Unravelling the potential of natural chelating agents in the control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms

Miguel M Leitão; Ariana S C Gonçalves; Joana Moreira; Carlos Fernandes; Fernanda Borges; Manuel Simões; Anabela Borges

doi:10.1016/j.ejmech.2024.117163

Unravelling the potential of natural chelating agents in the control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms

Eur J Med Chem. 2024 Dec 15:283:117163. doi: 10.1016/j.ejmech.2024.117163. Online ahead of print.

Authors

Miguel M Leitão¹, Ariana S C Gonçalves², Joana Moreira³, Carlos Fernandes³, Fernanda Borges³, Manuel Simões⁴, Anabela Borges⁵

Affiliations

¹ LEPABE-Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; ALICE-Associate Laboratory for Innovation in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; CIQUP-IMS-Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua Do Campo Alegre, 4169-007 Porto, Portugal.
² LEPABE-Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; ALICE-Associate Laboratory for Innovation in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; Environmental Health Department, Portuguese National Health Institute Doutor Ricardo Jorge, Porto, Portugal.
³ CIQUP-IMS-Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua Do Campo Alegre, 4169-007 Porto, Portugal.
⁴ LEPABE-Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; ALICE-Associate Laboratory for Innovation in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; DEQ-Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal.
⁵ LEPABE-Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; ALICE-Associate Laboratory for Innovation in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal; DEQ-Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465, Porto, Portugal. Electronic address: apborges@fe.up.pt.

PMID: 39700872
DOI: 10.1016/j.ejmech.2024.117163

Abstract

Iron is essential for the formation, maturation and dispersal of bacterial biofilms, playing a crucial role in the physiological and metabolic functions of bacteria as well as in the regulation of virulence. Limited availability of iron can impair the formation of robust biofilms by altering cellular motility, hydrophobicity and protein composition of the bacterial surface. In this study, the antibiofilm activity of two natural iron chelating agents, kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) and maltol (3-hydroxy-2-methyl-4-pyrone), were investigated against Staphylococcus aureus and Pseudomonas aeruginosa. In addition, the ability of these 2-hydroxy-4-pyrone derivatives in preventing and eradicating S. aureus and P. aeruginosa biofilms through the enhancement of the efficacy of two antibiotics (tobramycin and ciprofloxacin) was explored. The iron binding capacity of the kojic acid and maltol was confirmed by their affinity for iron (III) which was found to be about 90 %, comparable to the regular chelating agent ethylenediaminetetraacetic acid (EDTA, 89 %). The antibiofilm efficacy of 2-hydroxy-4-pyrone derivatives, alone and in combination with antibiotics, was evaluated by measuring the total biomass, metabolic activity, and culturability of biofilm cells. Furthermore, their impact on the membrane integrity of S. aureus biofilm cells was investigated using flow cytometry and epifluorescence microscopy with propidium iodide staining. It was also examined the ability of 2-hydroxy-4-pyrone derivatives and 2-hydroxy-4-pyrone derivate-antibiotic dual-combinations in inhibiting the production of virulence factors (total proteases, lipases, gelatinases and siderophores) by S. aureus. Regarding biofilm formation, the results showed that 2-hydroxy-4-pyrone derivatives alone reduced the metabolic activity of S. aureus biofilm cells by over 40 %. When combined with tobramycin, a 2-log (CFU cm^-2) reduction in S. aureus biofilm cells was observed. Moreover, the combination of maltol and kojic acid with ciprofloxacin prevented P. aeruginosa biomass production by 60 %, compared to 36 % with ciprofloxacin alone. In pre-established S. aureus and P. aeruginosa biofilms, selected compounds reduced the metabolic activity by over 75 %, and a 3-log (CFU cm^-2) reduction in the culturability of biofilm cells was noted when kojic acid and maltol were combined with antibiotics. Moreover, 2-hydroxy-4-pyrone derivatives alone and in combination with tobramycin, damaged the cell membranes of pre-established biofilms and completely inhibited total proteases production. Despite the increasing of reactive oxygen species production caused by the cellular treatment of maltol, both 2-hydroxy-4-pyrone derivatives showed good safe profile when tested in human hepatocarcinoma (HepG2) cells. The pre-treatment of HepG2 cells with both compounds was crucial to prevent the cellular damage caused by iron (III). This study demonstrates for the first time that the selected 2-hydroxy-4-pyrone derivatives significantly enhance the antibiofilm activity of tested antibiotics against S. aureus and P. aeruginosa, highlighting their potential as antibiotic adjuvants in preventing and eradicating biofilm-related infections.

Keywords: 2-Hydroxy-4-pyrone derivatives; Antibiofilm activity; Chelating activity; Dual combinations; Pseudomonas aeruginosa biofilms; Staphylococcus aureus biofilms.