Efficacy of polycationic peptides in preventing vascular graft infection due to Staphylococcus epidermidis

Andrea Giacomettia,*, Oscar Cirionia, Roberto Ghisellib, Luigi Goffib, Federico Mocchegianib, Alessandra Rivaa, Giorgio Scalisea and Vittorio Sabab

a Institute of Infectious Diseases and Public Health and b Department of General Surgery, INRCA IRCCS, University of Ancona, Ancona, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A rat model was used to investigate the efficacy of two polycationic peptides, ranalexin and buforin II, in the prevention of vascular prosthetic graft infection due to methicillin-resistant Staphylococcus epidermidis with intermediate resistance to glycopeptides. The in vitro activity of the peptides was compared with those of vancomycin and teicoplanin by MIC determination and time–kill study. Moreover, the efficacy of collagen-sealed peptide-soaked Dacron was evaluated in a rat model of graft infection. Graft infections were established in the dorsal subcutaneous tissue of 120 adult male Wistar rats. The in vivo study included a control group, one contaminated group that did not receive any antibiotic prophylaxis and four contaminated groups that received an antibiotic-soaked graft. Experiments demonstrated that the activities of buforin II and ranalexin were greater than those of vancomycin and teicoplanin. Particularly, rats with buforin II-coated Dacron grafts showed no evidence of staphylococcal infection while, for the rats with ranalexin-, vancomycin- and teicoplanin-coated Dacron grafts, the quantitative graft cultures demonstrated bacterial growth (1.9 x 102 ± 0.6 x 102 cfu/mL, 6.2 x 103 ± 1.9 x 103 cfu/mL and 5.1 x 104 ± 4.8 x 103 cfu/mL, respectively). The study demonstrated that the use of peptide-soaked Dacron graft can result in significant bacterial growth inhibition and indicates that these compounds may be potentially useful in prosthetic surgery.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Staphylococci are common causative organisms in nosocomial and community-acquired infection. Coagulase-negative staphylococci are among the most common pathogens that cause biomaterial infections. Staphylococcus epidermidis, a skin commensal, is the most frequent cause of late-appearing vascular graft infection in humans.1–4 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.1,3 Effective strategies for the prevention of prosthetic infection vary from device to device. The mainstay of prophylaxis is the perioperative administration of systemic antibiotics.3,5 For vascular grafts, antimicrobials bound in high concentrations to prosthetic grafts have been proposed as adjunctive prophylaxis.4,6–12 Since the emergence of methicillin-resistant (MR) staphylococci, glycopeptides have been the only uniformly active agents for staphylococcal infections. Vancomycin was introduced in the 1950s. For almost three decades following its introduction, resistance to vancomycin was reported only rarely and appeared to have little clinical significance. Nevertheless, in the 1980s the emergence of vancomycin resistance was described in coagulase-negative staphylococci (CNS),13–15 while in 1996 there was the world's first documented clinical infection due to Staphylococcus aureus with intermediate resistance to glycopeptides.16

In recent years several polycationic peptides, compounds with a broad spectrum of antimicrobial activity, have been isolated from a wide range of bacteria, plants, insects, fish, amphibians, birds, mammals and man.17–19 These compounds are amphipathic molecules: they have both a hydrophobic domain, comprising non-polar amino acid side chains, and a hydrophilic domain of polar and positively charged residues.19,20 The site for the antibacterial action of the peptides is the cytoplasmic membrane. Transient ion-channel pores are formed that span the membrane causing disruption in the absence of a specific target receptor.19–22 Very few in vivo studies of cationic peptide action have been published. Despite limited data, several compounds are under investigation in animal models and in the meantime, clinical trials have been proposed or are underway.19,21

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 from the skin of the American bullfrog Rana catasbeiana.23,24 Previous studies have demonstrated that these compounds have strong in vitro activity against Grampositive bacteria.23–27

In this study we used one strain of S. epidermidis with intermediate resistance to glycopeptides to investigate the in vitro activity of the two above-mentioned peptides and their in vivo efficacy, when bound to the Dacron graft, in preventing prosthesis infection in a rat model.


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

The strain of methicillin-resistant S. epidermidis with intermediate resistance to glycopeptides used in this study was isolated from a clinical specimen submitted for routine bacteriological investigation to the Institute of Infectious Diseases and Public Health, University of Ancona, Italy. This isolate was described using the acronym GISE (glycopeptide-intermediate S. epidermidis). Commercially available S. epidermidis ATCC 12228 was used as quality control strain in the in vitro investigations.

