Aspergillus fumigatus DBM 4057 biofilm formation is inhibited by chitosan, in contrast to baicalein and rhamnolipid

  • Eva Kvasničková
  • Vít Paulíček
  • Martina Paldrychová
  • Richard Ježdík
  • Olga Maťátková
  • Jan Masák
Original Paper

Abstract

The biofilms of filamentous-forming fungi are a novel and still insufficiently understood research topic. We have studied Aspergillus fumigatus, an ubiquitous opportunistic pathogenic fungus, as a representative model for a study of biofilm formation by filamentous fungi and for assessing the potential anti-biofilm activity of natural substances. The activity of antibiotic amphotericin B and selected natural substances: baicalein, chitosan and rhamnolipid was studied. The minimum suspension inhibitory concentrations (MIC) were determined and the biofilm susceptibility was investigated by determining the metabolic activity of sessile cells (XTT assay) and total biofilm biomass (crystal violet staining). Significant time-dependent differences in substances’ anti-biofilm activity were observed. Images of A. fumigatus biofilm were obtained by Cellavista automatic light microscope and spinning disc confocal microscopy. Baicalein and rhamnolipid were not found as suitable substances for inhibition of the A. fumigatus biofilm formation, as neither of the substances inhibited the sessile cells metabolic activity or the total biofilm biomass even at tenfold MIC after 48 h. In contrast, chitosan at 10 × MIC (25 µg mL−1), suppressed the biofilm metabolic activity by 90 % and the total biofilm biomass by 80 % even after 72 h of cultivation. Amphotericin B inhibited only 14 % of total biofilm biomass (crystal violet staining) and 35 % of metabolic activity (XTT assay) of adherent cells under the same conditions. Our results therefore suggest chitosan as potential alternative for treating A. fumigatus biofilm-associated infections.

