Untersuchungen zum therapeutischen Potential von Bakteriophagen der Gattungen PB1, phiKZ und LUZ24 gegen multi-resistente Isolate von Pseudomonas aeruginosa

One can hear and read it everywhere: we are in a period of antibiotic-crisis. Infections, often acquired in hospitals and termed nosocomial infections, are increasingly more difficult to treat due to a diverse repertoire of resistance mechanisms developed by bacteria. The focus of this dissertation is on bacteria belonging to the so called ESKAPE-group, causing the main part of nosocomial infections. Grampositive pathogens belonging to this group are Enterococcus faecium and Staphylococcus aureus, gramnegative pathogens are Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. During the course of this study bacteriophages (briefly: phages) against these pathogens were isolated from environmental samples. These natural predators of bacteria are able to lyse their hosts and are currently discussed as an alternative to antibiotics. The prevalence of phages could be assessed by screening 16 aquatic samples in the surroundings of the university hospital of Aachen. After successfully establishing all required methods, phages against all tested multi-resistant isolates from the ESKAPE-group could be isolated, except for S. aureus.Four phages (SL1, SL2, SL4 und SL5) were characterized in detail based on five multidrug-resistant P. aeruginosa strains (PA01, PA02, PA03, PA04 und PA05) as hosts with different resistance-mechanisms against antibiotics. To this end, all basic techniques for proper phenotypic and genetic characterization were established in laboratory. The morphology of phages was assessed via diverse staining approaches followed by transmission electron microscopy which enabled the classification of phages into the order of Caudovirales and into the families Myoviridae (SL1 and SL2) and Podoviridae (SL4). Only phage SL5 could not clearly be classified by transmission electron microscopy. In total, 24 clinical P. aeruginosa isolates were differentiated via individual enterobacterial repetitive intergenic consensus (ERIC-) sequences and then used to determine the host range of the isolated phages. Those bacterial strains also exhibited different resistance-profiles against diverse classes of antibiotics because, among others, of the possession of different carbapenemases. Phage SL1 lysed 58 %, phage SL2 66%, phage SL4 70 % and phage SL5 45 % of the tested strains. Further characterization of phages was done by de novo sequencing of the phage genomes using the MiSeq-Sequencing platform. The genomes were assembled via the SPAdes assembler and based on BLASTn-analysis the phages could be assigned to three different phage genera. Phage SL1 is genetically related to PB1-like phages, phage SL2 is a giant phage and belongs to the phiKZ-like phages, whereas phage SL4 is related to members of the LUZ24-like genus. Based on further bioinformatic analysis, the genomic features and particularly the differences to already known phages were assessed. Because of its genomic relationship to lysogenic phages and the identification of genes encoding for integrases, Phage SL5 was not further investigated in this study.In the planktonic infection assays the five strains (PA01-PA05) could be successfully suppressed or lysed during the testing period of 16 hours, with phage SL2 revealing the best potential. Four varying phage concentrations were tested each and even at a multiplicity of infection (MOI) of 10-6 bacteria could still be suppressed. Furthermore, development of resistance was observed only in a few cases (in contrast to most findings published in literature). The combination of all three phages in a phage cocktail led to the same result qualitatively. The data indicates no synergistic effect; however, they show that the phages did not interfere negatively with each other either. In order to increase the host-range and to minimize the risk of the development of phage resistance, an as of yet untested combination of those three phage genera for phage therapy can therefore be recommended.Anti-biofilm qualities of phages have been assessed by in vitro established biofilms of these five bacterial isolates. Significant reduction in biofilm mass (crystal-violet-assay) and a decrease of metabolic bacterial activity (ATP-activity-assay) were achieved with phage SL4. However, the therapeutical potential against biofilms was generally less strong pronounced compared to the lysis efficiency of planktonic bacteria. The newly isolated phages were further tested using the wax moth in vivo infection model of Galleria mellonella. Whereas larvae infected only with P. aeruginosa did not survive 24 hours, the lifespan of those larvae receiving phage therapy could be extended significantly. This could be achieved with single-phage applications as well as with a phage-cocktail.The results of this dissertation broaden the known spectrum of therapeutically applicable phages, with phage SL2 (a giant phage) and phage SL4 belonging to genera that had so far received little attention for this purpose. For a possible establishment of phage therapy against multidrug-resistant bacteria as alternative or supplementation to antibiotics more investigation will be needed, since the breadth of diversity of natural phages as well as their biological and therapeutic features have by far not been fully captured yet