Drugs

Buforin II, ranalexin and vancomycin were obtained from Sigma-Aldrich (Milan, Italy). Teicoplanin was obtained from Hoechst Marion Roussel S.p.A. (Milan, Italy). Laboratory 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 short periods.

Antimicrobial susceptibility testing

The antimicrobial susceptibilities of the strains to vancomycin, teicoplanin, buforin II and ranalexin were determined by the broth microdilution method described by the National Committee for Clinical Laboratory Standards (NCCLS).28 In addition, the strains were tested for susceptibility to vancomycin and teicoplanin by the NCCLS reference disc diffusion method using 30 µg vancomycin and teicoplanin discs.29

Moreover, the MICs of the four antimicrobial agents were also determined according to the procedures recently evaluated for testing antimicrobial peptides.30 Particularly, since cationic peptides bind polystyrene, polypropylene 96-well plates (Sigma-Aldrich) were substituted for polystyrene plates. The compounds were dissolved in distilled water at 20 times the required maximal concentration and serial dilutions were prepared in 0.01% acetic acid containing 0.2% bovine serum albumin in polypropylene tubes. The plates were incubated for 18 h at 37°C in air and shaken throughout the study. The MIC was considered the lowest peptide concentration that reduced growth by >50% of that in the control well.30 The viable count in each well was determined by performing six 10-fold dilutions and plating 10 µL of each dilution on to Mueller–Hinton (MH) agar (Becton Dickinson Italia, Milan, Italy) plates to obtain overnight cultures. Experiments were performed in triplicate.

Time–kill studies

To perform time–kill studies, the GISE strain and S. epidermidis ATCC 12228 were grown at 37°C in MH broth. When bacteria were in the log phase of growth the suspensions were centrifuged at 1000g for 15 min, the supernatants were discarded, and the bacteria were resuspended and diluted with sterile saline to achieve a concentration of 5 x 1010 cfu/mL saline. The organisms were resuspended in fresh MH broth at c. 5 x 109 cells/mL and exposed to buforin II, ranalexin, vancomycin or teicoplanin (final concentration 10 and 50 mg/L) for up to 24 h at 37°C. Throughout the experiments, triplicate samples (10 µL) were withdrawn after 0, 5, 10, 15, 20, 30, 60, 120, 180, 360, 720 and 1440 min incubation at 37°C. Up to seven 10-fold dilutions were made in MH broth from each sample and, finally, spread on to MH agar plates to obtain viable colonies. The limit of detection for this method was c. 10 cfu/mL. A bactericidal effect was defined as a >= 3 log10 (99.9% killing) reduction in cfu compared with the initial test inoculum.

Rat model

Adult male Wistar rats (weight range, 300–350 g) were studied. The study included a group with no graft contamination and no antibiotic prophylaxis (uncontaminated control), one contaminated group that did not receive any antibiotic prophylaxis (treated control) and four contaminated groups that received buforin II-, ranalexin-, vancomycin- and teicoplanin-soaked graft, respectively. Each group included 20 animals. Rats were anaesthetized with ether, the hair on the back was shaved and the skin cleansed with 10% povidone–iodine solution. One subcutaneous pocket was made on each side of the median line by a 1.5 cm incision. Aseptically, 1 cm2 sterile collagen-sealed Dacron grafts (Albograft, Sorin Biomedica Cardio, S.p.A., Saluggia VC, Italy) were implanted into the pockets. Before implantation, the Dacron graft segments were impregnated with 10 mg/L buforin II, 10 mg/L ranalexin, 50 mg/L vancomycin or 50 mg/L teicoplanin. Bonding of the antimicrobials was obtained immediately before implantation by soaking grafts for 20 min in a sterile solution of the above-mentioned agents. The pockets were closed by means of skin clips and sterile saline solution (1 mL) containing the GISE strain at a concentration of 2 x 107 cfu/mL was inoculated on to the graft surface using a tuberculin syringe to create a subcutaneous fluid-filled pocket.2 The animals were returned to individual cages and thoroughly examined daily. All grafts were explanted at 7 days following implantation.

Assessment of the 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. Quantification of viable bacteria was performed by culturing serial 10-fold dilutions (0.1 mL) of the bacterial suspension on blood agar plates. All plates were incubated at 37°C for 48 h and evaluated for the presence of the GISE strain. The organisms were quantified by counting the number of cfu per plate. The limit of detection for this method was c. 10 cfu/mL.

