{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,7]],"date-time":"2026-03-07T01:50:15Z","timestamp":1772848215328,"version":"3.50.1"},"reference-count":41,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2022,11,4]],"date-time":"2022-11-04T00:00:00Z","timestamp":1667520000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ph.D. of Eugenia Butucel","award":["49650"],"award-info":[{"award-number":["49650"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antioxidants"],"abstract":"<jats:p>Legionella pneumophila is responsible for causing Legionnaires\u2019 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.<\/jats:p>","DOI":"10.3390\/antiox11112186","type":"journal-article","created":{"date-parts":[[2022,11,7]],"date-time":"2022-11-07T02:43:51Z","timestamp":1667789031000},"page":"2186","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["The Effect Citrox BCL on Legionella pneumophila Mechanisms of Biofilm Formation, Oxidative Stress and Virulence"],"prefix":"10.3390","volume":"11","author":[{"given":"Eugenia","family":"Butucel","sequence":"first","affiliation":[{"name":"Bacteriology Branch, Veterinary Sciences Division, Agri-Food and Biosciences Institute, Belfast BT4 3SD, UK"},{"name":"Faculty of Bioengineering of Animal Resources, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1171-2623","authenticated-orcid":false,"given":"Igori","family":"Balta","sequence":"additional","affiliation":[{"name":"Faculty of Bioengineering of Animal Resources, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"McCleery","sequence":"additional","affiliation":[{"name":"Bacteriology Branch, Veterinary Sciences Division, Agri-Food and Biosciences Institute, Belfast BT4 3SD, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cosmin Alin","family":"Popescu","sequence":"additional","affiliation":[{"name":"Faculty of Agriculture, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1645-5064","authenticated-orcid":false,"given":"Tiberiu","family":"Iancu","sequence":"additional","affiliation":[{"name":"Faculty of Management and Rural Development, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2157-5009","authenticated-orcid":false,"given":"Ioan","family":"Pet","sequence":"additional","affiliation":[{"name":"Faculty of Bioengineering of Animal Resources, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9581-8208","authenticated-orcid":false,"given":"Adela","family":"Marcu","sequence":"additional","affiliation":[{"name":"Faculty of Bioengineering of Animal Resources, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolae-Marinel","family":"Horablaga","sequence":"additional","affiliation":[{"name":"Faculty of Agriculture, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7068-4223","authenticated-orcid":false,"given":"Lavinia","family":"Stef","sequence":"additional","affiliation":[{"name":"Faculty of Bioengineering of Animal Resources, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3011-3108","authenticated-orcid":false,"given":"Nicolae","family":"Corcionivoschi","sequence":"additional","affiliation":[{"name":"Bacteriology Branch, Veterinary Sciences Division, Agri-Food and Biosciences Institute, Belfast BT4 3SD, UK"},{"name":"Faculty of Bioengineering of Animal Resources, University of Life Sciences King Mihai I from Timisoara, 300645 Timisoara, Romania"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Mraz, A.L., and Weir, M.H. (2018). Knowledge to Predict Pathogens: Legionella pneumophila Lifecycle Critical Review Part I Uptake into Host Cells. Water, 10.","DOI":"10.3390\/w10020132"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"221","DOI":"10.4103\/2221-6189.272853","article-title":"Legionella and legionnaires\u2019 disease: An overview","volume":"8","author":"Jomehzadeh","year":"2019","journal-title":"J. Acute Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"477","DOI":"10.3389\/fcimb.2017.00477","article-title":"From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella","volume":"7","author":"Boamah","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_4","first-page":"159138","article-title":"The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids","volume":"1867","author":"Jurak","year":"2022","journal-title":"Biochim. Et Biophys. Acta (BBA)\u2014Mol. Cell Biol. Lipids"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3978","DOI":"10.1038\/s41598-020-60829-2","article-title":"In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts","volume":"10","author":"Ecale","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Falkinham, J.O. (2020). Living with Legionella and Other Waterborne Pathogens. Microorganisms, 8.","DOI":"10.3390\/microorganisms8122026"},{"key":"ref_7","unstructured":"Khweek, A., and Amer, A. (2019). Biofilm, a Cozy Structure for Legionella pneumophila Growth and Persistence in the Environment. Bacterial Biofilms, IntechOpen."