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Journal of Antimicrobial Chemotherapy 2004 54(4):772-779; doi:10.1093/jac/dkh407
JAC vol.54 no.4 © The British Society for Antimicrobial Chemotherapy 2004; all rights reserved

Pseudomonas aeruginosa-induced infection and degradation of human wound fluid and skin proteins ex vivo are eradicated by a synthetic cationic polymer

M. Werthén1, M. Davoudi2, A. Sonesson2, D. P. Nitsche3, M. Mörgelin3, K. Blom1 and A. Schmidtchen2,*

1 Mölnlycke Health Care AB, SE-40252 Göteborg; 2 Section for Dermatology, Department of Medical Microbiology, Dermatology and Infection, Biomedical Center, B14, Tornavägen 10, SE-22184 Lund; 3 Department of Cell and Molecular Biology, Biomedical Center, Lund University, SE-22184 Lund, Sweden

Received 23 April 2004; returned 19 June 2004; revised 29 June 2004; accepted 19 July 2004


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: Antimicrobial peptides are important effectors of innate immunity. Bacteria display multiple defence mechanisms against these peptides. For example, Pseudomonas aeruginosa releases potent proteinases that inactivate the human cathelicidin LL-37. Hence, in conditions characterized by persistent bacterial colonization, such as in P. aeruginosa-infected skin wounds, there is a need for efficient means of reducing bacterial load. Here, the effect of the cationic molecule polyhexamethylenebiguanide (PHMB) was evaluated.

Methods: Infection models in human wound fluid and human skin were established. Radial diffusion methods, bacterial growth and bactericidal assays were used for determination of effects of PHMB on bacteria in the presence of plasma, wound fluid or human skin. At the protein and tissue levels, SDS–PAGE, light microscopy and scanning electron microscopy were used to study the effects of P. aeruginosa infection before and after addition of PHMB.

Results: PHMB killed common ulcer-derived bacteria in the presence of human wound fluid. Furthermore, elastase-expressing P. aeruginosa completely degraded wound fluid proteins as well as human skin during infection ex vivo. The infection, and consequent protein degradation, was reversed by PHMB.

Conclusions: The ex vivo infection models presented here should be helpful in the screening of novel antimicrobials and constitute a prerequisite for future clinical studies.

Keywords: wound healing , bacteria , proteolysis , antimicrobials , polyhexamethylenebiguanide


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Multicellular organisms express a blend of multiple antimicrobial peptides (AMPs), which are ubiquitously distributed at biological boundaries prone to infection. These peptides, originally described in silk worms,1 occur in animals ranging from insects to mammals.2–5 At present, over 700 different peptide sequences are known (www.bbcm.univ.trieste.it/~tossi/search.htm). AMPs kill bacteria by permeating their membranes, and thus, the lack of a specific molecular microbial target minimizes the development of resistance. The fundamental principle of action of most peptides depends on the ability of these molecules to adopt a shape in which clusters of hydrophobic and cationic amino acids are organized in discrete sectors, creating an amphipathic {alpha}-helical, ß-sheet, extended coil or cyclic structure.6,7 Humans carry nearly 2 kg of microbes in the digestive system and about 200 g on the skin, indicating that various AMPs, such as the cathelicidin LL-37 and various defensins, control numerous microbes with pathogenic potential.8 Thus, peptide-based immune defences have remained an effective weapon of multicellular organisms during evolution and it is a general belief that resistance to AMPs seldom occurs.7,8 During recent years, it has become increasingly clear that microbial resistance to AMPs, or deficiencies amongst AMPs, may underlie significant diseases, such as atopic dermatitis, chronic leg ulcers, enteric infections and periodontitis.9–12 The growing problem of resistance to conventional antibiotics has spawned considerable interest in the development of novel AMPs and several peptides are currently in stage III clinical trials.7 Various strategies have been employed to enhance peptide efficiency, such as the introduction of stereoisomers composed of D-amino acids13 or cyclic D,L-{alpha}-peptides.14 Interestingly, polymeric biguanides represent a class of antibacterial agents that, like classical AMPs, function by disruption of bacterial membranes.15 The functional correspondence between these antibacterials is accompanied by structural similarities. Polymeric biguanides, such as polyhexamethylenebiguanide (PHMB), contain cationic biguanide groups interspersed between hydrophobic hexamethylene groups, a general structure shared with many linear or {alpha}-helical AMPs, such as LL-37 (Figure 1). Thus, PHMB could serve as an alternative to natural AMPs in diseases characterized by degradation and low levels of innate immune peptides, such as in infected skin ulcerations.11,16 Here, employing various experimental models, we show that PHMB completely blocks Pseudomonas aeruginosa-induced infection and degradation of human wound fluid and skin proteins ex vivo.



