JAC Advance Access originally published online on February 8, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):493-498; doi:10.1093/jac/dkl513
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Antimicrobial activity of the recombinant designer host defence peptide P-novispirin G10 in infected full-thickness wounds of porcine skin
1 Department of Plastic Surgery, Burn Centre, BG University Hospital Bergmannsheil, Ruhr University Bochum, Buerkle-de-la Camp Platz 1, 44789 Bochum, Germany 2 Novozymes A/S, Bagsvaerd, Denmark 3 Department for Microbiology, Ruhr University Bochum, Bochum, Germany
* Corresponding author. Tel: +49-234-302-3442; Fax: +49-234-302-6379; E-mail: lars.steinstraesser{at}ruhr-uni-bochum.de
Received 21 April 2006; returned 17 July 2006; revised 23 October 2006; accepted 8 November 2006
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Abstract |
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Objectives: The growing number of patients with impaired wound healing and the development of multidrug-resistant bacteria demand the investigation of alternatives in wound care. The antimicrobial activity of naturally occurring host defence peptides and their derivatives could be one alternative to the existing therapy options for topical treatment of wound infection. Therefore, the aim of this study was to investigate the antimicrobial activity of proline-novispirin G10 (P-novispirin G10) in vitro and in the infected porcine titanium wound chamber model.
Methods: The new derived designer host defence peptide P-novispirin G10 was tested in vitro against Gram-positive and Gram-negative bacterial strains. Additionally, cytotoxicity and haemolytic activities of P-novispirin G10 and protegrin-1 were measured. For in vivo studies, six wound chambers were implanted on each flank of Göttinger minipigs (n = 2, female, 6 months old, 15–20 kg). Eleven wound chambers were inoculated 8 days post-operatively with 5 x 108 of Staphylococcus aureus; one wound chamber remained uninfected as a system control. After wound infection had been established (4 days after inoculation), each wound chamber was topically treated with P-novispirin G10, protegrin-1 or carrier control. Wound fluid was harvested every hour for a total follow up of 3 h.
Results: P-novispirin G10 demonstrated broad-spectrum antimicrobial activity with moderate haemolytic and cytotoxic activities compared with protegrin-1. In the infected wound chamber model P-novispirin G10 demonstrated a 4 log10 reduction in bacterial counts.
Conclusions: This implicates the potential of P-novispirin G10 as an alternative in future antimicrobial wound care. However, more studies are necessary to further define clinical applications and potential side effects in greater detail.
Keywords: antimicrobial peptides , innate immunity , wound infection , animal model
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Introduction |
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Skin wound infection is of increasing concern in all parts of clinical wound management. The risk of wound infection increases as disturbances in local conditions favour bacterial growth rather than host defence. This can lead to impaired wound healing, resulting on the one hand in rising treatment costs and on the other hand in a traumatic and potentially life-threatening condition for the patient.1 Today's standard to defeat wound infection is still antibiotic therapy. However, the benefit of systemically delivered antibiotics to advance wound healing is still unclear,2 although the expansion of multidrug-resistant microbes is a growing threat. Therefore it is necessary to strike a new path to find alternatives for future wound care.
The use of host defence peptides as alternative treatments to defeat wound infections is an expanding field, which has shown its potential in both pre-clinical and clinical trials.3,4 A structurally diverse group of host defence peptides are the cathelicidins. Cathelicidins are produced as precursors and require proteolytic processing to liberate the mature antimicrobial peptide.5 The highly amphipathic ovispirin-1 constitutes the N-terminal 18 amino acids of the ovine cathelicidin SMAP-29, which is an 
-helical peptide of 29 amino acids in length.6 Both peptides show potent antimicrobial activity,7 but are also cytotoxic and haemolytic against human red blood cells. The backbone of ovispirin-1 was used to create modified, synthetically-derived peptides, which were called novispirins. The main goal of this series of peptides was to reduce the haemolytic activity while conserving the broad antimicrobial potential.8 The proline-novispirin G10 (P-novispirin G10) peptide described in this report is a variant of ovispirin-1 that displayed a more favourable therapeutic index in vitro.