Graphical Abstract

Keywords

Amphotericin B Aspergillus fumigatus Biofilm Chitosan Rhamnolipid 

References

  1. Abdel-Mawgoud A, Lépine F, Déziel E (2010) Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86:1323–1336CrossRefGoogle Scholar
  2. Andrews JM (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48:5–16CrossRefGoogle Scholar
  3. Beauvais A, Schmidt C, Guadagnini S, Roux P, Perret E, Henry C, Paris S, Mallet A, Prevost MC, Latge JP (2007) An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus. Cell Microbiol 9:1588–1600CrossRefGoogle Scholar
  4. Brakhage AA (2005) Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Curr Drug Targets 6:875–886CrossRefGoogle Scholar
  5. Bugli F, Posteraro B, Papi M, Torelli R, Maiorana A, Paroni Sterbini F, Posteraro P, Sanguinetti M, De Spirito M (2013) In vitro interaction between alginate lyase and amphotericin B against Aspergillus fumigatus biofilm determined by different methods. Antimicrob Agents Chemother 57:1275–1282CrossRefGoogle Scholar
  6. Cao Y, Dai B, Wang Y, Huang S, Xu Y, Cao Y, Gao P, Zhu Z, Jiang Y (2008) In vitro activity of baicalein against Candida albicans biofilms. Int J Antimicrob Agents 32:73–77CrossRefGoogle Scholar
  7. Chryssanthou E, Loebig A, Sjölin J (2008) Post-antifungal effect of amphotericin B and voriconazole against germinated Aspergillus fumigatus conidia. J Antimicrob Chemother 61:1309–1311CrossRefGoogle Scholar
  8. Colombo A, McGough D, Rinaldi M (1994) Discrepancies between MIC and MLC values of amphotericin B against isolates of Aspergillus species. Mycopathologia 128:129–133CrossRefGoogle Scholar
  9. Cota-Arriola O, Cortez-Rocha MO, Rosas-Burgos EC, Burgos-Hernández A, López-Franco YL, Plascencia-Jatomea M (2011) Antifungal effect of chitosan on the growth of Aspergillus parasiticus and production of aflatoxin B1. Polym Int 60:937–944CrossRefGoogle Scholar
  10. Dai BD, Cao YY, Huang S, Xu YG, Gao PH, Wang Y, Jiang YY (2009) Baicalein induces programmed cell death in Candida albicans. J Microbiol Biotechn 19:803–809Google Scholar
  11. Fanfair RN, Benedict K, Bos J, Bennett SD, Lo YC, Adebanjo T, Etienne K, Deak E, Derado G, Shieh WJ, Drew C, Zaki S, Sugerman D, Gade L, Thompson EH, Sutton DA, Engelthaler DM, Schupp JM, Brandt ME, Harris JR, Lockhart SR, Turabelidze G, Park BJ (2012) Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in 2011. N Engl J Med 367:2214–2225CrossRefGoogle Scholar
  12. Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O (2010) Antibiotic resistance of bacterial biofilms. Int J Antimicrob Ag 35:322–332CrossRefGoogle Scholar
  13. Hoskova M, Jezdik R, Schreiberova O, Chudoba J, Sir M, Cejkova A, Masak J, Jirku V, Rezanka T (2015) Structural and physiochemical characterization of rhamnolipids produced by Acinetobacter calcoaceticus, Enterobacter asburiae and Pseudomonas aeruginosa in single strain and mixed cultures. J Biotechnol 193:45–51CrossRefGoogle Scholar
  14. Kaur S, Singh S (2014) Biofilm formation by Aspergillus fumigatus. Med Mycol 52:2–9Google Scholar
  15. Kvasnickova E, Matatkova O, Cejkova A, Masak J (2015) Evaluation of baicalein, chitosan and usnic acid effect on Candida parapsilosis and Candida krusei biofilm using a Cellavista device. J Microbiol Methods 118:106–112CrossRefGoogle Scholar
  16. Latge JP (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12:310–350Google Scholar
  17. Lelievre L, Groh M, Angebault C, Maherault AC, Didier E, Bougnoux ME (2013) Azole resistant Aspergillus fumigatus: an emerging problem. Med Maladies Infect 43:139–145CrossRefGoogle Scholar
  18. Li DD, Zhao LX, Mylonakis E, Hu GH, Zou Y, Huang TK, Yan L, Wang Y, Jiang YY (2014) In vitro and in vivo activities of pterostilbene against Candida albicans biofilms. Antimicrob Agents Chemother 58:2344–2355CrossRefGoogle Scholar
  19. Maiorana A, Papi M, Bugli F, Torelli R, Maulucci G, Cacaci M, Posteraro B, Sanguinetti M, De Spirito M (2013) A fast and quantitative evaluation of the Aspergillus fumigatus biofilm adhesion properties by means of digital pulsed force mode. Appl Surf Sci 279:409–415CrossRefGoogle Scholar