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  		Figure 1.. Schematic drawing of the antimicrobial peptide LL-37 (a) and the compound polyhexamethylenebiguanide (b). The cationic peptide LL-37 contains clusters of hydrophobic and cationic amino acids (underlined and +) organized in discrete sectors, creating an amphipathic molecule. The synthetic polymeric biguanide has a similar overall organization.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Bacterial cultures and wound fluids

Various P. aeruginosa isolates,11,17–19 were obtained from patients with uninfected chronic venous ulcers. Sterile wound fluid was obtained from surgical drains after mastectomy. Collection was for 24 h after operation. Wound fluids were centrifuged, aliquotted and stored at –20°C. Chronic wound fluid was collected from patients with chronic venous leg ulcers with ulcer duration of more than 3 months. Venous insufficiency was routinely determined either by a handheld Doppler (5 MHz probe) or by colour duplex examination. The patients had a systolic index of >0.8. Patients showing signs of general or local infection, or patients with diabetes or immunological disorders, were excluded. Op-Site dressings (Smith & Nephew, Birmingham, UK) were applied to the wound and wound fluid was collected by gentle aspiration underneath the films after 2 h.20 Wound fluids were centrifuged, aliquotted and stored at –20°C until further use. Human skin was obtained in connection with skin transplant surgery. Informed consent was obtained from the patients. The use of this material was approved by the Ethics Committee at Lund University (LU 509-01, LU 708-01).

Infection of human wound fluid and skin biopsies by P. aeruginosa

For infection of human wound fluids, wound fluid from surgical wounds [1 mL acute wound fluid diluted 1:1 with Todd–Hewitt (TH) medium (Gibco)] was inoculated with 10 µL of overnight culture (in TH medium) of P. aeruginosa17 in the absence or presence of 10, 25, 50 or 100 mg/L PHMB (Cosmocil; Avecia, Manchester, UK). After 18 h, bacteria were pelleted by centrifugation and supernatants gently collected and stored at –20°C. In a separate experiment, 19 wound-derived P. aeruginosa isolates18 were cultured overnight (in TH medium) to an OD of ~0.8. TH medium (200 µL) containing acute wound fluid (50%) was inoculated by addition of the bacteria (2%) to 96-well plates with or without PHMB (200 mg/L). The absorbance was measured at 490 nm after 20 h in a Bio-Rad 550 micro-plate reader. For infection of human skin, 4 mm punch biopsies were made in the human skin samples, and immersed in 24-well plates (Becton Dickinson Biosciences, San Jose, CA, USA) in 400 µL of minimal essential medium (MEM) (Gibco BRL) with or without acute wound fluid (20%). Inoculation was made with 10 µL of overnight cultures of P. aeruginosa with or without the addition of PHMB. After 18 h, the supernatants were gently removed, bacteria were pelleted by centrifugation and supernatants were stored at –20°C. Skin biopsies were extracted by boiling for 10 min in 10% SDS (dissolved in water).17 After brief centrifugation to pellet unsoluble material (5 min in an Eppendorf centrifuge at 10 000 rpm), the supernatant was gently removed and stored at –20°C.