To acquire a better understanding of wound healing and to improve potential antimicrobial substances, a new porcine wound-infection model was established,9 which was used and modified in the presented study.
The aim was to compare the recombinantly produced designer host defence peptide P-novispirin G10 in vitro and in the infected porcine titanium wound chamber model.9
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Cloning, expression and purification of P-novispirin G10
P-novispirin G10 (PKNLRRIIRKGIHIIKKYG) was produced using standard protocols as described elsewhere.10
Protein synthesis was initiated by 1 mM IPTG (isopropyl ß-D-thiogalactopyranoside). Upon 3 h of induction, bacteria were harvested, resuspended in 1/10 volume buffer A (50 mM Tris–HCl, 1 mM EDTA, 100 mM NaCl, pH 8) and lysed by pressure disruption (1500 bar). P-novispirin G10 is located in inclusion bodies that were isolated by centrifugation (24 000 g, 2 h). Subsequently the resulting inclusion body pellet was washed twice in buffer B (50 mM Tris–HCl, 10 mM EDTA, 0.5% Triton X-100, 100 mM NaCl, pH 8). All standard protocols have been described elsewhere.10
For hot acid hydrolysis inclusion bodies were resuspended in 100 mM sodium phosphate (pH 2.3) and incubated overnight at 85°C. The sample was neutralized by adding 100 mM sodium phosphate (pH 12.3) and adjusted to pH 6 and 7 mS/cm prior to application to a CM-sephadex column (Amersham Biosciences). P-novispirin G10 was eluted with a 0–1 M NaCl gradient in 50 mM malonic acid, pH 6, over 10 column volumes. The fractions were analysed by MALDI TOF mass spectrometric analysis for P-novispirin identification and SDS–PAGE (Invitrogen NuPAGE, 12% Bis-Tris/MES) for high molecular weight impurity detection (Figure 1). The selected fractions were pooled and desalted on a Sephadex-G10 column (Amersham Biosciences) equilibrated with PBS pH 7.4 (50 mM phosphate buffer, 150 mM NaCl). Again the fractions were analysed by MALDI TOF mass spectrometric analysis and pooled. The P-novispirin G10 pool was analysed for purity by SDS–PAGE and RP-HPLC (Jupiter 5u C18 300 Å 150 x 2 mm, 50 min gradient from 8 to 80% acetonitrile in 0.1% triflouric acid, flow 0.15 mL/min, 
: 214 nm). The identity of the peptide was determined by automated N-terminal sequencing using a Procise automatic sequencer (Applied Biosystems Division, Perkin Elmer) and the concentration was determined by amino acid analysis performed on a Biochrom 20 Plus (Biochrom Ltd).
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Protegrin-1
The host defence peptide protegrin-1, which is naturally isolated from porcine leucocytes,11 was described by earlier publications to be efficient against bacteria strains in vitro and in vivo.12,13 Dr Peter Henklein produced protegrin-1 (molecular weight 2.0 kDa) by automated peptide synthesis (Charité, Berlin, Germany).
All bacteria were stored as a glycerol stock at –80°C. The following strains were used in this study. Gram-negative strains: Acinetobacter baumannii (ATCC 19606), Proteus mirabilis (ATCC 29906), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Gram-positive strains: C-MRSA (clinical isolate of a methicillin-resistant Staphylococcus aureus, which was susceptible to vancomycin; kindly provided by Professor Gatermann, Clinical Microbiology, Ruhr-Universität Bochum), S. aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228) and Enterococcus faecalis (ATCC 29212).
