Butucel, EugeniaBalta, IgoriMcCleery, DavidPopescu, Cosmin AlinIancu, TiberiuPet, IoanMarcu, AdelaHorablaga, Nicolae-MarinelStef, LaviniaCorcionivoschi, Nicolae2022-12-212022-12-212022-11-04Butucel, E., Balta, I., McCleery, D., Popescu, C.A., Iancu, T., Pet, I., Marcu, A., Horablaga, N.-M., Stef, L. and Corcionivoschi, N. (2022) ‘The Effect Citrox BCL on Legionella pneumophila Mechanisms of Biofilm Formation, Oxidative Stress and Virulence’, Antioxidants. MDPI AG. Available at: https://doi.org/10.3390/antiox11112186.2076-3921https://doi.org/10.3390/antiox11112186Publication history: Accepted - 1 November 2022; Published online - 4 November 2022Legionella pneumophila is responsible for causing Legionnaires’ disease and Pontiac fever, also known as legionellosis. The aim of this study was to investigate the mechanistic effect of a mixture of natural antimicrobials (Citrox BCL) in preventing L. pneumophila biofilm formation and reducing its in vitro virulence. The minimum inhibitory concentrations were detected at 0.06%, and the MBC was established at 0.125%. Based on the growth curve profile, the sub-inhibitory concentration of 0.02% was further used to study the mechanistic implications in the absence of a cytotoxic effect on A549 cells. At 24 h post-infection, Citrox BCL reduced (p = 0.005) the intracellular growth of L. pneumophila when the A549 cells or the bacteria were pre-treated with 0.02% Citrox BCL. This result was replicated when Citrox BCL was added during the 24 h infection assay leading to a reduction in intracellular growth (p = 0.003). Herein we show that at the sub-inhibitory concentration of 0.02%, Citrox CBL lowers the ROS levels in infected A549 cells and causes a 45% reduction in L. pneumophila EPS production, a reduction associated with the decline in biofilm formation. Overall, our results corroborate the low c-di-GMP production with the decrease in biofilm formation and low EPS levels. The low EPS levels seemed to be caused by the downregulation of the tatB and tatC gene expressions. Moreover, inhibition of pvcA and pvcB gene expressions, leading to lower siderophore levels, suggests that Citrox BCL reduces the ability of L. pneumophila to sequester iron and reduce biofilm formation through iron starvation.en© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/)L. pneumophilaCitrox BCLbiofilmvirulencesiderophoresArticle