  20. Martinez LR, Mihu MR, Tar M, Cordero RJB, Han G, Friedman AJ, Friedman JM, Nosanchuk JD (2010) Demonstration of antibiofilm and antifungal efficacy of chitosan against candidal biofilms, using an in vivo central venous catheter model. J Infect Dis 201:1436–1440CrossRefGoogle Scholar
  21. McFeeters H, McFeeters R (2013) Emerging approaches to inhibit Botrytis cinerea. Int J Mod Bot 2:127–144CrossRefGoogle Scholar
  22. Meletiadis J, te Dorsthorst DTA, Verweij PE (2003) Use of turbidimetric growth curves for early determination of antifungal drug resistance of filamentous fungi. J Clin Microbiol 41:4718–4725CrossRefGoogle Scholar
  23. Mowat E, Butcher J, Lang S, Williams C, Ramage G (2007) Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigatus. J Med Microbiol 56:1205–1212CrossRefGoogle Scholar
  24. Müller F-MC, Seidler M, Beauvais A (2011) Aspergillus fumigatus biofilms in the clinical setting. Med Mycol 49:S96–S100CrossRefGoogle Scholar
  25. Paolino KM, Henry JA, Hospenthal DR, Wortmann GW, Hartzell JD (2012) Invasive fungal infections following combat-related injury. Mil Med 177:681–685CrossRefGoogle Scholar
  26. Park Y, Kim M-H, Park S-C, Cheong H, Jang M-K, Nah J-W, Hahm K-S (2008) Investigation of the antifungal activity and mechanism of action of LMWS-chitosan. J Microbiol Biotechnol 18:1729–1734Google Scholar
  27. Rajendran R, Mowat E, McCulloch E, Lappin DF, Jones B, Lang S, Majithiya JB, Warn P, Williams C, Ramage G (2011) Azole resistance of Aspergillus fumigatus biofilms is partly associated with efflux pump activity. Antimicrob Agents Chemother 55:2092–2097CrossRefGoogle Scholar
  28. Ramage G, Rajendran R, Gutierrez-Correa M, Jones B, Williams C (2011) Aspergillus biofilms: clinical and industrial significance. FEMS Microbiol Lett 324:89–97CrossRefGoogle Scholar
  29. Reynolds TB, Fink GR (2001) Bakers’ yeast, a model for fungal biofilm formation. Science 291:878–881CrossRefGoogle Scholar
  30. Rezanka T, Kolouchova I, Cejkova A, Sigler K (2012) Natural products: Strategic tools for modulation of biofilm formation. In: Atta-ur-Rahman F (ed) Studies in natural products chemistry, vol 38. Elsevier, Amsterdam, pp 269–303Google Scholar
  31. Serpa R, França EJG, Furlaneto-Maia L, Andrade CGTJ, Diniz A, Furlaneto MC (2012) In vitro antifungal activity of the flavonoid baicalein against Candida species. J Med Microbiol 61:1704–1708CrossRefGoogle Scholar
  32. Shibuya K, Ando T, Hasegawa C, Wakayama M, Hamatani S, Hatori T, Nagayama T, Nonaka H (2004) Pathophysiology of pulmonary aspergillosis. J Infect Chemother 10:138–145CrossRefGoogle Scholar
  33. Silva WJd, Seneviratne J, Parahitiyawa N, Rosa EAR, Samaranayake LP, Cury AADB (2008) Improvement of XTT assay performance for studies involving Candida albicans biofilms. Braz Dent J 19:364–369Google Scholar
  34. Silva-Dias A, Palmeira-De-Oliveira A, Miranda IM, Branco J, Cobrado L, Monteiro-Soares M, Queiroz JA, Pina-Vaz C, Rodrigues AG (2014) Anti-biofilm activity of low-molecular weight chitosan hydrogel against Candida species. Med Microbiol Immun 203:25–33CrossRefGoogle Scholar
  35. Tekaia F, Latgé J-P (2005) Aspergillus fumigatus: saprophyte or pathogen? Curr Opin Microbiol 8:385–392CrossRefGoogle Scholar
  36. Villena GK, Fujikawa T, Tsuyumu S, Gutierrez-Correa M (2010) Structural analysis of biofilms and pellets of Aspergillus niger by confocal laser scanning microscopy and cryo scanning electron microscopy. Bioresour Technol 101:1920–1926CrossRefGoogle Scholar
  37. Warkentien T, Rodriguez C, Lloyd B, Wells J, Weintrob A, Dunne JR, Ganesan A, Li P, Bradley W, Gaskins LJ, Seillier-Moiseiwitsch F, Murray CK, Millar EV, Keenan B, Paolino K, Fleming M, Hospenthal DR, Wortmann GW, Landrum ML, Kortepeter MG, Tribble DR (2012) Invasive mold infections following combat-related Injuries. Clin Infect Dis 55:1441–1449CrossRefGoogle Scholar
  38. Watnick P, Kolter R (2000) Biofilm, city of microbes. J Bacteriol 182:2675–2679CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Department of BiotechnologyUniversity of Chemistry and Technology, PraguePrague 6Czech Republic

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