Grzegorz Gorczyca

Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus

Bacteria of the genus Staphylococcus are common pathogens responsible for a broad spectrum of human and animal infections and belong to the most important etiological factors causing food poisoning. Because of rapid increase in the prevalence of isolation of staphylococci resistant to many antibiotics, there is an urgent need for the development of new alternative chemotherapeutics. A number of studies have recently demonstrated the strong potential of peptidoglycan hydrolases (PHs) to control and treat infections caused by this group of bacteria. PHs cause rapid lysis and death of bacterial cells. The review concentrates on enzymes hydrolyzing peptidoglycan of staphylococci. Usually, they are characterized by high specificity to only Staphylococcus aureus cell wall components; however, some of them are also able to lyse cells of other staphylococci, e.g., Staphylococcus epidermidis-human pathogen of growing importance and also other groups of bacteria. Some PHs strengthen the bactericidal or bacteriostatic activity of common antibiotics, and as a result, they should be considered as component of combined therapy which could definitely reduced the development of bacterial resistance to both enzymes and antibiotics. The preliminary research revealed that most of these enzymes can be produced using heterologous, especially Escherichia coli expression systems; however, still much effort is required to develop more efficient and large-scale production technologies. This review discusses current state on knowledge with emphasis on the possibilities of application of PHs in the context of therapeutics for infections caused by staphylococci.

Preparation and characterization of genipin cross-linked porous chitosan–collagen–gelatin scaffolds using chitosan–CO2 solution

Novel porous scaffolds composed of chitosan, collagen and gelatin were prepared by the multistep procedure involving final freeze-drying and characterized. To eliminate the need for residual acid removal from the material after drying, carbon dioxide saturation process was used for chitosan blend formulation. The use of CO2 for chitosan dissolution made the scaffold preparation process more reproducible and economically sustainable. Genipin was applied to stabilize the structure of the scaffolds and those crosslinked at a level of 7.3% exhibited a homogenous porous structure (33.1%), high swelling capacity (27.6g/g for wound exudate like medium; 62.5 g/g for water), and were stable under cyclic compression. The values of other investigated parameters: dissolution degree (30%), lysozyme-induced degradation (5% after 168 h), good antioxidant properties (DPPH, ABTS, Fe(2+) assays) and especially very low in vitro cytotoxicity against fibroblasts (103%, MTT assay), were highly advantageous for possible biomedical applications of the novel materials.

Chitosan–protein scaffolds loaded with lysostaphin as potential antistaphylococcal wound dressing materials

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.

EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.