1887

Abstract

is an increasingly prevalent opportunistic fungal pathogen of various immunocompromised individuals. It has the ability to form filaments within the lungs, producing dense intertwined mycelial balls, which are difficult to treat. The aim of this study was to develop a suitable model of to examine the effects of antifungal challenge on these intertwined filamentous communities. NCPF 7367 growth conditions were optimized on both Thermanox coverslips and on flat-bottomed microtitre plates to establish optimal conidial seeding densities. Isolates were treated with itraconazole, voriconazole, amphotericin B and caspofungin and their overall killing efficiency was measured using an XTT formazan metabolic dye assay. This was compared with the CLSI (formerly NCCLS) methodology of broth microdilution of moulds (standard M38-A). It was shown that 1×10 conidia ml in RPMI 1640 was the optimum concentration of spores for biofilm formation. Filamentous growth characteristics were not observed until 10 h incubation, followed by an exponential increase in the biofilm biomass (hyphae and extracellular material) and cellular activity (metabolism). When susceptibility testing of biofilms was compared with that of planktonic cells by CLSI broth microdilution testing, all antifungal drugs were at least 1000 times less effective at reducing the overall metabolic activity of 90 % of the cells. Overall, this study showed that has the ability to form coherent multicellular biofilm structures that are resistant to the effects of antifungal drugs.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.47247-0
2007-09-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/56/9/1205.html?itemId=/content/journal/jmm/10.1099/jmm.0.47247-0&mimeType=html&fmt=ahah

