Chronic wounds are a growing socioeconomic problem in the western world. Knowledge on recalcitrant wounds relies on in vitro studies or clinical observations, and there is emerging evidence on the clinical impact of bacterial biofilm on skin healing. Chronic wounds are locked in the inflammatory state of wound healing, and there are multiple explanations for this arrest with the theory of exaggerated proteolysis as the most commonly accepted. Previously, there has not been enough focus on the different etiologies of chronic wounds compared to acute, healing wounds. There is an urgent need to group chronic wounds by its cause when searching for possible diagnostic or therapeutic targets. Good wound management should therefore consist of recognition of basic wound etiology, irrigation, and debridement in order to reduce microbial and necrotic load, frequently changed dressings, and appropriate antimicrobial and antibiofilm strategies based on precise diagnosis. Representative sampling is required for diagnosis and antimicrobial treatment of wounds. The present review aims at describing the impact of biofilm infections on wounds in relation to diagnosing, treatment strategies, including experimentally adjuvant approaches and animal models. 1. Introduction A practical classification of a nonhealing wound is one that fails to heal spontaneously within 3 months [1]. Emergence of chronic wounds is a substantial health problem as 1% of western population will suffer from it. Common chronic types of wounds are venous leg ulcers, ischemic wounds, diabetic foot ulcers, and pressure wounds [2]. Socioeconomically, management of chronic wounds reaches a total cost of 2–4% of the health budget in western countries [3]. This estimate is expected to rise as a natural consequence of an increasing population of the elderly and the diabetic and obesity epidemic. Complications to nonhealing wounds are vast, and patients are at risk of severe pain, septicaemia, hospitalization, and in some cases amputations. Microbiological findings in chronic wounds vary depending on the mode of sampling (swab versus biopsies) and the diagnostic method used (culturing, PCR methods, and microscopy preceded by PNA-FISH). The most common bacteriological findings in human chronic wounds are also present on the skin, in faeces and water: Staphylococcus aureus (SA), coagulase-negative staphylococci, Enterococcus faecalis, Proteus species, anaerobic bacteria, and Pseudomonas aeruginosa (PA) [4]. All the studies of chronic wounds so far agree on the almost universal presence of SA [5–8]. Also, most
References
[1]
A. E. Saltmarche, “Low level laser therapy for healing acute and chronic wounds—the extendicare experience,” International Wound Journal, vol. 5, no. 2, pp. 351–360, 2008.
[2]
M. Bitsch, I. Laursen, A. M. Engel et al., “Epidemiology of chronic wound patients and relation to serum levels of mannan-binding lectin,” Acta Dermato-Venereologica, vol. 89, no. 6, pp. 607–611, 2009.
[3]
F. Gottrup, “Optimizing wound treatment through health care structuring and professional education,” Wound Repair and Regeneration, vol. 12, no. 2, pp. 129–133, 2004.
[4]
K. Gj？dsb？l, J. J. Christensen, T. Karlsmark, B. J？rgensen, B. M. Klein, and K. A. Krogfelt, “Multiple bacterial species reside in chronic wounds: a longitudinal study,” International Wound Journal, vol. 3, no. 3, pp. 225–231, 2006.
[5]
T. R. Thomsen, M. S. Aasholm, V. B. Rudkj？bing et al., “The bacteriology of chronic venous leg ulcer examined by culture-independent molecular methods,” Wound Repair and Regeneration, vol. 18, no. 1, pp. 38–49, 2010.
[6]
G. A. James, E. Swogger, R. Wolcott et al., “Biofilms in chronic wounds,” Wound Repair and Regeneration, vol. 16, no. 1, pp. 37–44, 2008.
[7]
S. E. Dowd, Y. Sun, P. R. Secor et al., “Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing,” BMC Microbiology, vol. 8, article 43, 2008.
[8]
R. D. Wolcott, V. Gontcharova, Y. Sun, and S. E. Dowd, “Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and Titanium amplicon pyrosequencing and metagenomic approaches,” BMC Microbiology, vol. 9, article 226, 2009.
[9]
K. Kirketerp-M？ller, P. ？. Jensen, M. Fazli et al., “Distribution, organization, and ecology of bacteria in chronic wounds,” Journal of Clinical Microbiology, vol. 46, no. 8, pp. 2717–2722, 2008.