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"38","DOI":"10.3389\/fcimb.2018.00038","article-title":"Factors Mediating Environmental Biofilm Formation by Legionella pneumophila","volume":"8","author":"Amer","year":"2018","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1122","DOI":"10.1080\/21505594.2021.1903199","article-title":"Pathogenicity and Virulence of Legionella: Intracellular replication and host response","volume":"12","author":"Chauhan","year":"2021","journal-title":"Virulence"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"E22-E22","DOI":"10.1038\/sj.bdj.2010.1224","article-title":"Antimicrobial activity of Citrox\u00ae bioflavonoid preparations against oral microorganisms","volume":"210","author":"Hooper","year":"2011","journal-title":"Br. Dent. J."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Yehia, H.M., Al-Masoud, A.H., Elkhadragy, M.F., Korany, S.M., Nada, H.M.S., Albaridi, N.A., Alzahrani, A.A., and Al-Dagal, M.M. (2021). Improving the Quality and Safety of Fresh Camel Meat Contaminated with Campylobacter jejuni Using Citrox, Chitosan, and Vacuum Packaging to Extend Shelf Life. Animals, 11.","DOI":"10.3390\/ani11041152"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"107745","DOI":"10.1016\/j.foodcont.2020.107745","article-title":"The effect of natural antimicrobials against Campylobacter spp. and its similarities to Salmonella spp, Listeria spp., Escherichia coli, Vibrio spp., Clostridium spp. and Staphylococcus spp","volume":"121","author":"Balta","year":"2021","journal-title":"Food Control"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"108998","DOI":"10.1016\/j.ijfoodmicro.2020.108998","article-title":"The effect of natural antimicrobials on the Campylobacter coli T6SS+\/\u2212 during in vitro infection assays and on their ability to adhere to chicken skin and carcasses","volume":"338","author":"Balta","year":"2021","journal-title":"Int. J. Food Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Pinkerton, L., Linton, M., Kelly, C., Ward, P., Gradisteanu Pircalabioru, G., Pet, I., Stef, L., Sima, F., Adamov, T., and Gundogdu, O. (2019). Attenuation of Vibrio parahaemolyticus Virulence Factors by a Mixture of Natural Antimicrobials. Microorganisms, 7.","DOI":"10.3390\/microorganisms7120679"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"67","DOI":"10.2166\/washdev.2017.140","article-title":"Removing Legionella pneumophila and biofilms from water supply systems using plant essential oils","volume":"7","author":"Ceylan","year":"2017","journal-title":"J. Water Sanit. Hyg. Dev."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Gelmini, F., Testa, C., Angioletti, S., and Beretta, G. (2017). Essential Oils in the Environmental Bacteria Control: The Legionella Pneumophila case. J. Complement. Med. Altern. Healthc., 2.","DOI":"10.19080\/JCMAH.2017.02.555577"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"54","DOI":"10.2478\/ijser-2020-0005","article-title":"Antimicrobial activity of the volatile phase of essential oils and their constituents on","volume":"14","author":"Gobin","year":"2020","journal-title":"Sanit. In\u017eenirstvo Int. J. Sanit. Eng. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"16202","DOI":"10.1038\/s41598-021-95459-9","article-title":"The in vitro and in vivo anti-virulent effect of organic acid mixtures against Eimeria tenella and Eimeria bovis","volume":"11","author":"Balta","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2139","DOI":"10.3389\/fmicb.2018.02139","article-title":"A Novel Natural Antimicrobial Can Reduce the in vitro and in vivo Pathogenicity of T6SS Positive Campylobacter jejuni and Campylobacter coli Chicken Isolates","volume":"9","author":"Sima","year":"2018","journal-title":"Front. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Balta, I., Stef, L., Butucel, E., Gradisteanu Pircalabioru, G., Venig, A., Ward, P., Deshaies, M., Pet, I., Stef, D., and Koyun, O.Y. (2022). The Antioxidant Effect of Natural Antimicrobials in Shrimp Primary Intestinal Cells Infected with Nematopsis messor. Antioxidants (Basel), 11.","DOI":"10.3390\/antiox11050974"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1493","DOI":"10.1038\/s41467-022-29240-5","article-title":"cAMP and c-di-GMP synergistically support biofilm maintenance through the direct interaction of their effectors","volume":"13","author":"Liu","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1016\/j.bbrc.2004.03.091","article-title":"A putative twin-arginine translocation pathway in Legionella pneumophila","volume":"317","author":"Lebeau","year":"2004","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1080\/21505594.2017.1373925","article-title":"Differential expression of virulence genes in Legionella pneumophila growing in Acanthamoeba and human monocytes","volume":"9","author":"Mou","year":"2018","journal-title":"Virulence"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1264\/jsme2.ME16010","article-title":"Iron Availability Modulates the Persistence of Legionella pneumophila in Complex Biofilms","volume":"31","author":"Portier","year":"2016","journal-title":"Microbes Environ."