicated for 5 min to remove the adherent bacteria from the grafts. Quantitation of viable bacteria was done by preparing serial dilutions (0.1 ml) of the bacterial suspensions in 10 mM sodium HEPES buffer (pH 7.2) (Sigma-Aldrich) to minimize the carryover effect and by culturing each dilution on blood agar plates. All plates were incubated at 37°C for 48 h and were evaluated for the presence of the staphylococcal strains. The organisms were quantitated by counting the numbers of CFU per plate. The limit of detection for this method was approximately 5 × 10¹ CFU/cm² of graft tissue.

Statistical analysis. MICs are presented as the geometric means of three separate experiments. Quantitative culture results for all groups are presented as the mean ± standard deviation, and the statistical comparisons between groups were done by analysis of variance of the log-transformed data by the Tukey-Kramer honestly significant difference test. Significance was accepted when the P value was 0.05.

	RESULTS

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According to the broth microdilution method, the buforin II and ranalexin MICs for S. epidermidis ATCC 12228 and S. epidermidis Se56-99 were 2 and 4 mg/liter and 2 and 8 mg/liter, respectively. The two strains were similarly susceptible to rifampin (MICs, 0.25 mg/liter for both organisms), while they demonstrated different patterns of susceptibility to the beta-lactam antibiotics. Actually, S. epidermidis ATCC 12228 was susceptible to oxacillin and cefazolin (MICs, 0.5 and 2 mg/liter, respectively), while S. epidermidis Se56-99 was resistant (MICs, 8 and 32 mg/liter, respectively).

None of the animals included in the control group (no graft contamination) had anatomic or microbiological evidence of graft infection. On the contrary, all 30 rats included in groups MS1 and MR1 demonstrated evidence of graft infection, with quantitative culture results showing 3.1 × 10⁷ ± 6.0 × 10⁶ and 1.8 × 10⁷ ± 3.3 × 10⁶ CFU/cm² of graft, respectively, although there were no local signs of perigraft inflammation. Groups MS2 and MR2 (with buforin-coated Dacron grafts) and groups MS5 and MR5 (with buforin-coated Dacron grafts plus intraperitoneal cefazolin treatment) showed no evidence of staphylococcal infection, with negative quantitative culture results. For the 30 rats with ranalexin-coated Dacron grafts (groups MS3 and MR3), the quantitative graft cultures demonstrated bacterial growth (1.2 × 10³ ± 5.0 × 10² and 2.3 × 10³ ± 6.5 × 10² CFU/cm² of graft, respectively). On the contrary, none of the 30 rats with ranalexin-coated grafts plus intraperitoneal cefazolin treatment (groups MS6 and MR6) had evidence of infection. Cultures of the grafts from groups MS4 and MR4 (with rifampin-coated grafts) showed results similar to those for the animals treated with ranalexin-coated grafts (1.9 × 10³ ± 4.5 × 10² and 2.4 × 10³ ± 7.0 × 10² CFU/cm² of graft, respectively). Nevertheless, the results showed that the use of rifampin-coated grafts and cefazolin treatment (groups MS7 and MR7) was less effective than the use of ranalexin-coated grafts and cefazolin treatment. Actually, infection occurred in groups MS7 and MR7, although with low bacterial numbers (4.0 × 10² ± 1.0 × 10² and 8.0 × 10² ± 2.0 × 10² CFU/cm² of graft, respectively). The results for groups MS8 and MR8 (with intraperitoneal cefazolin treatment and a Dacron graft without antibiotic impregnation) confirmed the efficacy of preoperative cefazolin against the MS staphylococcal strains (6.5 × 10² ± 3.5 × 10² CFU/cm² of graft) and, on the contrary, its poor efficacy against the MR strains (1.5 × 10⁷ ± 4.7 × 10⁶ CFU/cm² of graft).

There were significant differences in the results for the quantitative bacterial graft cultures when the data obtained for all treated groups were compared with those obtained for the untreated groups. On the contrary, no statistically significant difference was observed between groups MR1 and MR8. Data on the quantitative culture results and from statistical comparisons of the groups are summarized in Table 1.