[10]
M. Fazli, T. Bjarnsholt, K. Kirketerp-M？ller et al., “Nonrandom distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds,” Journal of Clinical Microbiology, vol. 47, no. 12, pp. 4084–4089, 2009.
[11]
S. C. Davis, C. Ricotti, A. Cazzaniga, E. Welsh, W. H. Eaglstein, and P. M. Mertz, “Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo,” Wound Repair and Regeneration, vol. 16, no. 1, pp. 23–29, 2008.
[12]
T. Bjarnsholt, K. Kirketerp-M？ller, P. ？. Jensen et al., “Why chronic wounds will not heal: a novel hypothesis,” Wound Repair and Regeneration, vol. 16, no. 1, pp. 2–10, 2008.
[13]
W. K. Stadelmann, A. G. Digenis, and G. R. Tobin, “Physiology and healing dynamics of chronic cutaneous wounds,” American Journal of Surgery, vol. 176, no. 2A, supplement, pp. 26S–38S, 1998.
[14]
S. L. Drinkwater, A. Smith, and K. G. Burnand, “What can wound fluids tell us about the venous ulcer microenvironment?” The International Journal of Lower Extremity Wounds, vol. 1, no. 3, pp. 184–190, 2002.
[15]
A. N. Moor, D. J. Vachon, and L. J. Gould, “Proteolytic activity in wound fluids and tissues derived from chronic venous leg ulcers,” Wound Repair and Regeneration, vol. 17, no. 6, pp. 832–839, 2009.
[16]
N. J. Trengove, M. C. Stacey, S. Macauley et al., “Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors,” Wound Repair and Regeneration, vol. 7, no. 6, pp. 442–452, 1999.
[17]
M. Wlaschek and K. Scharffetter-Kochanek, “Oxidative stress in chronic venous leg ulcers,” Wound Repair and Regeneration, vol. 13, no. 5, pp. 452–461, 2005.
[18]
S. G. Jones, R. Edwards, and D. W. Thomas, “Inflammation and wound healing: the role of bacteria in the immuno-regulation of wound healing,” The International Journal of Lower Extremity Wounds, vol. 3, no. 4, pp. 201–208, 2004.
[19]
T. Bjarnsholt, T. Tolker-Nielsen, M. Givskov, M. Janssen, and L. H. Christensen, “Detection of bacteria by fluorescence in situ hybridization in culture-negative soft tissue filler lesions,” Dermatologic Surgery, vol. 35, no. 2, supplement, pp. 1620–1623, 2009.
[20]
S. Fexby, T. Bjarnsholt, P. ？. Jensen et al., “Biological trojan horse: antigen 43 provides specific bacterial uptake and survival in human neutrophils,” Infection and Immunity, vol. 75, no. 1, pp. 30–34, 2007.
[21]
A. Filloux and I. Vallet, “Biofilm: positioning and organisation of bacterial communities,” Medecine/Sciences, vol. 19, no. 1, pp. 77–83, 2003.
[22]
L. Yang, L. Jelsbak, R. L. Marvig et al., “Evolutionary dynamics of bacteria in a human host environment,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 18, pp. 7481–7486, 2011.
[23]
L. Hall-Stoodley, J. W. Costerton, and P. Stoodley, “Bacterial biofilms: from the natural environment to infectious diseases,” Nature Reviews Microbiology, vol. 2, no. 2, pp. 95–108, 2004.
[24]
W. Costerton, R. Veeh, M. Shirtliff, M. Pasmore, C. Post, and G. Ehrlich, “The application of biofilm science to the study and control of chronic bacterial infections,” Journal of Clinical Investigation, vol. 112, no. 10, pp. 1466–1477, 2003.
[25]
M. Alhede, K. N. Kragh, K. Qvortrup, et al., “Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm,” PloS One, vol. 6, no. 11, Article ID e27943, 2011.
[26]
M. Burm？lle, T. R. Thomsen, M. Fazli et al., “Biofilms in chronic infections—a matter of opportunity—monospecies biofilms in multispecies infections,” FEMS Immunology and Medical Microbiology, vol. 59, no. 3, pp. 324–336, 2010.
