Usnic Acid Activity on Oxidative and Nitrosative Stress of Azole-Resistant Candida albicans Biofilm

Abstract

Several studies report that (+)-usnic acid, a lichen secondary metabolite, inhibits growth of different bacteria and fungi; however, the mechanism of its antimicrobial activity remains unknown. In this study, we explored the ability of usnic acid, obtained from Usnea amblyoclada, as an antibiofilm agent against azole-resistant and azole-sensitive Candida albicans strains by studying the cellular stress and antioxidant response in biofilms. The biofilm inhibitory concentration of usnic acid (4 µg/mL) exhibited a significant biofilm inhibition, 71.08 % for azole-resistant and 87.84 % for azole-sensitive C. albicans strains. Confocal scanning laser microscopy showed that the morphology of mature biofilm was altered (reduced the biomass and thickness) in the presence of usnic acid. The antifungal effect was mediated by an oxidative and nitrosative stress, with a significant accumulation of intracellular and extracellular reactive oxygen species detected by confocal scanning laser microscopy and by nitro blue tetrazolium, respectively. In fact, azole-resistant and azole-sensitive C. albicans biofilms treated at the biofilm inhibitory concentration of usnic acid presented 30-fold and 10-fold increased reactive oxygen species measurements compared to basal levels, respectively, and important nitric oxide generation, showing 25-fold and 60-fold increased reactive nitrogen intermediates levels with respect to the controls, respectively. Nonenzymatic and enzymatic antioxidant defenses were increased in both strains compared to biofilm basal levels as response to the increase of oxidant metabolites. The present study shows for the first time that usnic acid can alter the prooxidant-antioxidant balance, which may be the cause of the irreversible cell damage and lead to cell death. Our results suggest that usnic acid could be an alternative for the treatment of Candida infections, which deserves further investigation.

Key words

Usnea amblyoclada - Usneaceae - usnic acid - Candida albicans - biofilms - prooxidant-antioxidant balance

