Isolation and characterization of bacteriophages specific for the spectrum of multidrug-resistant bacteria

Bakteriofagi (fagi) so virusi, ki okužujejo bakterije in tako v sklopu fagne terapije predstavljajo alternativo za boj proti večkatno odpornim bakterijam. V nalogi smo fage izolirali iz vodnih okoljskih vzorcev in odkrili fage specifične za 39 izmed 45 bakterijskih sevov treh bakterijskih vrst E. coli, K. pneumoniae in P. mirabilis. Fage smo na platformi podjeta JAFRAL izolirali v petih dneh. Za vsako izmed treh bakterijskih vrst smo si izbrali fag, ki lizira največ sevov bakterij in ima najvišji titer, in ga nagojili po načelih magistralne priprave fagnih produktov. Postopek priprave fagnih produktov je trajal 19–22 dni. Začeli smo s pomnoževanjem v manjšem obsegu, nato smo fage pomnožili v večjem obsegu, na litrski skali, nadaljevali s filtracijo lizatov gostiteljskih sevov in koncentriranjem s tangencialno pretočno filtracijo. Temu je sledilo odstranjevanje endotoksinov z afinitetno kromatografijo s sistemom Endotrap, na koncu pa smo fagne pripravke še dodatno očistili z metodo gelske izključitvene kromatografije. Postopki pomnoževanja so v večini primerov omogočili pridobivanje zadostnih količin bakteriofagov za klinično uporabo. Faga ZOO1 in P6 sta se izkazala kot potencialno primerna za uporabo v klinični medicini, saj je bila koncentracija fagov v pripravkih zadostna za priporočen dnevni odmerek, vrednosti endotoksinov v produktih pa znotraj kriterijev za zdravljenje okužb večkratno odpornih bakterij pri človeku s parenteralnim vnosom. Fagne pripravke smo tudi okarakterizirali s pregledom morfologije plakov, z ogledom strukturnih značilnosti s transmisijskim elektronskim mikroskopom in bioinformatsko analizo genomov. S slednjo smo potrdili, da gre za litične fage, ki nimajo zapisov za integraze in virulentne dejavnike. Faga ZOO1 in P6 tudi nimata zapisov za odpornosti proti antibiotikom in sta tako tudi s tega vidika primerna za uporabo v fagni terapiji, fag D13 pa za zdravljenje ni primeren, saj njegov genom vsebuje zapis za ß-laktamazo.Bacteriophages are viruses that infect bacteria and thus, as part of phage therapy, represent an alternative to combat multidrug-resistant bacteria. In this work, phages were isolated from aqueous environmental samples and phages specific for 39 of 45 bacterial strains belonging to three bacterial species E. coli, K. pneumoniae and P. mirabilis were found. Phages were isolated by the JAFRAL platform in five days. For each of the three bacterial species, the most successful phage was selected and propagated according to the principles of the magistral preparation of phage products. The process of preparation of phage products took 19–22 days. We started by small scale propagation, continued with large scale, followed by filtration of lysates and concentration by tangential flow filtration. Afterwards endotoxins were removed using affinity chromatography (Endotrap method), and finally further purification of phage products was performed by size exclusion chromatography. Cultivation procedures made it possible to obtain sufficient quantities of bacteriophages for clinical use. Two of the three selected phages, ZOO1 nad P6, were suitable for use in clinical medicine, as the concentration of phages in the phage products was sufficient for the recommended daily treatment dose and the endotoxin levels in the products met the criteria for treating multidrug-resistant bacterial infections in humans by parenteral administration. Magistral phage preparations were also characterized by transmission electron microscopy, plaque morphology examination, and bioinformatics analysis to confirm that they do not have any genes coding for integrase or virulent genes. In addition, analyzed genomes of phages ZOO1 anf P6 did not encode any antimicrobial resistance genes and are therefore potentially suitable for clinical use. Phage D13 on the other hand contains gene coding for ß-lactamase and is not suitable for use in phage therapy