References

  1. Antachopoulos C., Meletiadis J., Roilides E., Sein T., Walsh T. J. 2006; Rapid susceptibility testing of medically important zygomycetes by XTT assay. J Clin Microbiol 44:553–560 [CrossRef]
    [Google Scholar]
  2. Beauvais A., Schmidt C., Guadagnini S., Roux P., Perret E., Henry C., Paris S., Mallet A., Prévost M.-C., Latgé J. P. 2007; An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus . Cell Microbiol 9:1588–1600 [CrossRef]
    [Google Scholar]
  3. Bennett J. E., Powers J., Walsh T., Viscoli C., de Pauw B., Dismukes W., Galgiani J., Glauser M., Herbrecht R. other authors 2003; Forum report: issues in clinical trials of empirical antifungal therapy in treating febrile neutropenic patients. Clin Infect Dis 36:S117–S122 [CrossRef]
    [Google Scholar]
  4. Brakhage A. A. 2005; Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Curr Drug Targets 6:875–886 [CrossRef]
    [Google Scholar]
  5. Chandra J., Kuhn D. M., Mukherjee P. K., Hoyer L. L., McCormick T., Ghannoum M. A. 2001; Biofilm formation by the fungal pathogen Candida albicans : development, architecture, and drug resistance. J Bacteriol 183:5385–5394 [CrossRef]
    [Google Scholar]
  6. Christensen G. D., Simpson W. A., Younger J. J., Baddour L. M., Barrett F. F., Melton D. M., Beachey E. H. 1985; Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22:996–1006
    [Google Scholar]
  7. Cimon B., Symoens F., Zouhair R., Chabasse D., Nolard N., Defontaine A., Bouchara J. P. 2001; Molecular epidemiology of airway colonisation by Aspergillus fumigatus in cystic fibrosis patients. J Med Microbiol 50:367–374
    [Google Scholar]
  8. Costerton J. W., Lewandowski Z., Caldwell D. E., Korber D. R., Lappin-Scott H. M. 1995; Microbial biofilms. Annu Rev Microbiol 49:711–745 [CrossRef]
    [Google Scholar]
  9. de Almeida M. B., Bussamra M. H., Rodrigues J. C. 2006; Allergic bronchopulmonary aspergillosis in paediatric cystic fibrosis patients. Paediatr Respir Rev 7:67–72 [CrossRef]
    [Google Scholar]
  10. Denning D. W., Marinus A., Cohen J., Spence D., Herbrecht R., Pagano L., Kibbler C., Kcrmery V., Offner F. other authors 1998; An EORTC multicentre prospective survey of invasive aspergillosis in haematological patients: diagnosis and therapeutic outcome. EORTC Invasive Fungal Infections Cooperative Group. J Infect 37:173–180 [CrossRef]
    [Google Scholar]
  11. Ellis M., Richardson M., de Pauw B. 2000; Epidemiology. Hosp Med 61:605–609 [CrossRef]
    [Google Scholar]
  12. Filler S. G., Sheppard D. C. 2006; Fungal invasion of normally non-phagocytic host cells. PLoS Pathogens 2:e129 [CrossRef]
    [Google Scholar]
  13. Hawser S. P., Norris H., Jessup C. J., Ghannoum M. A. 1998; Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents. J Clin Microbiol 36:1450–1452
    [Google Scholar]
  14. Hawser S. P., Jessup C., Vitullo J., Ghannoum M. A. 2001; Utility of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT) and minimum effective concentration assays in the determination of antifungal susceptibility of Aspergillus fumigatus to the lipopeptide class compounds. J Clin Microbiol 39:2738–2741 [CrossRef]
    [Google Scholar]
  15. Herbrecht R., Denning D. W., Patterson T. F., Bennett J. E., Greene R. E., Oestmann J. W., Kern W. V., Marr K. A., Ribaud P. other authors 2002; Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 347:408–415 [CrossRef]
    [Google Scholar]
  16. Hope W. W., Walsh T. J., Denning D. W. 2005; Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis 5:609–622 [CrossRef]
    [Google Scholar]
  17. Kamai Y., Chiang L. Y., Lopes Bezerra L. M., Doedt T., Lossinsky A. S., Sheppard D. C., Filler S. G. 2006; Interactions of Aspergillus fumigatus with vascular endothelial cells. Med Mycol 44 (Suppl. 1):S115–S117 [CrossRef]
    [Google Scholar]
  18. Latge J. P. 2001; The pathobiology of Aspergillus fumigatus . Trends Microbiol 9:382–389 [CrossRef]
    [Google Scholar]
  19. Maertens J., Raad I., Petrikkos G., Boogaerts M., Selleslag D., Petersen F. B., Sable C. A., Kartsonis N. A., Ngai A. other authors 2004; Efficacy and safety of caspofungin for treatment of invasive aspergillosis in patients refractory to or intolerant of conventional antifungal therapy. Clin Infect Dis 39:1563–1571 [CrossRef]
    [Google Scholar]
  20. Martino R., Viscoli C. 2006; Empirical antifungal therapy in patients with neutropenia and persistent or recurrent fever of unknown origin. Br J Haematol 132:138–154 [CrossRef]
    [Google Scholar]
  21. Meletiadis J., Meis J. F., Mouton J. W., Verweij P. E. 2001a; Analysis of growth characteristics of filamentous fungi in different nutrient media. J Clin Microbiol 39:478–484 [CrossRef]
    [Google Scholar]
  22. Meletiadis J., Mouton J. W., Meis J. F., Bouman B. A., Donnelly J. P., Verweij P. E. 2001b; Colorimetric assay for antifungal susceptibility testing of Aspergillus species. J Clin Microbiol 39:3402–3408 [CrossRef]
    [Google Scholar]
  23. Meletiadis J., Mouton J. W., Meis J. F., Bouman B. A., Donnelly P. J., Verweij P. E. 2001c; Comparison of spectrophotometric and visual readings of NCCLS method and evaluation of a colorimetric method based on reduction of a soluble tetrazolium salt, 2,3-bis [2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide], for antifungal susceptibility testing of Aspergillus species. J Clin Microbiol 39:4256–4263 [CrossRef]
    [Google Scholar]
  24. Mensi M., Salgarello S., Pinsi G., Piccioni M. 2004; Mycetoma of the maxillary sinus: endodontic and microbiological correlations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 98:119–123 [CrossRef]
    [Google Scholar]
  25. NCCLS 2002 Reference Method for Broth Microdilution Antifungal Susceptibility Testing of Filamentous Fungi . NCCLS document M38-A Pennsylvania, PA: National Committee for Clinical Laboratory Standards;
    [Google Scholar]
  26. O'Toole G. A., Kolter R. 1998; Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28:449–461 [CrossRef]
    [Google Scholar]
  27. Ramage G., Vande Walle K., Wickes B. L., Lopez-Ribot J. L. 2001a; Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45:2475–2479 [CrossRef]
    [Google Scholar]
  28. Ramage G., Vande Walle K., Wickes B. L., Lopez-Ribot J. L. 2001b; Biofilm formation by Candida dubliniensis . J Clin Microbiol 39:3234–3240 [CrossRef]
    [Google Scholar]
  29. Ramage G., Vandewalle K., Wickes B. L., Lopez-Ribot J. L. 2001c; Characteristics of biofilm formation by Candida albicans . Rev Iberoam Micol 18:163–170
    [Google Scholar]
  30. Ramage G., Bachmann S., Patterson T. F., Wickes B. L., Lopez-Ribot J. L. 2002a; Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother 49:973–980 [CrossRef]
    [Google Scholar]
  31. Ramage G., Saville S. P., Wickes B. L., Lopez-Ribot J. L. 2002b; Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule. Appl Environ Microbiol 68:5459–5463 [CrossRef]
    [Google Scholar]
  32. Ramage G., VandeWalle K., Bachmann S. P., Wickes B. L., Lopez-Ribot J. L. 2002c; In vitro pharmacodynamic properties of three antifungal agents against preformed Candida albicans biofilms determined by time-kill studies. Antimicrob Agents Chemother 46:3634–3636 [CrossRef]
    [Google Scholar]
  33. Ramage G., Saville S. P., Thomas D. P., Lopez-Ribot J. L. 2005; Candida biofilms: an update. Eukaryot Cell 4:633–638 [CrossRef]
    [Google Scholar]
  34. Rivera A., Hohl T., Pamer E. G. 2006; Immune responses to Aspergillus fumigatus infections. Biol Blood Marrow Transplant 12:47–49
    [Google Scholar]
  35. Shibuya K., Ando T., Hasegawa C., Wakayama M., Hamatani S., Hatori T., Nagayama T., Nonaka H. 2004; Pathophysiology of pulmonary aspergillosis. J Infect Chemother 10:138–145 [CrossRef]
    [Google Scholar]
  36. Shoseyov D., Brownlee K. G., Conway S. P., Kerem E. 2006; Aspergillus bronchitis in cystic fibrosis. Chest 130:222–226 [CrossRef]
    [Google Scholar]
  37. Singh N. 2005; Invasive aspergillosis in organ transplant recipients: new issues in epidemiologic characteristics, diagnosis, and management. Med Mycol 43 (Suppl. 1):S267–S270 [CrossRef]
    [Google Scholar]
  38. Tunney M. M., Ramage G., Field T. R., Moriarty T. F., Storey D. G. 2004; Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa . Antimicrob Agents Chemother 48:1879–1881 [CrossRef]
    [Google Scholar]
  39. Villena G. K., Gutierrez-Correa M. 2006; Production of cellulase by Aspergillus niger biofilms developed on polyester cloth. Lett Appl Microbiol 43:262–268 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.47247-0
Loading
/content/journal/jmm/10.1099/jmm.0.47247-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error