[27]
T. H？gsberg, T. Bjarnsholt, J. S. Thomsen, and K. Kirketerp-M？ller, “Success rate of split-thickness skin grafting of chronic venous leg ulcers depends on the presence of Pseudomonas aeruginosa: a retrospective study,” PLoS ONE, vol. 6, no. 5, Article ID e20492, 2011.
[28]
G. Zhao, P. C. Hochwalt, M. L. Usui et al., “Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: a model for the study of chronic wounds,” Wound Repair and Regeneration, vol. 18, no. 5, pp. 467–477, 2010.
[29]
A. K. Seth, M. R. Geringer, A. N. Gurjala, et al., “Treatment of Pseudomonas aeruginosa biofilm-infected wounds with clinical wound care strategies: a quantitative study using an in vivo rabbit ear model,” Plastic and Reconstructive Surgery, vol. 129, no. 2, pp. 262e–274e, 2012.
[30]
T. C. R. Conibear, S. L. Collins, and J. S. Webb, “Role of mutation in Pseudomonas aeruginosa biofilm development,” PLoS ONE, vol. 4, no. 7, Article ID e6289, 2009.
[31]
K. Lewis, “Persister cells,” Annual Review of Microbiology, vol. 64, pp. 357–372, 2010.
[32]
J. W. Costerton, Z. Lewandowski, D. E. Caldwell, D. R. Korber, and H. M. Lappin-Scott, “Microbial biofilms,” Annual Review of Microbiology, vol. 49, pp. 711–745, 1995.
[33]
M. L. Fernandez, J. A. Broadbent, G. K. Shooter, J. Malda, and Z. Upton, “Development of an enhanced proteomic method to detect prognostic and diagnostic markers of healing in chronic wound fluid,” British Journal of Dermatology, vol. 158, no. 2, pp. 281–290, 2008.
[34]
A. B. Wysocki, “Wound fluids and the pathogenesis of chronic wounds,” Journal of Wound, Ostomy and Continence Nursing, vol. 23, no. 6, pp. 283–290, 1996.
[35]
D. R. Yager and B. C. Nwomeh, “The proteolytic environment of chronic wounds,” Wound Repair and Regeneration, vol. 7, no. 6, pp. 433–441, 1999.
[36]
F. Grinnell and M. Zhu, “Fibronectin degradation in chronic wounds depends on the relative levels of elastase, α1-proteinase inhibitor, and α2-macroglobulin,” Journal of Investigative Dermatology, vol. 106, no. 2, pp. 335–341, 1996.
[37]
V. Falanga, “Growth factors and chronic wounds: the need to understand the microenvironment,” Journal of Dermatology, vol. 19, no. 11, pp. 667–672, 1992.
[38]
H. Tr？strup, R. Lundquist, L. H. Christensen et al., “S100A8/A9 deficiency in nonhealing venous leg ulcers uncovered by multiplexed antibody microarray profiling,” British Journal of Dermatology, vol. 165, no. 2, pp. 292–301, 2011.
[39]
I. S. Thorey, J. Roth, J. Regenbogen et al., “The Ca2+-binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes,” Journal of Biological Chemistry, vol. 276, no. 38, pp. 35818–35825, 2001.
[40]
R. Zillmer, H. Tr？strup, T. Karlsmark, P. Ifversen, and M. S. Agren, “Duration of wound fluid secretion from chronic venous leg ulcers is critical for interleukin-1alpha, interleukin-1beta, interleukin-8 levels and fibroblast activation,” Archives of Dermatological Research, vol. 303, no. 8, pp. 601–606, 2011.
[41]
B. Bucalo, W. H. Eaglstein, and V. Falanga, “Inhibition of cell proliferation by chronic wound fluid,” Wound Repair and Regeneration, vol. 1, no. 3, pp. 181–186, 1993.
[42]
P. Svedman, “Irrigation treatment of leg ulcers,” The Lancet, vol. 2, no. 8349, pp. 532–534, 1983.
[43]
G. S. Schultz, R. G. Sibbald, V. Falanga et al., “Wound bed preparation: a systematic approach to wound management,” Wound Repair and Regeneration, vol. 11, supplement 1, pp. S1–S28, 2003.