	DISCUSSION

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The vascular surgery patient's own endogenous flora is the most likely source of S. epidermidis organisms that colonize the graft. This organism has been recovered from the skin, subcutaneous fat, lymph nodes, and arterial walls of greater than one-third of individuals undergoing vascular reconstruction, despite the use of aseptic vascular surgical technique and prophylactic antibiotics (3). The success of surgical prophylaxis on the prevention of graft infections is dependent on the pharmacokinetics of antibiotic penetration into tissue and maintenance of adequate levels in tissue for the duration of the vascular surgical procedure. Nevertheless, errors in sterilization procedures and increases in the incidence and resistance of S. epidermidis can predispose an individual to prosthesis infection. In particular, after the initial success with beta-lactams as preoperative antibiotic prophylaxis, resistance to these drugs began to emerge. Since the emergence of MR staphylococci, glycopeptides have been the only uniformly effective treatments for staphylococcal infections. However, the recent emergence of glycopeptide resistance in coagulase-negative staphylococci heightens concern about the need for other antibiotics in prophylactic regimens (18, 21). Polycationic peptides are known to have variable antibacterial, antifungal, and antiprotozoan activities in vitro. These compounds provide a basic host defense system that combats infections: for these reasons, in this study we investigated the in vivo efficacies of two polycationic peptides, ranalexin and buforin II, spontaneously bound to an albumin-sealed Dacron graft for the prevention of S. epidermidis infection of the graft in a rat model.

Buforin II and ranalexin showed similar in vitro activities against the two staphylococcal strains tested. In addition, rifampin exerted equal in vitro activity against the two strains. Actually, in the present study S. epidermidis Se56-99 was chosen because it has been demonstrated to be oxacillin resistant and is as susceptible to rifampin as the standard control strain S. epidermidis ATCC 12228. Cefazolin, a narrow-spectrum cephalosporin, was chosen as the intraperitoneal prophylactic agent because of its minimal toxicity over a wide range of doses. Moreover, it is a commonly used antibiotic for surgical prophylaxis in vascular surgery patients and achieved adequate levels in tissue after administration of a single dose, with maintenance of those levels for 2 to 3 h. In order to evaluate the presence of a positive interaction with the drugs bonded to the Dacron graft, cefazolin was also tested against MR S. epidermidis, although we presumed that it would be ineffective when used alone. Indeed, recent studies demonstrated that the polycationic peptides present properties of synergy with lipophilic and amphiphilic agents such as rifampin, macrolides, fusidic acid, and novobiocin. Actually, those reports indicated that they allow maximal entry of several hydrophobic substrates into the cell (11, 12, 23). Moreover, recent investigations demonstrated a positive interaction between peptides and beta-lactams: it might be due to increased access of the peptides to the cytoplasmic membrane following breakdown of the peptidoglycan by beta-lactams. On the other hand, the peptides, by triggering the activities of bacterial murein hydrolases, might cause degradation of the peptidoglycan and enhance the activities of the beta-lactams (10, 11, 12).

Taken together, the results of this study demonstrated that the use of preoperative intraperitoneal cefazolin or an antibiotic-soaked Dacron graft can result in significant bacterial growth inhibition even if high concentrations of organisms are topically inoculated into the Dacron prostheses. Statistical analysis demonstrated that any prophylactic antibiotic treatment was useful; nevertheless, only buforin II was able to inhibit the bacterial growth completely, even though buforin II bonded to the Dacron graft was used alone. On the other hand, ranalexin was also shown to be highly effective. Actually, ranalexin was demonstrated to be as effective as rifampin and, when combined with intraperitoneal cefazolin, produced complete suppression of both MS and MR staphylococcal strains.

Similar to the other agents tested, neither buforin II nor ranalexin showed any noteworthy toxicity. Actually, none of the animals included in any group died or had clinical evidence of drug-related adverse effects, such as local signs of perigraft inflammation, anorexia, vomiting, diarrhea, or behavioral alterations.

Buforin II and ranalexin are polycationic peptides derived from amphibian tissues: the first was derived from buforin I, a potent peptide isolated from the stomach tissue of an Asian toad, Bufo bufo gargarizans, and the second was isolated from the skin of the American bullfrog (Rana catasbeiana) (8, 16). The strong in vivo antibacterial efficacies of the two peptides chosen for this study well suit the remarkable resistance of frogs and toads to infection after external injury, despite the contaminated environments in which these animals live (5, 11). The widespread use of several antimicrobial agents both in therapeutic regimens and in prophylactic regimens resulted in a dramatic increase in the prevalence of multidrug-resistant organisms, such as MR staphylococci. In fact, the short doubling times and genetic plasticity of bacteria permit these organisms to rapidly prove whether specific mutations enhance their ability to grow in inhospitable environments. Mutations that confer resistance help bacteria survive attacks from antibiotics used clinically. Nevertheless, as a consequence of the mode of action of the peptides, the emergence of peptide-resistant mutants should be an unlikely event, since alteration of the membrane structure to prevent insertion and channel formation is far more difficult than remodeling of target enzymes. Today, despite the speculated modes of action of the peptides, proof of their clinical benefits are lacking. However, the antistaphylococcal in vitro activity and the prophylactic in vivo efficacy demonstrated in the present study make these molecules potentially useful for preoperative antimicrobial chemoprophylaxis. Future research based on animal and human models is needed to elucidate their in vivo efficacies and toxicities and their utility in clinical practice.