Preučevanje reverzibilne vezave bakteriofagov na bakterije

Tovornik, Natalija (author), Podgornik, Aleš (mentor)

Preučevanje bakteriofagov (fagov) je v zadnjih letih zopet postalo aktualno, med drugim tudi zaradi povečane odpornosti nekaterih bakterij na antibiotike. Zaradi njihove zmožnosti specifičnega napadanja določene vrste ali seva bakterije, predstavljajo dobro alternativo uporabi antibiotikov. Fag T4 specifično napada bakterijo E. coli in fag PC5 bakterijo C. jejuni. Vsi fagi se v prvi stopnji na gostitelja vežejo reverzibilno, šele nato pride do ireverzibilne vezave in posledično morebitne infekcije celice. Preučevanje reverzibilne vezave obeh bakteriofagov oz. desorpcije in hitrost adsorpcije faga T4, smo zasledovali s spremljanjem titra prostih fagov, ki smo jih nadalje analizirali z metodo štetja plakov »spot assay«. Hitrost adsorpcije smo določili z izračunom adsorpcijske konstante, ki je znašala 3,18 × 10-9 ml/min. Pri fagu PC5 smo določili desorpcijo znatnega števila reverzibilno vezanih fagov, kar je omogočilo analizo s pomočjo Langmuirjeve adsorpcijske izoterme. Vezava faga T4 se je izkazala kot bistveno močnejša in pod pogoji redčenja v hladnem mediju nismo zaznali desorpcije, zato smo reverzibilno vezavo faga T4 preučili še z različnimi mediji, ki bi vplivali na različne tipe interakcij, kot sta dodatek 10 % etanola in dodatek 3 M NaCl, preverili pa smo tudi vpliv ultrazvoka. Z dodajanjem etanola smo dosegli povprečno koncentracijo desorbiranih fagov 1,98 × 104 PFU/ml, kar predstavlja 0,17 % začetnih fagov. Z dodajanjem NaCl povprečno koncentracijo 3,25 × 103 PFU/ml (0,02 % začetnih fagov) in z obdelavo v ultrazvočni kopeli povprečno koncentracijo 3,75 × 103 PFU/ml (0,04 % začetnih fagov). V vseh primerih je bil delež desorbiranih fagov izredno nizek, zaradi česar ne moremo trditi, da gre za reverzibilno desorpcijo.

• Language: Slovenian
• Keywords: bakteriofag PC5, Campylobacter jejuni, bakteriofag T4, E. coli, reverzibilna vezava, desorpcija, adsorpcija, adsorbcijska izoterma, kloroform, «spot assay«
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [N. Tovornik]
• Year: 2017
• PID: 20.500.12556/RUL-96734
• UDC: 606:602.3:578.347(043.2)
• COBISS.SI-ID: 8760441
• Publication date in RUL: 13.10.2017

TOVORNIK, Natalija, 2017, Preučevanje reverzibilne vezave bakteriofagov na bakterije [online]. Master’s thesis. N. Tovornik. [Accessed 5 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=96734

Secondary language

• Language: English
• Title: Study of bacteriophage reversible binding on bacteria

Abstract

Over the last several years bacteriophages (phages) related research became more interesting again. One of the reasons is the problem of antibiotic resistance that some bacteria has developed. Because of their natural ability for selective targeting to bacterial species or strains they represent a good therapeutic option in treatment of bacterial infections. Phage T4 specific targets E. coli and phage PC5 attacks C. jejuni. Phage adsorption involves reversible and irreversible interactions with host cells. Study of both phages reversible binding or desorption and speed of phage T4 adsorption was followed with free phages concentration and analyzed with »spot assay« method. Adsorption constant (adsorption speed) of T4 phage was 3,18 × 10-9 ml/min. We determined significant number of phage PC5 desorption which enabled analysis with Langmuir adsorption isotherm. It turns out that phage T4 binding on bacteria is much stronger and therefore we have not detected any desorption with 100-fold dilution in cold medium. We considered phage T4 reversible binding using different desorption media which could have an effect on different interaction types, like addition of 10 % ethanol and addition of 3 M NaCl. We also verified influence of ultrasonic treatment. We reached desorption of reversibly bonded phages in average concentration 1,98 × 104 PFU/ml with ethanol addition, (the number represent 0,17 % of initial phages), 3,25 × 103 PFU/ml with NaCl addition (0,02 % of initial phages) and 3,75 × 103 PFU/ml with ultrasonic treatment (0,04 % of initial phages). In all cases the percent of desorbed phages was extremely low, therefore we cannot absolutely talk about reversible desorption.

• Keywords: bacteriophage PC5, Campylobacter jejuni, bacteriophage T4, E. coli, reversible binding, desorption, adsorption, adsorption isotherm, chloroform, «spot assay«