[44]
S. J. Weiss, “Tissue destruction by neutrophils,” New England Journal of Medicine, vol. 320, no. 6, pp. 365–376, 1989.
[45]
L. E. Edsberg, J. T. Wyffels, M. S. Brogan, and K. M. Fries, “Analysis of the proteomic profile of chronic pressure ulcers,” Wound Repair and Regeneration, vol. 20, no. 3, pp. 378–401, 2012.
[46]
F. Werdin, M. Tennenhaus, H. E. Schaller, and H. O. Rennekampff, “Evidence-based management strategies for treatment of chronic wounds,” Eplasty, vol. 9, article e19, 2009.
[47]
F. E. Brolmann, D. T. Ubbink, E. A. Nelson, K. Munte, C. M. van der Horst, and H. Vermeulen, “Evidence-based decisions for local and systemic wound care,” The British Journal of Surgery, vol. 99, no. 9, pp. 1172–1183, 2012.
[48]
F. Gottrup and J. Apelqvist, “The challenge of using randomized trials in wound healing,” British Journal of Surgery, vol. 97, no. 3, pp. 303–304, 2010.
[49]
J. E. Jones and E. A. Nelson, “Skin grafting for venous leg ulcers,” Cochrane Database of Systematic Reviews, no. 2, Article ID CD001737, 2007.
[50]
W. H. Eaglstein and V. Falanga, “Tissue engineering and the development of Apligraf, a human skin equivalent,” Cutis; Cutaneous Medicine for the Practitioner, vol. 62, no. 1, supplement, pp. 1–8, 1998.
[51]
M. S. Gohel, J. R. Barwell, M. Taylor et al., “Long term results of compression therapy alone versus compression plus surgery in chronic venous ulceration (ESCHAR): randomised controlled trial,” British Medical Journal, vol. 335, no. 7610, pp. 83–87, 2007.
[52]
P. Kranke, M. Bennett, I. Roeckl-Wiedmann, and S. Debus, “Hyperbaric oxygen therapy for chronic wounds,” Cochrane Database of Systematic Reviews, no. 2, Article ID CD004123, 2004.
[53]
M. Cruciani, B. A. Lipsky, C. Mengoli, and F. de Lalla, “Granulocyte-colony stimulating factors as adjunctive therapy for diabetic foot infections,” Cochrane Database of Systematic Reviews, no. 3, Article ID CD006810, 2009.
[54]
F. Gottrup and B. Jorgensen, “Maggot debridement: an alternative method for debridement,” Eplasty, vol. 11, article e33, 2011.
[55]
L. Gilead, K. Y. Mumcuoglu, and A. Ingber, “The use of maggot debridement therapy in the treatment of chronic wounds in hospitalised and ambulatory patients,” Journal of Wound Care, vol. 21, no. 2, pp. 78, 80, 82–85, 2012.
[56]
F. Gottrup and T. Karlsmark, “Current management of wound healing,” Giornale Italiano di Dermatologia e Venereologia, vol. 144, no. 3, pp. 217–228, 2009.
[57]
S. J. Palfreyman, E. A. Nelson, R. Lochiel, and J. A. Michaels, “Dressings for healing venous leg ulcers,” Cochrane Database of Systematic Reviews, vol. 3, Article ID CD001103, 2006.
[58]
H. Vermeulen, J. M. van Hattem, M. N. Storm-Versloot, and D. T. Ubbink, “Topical silver for treating infected wounds,” Cochrane Database of Systematic Reviews, no. 1, Article ID CD005486, 2007.
[59]
J. L. Richard, C. Parer-Richard, J. P. Daures et al., “Effect of topical basic fibroblast growth factor on the healing of chronic diabetic neuropathic ulcer of the foot: a pilot, randomized, double-blind, placebo-controlled study,” Diabetes Care, vol. 18, no. 1, pp. 64–69, 1995.
[60]
V. Falanga, W. H. Eaglstein, B. Bucalo, M. H. Katz, B. Harris, and P. Carson, “Topical use of human recombinant epidermal growth factor (h-EGF) in venous ulcers,” Journal of Dermatologic Surgery and Oncology, vol. 18, no. 7, pp. 604–606, 1992.