• References

• 1 Ta CA, Freundorfer M, Mah TF, Otárola-Rojas M, Garcia M, Sanchez-Vindas P, Poveda L, J. Alan Maschek JA, Baker BJ, Adonizio AL, Downum K, Durst T, Arnason JT. Inhibition of bacterial quorum sensing and biofilm formation by extracts of neotropical rainforest plants. Planta Med 2014; 80: 343-350
• 2 Messier C, Epifano F, Genovese S, Grenier D. Inhibition of Candida albicans biofilm formation and yeast-hyphal transition by 4-hydroxycordoin. Phytomedicine 2011; 18: 380-383
• 3 Cao YY, Dai BD, Wang Y, Huang S, Xu YG, Cao YB, Gao PH, Zhu ZY, Jiang YY. In vitro activity of baicalein against Candida albicans biofilms. Int J Antimicrob Agents 2008; 32: 73-77
• 4 Walencka E, Rozalska S, Wysokinska H, Rozalski M, Kuzma L, Rozalska B. Salvipisone and aethiopinone from Salvia sclarea hairy roots modulate staphylococcal antibiotic resistance and express anti-biofilm activity. Planta Med 2007; 73: 545-551
• 5 Rodriguez JM, Estrabou C. Usnea amblyoclada «Barba de piedra» (Ascomycetes liquenizados) en Argentina. Bol Soc Argent Bot 2008; 43: 221-225
• 6 Correche ER, Carrasco M, Escudero ME, Velazquez L, de Guzmán AMS, Giannini F, Enriz RD, Jáuregui EA, Cenal JP, Giordano OS. Study of the cytotoxic and antimicrobial activities of usnic acid and derivatives. Fitoterapia 1998; 69: 493-501
• 7 Ingólfsdóttir K. Usnic acid. Phytochemistry 2002; 61: 729-736
• 8 Pires RH, Lucarini R, Soares Mendes-Giannini MJ. Effect of usnic acid on Candida orthopsilosis and C. parapsilosis . Antimicrob Agents Chemother 2012; 56: 595-597
• 9 Francolini I, Norris P, Piozzi A, Donelli G, Stoodley P. Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces. Antimicrob Agents Chemother 2004; 48: 4360-4365
• 10 Nithyanand P, Beema Shafreen RM, Muthamil S, Karutha Pandian S. Usnic acid inhibits biofilm formation and virulent morphological traits of Candida albicans . Microbiol Res 2015; 179: 20-28
• 11 Pompilio A, Pomponio S, Di Vincenzo V, Crocetta V, Nicoletti M, Piovano M, Juan A Garbarino JA, Di Bonaventura G. Antimicrobial and antibiofilm activity of secondary metabolites of lichens against methicillin-resistant Staphylococcus aureus strains from cystic fibrosis patients. Future Microbiol 2013; 8: 281-292
• 12 Cannon R, Lamping E, Holmes A, Niimi K, Baret P, Keniya M, Tanabe K, Niimi M, Goffeau A, Monk C. Efflux mediated antifungal drug resistance. Clin Microbiol Rev 2009; 22: 291-321
• 13 Delattin N, Cammue BP, Thevissen K. Reactive oxygen species inducing antifungal agents and their activity against fungal biofilms. Future Med Chem 2014; 6: 77-90
• 14 Maurya IK, Pathak S, Sharma M, Sanwal H, Chaudhary P, Tupe S, Deshpande M, Chauhan VS, Prasad R. Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans . Peptides 2011; 32: 1732-1740
• 15 Peralta MA, da Silva MA, Ortega MG, Cabrera JL, Paraje MG. Antifungal activity of a prenylated flavonoid from Dalea elegans against Candida albicans biofilms. Phytomedicine 2015; 22: 975-980
• 16 Gupta VK, Verma S, Gupta S, Singh A, Pal A, Srivastava SK, Srivastava PK, Singh SC, Darokar MP. Membrane-damaging potential of natural L-(−)-usnic acid in Staphylococcus aureus . Eur J Clin Microbiol Infect Dis 2012; 31: 3375-3383
• 17 Vandenbosch D, Braeckmans K, Nelis HJ, Coenye T. Fungicidal activity of miconazole against Candida spp. biofilms. J Antimicrob Chemother 2010; 65: 694-700
• 18 Arce Miranda JE, Sotomayor CE, Albesa I, Paraje MG. Oxidative and nitrosative stress in Staphylococcus aureus biofilm. FEMS Microbiol Lett 2011; 1: 23-29
• 19 Mahl CD, Behling CS, Hackenhaar FS, de Carvalho e Silva MN, Putti J, Salomon TB, Alves SH, Fuentefria A, Benfato MS. Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage. Diagn Microbiol Infect Dis 2015; 82: 203-208
• 20 Da Silva Dantas A, Day A, Ikeh M, Kos I, Achan B, Quinn J. Oxidative stress responses in the human fungal pathogen, Candida albicans . Biomolecules 2015; 5: 142-165
• 21 Maciazg-Dorszynska M, Wezgrzyn G, Guzow-Krzeminska B. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis. FEMS Microbiol Lett 2014; 353: 57-62
• 22 Mah T. Biofilm-specific antibiotic resistance. Future Microbiol 2012; 7: 1061-1072
• 23 White T, Holleman S, Dy F, Mirels L, Stevens D. Resistance mechanisms in clinical isolates of Candida albicans . Antimicrob Agents Chemother 2002; 46: 1704-1713
• 24 Páez PL, Becerra MC, Albesa I. Antioxidative mechanisms protect resistant strains of Staphylococcus aureus against ciprofloxacin oxidative damage. Fundam Clin Pharmacol 2010; 24: 771-776
• 25 Rashid MA, Majid MA, Quader MA. Complete NMR assignments of (+)-usnic acid. Fitoterapia 1999; 70: 113-115
• 26 CLSI – Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution antifungal Susceptibility Testing of Yeasts. Approved Standard. 2nd Edition. CLSI Document M27-A2 Wayne, PA: CLSI; 2002
• 27 Pierce CG, Uppuluri P, Tristan AR, Wormley Jr. FL, Mowat E, Ramage G, Lopez-Ribot JL. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 2008; 3: 1494-1500
• 28 Ramage G, López-Ribot JL. Techniques for antifungal susceptibility testing of Candida albicans biofilms. Methods Mol Med 2005; 118: 71-79
• 29 Angel Villegas N, Baronetti J, Albesa I, Polifroni R, Parma A, Etcheverría A, Becerra M, Padola N, Paraje MG. Relevance of biofilms in the pathogenesis of shiga-toxin-producing Escherichia coli infection. Scientific World J 2013; 2013: 607258
• 30 Zhao T, Liu Y. N-acetylcysteine inhibit biofilms produced by Pseudomonas aeruginosa . BMC Microbiol 2010; 10: 140-147
• 31 CLSI – Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution antifungal Susceptibility Testing of Yeasts. Approved Standard. 3rd Edition. CLSI Document M27-A3 Wayne, PA: CLSI; 2008
• 32 Weissman L, Garty J, Hochman A. Rehydration of the lichen Ramalina lacera results in production of reactive oxygen species and nitric oxide and a decrease in antioxidants. Appl Environ Microbiol 2005; 71: 2121-2129