Bacterial growth inhibition assay
A modified CLSI (formerly NCCLS) standardized antimicrobial susceptibility test was performed as described previously.12,14,15 This method was used to monitor growth inhibition and to define the minimal effective concentration (MEC) for each bacterial strain. MEC was defined as the lowest concentration (mg/L) of the drug that represented a clear zone of growth inhibition. The results were compared with those obtained for the naturally occurring porcine antimicrobial peptide protegrin-112,17 and commonly used antibiotics. Therefore, imipenem (Merck, Darmstadt, Germany) was utilized against P. aeruginosa. Ampicillin (Roth, Karlsruhe, Germany) was used against P. mirabilis, E. coli, A. baumannii and S. aureus. E. faecalis, S. epidermidis and C-MRSA were treated with vancomycin (Merck, Darmstadt, Germany).
The assay was prepared as described previously.14,18 Human blood erythrocytes were incubated with different peptide concentrations. After 30 min at 37°C the tubes were centrifuged and the optical density (OD) was measured at 540 nm and the haemolytic activity was calculated. All tests were performed in triplicate.
Cell culture and cytotoxic activity
The protocols used to prepare and culture primary human keratinocytes and to measure the cytotoxic effect of P-novispirin G10 against these cells have been described in a detailed fashion previously.14
The research protocol described below complied with all regulations related to animal use and other federal statutes. It was conducted in compliance with the principles in the ‘Guide for the Care and Use of Laboratory Animals’ from the German Animal Welfare Act. The animals were housed at the animal facility of the ‘Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin’ BGFA, Ruhr-University, Bochum. Göttinger minipigs (n = 2, female, 6 months old, 15–20 kg weight; Fa. Ellegaard, Dalmose, Denmark) were chosen for the wound chamber models. The pigs were kept for at least 2 weeks in the stable, fed a standard porcine diet and housed at 20–30°C in an atmosphere of 
65% humidity with a light cycle of 12 h on and 12 h off. The wound chambers (BO-chamber®, WiMed, Bochum, Germany) are made of one piece of titanium and have a screw titanium top cover. The wound chambers have a height of 14 mm, an inner diameter of 25 mm and an outer diameter of 32 mm without and 50 mm including the base plate. The infliction of full-thickness skin wounds and the insertion of 12 titanium wound chambers was performed as described previously.9 Dressings were changed every second day under ketamine (10 mg/kg)/midazolam (1 mg/kg)-sedation and the wound fluid was collected to determine bacterial contamination. Afterwards, the wounds were cleaned (removal of blood clots and purulence) and dressed again.
On day 8, all wound chambers were inoculated with 5 x 108 cfu of S. aureus, apart from one that remained bacteria-free. Wound fluid was further collected every second day to analyse the amount of bacterial counts/mL.
The 3 h time-course started 4 days after inoculation of bacteria when a stable wound infection had been established. Before starting, wound fluid was collected and each wound chamber was cleaned as described above.
The wound chambers were randomized except for one that remained bacteria-free and one that received no treatment. The following treatments were applied: 1 mL of P-novispirin G10 (1, 10, 100 and 1000 µg/mL), protegrin 1 (100 µg/mL) or 0.01% acetic acid containing 0.1% BSA (carrier control). Fifty microlitres was harvested from each wound chamber to determine the bacterial concentration at t0. Wound fluid was further taken every hour with a total follow up of 3 h. All samples were cooled down and stored at 4°C until further processing. The amount of bacteria (cfu) per mL of wound fluid was determined by plating serial dilutions of each sample on Mueller–Hinton agar with 5% sheep blood (Becton Dickinson, Heidelberg, Germany). After finishing the experiment, the animals were euthanized by an intravenous injection of T61® (Bayer, Leverkusen, Germany) at a dose of 1 mL/5 kg of body weight.
Data were analysed by analysis of variance (ANOVA) and independent sample t-test when the data had a normal distribution (SPSS, Chicago, USA). Results were considered to be significant at P < 0.05. All values are means ± SEM.