Gelatin zymography

Substrate gel zymography was performed essentially as described previously11 with 1 mg of bovine gelatin per mL of gel. To visualize gelatinases, supernatants obtained from experiments with wound fluid and skin biopsies were mixed with sample buffer (0.4 M Tris–HCl, 20% glycerol, 5% SDS, 0.03% Bromophenol Blue, pH 6.8) and electrophoresed on 10% polyacrylamide gels. To remove SDS, gels were incubated with 2.5% Triton X-100 for 1 h. Incubation was then performed for 18 h at 37°C in buffer containing 50 mM Tris–HCl, 200 mM NaCl, 5 mM CaCl2, 1 mM ZnCl2 (pH 7.5). Gels were stained with Coomassie Blue G-250 in 30% methanol/10% acetic acid for 1 h and destained in the same solution without the dye. Gelatinase-containing bands were visualized as clear bands against a dark background.

SDS–PAGE

For detection of proteins, SDS–PAGE was performed on 10% polyacrylamide gradient gels (Hoefer system; Pharmacia, Sweden). Wound fluid supernatants and extracts from the skin infection experiments were dissolved in 25 µL of 5% (w/v) SDS, 20% (v/v) glycerol, 4 mM EDTA, 0.04% Bromophenol Blue, 125 mM Tris–HCl, pH 6.8. ß-Mercaptoethanol was added to a final concentration of 10% (v/v). Samples were boiled for 3 min and electrophoresed for ~16 h. The gels were developed with Coomassie Blue stain as described above.

Antimicrobial assay

For antimicrobial assays, P. aeruginosa was grown to mid-log phase in TH medium.11 Bacteria were washed and diluted in 10 mM Tris–HCl, pH 7.5, containing 5 mM glucose with or without acute wound fluid (10%). Bacteria (50 µL; 2 x 106 cfu/mL) were incubated with PHMB at concentrations ranging from 0 to 100 mg/L. Incubations were carried out at 37°C for 2 h. To quantify the bactericidal activity, serial dilutions (in 10 mM Tris, pH 7.5, 5 mM glucose) of the incubation mixture were plated onto TH agar, incubated at 37°C overnight, and the number of cfu was determined.

Radial diffusion assay

Radial diffusion assays (RDAs) were performed essentially as described previously.21 Briefly, bacteria were grown to mid-log phase in 10 mL of (3%, w/v) trypticase soy broth (TSB) (Becton-Dickinson, Cockeysville, MD, USA). The bacteria were washed once with 10 mM Tris, pH 7.4, and 4 x 106 cfu was added to 5 mL of the underlay agarose gel, consisting of 0.03% (w/v) TSB, 1% (w/v) low-electroendosmosis type (low-EEO) agarose (Sigma, St Louis, MO, USA) and 0.02% (v/v) Tween 20 (Sigma). The underlay was poured into a 85 mm diameter Petri dish. After agarose solidification, eight 4 mm diameter wells were punched per plate. Three microlitres of LL-37 and PHMB (in 10 mM Tris, pH 7.4), respectively, was dissolved in 3 µL of 10 mM Tris–HCl, pH 7.4, citrate-plasma, acute or chronic wound fluid and added to each well. Plates were incubated at 37°C for 3 h to allow diffusion of antimicrobials. The bacteria-containing underlay was then covered with 5 mL of molten overlay (6% TSB and 1% low-EEO agarose in dH2O).

The antibacterial activity of a substance was visualized as a zone of clearing around each well after 18–24 h of incubation at 37°C.

Histological examination

Human skin biopsies were co-cultivated with P. aeruginosa in microtitre plates with or without the addition of 200 mg/L PHMB. Alternatively, biopsies were pre-infected for 2 h in a suspension of P. aeruginosa in PBS (2 x 109 cfu/mL). Non-bound bacteria were removed by washing three times in PBS prior to cultivation in MEM. Incubation at 37°C, 5% CO2 was carried out for 6 and 16 h, respectively. Samples as well as non-inoculated controls were cultivated with and without the addition of 200 mg/L PHMB. Afterwards biopsies from both treatments were washed three times in PBS and subsequently fixed in PBS containing 4% formaldehyde (18 h, 4°C) and processed for paraffin sectioning.22 Five micrometre sections were successively stained with Mayer's haematoxylin (Histolab AB, Gothenburg, Sweden) and eosin Y (Surgipath Inc., Richmond, VA, USA).

