Photodynamic inactivation of ESKAPE group bacterial pathogens in planktonic and biofilm cultures using metallated porphyrin-doped conjugated polymer nanoparticles

LUIS IBARRA; ANA WENDEL; RODRIGO PALACIOS; SOL ROMINA MARTINEZ; VIRGINIA FORCONE; MARIANA SPESIA; RODRIGO PONZIO; CARLOS CHESTA

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Jornada; II Jornada de Jovenes Nanobiocientificxs; 2020

Microorganisms, particularly bacteria, have been natives of the earth for 3.5millions of years and their evolution demonstrates that they can adapt to manydifferent environments. The antibiotic development has seemed to give battleagainst pathogens. However, in 2017 the World Health Organization (WHO) published itsfirst-ever list of antibiotic-resistant priority pathogens. The most criticalgroup of multidrug resistant bacteria were Escherichiacoli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and themethicillin-resistant Staphylococcusaureus (MRSA). In response to this latent threat, scientists developedalternative therapies such as photodynamic inactivation (PDI), which emergedlate in the 20th century as a promising treatment to kill pathogens.In this work, we efficiently combine photoactive dye doped polymericnanoparticles (CPNs) with light to kill some multidrug pathogens included inthe WHO list. Initial screening with different CPNs concentrations (0.3 to 6.5 ppm) andirradiation doses was performed in 9 pathogenic strains to explore theefficiency of the PDI treatment in planktonic cells. Bactericidal effect wasobserved in MRSA strains using shortirradiation period/ low light dose (10-15 min/ 9.6-14.4 J/cm2). TheGram-negative species required higher light (28.8 J/cm2) and CPNs(16.5 ppm) doses. Bacterial CPNs uptake was initially studied using bright-field andfluorescent microscopy taking advantage of the intrinsic NPs fluorescence. Further studies on CPNs uptake for different bacterial species were performed using flowcytometry. Restults from both techniques indicate that CPNs superficially bindand slightly penetrate the bacterial envelope. To further explore PDI treatmentstrategies using our CPNs we tested their performance on mature bacterial biofilms.In both Gram-positive and negative bacteria, the crystal violet assay displayeda similar antibiofilm trend after PDI treatment. At the same time, the effectachieved on the metabolic activity of the cellular cluster after PDI treatmentwas larger in Gram-negative cells than Gram-positive strains; showing a drop inthe activity between 67 to 84 % respect to the control. To further test theoverall effect on the biofilm matrix and the eradication effect of our PDItherapy we used confocal microscopy. For these experiments, E. coli and S. aureus were selected and both strains exhibited substantialmatrix disruption. Notably, almost complete biofilm eradication was achievedfor the E. coli strain. Overall, ourresults demonstrated that PDI protocols using NPs plus blue light are anefficient tool not only to kill superbugs as sessile cells but also to disruptand eradicate mature biofilms of relevant bacterial species.