Vpliv različnih postopkov liofilizacije in shranjevanja bakteriofagov bakterijskih vrst Staphylococcus aureus, Pseudomonas aeruginosa in Cutibacterium acnes na infektivnost

Janežič, Anja (author), Vodovnik, Maša (mentor), Smrekar, Frenk (comentor)

Alternativo zdravljenju bakterijskih infekcij z antibiotiki predstavlja bakteriofagna terapija. Liofilizacija je učinkovita metoda za ohranjanje stabilnosti bakteriofagov tekom dolgotrajnejšega shranjevanja. V nalogi smo želeli raziskati vplive različnih kriprotektantov na ohranjanje infektivnosti fagov med liofilizacijo, ugotoviti ali liofilizirani fagi po treh mesecih ohranijo višjo raven infektivnosti kot neliofilizirani in kakšen vpliv ima temperatura skladiščenja na ohranjanje infektivnosti fagov. V ta namen smo izbrali tri fage baterijskih vrst Staphylococcus aureus (fag JSAP1), Pseudomonas aeruginosa (fag JPAP1) and Cutibacterium acnes (fag JCAP1). Z namnoževanjem v manjšem obsegu smo najprej določili parametre gojenja. Sledila je filtracija bakteriofagnih lizatov, koncentriranje in diafiltracija s tangencialno pretočno filtracijo ter čiščenje z gelsko filtracijo oz. velikostno izključitveno kromatografijo. Nato smo pripravili različne mešanice bakteriofagov. Mešanice smo zamrznili in liofilizirali. Ugotovili smo, da so fagi v večji meri preživeli v mešanicah, ki so vsebovale sladkorje, kot pa v mešanicah, v katerih je bil krioprotektant PEG 6000. Nato smo izvedli trimesečno študijo stabilnosti z uporabo mešanic, ki so vsebovale pufer (PBS ali SMB) ali destilirano vodo ter 0,5 M saharozo, 0,5 M trehalozo, 0,25 M saharozo in 0,25 M trehalozo oziroma niso vsebovale nobenega krioprotektanta. Ugotovili smo, da so fagi v mešanicah, ki niso vsebovale krioprotektantov med liofilizacijo popolnoma izgubili infektivnost. Ugotovili smo, da se je raven infektivnosti fagov v večji meri ohranila v mešanicah, ki niso bile liofilizirane kot pa v mešanicah, ki so bile liofilizirane, ter da se je infektivnost fagov v večji meri ohranila v mešanicah, ki so bile shranjene pri temperaturi 5+/-3 °C kot pa v mešanicah, ki so bile shranjene pri temperaturi 25 °C.

• Language: Slovenian
• Keywords: bakteriofagi, fagi, liofilizacija, stabilnost, fagna terapija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [A. Janežič]
• Year: 2024
• PID: 20.500.12556/RUL-158538
• UDC: 579.25(043.2)
• COBISS.SI-ID: 200066563
• Publication date in RUL: 15.06.2024

Secondary language

• Language: English
• Title: The effect of different lyophilization and storage protocols on the infectivity of Staphylococcus aureus, Pseudomonas aeruginosa and Cutibacterium acnes bacteriophages

Abstract

Bacteriophage therapy presents a promising alternative to the treatment of bacterial infections with antibiotics. Freeze-drying is an effective method for maintaining the stability of bacteriophages during long-term storage. The aim of our work was to investigate the effects of various cryoprotectants on infectivity perservation during and after freeze-drying of Staphylococcus aureus (JSAP1), Pseudomonas aeruginosa (JPAP1) and Cutibacterium acnes (JCAP1). Furthermore, we aimed to determine whether freeze-dried phages maintain a higher level of infectivity after three months storage than phages stored in suspensions and how the storage temperature influences phage titers. Initially, we determined the parameters of phage cultivation on a small scale and then continued with phage cultivation on a large scale. This was followed by filtration of bacteriophage lysates, concentration and diafiltration with tangential flow filtration and purification with size exclusion chromatography. We prepared various formulations of bacteriophages. The formulations were frozen and freeze dried. The results revealed a higher level of infectivity in freeze-dried formulations containing sugar cryoprotectants than in formulations containing PEG 6000 as a cryoprotectant. A three-month stability study was conducted. We found out that the level of infectivity of phages is maintained to a greater extent in formulations that have not been lyophilized compared to lyophilized formulations and we also found out that the level of infectivity of phages is maintained to a greater extent in formulations that were stored at 5+/-3°C compared to formulations stored at 25 °C.

• Keywords: Bacteriopgahes, phages, lyophilization, stability, phage therapy