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Release, purification and characterization
The identity of purified P-novispirin G10 was confirmed by automatic Edman degradation and MALDI TOF mass spectrometric analysis. The determined amino acid sequence was identical with the theoretical sequence and the measured mono isotopic mass matched within 0.01%. The purity was analysed by RP-HPLC to be higher than 90% at = 214 nm. No significant impurities were detected by SDS–PAGE analysis or MALDI TOF mass spectrometric analysis.
Haemolytic and cytotoxic properties
P-novispirin G10 showed low haemolytic activity. Only 33% of human erythrocytes were lysed at concentrations of 2.5 mg/mL hence a value for the EC50 could not be determined. In contrast, haemolytic activity for protegrin-1 revealed an EC50 of 25 µg/mL (Figure 2a).
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P-novispirin G10 showed reduced cytotoxicity at peptide amounts lower than 100 µg/mL compared with protegrin-1, which was strongly cytotoxic even at the lowest peptide concentrations. The correlation between the amount of peptide used and the ratio of proliferating cells showed a typical, sigmoid progression for each peptide after 12 h of incubation (Figure 2b).
P-novispirin G10 showed potent activity against all tested bacterial strains. For all strains except C-MRSA and S. epidermidis, P-novispirin G10 was significantly (P < 0.05) more effective than the corresponding antibiotics. With the use of P-novispirin G10 MECs of 11.4 µg/mL for C-MRSA, 4.6 µg/mL for S. aureus, 5.1 µg/mL for S. epidermidis and 10 µg/mL for E. faecalis were found (Figure 3a). MECs of 1.6 µg/mL for E. coli, 3.6 µg/mL for P. aeruginosa, 4.6 µg/mL for A. baumannii and 11.3 µg/mL for P. mirabilis were detected for Gram-negative bacteria (Figure 3b). C-MRSA, E. faecalis, S. aureus and P. mirabilis were significantly (P < 0.05) more susceptible to protegrin-1 compared with P-novispirin G10. The following MECs were determined for the corresponding antibiotics: 2 µg/mL for C-MRSA (vancomycin), 10.5 µg/mL for S. aureus (ampicillin), 4.5 µg/mL for S. epidermidis (vancomycin), 28.9 µg/mL for E. faecalis (vancomycin), 19 µg/mL for E. coli (ampicillin), 7.7 µg/mL for P. aeruginosa (imipenem), 11.8 µg/mL for A. baumannii (ampicillin) and 27.6 µg/mL for P. mirabilis (ampicillin).
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In vivo activity of P-novispirin G10
The titanium wound chambers healed in without any sign of foreign body reaction. The wound bed contained proliferated granulation tissue shown by punch biopsies taken from the middle of each wound chamber at the end of the trial (data not shown). A stable infection could be established 48 h post-inoculation and during the whole experiment. Non-infected wounds showed no signs of bacterial cross-contamination. The activity of P-novispirin G10 was analysed within wound chambers infected with S. aureus. Four different concentrations (1 mg/mL, and 100, 10 and 1 µg/mL) were used for P-novispirin G10 and were compared with protegrin-1 (at a concentration of 100 µg/mL) and carrier control. Each tested peptide concentration showed a decrease in bacterial load in measured wound fluid. At 1 mg/mL P-novispirin G10 the bacterial counts decreased rapidly within the first hour and remained stable at background level (P < 0.05). Overall a reduction in bacterial counts of more than 99.99% could be determined for 1 mg/mL P-novispirin G10 after 3 h. No apparent dose dependency was observed for the lower concentrations of P-novispirin G10, but within all cases more than 90% of the bacterial amount measured for t0 was killed. The carrier control showed no reduction in bacterial load (Figure 4).
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Discussion |
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The emergence of virulent, antibiotic-resistant strains of bacteria has created a pressing need for alternative therapies for infection control.18 In light of this, the field of host defence peptide research is promising.