[61]
S. M. Chen, S. I. Ward, O. O. Olutoye, R. F. Diegelmann, and I. K. Cohen, “Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors,” Wound Repair and Regeneration, vol. 5, no. 1, pp. 23–32, 1997.
[62]
M. C. Robson, “The role of growth factors in the healing of chronic wounds,” Wound Repair and Regeneration, vol. 5, no. 1, pp. 12–17, 1997.
[63]
P. A. M. Everts, J. T. A. Knape, G. Weibrich et al., “Platelet-rich plasma and platelet gel: a review,” Journal of Extra-Corporeal Technology, vol. 38, no. 2, pp. 174–187, 2006.
[64]
K. M. Lacci and A. Dardik, “Platelet-rich plasma: support for its use in wound healing,” Yale Journal of Biology and Medicine, vol. 83, no. 1, pp. 1–9, 2010.
[65]
T. M. Bielecki, T. S. Gazdzik, J. Arendt, T. Szczepanski, W. Król, and T. Wielkoszynski, “Antibacterial effect of autologous platelet gel enriched with growth factors and other active substances: an in vitro study,” Journal of Bone and Joint Surgery B, vol. 89, no. 3, pp. 417–420, 2007.
[66]
D. J. Margolis, T. Crombleholme, and M. Herlyn, “Clinical protocol: phase I trial to evaluate the safety of H5.020CMV.PDGF-B for the treatment of a diabetic insensate foot ulcer,” Wound Repair and Regeneration, vol. 8, no. 6, pp. 480–493, 2000.
[67]
P. J. Kim and J. S. Steinberg, “Wound care: biofilm and its impact on the latest treatment modalities for ulcerations of the diabetic foot,” Seminars in Vascular Surgery, vol. 25, no. 2, pp. 70–74, 2012.
[68]
B. Jorgensen, T. Karlsmark, H. Vogensen, L. Haase, and R. Lundquist, “A pilot study to evaluate the safety and clinical performance of Leucopatch, an autologous, additive-free, platelet-rich fibrin for the treatment of recalcitrant chronic wounds,” The International Journal of Lower Extremity Wounds, vol. 10, no. 4, pp. 218–223, 2011.
[69]
S. M. O'Connell, T. Impeduglia, K. Hessler, X. J. Wang, R. J. Carroll, and H. Dardik, “Autologous platelet-rich fibrin matrix as cell therapy in the healing of chronic lower-extremity ulcers,” Wound Repair and Regeneration, vol. 16, no. 6, pp. 749–756, 2008.
[70]
W. C. Krupski, L. M. Reilly, S. Perez, K. M. Moss, P. A. Crombleholme, and J. H. Rapp, “A prospective randomized trial of autologous platelet-derived wound healing factors for treatment of chronic nonhealing wounds: a preliminary report,” Journal of Vascular Surgery, vol. 14, no. 4, pp. 526–536, 1991.
[71]
J. Cha and V. Falanga, “Stem cells in cutaneous wound healing,” Clinics in Dermatology, vol. 25, no. 1, pp. 73–78, 2007.
[72]
D. M. Castilla, Z. J. Liu, R. Tian, Y. Li, A. S. Livingstone, and O. C. Velazquez, “A novel autologous cell-based therapy to promote diabetic wound healing,” Annals of Surgery, vol. 256, no. 4, pp. 560–572, 2012.
[73]
N. M. Argolo Neto, R. J. Del Carlo, B. S. Monteiro, et al., “Role of autologous mesenchymal stem cells associated with platelet-rich plasma on healing of cutaneous wounds in diabetic mice,” Clinical and Experimental Dermatology, vol. 37, no. 5, pp. 544–553, 2012.
[74]
A. Nather, S. B. Chionh, A. Y. Y. Han, P. P. L. Chan, and A. Nambiar, “Effectiveness of vacuum-assisted closure (VAC) therapy in the healing of chronic diabetic foot ulcers,” Annals of the Academy of Medicine Singapore, vol. 39, no. 5, pp. 353–358, 2010.
[75]
C. M. Mou？s, F. Heule, and S. E. R. Hovius, “A review of topical negative pressure therapy in wound healing: sufficient evidence?” American Journal of Surgery, vol. 201, no. 4, pp. 544–556, 2011.
