Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Chitosan–protein scaffolds loaded with lysostaphin as potential antistaphylococcal wound dressing materials P. Szweda1, G. Gorczyca 1, R. Tylingo2, J. Kurlenda3, J. Kwiecinski4 and S. Milewski1 1 2 3 4
sk, Poland Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, Gdan sk, Poland Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, Gdan State Higher Vocational School in Koszalin, Koszalin, Poland Department of Rheumatology and Inflammation Research, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
Keywords carbon dioxide, chitosan, collagen, lysostaphin, wound dressing. Correspondence Piotr Szweda, Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, sk, Narutowicza Str. 11/12, 80-233 Gdan Poland. E-mail: [email protected] 2014/0456: received 4 March 2014, revised 15 May 2014 and accepted 3 June 2014 doi:10.1111/jam.12568
Abstract Aims: The development of technology for preparing chitosan–protein scaffolds loaded with lysostaphin, which potentially could be used as dressing for wound treatment and soft tissue infections caused by Staphylococcus aureus. Methods and Results: The unique technology of chitosan solubilization using gaseous CO2 instead of organic or inorganic acids was used for the incorporation of lysostaphin, the enzyme that exhibits bactericidal activity against staphylococci, within the structure of chitosan–protein sponges. The developed chitosan–protein scaffolds loaded with lysostaphin revealed high antistaphylococcal activity, which has been confirmed with a large (n = 143) collection of clinical (skin and wound infections) and animal (bovine mastitis) isolates of these bacteria, including MRSA. No change of bactericidal activity of the lyophilized materials has been observed during half-year storage at 4°C. Conclusions: The developed materials are potential candidates for preparing biologically active, antistaphylococcal wound dressing materials. Significance and Impact of the Study: Staphylococci belong to the most popular and most burdensome aetiological factors of wound and soft tissues infections. The developed chitosan-protein scaffolds loaded with lysostaphin could be a possible solution to problems associated with treatment of these infections.
Introduction Fast and effective treatment of wounds constitutes one of the most important challenges of modern medicine. In the United States, chronic wounds alone affect around 65 million patients per year, with annual treatment costs rising up to 25 billion USD (Sen et al. 2009). The most important factors affecting the wound healing, significantly delaying this process, are bacterial and fungal infections, and the most common micro-organisms isolated from both acute and chronic wounds of various aetiologies are as follows: Staphylococcus aureus, Pseudomonas aeruginosa, b-haemolytic streptococci and also Candida albicans. Infections caused by Staph. aureus are especially difficult to treat. This group of bacteria has evolved resistance to a plethora of antibiotics currently in 634
use for human therapies and has also developed biofilmforming ability, which significantly reduces antibacterial activity of antibiotics and disinfectants. The most worrisome problem is the rapidly growing number of methicillin-resistant staphylococci, which are designated as MRSA (Methicillin-Resistant Staph. aureus). The MRSA isolates, including strains isolated from wounds, often exhibit multi-drug resistance (MDR) and are simultaneously not susceptible to many antibiotics from different chemical groups with different mechanisms of activity (Fry 2013; Goyal et al. 2013). Therefore, there is a great need for novel, nonantibiotic chemotherapeutics with marked antistaphylococcal activity. In the case of wound healing, it is also very important to develop an effective delivery method for antimicrobial agents at the infection site.
Journal of Applied Microbiology 117, 634--642 © 2014 The Society for Applied Microbiology
P. Szweda et al.
The aim of the present study was to develop chitosan– protein scaffolds loaded with lysostaphin, which could be used as bactericidal, antistaphylococcal, wound dressing materials. Many previous reports revealed that using natural biopolymers as components of dressings provides favourable conditions for cell proliferation, tissue regeneration and finally healing of the wound (Ma 2008; Cui et al. 2010, 2011; Gorczyca et al. 2013). Our choice of lysostaphin as an antistaphylococcal agent was not accidental. Lysostaphin is an enzyme with bactericidal activity against Staph. aureus and other staphylococcal species (Schindler and Schuhardt 1964). The target of the lysostaphin activity is the pentaglycine interpeptide bridges of the unique staphylococcal peptidoglycan (Schleifer and Kandler 1972; Iversen and Grov 1973). Other Gram-positive and Gram-negative bacteria, including the normal microbiota of human and animals’ skin, are not susceptible to this enzyme (Schindler and Schuhardt 1964). The unique biological activity of lysostaphin presents numerous possibilities for applications of this enzyme as an antistaphylococcal agent in human and animal therapies. The high therapeutic potential of the enzyme has been confirmed in many animal models of staphylococcal infections, including endocarditis (Climo et al. 1998) and ocular infections (Dajcs et al. 2000). Several reports have also shown that lysostaphin is a promising agent in the eradication of staphylococci biofilms from biotic and abiotic surfaces including medical devices such as catheters (Wu et al. 2003; Walencka et al. 2005, 2006). Recently, lysostaphin has been successfully tested as a bactericidal, antistaphylococcal agent in wound dressing materials (Cui et al. 2011; Miao et al. 2011) and in meshes used in herniorrhaphy (Belyansky et al. 2011). Herein, we have described a novel technology for the preparation of chitosan–protein scaffolds loaded with lysostaphin. The in vitro analysis of antistaphylococcal activity of the produced materials confirmed their high potential for use as wound dressing and treatment of soft tissue infections caused by Staph. aureus. Moreover, in our opinion, the prepared biopolymer matrix has potential application as a universal vehicle to deliver any other protein or peptide with antimicrobial activity to the infected tissue location. Materials and methods Reagents Chitosan and all buffers were purchased from Sigma (Seelze, Germany). Genipin was bought from Challenge Bioproducts (Yun-Lin Hsien, Taiwan), and media for growing bacteria, LB broth, LA agar and Baird-Parker agar, were supplied by BioMaxima (Gdansk, Poland).
