Collagen and hyaluronan at wound sites influence early polymicrobial biofilm adhesive events

BMC Microbiology

J Scott Weese¹  Suresh Neethirajan²  Eric Birkenhauer² 

[1] Department of Pathobiology, Ontario Veterinary College, University of Guelph, Ontario N1G 2 W1, Canada;BioNano Laboratory, School of Engineering, University of Guelph, Guelph, Ontario N1G 2 W1, Canada

关键词: Atomic force microscopy;    Wound biofilms;    Chronic wound infections;    Microtiter plate assay;    Hyaluronan;    Collagen;    Polymicrobial biofilms;    MRSA;    Pseudomonas aeruginosa;   

Others  :  1140753
DOI  :  10.1186/1471-2180-14-191

 received in 2013-11-03, accepted in 2014-07-12,  发布年份 2014

【 摘 要 】

Background

Wounds can easily become chronically infected, leading to secondary health complications, which occur more frequently in individuals with diabetes, compromised immune systems, and those that have suffered severe burns. When wounds become chronically infected, biofilm producing microbes are often isolated from these sites. The presence of a biofilm at a wound site has significant negative impact on the treatment outcomes, as biofilms are characteristically recalcitrant to removal, in part due to the formation of a protective matrix that shield residents organisms from inimical forces. Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) are two of the organisms most prevalently isolated from wound sites, and are of particular concern due to their elevated levels of antibiotic resistance, rapid growth, and exotoxin production. In order to understand the biofilm forming abilities of these microbes in a simulated wound environment we used a microtiter plate assay to assess the ability of these two organisms to bind to proteins that are typically found at wound sites: collagen and hyaluronan.

Results

Collagen and hyaluronan were used to coat the wells of 96-well plates in collagen:hyaluronan ratios of 0:1, 3:1, 1:1, 1:3, and 1:0 . P. aeruginosa and MRSA were inoculated as mono- and co-cultures (1:1 and a 3:1 MRSA: P. aeruginosa). We determined that coating the wells with collagen and/or hyaluronan significantly increased the biofilm biomass of attached cells compared to an uncoated control, although no one coating formulation showed a significant increase compared to any other combination. We also noted that the fold-change increase for MRSA upon coating was greater than for P. aeruginosa.

Conclusions

Our study suggests that the presence of collagen and/or hyaluronan at wound sites may be an important factor that influences the attachment and subsequent biofilm formation of notorious biofilm-formers, such as MRSA and P. aeruginosa. Understanding the kinetics of binding may aid in our comprehension of recalcitrant wound infection development, better enabling our ability to design therapies that would prevent or mitigate the negative outcomes associated with such infections.