Scanning electron microscopy

For scanning electron microscopy (SEM) human skin biopsies were incubated with P. aeruginosa in the presence or absence of PHMB as described above. Specimens were then fixed overnight at 4°C in PBS containing 4% formalin and 2.5% glutaraldehyde. This was followed by dehydration in an ascending ethanol series (50%, 70%, 96%, 99.5%), 2 x 15 min at each step, and critical point drying in a Balzers critical point dryer, using absolute ethanol as the intermediate solvent. Specimens were mounted on aluminium stubs, palladium/gold-coated and examined in a Jeol J-330 scanning electron microscope.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
In initial studies, bactericidal assays were performed to assess the efficiency of PHMB in 10 mM Tris buffer and in the presence of human acute wound fluid against an elastase-expressing P. aeruginosa isolate, originally derived from a patient with a chronic venous ulcer.17 PHMB killed the bacteria at ≥1 mg/L in 10 mM Tris whereas addition of the acute wound fluid (at 10%) increased the bactericidal concentration of PHMB to ~10 mg/L (not shown). In radial diffusion assays, PHMB was effective in the presence of plasma, acute wound fluid or fluid from chronic ulcers (Figure 2, right panel). The antibacterial activity of the peptide LL-37 was inhibited by both wound fluids (Figure 2, left panel). Next, we performed growth assays in human wound fluid infected by elastase-producing P. aeruginosa. Under these conditions, LL-37 is completely degraded by P. aeruginosa elastase enzyme and thus, exerted no bactericidal activity.11 As demonstrated in Figure 3(a), P. aeruginosa growth was inhibited by PHMB at concentrations of ≥50 mg/L. To determine whether the reduction in bacterial growth was due to bacteriostatic or bactericidal effects, the number of cfu was determined and the extent of bacterial survival (relative to the control) was calculated. No bacteria were detected at PHMB concentrations of ≥50 mg/L indicating that the antibacterial agent exerted a bactericidal effect in the acute wound fluid (Figure 3b). Colony counting also indicated that 25 mg/L PHMB was effective (Figure 3b), and the reduction in cfu was >90%, thus contrasting with the results obtained by absorbance measurements (Figure 3a). This could be due to the high absorbance levels obtained at stationary phase, yielding a non-linear correlation with bacterial density, or an increase in the amounts of non-viable bacteria at 25 mg/L PHMB. Next, we investigated the effects of P. aeruginosa infection on wound fluid proteins. The infected acute wound fluids and corresponding controls were analysed by SDS–PAGE. In comparison with the controls (Figure 3c), P. aeruginosa infection resulted in a pronounced degradation of wound fluid proteins. PHMB at ≥50 mg/L completely abolished the degradation (Figure 3c, left panel), and these findings were in perfect agreement with the results obtained from the growth assays (Figure 3a and b). Enzyme detection by gelatin zymography identified major enzymes in the uninfected wound fluid that migrated at positions corresponding to matrix metalloproteinase (MMP)-9 and -2 (Figure 3c, right panel). During infection, however, P. aeruginosa released a potent metalloproteinase, elastase, previously characterized at the amino acid and gene level.11 Interestingly, no endogenous MMPs were detected in the infected wound fluids, suggesting that these are inactivated (or degraded) by the bacterial proteinase (Figure 3c, right panel). PHMB-mediated eradication of the bacteria normalized the enzyme pattern (Figure 3c, right panel). PHMB did not affect the activity of P. aeruginosa elastase in an azocasein assay (not shown).17



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  		Figure 2.. Effects of PHMB and the antibacterial peptide LL-37 on P. aeruginosa. LL-37 and PHMB were tested in radial diffusion assays using P. aeruginosa in the presence of buffer, plasma, acute wound fluid or chronic wound fluid. The final concentration of the molecules was 25 and 100 µM.

