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
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Abstract |
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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, SDSPAGE, 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
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Introduction |
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Materials and methods |
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Various P. aeruginosa isolates,11,1719 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 ToddHewitt (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 TrisHCl, 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 TrisHCl, 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.
SDSPAGE
For detection of proteins, SDSPAGE 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 TrisHCl, 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 TrisHCl, 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 TrisHCl, 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 1824 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.
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Results |
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Discussion |
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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.
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Acknowledgements |
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Footnotes |
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References |
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2 . Selsted, M. E. & Quellette, A. J. (1995). Defensins in granules of phagocytic and non-phagocytic cells. Trends in Cell Biology 5, 1149.[CrossRef][ISI][Medline]
3 . Schröder, J. M. & Harder, J. (1999). Human ß-defensin-2. International Journal of Biochemistry and Cell Biology 31, 64551.[CrossRef][ISI][Medline]
4 . Lehrer, R. I. & Ganz, T. (1999). Antimicrobial peptides in mammalian and insect host defence. Current Opinion in Immunology 11, 237.[CrossRef][ISI][Medline]
5 . Boman, H. G. (2000). Innate immunity and the normal microflora. Immunological Reviews 173, 516.[CrossRef][ISI][Medline]
6 . Gennaro, R. & Zanetti, M. (2000). Structural features and biological activities of the cathelicidin-derived antimicrobial peptides. Biopolymers 55, 3149.[CrossRef][ISI][Medline]
7 . Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature 415, 38995.[CrossRef][ISI][Medline]
8 . Boman, H. G. (2003). Antibacterial peptides: basic facts and emerging concepts. Journal of Internal Medicine 254, 197215.[CrossRef][ISI][Medline]
9
.
Ong, P. Y., Ohtake, T., Brandt, C. et al. (2002). Endogenous antimicrobial peptides and skin infections in atopic dermatitis. New England Journal of Medicine 347, 115160.
10 . Pütsep, K., Carlsson, G., Boman, H. G. et al. (2002). Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet 360, 11449.[CrossRef][ISI][Medline]
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, 15768.[CrossRef][ISI][Medline]
12 . Islam, D., Bandholtz, L., Nilsson, J. et al. (2001). Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator. Nature Medicine 7, 1805.[CrossRef][ISI][Medline]
13
.
Sajjan, U. S., Tran, L. T., Sole, N. et al. (2001). P-113D, an antimicrobial peptide active against Pseudomonas aeruginosa, retains activity in the presence of sputum from cystic fibrosis patients. Antimicrobial Agents and Chemotherapy 45, 343744.
14
.
Fernandez-Lopez, S., Kim, H. S., Choi, E. C. et al. (2001). Antibacterial agents based on the cyclic D,L--peptide architecture. Nature 412, 4525.[CrossRef][ISI][Medline]
15 . Broxton, P., Woodcock, P. M., Heatley, F. et al. (1984). Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli. Journal of Applied Bacteriology 57, 11524.[ISI][Medline]
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, 37989.
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, 4755.[CrossRef][ISI][Medline]
18 . Schmidtchen, A., Wolff, H. & Hansson, C. (2001). Differential proteinase expression by Pseudomonas aeruginosa derived from chronic leg ulcers. Acta Dermato-Venereologica 81, 4069.[CrossRef][ISI][Medline]
19
.
Schmidtchen, A., Frick, I. M. & Björck, L. (2001). Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial -defensin. Molecular Microbiology 39, 70813.[CrossRef][ISI][Medline]
20
.
Grinnell, F. & Zhu, M. (1996). Fibronectin degradation in chronic wounds depends on the relative levels of elastase, 1-proteinase inhibitor, and
2-macroglobulin. Journal of Investigative Dermatology 106, 33541.[Abstract]
21 . Lehrer, R. I., Rosenman, M., Harwig, S. S. et al. (1991). Ultrasensitive assays for endogenous antimicrobial polypeptides. Journal of Immunological Methods 137, 16773.[CrossRef][ISI][Medline]
22
.
Aszodi, A., Chan, D., Hunziker, E. et al. (1998). Collagen II is essential for the removal of the notochord and the formation of intervertebral discs. Journal of Cell Biology 143, 1399412.
23 . Oren, Z., Lerman, J. C., Gudmundsson, G. H. et al. (1999). Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochemical Journal 341, 50113.[CrossRef][ISI][Medline]
24 . Jones, M. V., Herd, T. M. & Christie, H. J. (1989). Resistance of Pseudomonas aeruginosa to amphoteric and quaternary ammonium biocides. Microbios 58, 4961.[ISI][Medline]
25 . Gilbert, P., Das, J. R., Jones, M. V. et al. (2001). Assessment of resistance towards biocides following the attachment of micro-organisms to, and growth on, surfaces. Journal of Applied Microbiology 91, 24854.[CrossRef][ISI][Medline]
26 . Larkin, D. F., Kilvington, S. & Dart, J. K. (1992). Treatment of Acanthamoeba keratitis with polyhexamethylene biguanide. Ophthalmology 99, 18591.[ISI][Medline]
27 . Hansson, C., Hoborn, J., Möller, A. et al. (1995). The microbial flora in venous leg ulcers without clinical signs of infection. Repeated culture using a validated standardised microbiological technique. Acta Dermato-Venereologica 75, 2430.[ISI][Medline]
28 . Davies, C. E., Wilson, M. J., Hill, K. E. et al. (2001). Use of molecular techniques to study microbial diversity in the skin: chronic wounds reevaluated. Wound Repair and Regeneration 9, 33240.[CrossRef][ISI][Medline]
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