Antimicrobial Activity of Bacteriophages in Multidrug-Resistant and Biofilm Associated Infections. Summary of the Doctoral Thesis

Abstract

In recent years multi-drug resistant bacterial infections are on the rise. Various virulence factors play an important role in the pathogenesis of bacterial infections. One of them, which leads to recurrent and prolonged infection, is the ability of bacteria to form biofilms. Multiresistant and biofilm-associated infections are observed in patients with risk factors such as artificially implanted devices, chronic disease, immunosuppression, severe tissue structural damage due to trauma. The use of antibiotics in the treatment of these infections does not always ensure a successful outcome, so alternative treatments such as bacteriophage therapy are being evaluated and used. Bacteriophages or phages are bacterial viruses that are able to enter a specific bacterium, multiply in it and, at the end of their life cycle, lyse it. Phages are unaffected by bacterial resistance to antibiotics and have the potential to break down bacterial biofilms. Bacteria have the ability to acquire resistance to phages, therefore phage therapy is often combined with antibiotics. The interaction of these agents with planktonic and biofilm-forming cells is highly variable due to differences in the rates of interaction between phages and the bacterium, as well as the mechanisms of action of the antibiotics. The aim of this study is to evaluate the antimicrobial effect of bacteriophages and their interaction with antibiotics in vitro of selected multidrug-resistant and biofilm-forming bacteria and in the treatment of life-threatening infections. The dissertation is organised as a collection of four consecutive peer-reviewed articles. The structure of the study consists of three parts.In the first part investigation of the outcomes of S. aureus colonisation and biofilm formation in peritoneal dialysis patients over a two-year period was performed. Phenotypic differences in biofilm formation between different isolates of S. aureus, from the same individual were assessed. Bacteriophage lytic effect in isolated cultures was detected, and phage adaptation procedure to overcome bacteriophage resistance was performed. In the second part, the biofilm-forming capacity of uropathogenic E. coli isolates was investigated, the antibiotic and phage biofilm eradication capacity was determined. Bacteriophage adaptation was performed to overcome resistance and assess the lytic effect of adapted phages in planktonic and biofilm-forming cells. In the third part, the clinical efficacy of bacteriophages in patients with complicated multidrug-resistant P. aeruginosa osteomyelitis and Left Ventricular Assist Device (LVAD) associated infection was evaluated. Patients received local and systemic phage therapy supplemented with antibiotics and underwent wound revision. Clinical outcome and tolerability of therapy were analysed. This was preceded by bacteriophage selection, phage preparation and evaluation for clinical use. The effect of bacteriophages in biofilm models was also evaluated in the laboratory, the interaction of bacteriophages with the antibiotics used was determined and the development of phage resistance was assessed. The results show that biofilm-forming strains are common in the patient population studied and that their biofilm-forming capacity can vary widely. The biofilm-forming phenotype of the same bacterium may differ even within the same individual. The antibacterial effect of bacteriophages in the isolated bacteria was of broad lytic efficiency. In phage resistant S. aureus and E. coli isolates adaptation procedure led to overcoming phage resistance. After bacteriophage adaptation in E. coli culture, a strong biofilm eradication effect of the adapted bacteriophage was also observed. Treatment of patients with bacteriophages resulted in clinical recovery in both clinical cases. Complete eradication of P. aeruginosa was achieved in the patient with LVAD infection, whereas the patient with osteomyelitis had a relapse of infection after nine months in another bone segment, which was successfully treated with antibiotics. Different bacteriophages were used to treat the patients and their lytic and biofilm eradication effects were studied in combination with antibiotics. The bacteriophage BFC1.10 showed an additive effect with ceftazidime-avibactam, which rendered the causative organism sensitive to the antibiotic used. In contrast, phages PNM and PT07 had a better biofilm eradication effect when used first and then switched to ceftazidime-avibactam. Bacteriophage resistance to phages PNM and PT07 in P. aeruginosa culture developed as early as 12 h after the start of the experiment in the biofilm model. The development of resistance was different using different strains of the bacterium. The study shows that bacteriophages have a high potential to treat multidrug-resistant and biofilm-associated infections in both laboratory and human settings. Bacteriophage therapy can be complemented with antibiotics, but the interaction and sequence of administration of specific agents needs to be assessed as this has a significant impact on the efficacy of therapy. Although bacteriophage resistance is common in phage therapy, it can be overcome by using phage cocktails, an adaptation procedure or by combining phage therapy with antibiotics, even if the bacterium is moderately sensitive to the antibiotic. Further studies, especially clinical studies, are needed to evaluate phage therapy more precisely.