 


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  		Figure 3.. Inoculation of acute wound fluids by P. aeruginosa and the effect of PHMB. (a) Wound fluid was inoculated with 10 µL of overnight cultures of P. aeruginosa in the absence (filled squares) or presence of PHMB (filled triangles, 10 mg/L; open squares, 25 mg/L; open diamonds, 50 mg/L; open circles, 100 mg/L). Wound fluid with PHMB (100 mg/L) (filled diamonds) or with no addition of PHMB (filled circles) were included as controls. Bacterial growth was determined by measurement of absorbance at 490 nm.23 The data points represent mean values (n=3) of an experiment that was performed three times. The standard deviation was <10%. (b) Bacterial numbers were determined after 20 h by plate counting. The inset illustrates that PHMB (rightmost plate, 100 mg/L) exerted a bactericidal effect (control without PHMB is shown to the left). (c) The infected acute wound fluids and controls were analysed by SDS–PAGE (left panel) and gelatin zymography (right panel). Wound fluid without addition of PHMB (0 mg/L) and with 100 mg/L PHMB were included as controls. Increasing amounts of PHMB (0–100 mg/L) was added to the wound fluids infected by P. aeruginosa (P. aer). The position of P. aeruginosa elastase is indicated to the right on the zymogram. Molecular weight markers are indicated on the left (in kDa).

 
Next, we examined whether the antibacterial agent was effective in the presence of human skin. Additionally, it was of interest to study whether PHMB affected skin proteins during incubation. Thus, using a previously established ex vivo infection model,17 4 mm biopsies, cultured in MEM in the absence (Figure 4a) or presence (Figure 4b) of wound fluid (20%), were infected with elastase-producing P. aeruginosa. The number of cfu was determined after 6, 12 and 18 h. Analogously to the previous experiments in wound fluid, PHMB was lethal to the bacteria and no surviving P. aeruginosa was detected at 200 mg/L PHMB. A significant degradation of wound fluid as well as human skin proteins was noted during infection with P. aeruginosa and the degradation was abolished by PHMB (Figure 4c, left panel). The results using gelatin zymography were similar to those obtained with P. aeruginosa-infected wound fluids (Figure 4c, right panel).



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  		Figure 4.. Infection of human skin biopsies and analysis of tissue degradation. (a) Human skin punch biopsies (4 mm) were immersed in 24-well plates in 400 µL of MEM. Inoculation was made with 10 µL of overnight culture of P. aeruginosa with or without the addition of PHMB (filled squares, 0 mg/L; filled circles, 25 mg/L; filled triangles, 50 mg/L; open circles, 100 mg/L; filled diamonds, 200 mg/L). After 6, 12 and 18 h, bacterial cells were determined by plating and colony counting. The inset illustrates the effect of 200 mg/L PHMB (right plate) compared with the control (left plate). (b) A similar experiment to that in (a) but with the addition of 20% acute wound fluid and using a PHMB concentration of 200 mg/L (filled squares, 0 mg/L; filled diamonds, 200 mg/L). (c) In a separate but identical experiment to that in (b), supernatants (medium) and extracts of the skin biopsies (extract) were analysed by SDS–PAGE (10% gels) (left panel). Zymographic analysis was performed on the supernatants (right panel). The PHMB concentration is indicated at the bottom. PHMB at bactericidal concentrations eradicated P. aeruginosa-induced degradation of wound fluid proteins and skin components.

