MECHANISM OF ACTION OF AMPHIPHILIC CATIONIC PORPHYRIN TMPyP3-C17H35 ON LEGIONELA

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.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, porphyri