Bacteria of the genus Listeria form a biofilm that, protects them from the harmful effects of the environment, and the bacteria within the biofilm are more resistant to external influences than planktonic bacteria. The content of the Master's thesis is related to the project of the Public Research Agency of the Republic of Slovenia (ARRS) "Bactericidal nanoparticles: a proof-of-concept approach for bimodal chemo-mechanical eradication of persistent biofilms". The aim of the Master's thesis was to test a method for removing the biofilm of Listeria innocua bacteria using three different nanoparticles by incubation in a magnetic field. We tested the effect of nanoparticles on a three-times-washed biofilm of Listeria innocua strain ŽM39, expressesing the fluorescent protein DsRedExpress. We analyzed the fluorescence and the number of live bacteria in the biofilm after each rinse. Then we tested the suitability of the dyes for monitoring the effect of nanocparticles on the biofilm of wild-type Listeria innocua strain ŽM39. After choosing the most suitable dye, we also monitored the effect of the nanoparticles on the biofilm of the bacteria Listeria innocua strain ŽM39, washed three times, with the addition of this dye. We also analyzed the fluorescence of the nanoparticles at different wavelenghts. We checked the growth of the bacteria Listeria innocua and Listeria monocytogenes ATCC 19115. Finally, we analyzed all the dyes on the intact and thrice-washed bacterial biofilm. We found that none of the nanoparticles used could successfully remove the biofilm of the Listeria innocua strain ŽM39 and that the viability of the bacteria decreases with each washing of the bacterial biofilm. The most useful dye turned out to be the dye 9-(diethylamino)-3-((1-(2-(methyl(tetradyl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)- 5H-benzo[a]phenoxazin-5-one (dye 10*) because it has the lowest fluorescence intensity of the blank. When monitoring the action of nanoparticles on a three-times-washed biofilm of Listeria innocua strain ŽM39 with the addition of dye 10*, similar values were obtained for all used nanoparticles, but the nanoparticle Hex showed to be the most effective. The nanoparticles had the strongest fluorescence at excitation wavelength of 563 nm and emission wavelength of 581 nm. The results of the analysis of all dyes showed that the dye (5-(4-(((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-3-yl)oxy)methyl)-1H-1,2, 3-triazol-1 yl)pentyl)triphenylphosphonium bromide (dye 7*) has the most negative effect on the growth of bacteria both on the intact biofilm and on the washed one. We have shown that fluorescent dyes or reporter proteins can be used to track Listeria strains when analyzing the effect of nanoparticles on biofilms.
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