 
As demonstrated by histological analysis of the infection model (Figure 5), P. aeruginosa deeply invades the dermis of skin biopsies within 6 h of incubation (Figure 5a). Progression of infection was observed after 16 h of incubation (Figure 5b). The presence of PHMB (200 mg/L) prevented infection of the skin biopsies (Figure 5c). Control cultures without P. aeruginosa did not show any signs of bacterial infection (Figure 5d) excluding the possibility of infection by skin-derived contaminating bacteria. To test whether PHMB is effective against bacteria that have already adhered to the dermis, skin biopsies were preinfected with P. aeruginosa and treated by addition of PHMB (200 mg/L). Comparison with an untreated sample (Figure 5e) revealed that the extent of infection was significantly decreased when PHMB was added (Figure 5f). Similar observations were made when skin biopsies infected with P. aeruginosa were analysed by SEM (Figure 6). Incubation of biopsies with bacteria led to severe destruction of exposed layers in the dermis, where the collagen network architecture was no longer visible (Figure 6c and e). Upon treatment with PHMB the collagen fibril bundles in the dermis appeared normal (Figure 6d and f) as compared with the control (Figure 6a). PHMB itself apparently did not alter collagen fibril architecture in the tissue (Figure 6b).



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  		Figure 5.. Histological examination of P. aeruginosa-infected skin biopsies and the effect of PHMB. Four-millimetre biopsies of human skin were cultivated in cell culture medium initially inoculated with P. aeruginosa (a–c). In the absence of PHMB (a and b) during the first 6 h (a) the bacteria were able to infect the dermis. Arrowheads indicate deep invasion of single bacteria into the tissue. Infection of the skin samples was demonstrated after 16 h of incubation (b). Biopsies cultivated for 16 h in the presence of PHMB (200 mg/L) showed no signs of bacterial infection (c) and were identical to uninfected controls (d). Pre-incubation with bacteria prior to cultivation in cell culture medium: a massive infection occurred after 16 h in the absence of PHMB (e). The tissue was considerably less infected in the presence of PHMB (f). The bar corresponds to 10 µm.

 


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  		Figure 6.. SEM of human skin biopsies infected by P. aeruginosa. Human skin biopsies were infected with P. aeruginosa in the absence (c and e) or presence (d and f) of PHMB and compared with bacteria-free control samples incubated without (a) or with (b) PHMB. Bacteria were incubated in the presence of PHMB (d), or PHMB was added after a 6 h pre-incubation of the biopsies with bacteria (f). PHMB strongly prevented bacterial infection (d and f), whereas P. aeruginosa infected the biopsies in the absence of PHMB (c and e). PHMB alone does not appear to alter tissue structure (b) as compared with a control specimen (a). The scale bar corresponds to 5 µm.

 
Having shown that PHMB was effective against a previously well-characterized P. aeruginosa isolate, we examined the susceptibility of other wound-derived P. aeruginosa isolates. Hence, wound fluid was inoculated with 19 isolates derived from patients with chronic venous ulcers18 and as demonstrated in Figure 7, all isolates were susceptible to PHMB at ≥200 mg/L.



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  		Figure 7.. Antibacterial effects of PHMB against wound-derived P. aeruginosa isolates. Acute wound fluid (50%) was inoculated with 19 isolates of ulcer-derived P. aeruginosa with or without the addition of PHMB (200 mg/L). The bacterial growth after 20 h of incubation was determined by absorbance measurement at 490 nm. PHMB inhibited the growth of all isolates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
It is becoming increasingly clear, that bacterial evasion of innate immune defences underlies infections at epithelial surfaces. The current work originated from the observation that several metalloproteinases of the thermolysin family, such as P. aeruginosa elastase, Enterococcus faecalis gelatinase and Proteus mirabilis 50 kDa proteinase, as well as the cysteine proteinase of Streptococcus pyogenes, rapidly degrade the human AMP LL-37 and that this degradation results in the loss of LL-37 binding to bacteria and hence, increased bacterial survival.11 The observation that the bacterial metalloproteinases preferably cleaved AMPs at positions adjacent to hydrophobic amino acids combined with the fact that AMPs are composed of ~50% hydrophobic amino acids, suggests that one of many functions of bacterial proteinases is to counteract innate immune peptides.11