Statistical analysis

MIC values are presented as the mode of three separate experiments. Quantitative culture results regarding the in vivo experiments were presented as mean ± S.D. of the mean; comparisons of the results were performed by analysis of variance (ANOVA) on the log-transformed data. Significance was accepted when the P value was <=0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro studies

According to the broth microdilution method recommended by the NCCLS, vancomycin exhibited the greatest potency against the strain tested, with MICs of 0.25 and 8 mg/L for S. epidermidis ATCC 12228 and the GISE strain, while teicoplanin showed MICs of 0.25 and 16 mg/L. Interestingly, the two strains were similarly susceptible to the polycationic peptides: buforin II showed MICs of 8 and 16 mg/L for S. epidermidis ATCC 12228 and the GISE strain, while ranalexin demonstrated MICs of 16 mg/L for both strains. The different pattern of susceptibility was confirmed by the disc diffusion test: S. epidermidis ATCC 12228 showed zone sizes of 15 and 18 mm for teicoplanin and vancomycin, respectively, while the intermediate resistance of the GISE strain to the glycopeptides was demonstrated by zone sizes of 11 mm for both vancomycin and teicoplanin. When the broth dilution method was performed according to the modified procedure recently described for testing antimicrobial peptides,30 the two strains demonstrated different susceptibility patterns for the polycationic peptides. Actually, both S. epidermidis ATCC 12228 and the GISE strain showed MICs of 2 mg/L for buforin II and ranalexin. In contrast, the MICs of the glycopeptides were not substantially modified by using the second method (vancomycin, 0.25 and 4 mg/L for S. epidermidis ATCC 12228 and the GISE strain; teicoplanin, 0.25 and 8 mg/L for S. epidermidis ATCC 12228 and the GISE strain).

Killing by buforin II and ranalexin was shown to be very rapid. At a concentration of 10 mg/L they produced a 4 log10 cfu/mL decrease from the initial inoculum after a 10 and 15 min exposure period, respectively, while at a concentration of 50 mg/L no viable cell was detected after a 5 min exposure period. On the other hand, a 3 log10 cfu/mL decrease from the initial inoculum was produced by vancomycin and teicoplanin only after 60 min exposure at the highest concentration. The time–kill study for the two staphylococcal strains tested is summarized in the FigureGo.



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Figure. Time–kill study of buforin II, ranalexin, vancomycin and teicoplanin against Staphylococcus epidermidis with intermediate resistance to glycopeptides: –x–, control; –{blacksquare}–, buforin II (10 mg/L); –{blacktriangleup}–, ranalexin (10 mg/L); –{diamondsuit}–, vancomycin (10 mg/L); –•–, teicoplanin (10 mg/L); –{square}–, buforin II (50 mg/L); –{triangleup}–, ranalexin (50 mg/L); –{diamond}–, vancomycin (50 mg/L); –{circ}–, teicoplanin (50 mg/L).

 
In vivo studies

None of the animals included in the uncontaminated control group had microbiological evidence of graft infection. In contrast, all 20 rats included in the untreated control group demonstrated evidence of graft infection, with quantitative culture results showing 4.9 x 106 ± 9.7 x 105 cfu/mL. The group with buforin II-coated Dacron grafts showed no evidence of staphylococcal infection (<10 cfu/ mL). In contrast, for the rats with ranalexin-, vancomycin- or teicoplanin-coated Dacron grafts the quantitative graft cultures demonstrated bacterial growth (TableGo).


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Table. Efficacy of buforin II, ranalexin, vancomycin and teicoplanin against a glycopeptide-intermediate S. epidermidis strain causing graft infection in a rat model
 
