EFFICACY OF BACTERIOPHAGES FOR TREATEMENT OF EXTRACELULAR AND INTRACELULAR BACTERIAL INFECTIONS OF IMPLANTANTS

Goal of the proposed project is to investigate possibility of bacteriophage therapy in periprosthetic joint infections (PJI) treatment. The number of joint implants is increasing every year, as they nearly eliminate pain and contribute significantly to better mobility of the patients affected with end stage joint derangements. Along with increasing number of patients with joint replacements, the associated complications increase as well. They occur in slightly less than 10% of patients, with the largest proportion representing aseptic failures of the joint prostheses. Rarer but significantly more dangerous complications are PJI since they pose a great health risk for patients as they cause pain, reduced mobility, and failure of the artificial joint, in the worst cases even loss of patient's limb or even death. Chronic PJI are the most difficult ones. Their treatment is complicated due low bacterial cell concentration or their particular physiological state with low metabolic activity, making proper diagnosis challenging. In many chronical PJI bacteria grow in biofilms but in some cases they probably also internalize human cells (e.g. osteoblasts), in both cases causing significantly reduced antibiotic efficacy. During the proposed project we will focus on a treatment of bacterial strains of S. aureus, S. epidermitis and C. acnes, being the most common source of PJI. Appropriate bacteriophages that are able to efficiently lyse selected bacterial strains during their exponential growth will be chosen. All selected bacteria are also able to form biofilm, consisting of extracellular polysaccharides and bacterial cells in different physiological states. To investigate efficiency of bacteriophage treatment on bacteria in defined physiological state, a continuous cultivation system will be used allowing to accurately estimated bacteriophage growth parameters such as burst size, latent period and adsorption constant and through them indirectly estimate efficiency of phage therapy. Obtained results will be compared with results treating with bacteriophages biofilms formed under different conditions. Furthermore, efficiency of bacteriophages on bacteria being in viable but non culturable (VBNC) physiological state will be examined. Substantial efforts will be dedicated to investigate effect of bacteriophages on bacteria internalizing human cells, involved in prosthetic joints osteointegration like mesenchymal stem/ stromal cells and primary osteoblasts. Penetration of bacteriophages into primary human cells will be investigated as well as the effect of bacterial physiological state on internalization of primary human cells. Finally, effect of bacteriophage treatment on internalized bacterial cells will be investigated. Throughout the project the following goals will be accomplished: · - To determine effect of physiological state (including VBNC) of planktonic bacteria cells S. aureus, S. epidermitis in C. acnes on lytic bacteriophage efficiency · - To determine efficiency on bacteriophages for treatment of biofilms formed by selected bacteria species · - To determine bacteriophages being able to penetrate into primary human cells · - To determine effect of bacteria physiological state on their ability to penetrate into primary human cells (intracellular infection) · - To determine bacteriophage efficiency in treatment of primary human cells intracellular infection - Based on obtained results to propose an algorithm of PJI treatment using bacteriophages that can serve as basis for further clinical tests.