Several lines of evidence suggest that cationic polymeric molecules, such as PHMB, are interesting as topical antimicrobials. First, the molecule resembles many AMPs with respect to molecular size, amphipathicity and cationicity (see Figure 1). Secondly, its mode of action on bacterial membranes is similar to that of many AMPs,15 and thus, PHMB is not likely to induce problems with resistant mutants, although PHMB adaptation (yielding higher MIC values) has indeed been described. This tolerance was lost gradually after removal of PHMB.24 Analogously, experiments carried out with AMPs indicate that it is quite difficult (although possible) to isolate mutants with an altered membrane composition. Furthermore, the changes introduced (usually in membrane composition) tend to reduce bacterial viability, thus minimizing the risk of spreading resistant bacteria in nature.8 Thirdly, the results presented herein clearly demonstrate that being proteolysis resistant, PHMB exerts bactericidal effects on various wound-derived P. aeruginosa isolates irrespective of the presence of bacterial proteinase. Additionally, the agent exerts potent effects in the presence of human wound fluid. Furthermore, at the protein level, PHMB did not affect the activity of endogenous MMPs in wound fluid and induced no alteration in the protein patterns of non-infected wound fluids. Furthermore, SEM analysis demonstrated that PHMB did not appear to affect connective tissue components of dermis, such as collagen fibres and elastin. Experiments with preinfected skin biopsies indicated that PHMB was active against bacteria that have adhered to the tissue. This is a prerequisite for an application in wound treatment. It is of note that it was beyond the scope of this work to investigate other factors of importance for PHMB efficiency in vivo, such as the presence of P. aeruginosa biofilms, which may attenuate PHMB effects.25 In this context, it is interesting that PHMB (at 200 mg/L) has been used successfully in the treatment of acanthamoebal keratitis.26 We did not address whether PHMB affects the wound healing process itself. However, preliminary results indicate that epithelial closure in a pig wound healing model is not affected by PHMB at doses of 200 mg/L (not shown).

In conclusion, chronic ulcers are constantly colonized or infected by various bacteria such as P. aeruginosa, Staphylococcus aureus, E. faecalis and P. mirabilis;27,28 and clinical and experimental data support the view that these, and other pathogens, may contribute to the non-healing state of chronic ulcers.28 The ex vivo infection models presented herein should be helpful in the screening of novel antimicrobials and constitute a prerequisite for future clinical studies.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Dr Anders Alvmark and his staff at the Department of Surgery, Landskrona and Dr Katarina Lundqvist for providing the acute and chronic wound fluids, respectively. This work was supported by grants from the Swedish Medical Research Council (projects 13471), the Royal Physiographic Society in Lund, the Welander-Finsen, Crafoord, Alfred Österlund, Groschinsky, Åhlen, Lundgren, Lion and Kock Foundations, and Mölnlycke Health Care AB.


   Footnotes
 
* Corresponding author. Tel: +46-46-2224522; Fax: + 46-46-157756; Email: artur.schmidtchen{at}derm.lu.se


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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11 . Schmidtchen, A., Frick, I. M., Andersson, E. et al. (2002). Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37. Molecular Microbiology 46, 157–68.[CrossRef][Web of Science][Medline]

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16 . Heilborn, J. D., Nilsson, M. F., Kratz, G. et al. (2003). The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium. Journal of Investigative Dermatology 120, 379–89.[CrossRef][Web of Science][Medline]

17 . Schmidtchen, A., Holst, E., Tapper, H. et al. (2003). Elastase-producing Pseudomonas aeruginosa degrade plasma proteins and extracellular products of human skin and fibroblasts, and inhibit fibroblast growth. Microbial Pathogenesis 34, 47–55.[CrossRef][Web of Science][Medline]

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19 . Schmidtchen, A., Frick, I. M. & Björck, L. (2001). Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial {alpha}-defensin. Molecular Microbiology 39, 708–13.[CrossRef][Web of Science][Medline]

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