There were significant differences in the results from the quantitative bacterial graft cultures when the data obtained from the antibiotic-treated groups were compared with those obtained from the contaminated control group (P < 0.05), with the exception of the teicoplanin-treated group (P = 0.159). When data obtained from each antibiotic-treated group were compared with those obtained from any other antibiotic-treated group, the differences were always statistically significant for buforin II, while ranalexin produced significant differences only when compared with teicoplanin (P = 0.013). Finally, no statistically significant differences were observed between the two glycopeptides tested (P = 0.414).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experience demonstrates that every new antimicrobial agent introduced into clinical practice is plagued by the emergence of organisms resistant to its effect. The peptide antimicrobials were discovered some time ago. Some of them, such as the gramicidins and the polymyxins, have been used extensively for topical therapy.19 Other peptides, namely the glycopeptides vancomycin and teicoplanin, are used as parenteral antibiotic therapy to treat infections caused by Gram-positive bacteria, especially staphylococcal infections since the emergence of methicillin-resistant staphylococci.15 Recently, vancomycin and teicoplanin have been administered as perioperative antibiotic prophylaxis. In the last decade several reports have described a decrease in vancomycin susceptibility among clinical staphylococcal isolates, accompanied, in most cases, by decreased susceptibility to teicoplanin.15 Glycopeptide antibiotics carry net negative charges situated on their sugars; in contrast, 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.19

Interestingly, the selective antibiotic activity of the cationic peptides is determined by their mode of interaction with the bacterial surface: their positively charged residues interact with the negatively charged lipids of the bacterial membranes. On the other hand, the low anionic lipid content of eukaryotic cells leads to the selectivity of activity of the peptides for bacteria.19–22

The vascular surgery patient's own endogenous flora is the most likely source of S. epidermidis that colonize the graft. This organism has been recovered from the skin, subcutaneous fat, lymph nodes and arterial wall of more than one-third of individuals undergoing vascular reconstruction, in spite of the use of aseptic vascular surgical technique and prophylactic antibiotics.3 The success of surgical prophylaxis in the prevention of graft infections is dependent on the pharmacokinetics of antibiotic tissue penetration with its maintenance of adequate tissue levels for the duration of the vascular surgical procedure. In spite of all precautions, infection of prostheses still occurs. The recent emergence of glycopeptide resistance in CNS heightens concern about the need for other antibiotics to use in prophylactic regimens.13,14,31,32

Buforin II and ranalexin showed similar in vitro activity against the control strain S. epidermidis ATCC 12228 and the GISE clinical isolate, using either the method outlined by the NCCLS or the new method described for the cationic compounds, even if the second method produced lower MICs. The methods differ primarily for two reasons: the different plastic materials utilized and the different definition of the MIC. Therefore, since many cationic peptides bind avidly to the negatively charged surface of several target cells and plastic materials, such as polystyrene microtitre plates, the use of inadequate techniques may underestimate their antimicrobial potency. The broth microdilution method and the disc diffusion method recommended by the NCCLS demonstrated that both the glycopeptides exerted intermediate activity against the GISE strain, although there are still differences between the current NCCLS interpretative standards and the recommendations made to define categories of susceptibility to glycopeptides in some other countries.15

In the time–kill study, buforin II and ranalexin at concentrations of 10 and 50 mg/L killed bacteria much more quickly than the glycopeptides, an observation that could be ascribed to their physical mechanism of action.

Finally, taken together, the results of the in vivo study demonstrated that the use of antimicrobial-soaked Dacron grafts can result in significant bacterial growth inhibition even if high concentrations of organisms are topically inoculated on to the Dacron prostheses. Statistical analysis demonstrated that any antibiotic prophylactic treatment was useful, nevertheless only buforin II was able to inhibit bacterial growth completely. On the other hand, ranalexin was shown to be more effective than the glycopeptides, too, producing a 4 log10 decrease in the number of cfu/mL in comparison with the untreated control group.

Similar to the glycopeptides, the polycationic peptides did not show a noteworthy toxicity when coated on to the graft (data not shown). 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, diarrhoea or behavioural alterations.

The strong in vitro activity and the prophylactic in vivo efficacy demonstrated against a staphylococcal strain with decreased susceptibility to the glycopeptides used in the present study make polycationic peptides potentially useful for future topical antimicrobial treatments, such as perioperative chemoprophylaxis in prosthetic surgery. Further studies are needed to assess fully the efficacy and safety of these compounds.

This study was approved by the Animal Research Ethics Committee of the INRCA IRRCS, University of Ancona, Ancona, Italy.


    Notes
 
* Corresponding address. Clinica delle Malattie Infettive, c/o Azienda Ospedaliera Umberto I, Piazza Cappelli 1, 60121 Ancona, Italy. Tel: +39-071-5963467; Fax: +39-071-5963468; E-mail: cmalinf{at}popcsi.unian.it Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 9 February 2000; returned 30 May 2000; revised 19 June 2000; accepted 1 August 2000