[76]
D. T. Ubbink, S. J. Westerbos, E. A. Nelson, and H. Vermeulen, “A systematic review of topical negative pressure therapy for acute and chronic wounds,” British Journal of Surgery, vol. 95, no. 6, pp. 685–692, 2008.
[77]
N. A. Cullum, D. Al-Kurdi, and S. E. Bell-Syer, “Therapeutic ultrasound for venous leg ulcers,” Cochrane Database of Systematic Reviews, vol. 6, Article ID CD001180, 2010.
[78]
D. Al-Kurdi, S. E. Bell-Syer, and K. Flemming, “Therapeutic ultrasound for venous leg ulcers,” Cochrane Database of Systematic Reviews, no. 1, Article ID CD001180, 2008.
[79]
K. A. Flemming, N. A. Cullum, and E. A. Nelson, “A systematic review of laser therapy for venous leg ulcers,” Journal of Wound Care, vol. 8, no. 3, pp. 111–114, 1999.
[80]
H. Anwar, “Association of a 38 kDa bovine serum protein with the outer membrane of Bordetella pertussis,” FEMS Microbiology Letters, vol. 95, no. 3, pp. 305–309, 1990.
[81]
S. M. Moskowitz, J. M. Foster, J. Emerson, and J. L. Burns, “Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis,” Journal of Clinical Microbiology, vol. 42, no. 5, pp. 1915–1922, 2004.
[82]
U. Furustrand Tafin, I. Majic, C. Zalila Belkhodja, et al., “Gentamicin improves the activities of daptomycin and vancomycin against Enterococcus faecalis in vitro and in an experimental foreign-body infection model,” Antimicrobial Agents and Chemotherapy, vol. 55, no. 10, pp. 4821–4827, 2011.
[83]
W. Hengzhuang, H. Wu, O. Ciofu, Z. Song, and N. Hoiby, “Pharmacokinetics/pharmacodynamics of colistin and imipenem on mucoid and nonmucoid Pseudomonas aeruginosa biofilms,” Antimicrobial Agents and Chemotherapy, vol. 55, no. 9, pp. 4469–4474, 2011.
[84]
W. Hengzhuang, H. Wu, O. Ciofu, Z. Song, and N. Hoiby, “In vivo pharmacokinetics/pharmacodynamics of colistin and imipenem in Pseudomonas aeruginosa biofilm infection,” Antimicrobial Agents and Chemotherapy, vol. 56, no. 5, pp. 2683–2690, 2012.
[85]
W. Zimmerli and C. Moser, “Pathogenesis and treatment concepts of orthopaedic biofilm infections,” FEMS Immunology and Medical Microbiology, vol. 65, no. 2, pp. 158–168, 2012.
[86]
O. C. El Helou, E. F. Berbari, B. D. Lahr et al., “Efficacy and safety of rifampin containing regimen for staphylococcal prosthetic joint infections treated with debridement and retention,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 29, no. 8, pp. 961–967, 2010.
[87]
N. H？iby, T. Bjarnsholt, M. Givskov, S. Molin, and O. Ciofu, “Antibiotic resistance of bacterial biofilms,” International Journal of Antimicrobial Agents, vol. 35, no. 4, pp. 322–332, 2010.
[88]
G. Herrmann, L. Yang, H. Wu et al., “Colistin-tobramycin combinations are superior to monotherapy concerning the killing of biofilm Pseudomonas aeruginosa,” Journal of Infectious Diseases, vol. 202, no. 10, pp. 1585–1592, 2010.
[89]
S. J. Pamp, M. Gjermansen, H. K. Johansen, and T. Tolker-Nielsen, “Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes,” Molecular Microbiology, vol. 68, no. 1, pp. 223–240, 2008.
[90]
N. H？iby, O. Ciofu, H. K. Johansen et al., “The clinical impact of bacterial biofilms,” International Journal of Oral Science, vol. 3, no. 2, pp. 55–65, 2011.
[91]
S. E. Dowd, R. D. Wolcott, J. Kennedy, C. Jones, and S. B. Cox, “Molecular diagnostics and personalised medicine in wound care: assessment of outcomes,” Journal of Wound Care, vol. 20, no. 5, pp. 232–239, 2011.