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Stratakos, A.C., Linton, M., Ward, P., Campbell, M., Kelly, C., and Pinkerton, L. (2019). The antimicrobial effect of a commercial mixture of natural antimicrobials against Escherichia coli O157:H7. Foodborne Pathog. Dis., 16.","DOI":"10.1089\/fpd.2018.2465"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"63","DOI":"10.3389\/fcimb.2017.00063","article-title":"PilY1 Promotes Legionella pneumophila Infection of Human Lung Tissue Explants and Contributes to Bacterial Adhesion, Host Cell Invasion, and Twitching Motility","volume":"7","author":"Hoppe","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4405","DOI":"10.1128\/IAI.00575-08","article-title":"Mycoplasma pneumoniae infection induces reactive oxygen species and DNA damage in A549 human lung carcinoma cells","volume":"76","author":"Sun","year":"2008","journal-title":"Infect. Immun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1007\/s00284-004-4342-3","article-title":"Siderophore activity among members of the Legionella genus","volume":"49","author":"Starkenburg","year":"2004","journal-title":"Curr. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/0076-6879(94)35151-1","article-title":"Detection, isolation, and characterization of siderophores","volume":"235","author":"Payne","year":"1994","journal-title":"Methods Enzymol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1128\/JB.182.3.749-757.2000","article-title":"Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila","volume":"182","author":"Liles","year":"2000","journal-title":"J. Bacteriol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1204","DOI":"10.3389\/fmicb.2018.01204","article-title":"Enhanced Biofilm Formation by Ferrous and Ferric Iron Through Oxidative Stress in Campylobacter jejuni","volume":"9","author":"Oh","year":"2018","journal-title":"Front. Microbiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1016\/j.ab.2004.03.049","article-title":"Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells","volume":"331","author":"Riemer","year":"2004","journal-title":"Anal. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1016\/j.watres.2007.07.008","article-title":"Antibacterial activities of plant essential oils against Legionella pneumophila","volume":"42","author":"Chang","year":"2008","journal-title":"Water Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"6031","DOI":"10.1128\/AEM.01275-14","article-title":"Reduction of Legionella spp. in water and in soil by a citrus plant extract vapor","volume":"80","author":"Laird","year":"2014","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1146\/annurev.micro.61.080706.093426","article-title":"Roles of cyclic diguanylate in the regulation of bacterial pathogenesis","volume":"61","author":"Tamayo","year":"2007","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1413","DOI":"10.1016\/j.bbrc.2005.04.060","article-title":"Legionella pneumophila Philadelphia-1 tatB and tatC affect intracellular replication and biofilm formation","volume":"331","author":"Maes","year":"2005","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e01043-19","DOI":"10.1128\/AEM.01043-19","article-title":"Cyclic di-GMP Increases Catalase Production and Hydrogen Peroxide Tolerance in Vibrio cholerae","volume":"85","author":"Fernandez","year":"2019","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1111\/2049-632X.12132","article-title":"The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance of Pseudomonas aeruginosa","volume":"70","author":"Djapgne","year":"2014","journal-title":"Pathog. Dis."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Mraz, A.L., and Weir, M.H. (2022). Knowledge to Predict Pathogens: Legionella pneumophila Lifecycle Systematic Review Part II Growth within and Egress from a Host Cell. Microorganisms, 10.","DOI":"10.3390\/microorganisms10010141"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"454","DOI":"10.3389\/fcimb.2017.00454","article-title":"The Flagellar Regulon of Legionella\u2014A Review","volume":"7","author":"Appelt","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1038\/s41396-020-00774-0","article-title":"Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms","volume":"15","author":"Personnic","year":"2021","journal-title":"ISME J."}],"container-title":["Antioxidants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-3921\/11\/11\/2186\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:10:41Z","timestamp":1760145041000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-3921\/11\/11\/2186"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,4]]},"references-count":41,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2022,11]]}},"alternative-id":["antiox11112186"],"URL":"https:\/\/doi.org\/10.3390\/antiox11112186","relation":{},"ISSN":["2076-3921"],"issn-type":[{"value":"2076-3921","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,4]]}}}