Lysostaphin in dressing materials
Fish proteins used in this work were extracted from salmon skin (Salmo salar). The extraction was performed according to the procedures described earlier by Kolodziejska et al. (2008). Preparing of recombinant lysostaphin The recombinant lysostaphin was produced in the cells of Escherichia coli TOP10F’ strain (Invitrogen, Carlsbad, CA), transformed with the plasmid pBAD2Lys and constructed earlier in our laboratory (Szweda et al. 2007). The production of the enzyme was carried out in a 5-l bioreactor (Biostat C; Braun Co., Germany) according to the procedure optimized by Szweda et al. (2014a). Enzyme purification was conducted with the use of metal affinity chromatography on a Ni-NTA His-Bind Resign column (Novagen, Madison, WI). Preparation of chitosan–protein sponges loaded with lysostaphin Chitosan–protein sponges were formulated according to a previously described method (Gorczyca et al. 2013). Briefly, 15 g of chitosan powder was suspended in 100 ml of 05 mol l 1 acetic acid. The dissolved chitosan was precipitated from the suspension by dropwise addition of 05 mol l 1 sodium hydroxide, followed by centrifugation at 4000 g for 30 min. The resulting precipitate was washed twice with distilled water, centrifuged again at 4000 g for 30 min, suspended in distilled water in an amount necessary to obtain a total weight of 80 g, homogenized and saturated with CO2 for 3 h at room temperature with the use of a hollow shaft stirrer to obtain a clear aqueous chitosan solution. The genipin ethanolic solution and lysostaphin in phosphate buffer (pH 74) were next added to final concentrations, as reported in Table 1. The mixture was stirred for 10 min and cooled to
ORIGINAL ARTICLE
Chitosan–protein scaffolds loaded with lysostaphin as potential antistaphylococcal wound dressing materials P. Szweda1, G. Gorczyca 1, R. Tylingo2, J. Kurlenda3, J. Kwiecinski4 and S. Milewski1 1 2 3 4
sk, Poland Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, Gdan sk, Poland Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, Gdan State Higher Vocational School in Koszalin, Koszalin, Poland Department of Rheumatology and Inflammation Research, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
Keywords carbon dioxide, chitosan, collagen, lysostaphin, wound dressing. Correspondence Piotr Szweda, Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, sk, Narutowicza Str. 11/12, 80-233 Gdan Poland. E-mail: [email protected] 2014/0456: received 4 March 2014, revised 15 May 2014 and accepted 3 June 2014 doi:10.1111/jam.12568
Abstract Aims: The development of technology for preparing chitosan–protein scaffolds loaded with lysostaphin, which potentially could be used as dressing for wound treatment and soft tissue infections caused by Staphylococcus aureus. Methods and Results: The unique technology of chitosan solubilization using gaseous CO2 instead of organic or inorganic acids was used for the incorporation of lysostaphin, the enzyme that exhibits bactericidal activity against staphylococci, within the structure of chitosan–protein sponges. The developed chitosan–protein scaffolds loaded with lysostaphin revealed high antistaphylococcal activity, which has been confirmed with a large (n = 143) collection of clinical (skin and wound infections) and animal (bovine mastitis) isolates of these bacteria, including MRSA. No change of bactericidal activity of the lyophilized materials has been observed during half-year storage at 4°C. Conclusions: The developed materials are potential candidates for preparing biologically active, antistaphylococcal wound dressing materials. Significance and Impact of the Study: Staphylococci belong to the most popular and most burdensome aetiological factors of wound and soft tissues infections. The developed chitosan-protein scaffolds loaded with lysostaphin could be a possible solution to problems associated with treatment of these infections.