【 授权许可】

   
2014 Birkenhauer et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Canadian Association of Wound Care Statistics 2013. Retrieved from http://cawc.net/en/index.php/public/facts-stats-and-tools/statistics/ webcite
• [2]Dealey C, Posnett J, Walker A: The cost of pressure ulcers in the United Kingdom. J Wound Care 2012, 21:261-266.
• [3]Wu S, Marston W, Armstrong D: Wound care: the role of advanced wound healing technologies. J Am Podiatr Med Assoc 2010, 100:385-394.
• [4]Asada M, Nakagami G, Minematsu T, Nagase T, Akase T, Huang L, Yoshimura K, Sanada H: Novel models for bacterial colonization and infection of full-thickness wounds in rats. Wound Repair Regen 2012, 20:601-610.
• [5]Seth A, Geringer M, Hong S, Leung K, Mustoe T, Galiano R: In vivo modeling of biofilm-infected wounds: a review. J Surg Res 2012, 178:330-338.
• [6]Woods J, Boegli L, Kirker K, Agostinho A, Durch A, deLancey Pulcini E, Stewart P, James G: Development and application of a polymicrobial, in vitro, wound biofilm model. J Appl Microbiol 2012, 112:998-1006.
• [7]Wolcott R, Ehrlich G: Biofilms and chronic infections. JAMA 2008, 299:2682-2684.
• [8]Fux C, Costerton J, Stewart P, Stoodley P: Survival strategies of infectious biofilms. Trends Microbiol 2005, 13:34-40.
• [9]Kim J, Park H, Chung S: Microfluidic approaches to bacterial biofilm formation. Molecules 2012, 17:9818-9834.
• [10]Peters B, Jabra-Rizk M, O'May G, Costerton W, Shirtliff M: Polymicrobial interactions: impact on pathogenesis and human disease. Clin Microbiol Rev 2012, 25:193-213.
• [11]Wolcott R, Costerton J, Raoult D, Cutler S: The polymicrobial nature of biofilm interaction. Clin Microbiol Infect 2013, 19:107-112.
• [12]Pastar I, Nusbaum A, Gil J, Patel S, Chen J, Valdes J, Stojadinovic O, Plano L, Tomic-Canic M, Plano L: Interactions of methicillin resistant Staphylococcus aureus usa300 and Pseudomonas aeruginosa in polymicrobial wound infection. PLoS One 2013, 8:1-11.
• [13]Percival S, Emanuel C, Cutting K, Williams D: Microbiology of the skin and the role of biofilms in infection. Int Wound J 2011, 9:14-32.
• [14]Seth A, Geringer M, Hong S, Leung K, Galiano R, Mustoe T: Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model. PLoS One 2012, 7:1-9.
• [15]Singh P, Parsek M, Greenberg P, Welsh M: A component of innate immunity prevents bacterial biofilm development. Nature 2002, 417:552-555.
• [16]Brauer L, Schicht M, Worlitzsch D, Bensel T, Sawers R, Paulsen F: Staphylococcus aureus and Pseudomonas aeruginosa express and secrete human surfactant proteins. PLoS One 2013., 8doi:10.1371/journal.pone.0053705
• [17]Harrison-Balestra C, Cazzaniga A, Davis S, Mertz P: A wound-isolated Pseudomonas aeruginosa grows a biofilm in vitro within 10 hours and is visualized by light micrscopy. Am Soc Dermatol Surg 2003, 26:631-635.
• [18]Ciofu O, Mandsberg L, Wang H, Hoiby N: Phenotypes selected during chronic lung infection in cystic fibrosis patients: implications for the treatment of Pseudomonas aeruginosa biofilm infections. FEMS Immunol Med Microbiol 2012, 65:215-225.
• [19]Hoiby N, Johansen H, Moser C, Song Z, Ciofu O, Kharazmi A: Pseudomonas aeruginosa and the in vitro and in vivo biofilm mode of growth. Microbes Infect 2001, 3:23-35.
• [20]Su S, Hassett D: Anaerobic Pseudomonas aeruginosa and other obligately anaerobic bacterial biofilms growing in the thick airway mucus of chronically infected cystic fibrosis patients: an emerging paradigm or "old hat"? Expert Opin Ther Targets 2012, 16:859-873.
• [21]Scales B, Huffnagle G: The microbiome in wound repair and tissue fibrosis. J Pathol 2013, 229:323-331.
• [22]Agostinho A, Hartman A, Lipp C, Parker A, Stewart P, James G: An in vitro model for the growth and analysis of chronic wound MRSA biofilms. J Appl Microbiol 2011, 111:1275-1282.
• [23]Athanasopoulos A, Economopoulou M, Orlova V, Sobke A, Schneider D, Weber H, Augustin H, Eming S, Schubert U, Linn T, Nawroth P, Hussain M, Hammes H, Herrmann M, Preissner K, Chavakis T: The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms. Blood 2006, 107:2720-2727.
• [24]Mulcahy M, Geoghegan J, Monk I, O'Keeffe K, Walsh E, Foster T, McLoughlin R: Nasal colonisation by Staphylococcus aureus depends upon clumping factor b binding to the squamous epithelial cell envelope protein loricrin. PLoS Pathog 2012., 8doi:10.1371/journal.ppat.1003092. Epub 2012 Dec 27
• [25]Palmer J, Flint S, Brooks J: Bacterial cell attachment, the beginning of a biofilm. J Ind Microbiol Biotechnol 2007, 34:577-588.
• [26]Shevchenko R, James S, James S: A review of tissue-engineered skin bioconstructs available for skin reconstruction. J R Soc Interface 2010, 7:229-258.
• [27]Huang G, Zhou L, Zhang Q, Chen Y, Sun W, Xy F, Lu T: Microfluidic hydrogels for tissue engineering. Biofabrication 2011., 3doi: 10.1088/1758-5082/3/1/012001. Epub 2011 Mar 4
