Title MEHANIZAM DJELOVANJA AMFIPATSKOG KATIONSKOG
PORFIRINA TMPyP3-C17H35 NA LEGIONELE
Title (english) MECHANISM OF ACTION OF AMPHIPHILIC CATIONIC
PORPHYRIN TMPyP3-C17H35 ON LEGIONELA
Author Andrija Lesar
Mentor Ivana Gobin (mentor)
Mentor Nela Malatesti (komentor)
Committee member Darinka Vučković (predsjednik povjerenstva)
Committee member Dijana Tomić Linšak (član povjerenstva)
Granter University of Rijeka Faculty of Medicine (Department of Microbiology and Parasitology) Rijeka
Granter University of Rijeka (Faculty of Biotechnology and Drug Development) Rijeka
Defense date and country 2021-02-22, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Public Health and Health Care Health Ecology
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Clinical Medical Sciences Medical Microbiology
Universal decimal classification (UDC ) 614 - Public health and hygiene. Accident prevention
Thesaurus (MESH - Medical Subject Headings )
Legionella pneumophila
Abstract Ciljevi istraživanja: Utvrditi mehanizam antimikrobnog fotodinamičkog učinka amfipatskog
kationskog porfirina TMPyP3-C17H35 na oportunističkog patogena vodoopskrbnih sustava –
legionelu. Zatim, ispitati brzinu fotoinaktivacije legionele u AYE bujonu i sterilnoj
vodovodnoj vodi te ispitati utječe li tvrdoća vode na fotoinaktivaciju kao i na inhibicije
stvaranja ili razaranja bakterijskog biofilma.
Metode: Kao modelni organizam korištena je Legionella pneumophila ATCC BAA–74, a
ispitivao se antimikrobni potencijal kationskog amfipatskog porfirina (5-(4-
oktadekanamidofenil)-10,15,20-tri(N-metilpiridin-3-il)porfirin klorid (TMPyP3-C17H35). In
vitro metodama određene su minimalna baktericidna koncentracija (MBK), minimalna
inhibitorna koncentracija (MIK) u AYE bujonu uz fotoinaktivaciju pokrenutu crvenim
svjetlom i minimalna efektivna koncentracija (MEK) uz fotoinaktivaciju pokrenutu ljubičastim
svjetlom. Ispitana je adhezija porfirina na bakterijske stanice fotoinaktivacijskim testom u AYE
bujonu i sterilnoj vodovodnoj vodi te je ispitana dinamika učinka porfirina TMPyP3-C17H35.
Mehanizam djelovanja porfirina TMPyP3-C17H35 ispitan je određivanjem curenja
unutarstaničnog sadržaja te elektronskom mikroskopijom. Vijabilnost legionela nakon
tretmana određivana je kokultivacijom s amebom Acanthamoeba castellanii i fluorescentnim
bojenjem „LIVE/DEAD“. Ispitano je i utječe li tvrdoća vode na adheziju L. pneumophila na
polistiren, na sposobnost stvaranja biofilma, kao i na sposobnost razaranja biofilma.
Rezultati: Rezultati ovog istraživanja pokazuju da ispitivani porfirin TMPyP3-C17H35 ima
antimikrobni fotodinamički učinak na legionele te da njegovom primjenom dolazi do
fotoinaktivacije legionele u bujonu i sterilnoj vodi, također je dokazano da ispitivani porfirin
uspješno adherira na stanice legionela. Rezultati nadalje pokazuju da nakon fotoinaktivacije
ispitivanim porfirinom dolazi do značajnih oštećenja legionela i bakterija više nije kultivabilna
ni vijabilna jer se ne može izolirati ni kokultivacijom s amebama, također je dokazan dobar
inhibitorni učinak na biofilm.
Zaključak: Ovim istraživanjem je dokazan dobar potencijal fotodinamičke inaktivacije
legionela korištenim fotosenzibilizatorom, porfirinom TMPyP3-C17H35 i njegova moguća
praktična primjena u suzbijanju legionela u vodoopskrbnim sustavima.
Abstract (english) Research objectives: To determine the mechanism of antimicrobial photodynamic effect of
amphipathic cationic porphyrin TMPyP3-C17H35 on opportunistic premise plumbing pathogen
- Legionella. Furthermore, to examine the rate of Legionella photoinactivation in AYE broth
and sterile tap water and to examine whether water hardness affects photoinactivation as well
as inhibitions of bacterial biofilm formation or destruction.
Methods: Legionella pneumophila ATCC BAA – 74 was used as a model organism, and the
antimicrobial potential of cationic amphipathic porphyrin (5-(4-octadecanamidophenyl)-
10,15,20-tri(N-methylpyridinium-3-yl)porphyrin chloride (TMPyP3-C17H35) has been
researched. Minimum bactericidal concentration (MBK), minimum inhibitory concentration
(MIC) in AYE broth with red light initiated photoinactivation and minimum effective
concentration (MEK) by purple light initiated photoinactivation were determined with in vitro
methods.
The adhesion of porphyrins to bacterial cells was examined as well as photoinactivation test in
AYE broth and sterile tap water, where the dynamics of the antibacterial effect of porphyrin
TMPyP3-C17H35 was examined. The mechanism of action of porphyrin TMPyP3-C17H35 was
examined by determining cellular leakage and by electron microscopy. The viability of
Legionella after treatment was determined by co-cultivation with the amoeba Acanthamoeba
castellanii and "LIVE/DEAD" fluorescent staining technique. Whether the hardness of water
affects the adhesion of L. pneumophila to polystyrene, the ability to form a biofilm, as well as
the ability to destroy the biofilm was also determined.
Results: The results of this study show that the tested porphyrin TMPyP3-C17H35 has an
antimicrobial photodynamic effect on legionella and that its application leads to
photoinactivation of legionella in broth and sterile water, it was also proven that the tested
porphyrin successfully adheres to Legionella cells. The results further show that after
photoinactivation with the tested porphyrin there is significant damage to legionella and the
bacterium is no longer culturable or viable because it cannot be isolated by cocultivation with
amoebae, and a good inhibitory effect on biofilm has also been demonstrated.
Conclusion: This study demonstrated the good potential of photodynamic inactivation of
legionella by the used photosensitizer, porphyrin TMPyP3-C17H35 and its possible practical
application in the control of legionella in water supply systems.
Key words: disinfection, photodynamic inactivation, Legionella, Opportunistic Premise
Plumbing Pathogens, porphyrin
Keywords
dezinfekcija
fotodinamička inaktivacija
Legionella
oportunistički patogeni vodoopskrbnih sustava
porfirini
Keywords (english)
disinfection
photodynamic inactivation
Legionella
Opportunistic Premise Plumbing Pathogens
porphyrins
Language croatian
URN:NBN urn:nbn:hr:184:772323
Promotion 2022
Study programme Title: Biomedicine Postgraduate (doctoral) study programme Study programme type: university Study level: postgraduate Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
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Created on 2022-01-24 08:29:55