It appears that the array of host defence peptides in the innate immune response plays an important role in the protective barrier function of the epithelia. For example, expression of host defence peptides was strongly induced in keratinocytes on contact with bacteria or pro-inflammatory cytokines.19 In localized insults, such as thermal injury, it has been demonstrated that the confined expression of host defence peptides is reduced, presumably rendering the site of injury more vulnerable to bacterial colonization.20,21 Many of these peptides demonstrate broad-spectrum antimicrobial activity, eliminating Gram-positive and Gram-negative bacteria, fungi and enveloped viruses.22,23 In addition, they achieve bacterial killing much more rapidly than any commercially available antibiotic.12 Recently, a new family of synthetic, -helical host defence peptides called ovispirins was described.24–26 Although some of these peptides largely retained the antimicrobial activity of naturally occurring peptides, they manifested appreciable cytotoxicity.
Novispirin G10 was as effective in the infected burn model as protegrin-1,27 a naturally occurring host defence peptide with superior antimicrobial properties but greater cytotoxicity. Our in vitro studies were largely carried out using a quantitative and highly sensitive two-layer radial diffusion assay. In these studies, P-novispirin G10 was less active against Gram-positive bacteria, but showed similar activity against the tested Gram-negative strains compared with protegrin-1.
In studies examining the cytotoxic and haemolytic activities of the peptides, P-novispirin G10 demonstrated minimal damage to host cells, in contrast to protegrin-1, which exhibited considerable cytotoxic effects. However, the highest concentration of P-novispirin G10 used in vivo was about the same concentration that started demonstrating cytotoxicity within the in vitro assay. Moreover, higher concentrations of host defence peptides are required in vivo to attain the growth-inhibitory concentrations in the more aggressive wound milieu full of proteases. Recent studies with other naturally occurring peptides such as defensins (HRP and NP-2) and pig host defence peptides from leucocytes (PR-39, prophenin-PF-2 and protegrin PG-2) have also recently demonstrated time- and concentration-dependent cytotoxicity.17,28 P-novispirin G10 is devoid of disulphide bridges or unusual residues, which allows facile and less costly chemical synthesis. Finally, the rapidity of killing by P-novispirin G10 should provide an advantage for topical applications, allowing bacterial killing before the peptide is cleared or otherwise inactivated, e.g. by local proteases.
To evaluate the effectiveness of P-novispirin G10 in vivo, we used the porcine infected wound chamber model using an S. aureus (ATCC 25923) strain. P-novispirin G10 showed high activity against wound-infecting bacteria. In all cases bacterial counts were reduced compared with carrier control within the first 3 h. Our study revealed a rapid decrease in bacterial counts of up to 4 log10 after topical application of P-novispirin G10.
Wound chambers that remained untreated or bacteria-free demonstrated that there was no communication between neighbouring wound chambers, which indicates that each wound chamber is a separate wound that can be individually observed.
Overall, these studies demonstrate that P-novispirin G10 was effective in vitro and in vivo against clinically relevant strains. The potency of P-novispirin G10 coupled with its simple structure may also make it a candidate for large-scale industrial production. P-novispirin G10 will also be a candidate for local transient gene delivery into infected wounds to advance wound healing. Such a drug delivery system could allow local production of this powerful host defence peptide within the wound, exactly where it will be beneficial.29 Within this present study we analysed the antimicrobial effect against the soluble bacterial counts of the wound fluid. However, we have not determined the antimicrobial effect of novispirin in the wound tissue. Furthermore, insight into mechanisms of endogenous host defence peptide up-regulation may allow the development of compounds that elicit epithelial defence reactions by stimulating locally increased synthesis of endogenous host defence peptides. Host defence peptides represent promising candidates for further investigation of effective topical therapeutics.
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None to declare.
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Acknowledgements |
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We sincerely thank Janine Mertens-Rill, Andrea Rittig and Susanne Friedrich for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, Ste1099) and by the medical faculty of the Ruhr University Bochum (FoRUM, F321/01).
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References |
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