• [28]Menon G: New insights into skin structure: scratching the surface. Adv Drug Deliv Rev 2002, 54:3-17.
• [29]Sorokin L: The impact of the extracellular matrix on inflammation. Nat Rev Immunol 2010, 10:712-723.
• [30]Beele H: Artificial skin: past, present and future. Int J Artif Organs 2002, 25:163-173.
• [31]Frenkel J: The role of hyaluronan in wound healing. Int Wound J 2012. doi:10.1111/j.1742-481X.2012.01057.x
• [32]Nijssen A, Schut TCB, Heule F, Caspers PJ, Hayes DP, Neumann MH, Puppels GJ: Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy. J Investig Dermatol 2002, 119:64-69.
• [33]Taguchi T, Ikoma T, Tanaka J: An improved method to prepare hyaluronic acid and type II collagen composite matrices. J Biomed Mater Res 2002, 61:330-336.
• [34]Doktycz M, Sullivan C, Hoyt P, Pelletier D, Wu S, Allison D: AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces. Ultramicroscopy 2003, 97:209-216.
• [35]O'Toole G: Microtiter dish biofilm formation assay. J Vis Exp 2011, 47:2437.
• [36]Singh A, Walker M, Rousseau J, Weese J: Characterization of the biofilm forming ability of Staphylococcus pseudintermedius from dogs. BMC Vet Res 2013., 9doi: 10.1186/1746-6148-9-93
• [37]Biswas L, Biswas R, Schlag M, Bertram R, Gotz F: Small-colony variant selection as a survival strategy for Staphylococcus aureus in the presence of Pseudomonas aeruginosa. Appl Environ Microbiol 2009, 75:6910-6912.
• [38]Wang F, Garza L, Kang S, Varani J, Orringer J, Fisher G, Voorhees J: In vivo stimulation of de novo collagen production caused by cross-linked hyaluronic acid dermal filler injections in photodamaged human skin. Arch Dermatol 2007, 143:153-163.
• [39]Werthen M, Henriksson L, Jensen P, Sternberg C, Givskov M, Bjarnsholt T: An in vitro model of bacterial infections in wounds and other soft tissues. APMIS 2010, 118:156-164.
• [40]Grenho L, Manso M, Monteiro F, Ferraz M: Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa onto nanohydroxyapatite as a bone regeneration material. J Biomed Mater Res 2012, 100:1823-1830.
• [41]Gross M, Cramton S, Gotz F, Peschel A: Key role of teichoic acid net charge in Staphylococcus aureus colonization of artificial surfaces. Infect Immun 2001, 69:3423-3426.
• [42]Cerqueira L, Oliveira J, Nicolau A, Azevedo N, Vieira J: Biofilm formation with mixed cultures of Pseudomonas aeruginosa/Escherichia coli on silicone using artificial urine to mimic urinary catheters. Biofouling 2013, 29:829-840.
• [43]Yang L, Liu Y, Markussen T, Hoiby N, Tolker-Nielsen T, Molin S: Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa. FEMS Immunol Med Microbiol 2011, 62:339-347.
• [44]Bucior I, Pielage J, Engel J: Pseudomonas aeruginosa pili and flagella mediate distinct binding and signaling events at the apical and basolateral surface of airway epithelium. PLoS Pathog 2012., 8doi:10.1371/journal.ppat.1002616. Epub 2012 Apr 5
• [45]De La Fuente L, Montanes E, Meng Y, Li Y, Burr T, Hoch H, Wu M: Assessing adhesion forces of type i and type iv pili of xylella fastidiosa bacteria by use of a microfluidic flow chamber. Appl Environ Microbiol 2007, 73:2690-2696.
• [46]Bahar O, De La Fuente L, Burdman S: Assessing adhesion, biofilm formation and motility of acidovorax citrulli using microfluidic flow chambers. FEMS Microbiol Lett 2010, 312:33-39.
• [47]Otto M: Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity. Annu Rev Med 2013, 64:175-188.
• [48]Inclan YF, Huseby MJ, Engel JN: FimL regulates cAMP synthesis in Pseudomonas aeruginosa. PLoS One 2011, 6:e15867.
• [49]Tenover FC, McDougal LK, Goering RV, Killgore G, Projan SJ, Patel JB, Dunman PM: Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States. J Clin Microbiol 2006, 44:108-118.
• [50]Qin Z, Yang L, Qu D, Molin S, Molin S: Pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by Staphylococcus epidermidis. Microbiology 2009, 155:2148-2156.
• [51]Malic S, Hill K, Hayes A, Percival S, Thomas D, Williams D: Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH). Microbiology 2009, 155:2603-2611.
• [52]Pihl M, Arvidsson A, Skepo M, Nilsson M, Givskov M, Tolker-Nielsen T, Svensater G, Davies J: Biofilm formation by Staphylococcus epidermidis on peritoneal dialysis catheters and the effects of extracellular products from Pseudomonas aeruginosa. Pathog Dis 2013, 67:192-198.
• [53]Lopes S, Machado I, Pereira M: Role of planktonic and sessile extracellular metabolic byproducts on Pseudomonas aeruginosa and Escherichia coli intra and interspecies relationships. J Ind Microbiol Biotechnol 2011, 38:133-140.
• [54]Filipiak W, Sponring A, Baur M, Filipiak A, Ager C, Wiesenhofer H, Nagl M, Troppmair J, Amann A: Molecular analysis of volatile metabolites released specifically by Staphylococcus aureus and Pseudomonas aeruginosa. BMC Microbiol 2012., 12doi:10.1186/1471-2180-12-113