Introduction Fast and effective treatment of wounds constitutes one of the most important challenges of modern medicine. In the United States, chronic wounds alone affect around 65 million patients per year, with annual treatment costs rising up to 25 billion USD (Sen et al. 2009). The most important factors affecting the wound healing, significantly delaying this process, are bacterial and fungal infections, and the most common micro-organisms isolated from both acute and chronic wounds of various aetiologies are as follows: Staphylococcus aureus, Pseudomonas aeruginosa, b-haemolytic streptococci and also Candida albicans. Infections caused by Staph. aureus are especially difficult to treat. This group of bacteria has evolved resistance to a plethora of antibiotics currently in 634
use for human therapies and has also developed biofilmforming ability, which significantly reduces antibacterial activity of antibiotics and disinfectants. The most worrisome problem is the rapidly growing number of methicillin-resistant staphylococci, which are designated as MRSA (Methicillin-Resistant Staph. aureus). The MRSA isolates, including strains isolated from wounds, often exhibit multi-drug resistance (MDR) and are simultaneously not susceptible to many antibiotics from different chemical groups with different mechanisms of activity (Fry 2013; Goyal et al. 2013). Therefore, there is a great need for novel, nonantibiotic chemotherapeutics with marked antistaphylococcal activity. In the case of wound healing, it is also very important to develop an effective delivery method for antimicrobial agents at the infection site.
Journal of Applied Microbiology 117, 634--642 © 2014 The Society for Applied Microbiology
P. Szweda et al.
The aim of the present study was to develop chitosan– protein scaffolds loaded with lysostaphin, which could be used as bactericidal, antistaphylococcal, wound dressing materials. Many previous reports revealed that using natural biopolymers as components of dressings provides favourable conditions for cell proliferation, tissue regeneration and finally healing of the wound (Ma 2008; Cui et al. 2010, 2011; Gorczyca et al. 2013). Our choice of lysostaphin as an antistaphylococcal agent was not accidental. Lysostaphin is an enzyme with bactericidal activity against Staph. aureus and other staphylococcal species (Schindler and Schuhardt 1964). The target of the lysostaphin activity is the pentaglycine interpeptide bridges of the unique staphylococcal peptidoglycan (Schleifer and Kandler 1972; Iversen and Grov 1973). Other Gram-positive and Gram-negative bacteria, including the normal microbiota of human and animals’ skin, are not susceptible to this enzyme (Schindler and Schuhardt 1964). The unique biological activity of lysostaphin presents numerous possibilities for applications of this enzyme as an antistaphylococcal agent in human and animal therapies. The high therapeutic potential of the enzyme has been confirmed in many animal models of staphylococcal infections, including endocarditis (Climo et al. 1998) and ocular infections (Dajcs et al. 2000). Several reports have also shown that lysostaphin is a promising agent in the eradication of staphylococci biofilms from biotic and abiotic surfaces including medical devices such as catheters (Wu et al. 2003; Walencka et al. 2005, 2006). Recently, lysostaphin has been successfully tested as a bactericidal, antistaphylococcal agent in wound dressing materials (Cui et al. 2011; Miao et al. 2011) and in meshes used in herniorrhaphy (Belyansky et al. 2011). Herein, we have described a novel technology for the preparation of chitosan–protein scaffolds loaded with lysostaphin. The in vitro analysis of antistaphylococcal activity of the produced materials confirmed their high potential for use as wound dressing and treatment of soft tissue infections caused by Staph. aureus. Moreover, in our opinion, the prepared biopolymer matrix has potential application as a universal vehicle to deliver any other protein or peptide with antimicrobial activity to the infected tissue location. Materials and methods Reagents Chitosan and all buffers were purchased from Sigma (Seelze, Germany). Genipin was bought from Challenge Bioproducts (Yun-Lin Hsien, Taiwan), and media for growing bacteria, LB broth, LA agar and Baird-Parker agar, were supplied by BioMaxima (Gdansk, Poland).
Lysostaphin in dressing materials
Fish proteins used in this work were extracted from salmon skin (Salmo salar). The extraction was performed according to the procedures described earlier by Kolodziejska et al. (2008). Preparing of recombinant lysostaphin The recombinant lysostaphin was produced in the cells of Escherichia coli TOP10F’ strain (Invitrogen, Carlsbad, CA), transformed with the plasmid pBAD2Lys and constructed earlier in our laboratory (Szweda et al. 2007). The production of the enzyme was carried out in a 5-l bioreactor (Biostat C; Braun Co., Germany) according to the procedure optimized by Szweda et al. (2014a). Enzyme purification was conducted with the use of metal affinity chromatography on a Ni-NTA His-Bind Resign column (Novagen, Madison, WI). Preparation of chitosan–protein sponges loaded with lysostaphin Chitosan–protein sponges were formulated according to a previously described method (Gorczyca et al. 2013). Briefly, 15 g of chitosan powder was suspended in 100 ml of 05 mol l 1 acetic acid. The dissolved chitosan was precipitated from the suspension by dropwise addition of 05 mol l 1 sodium hydroxide, followed by centrifugation at 4000 g for 30 min. The resulting precipitate was washed twice with distilled water, centrifuged again at 4000 g for 30 min, suspended in distilled water in an amount necessary to obtain a total weight of 80 g, homogenized and saturated with CO2 for 3 h at room temperature with the use of a hollow shaft stirrer to obtain a clear aqueous chitosan solution. The genipin ethanolic solution and lysostaphin in phosphate buffer (pH 74) were next added to final concentrations, as reported in Table 1. The mixture was stirred for 10 min and cooled to
