Skip to main content

Advertisement

SpringerLink
Log in

Antifolates inhibit Cryptococcus biofilms and enhance susceptibility of planktonic cells to amphotericin B

  • Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

The Cryptococcus neoformans species complex contains the most important agents of fungal meningoencephalitis. Therapeutic choices are limited and issues related to toxicity and resistance to antifungals have been described. The present study evaluated the inhibitory effect of the antifolate combinations sulfamethoxazole–trimethoprim (SMX/TMP) and sulfadiazine–pyrimethamine (SDZ/PYR) against planktonic cells and biofilms of C. neoformans and C. gattii. The influence of the antifolate combinations on the amphotericin minimum inhibitory concentration (MIC) of planktonic cells was also investigated. In addition, the effect of these combinations on the cellular ergosterol content of planktonic cells was studied. Strains of C. neoformans (n = 15) and C. gattii (n = 15) obtained from environmental or clinical sources were evaluated by the broth microdilution method. SMX/TMP and SDZ/PYR showed antifungal activity against free living cells and sessile cells of Cryptococcus spp. Moreover, planktonic cells showed increased susceptibility to amphotericin B after pre-incubation with sub-inhibitory concentrations of SMX/TMP or SDZ/PYR. The drug combinations SMX/TMP and SDZ/PYR were able to prevent the biofilm formation and showed inhibitory effect against mature biofilms of both species. Additionally, the study showed that antifolate drugs reduced the ergosterol content in C. neoformans and C. gattii planktonic cells. Our results highlight the antifungal potential of antifolate drugs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Lin X, Heitman J (2006) The biology of the Cryptococcus neoformans species complex. Annu Rev Microbiol 60:69–105

    Article  PubMed  CAS  Google Scholar 

  2. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC (2010) Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 50(3):291–322

    Article  PubMed  Google Scholar 

  3. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23(4):525–530

    Article  PubMed  Google Scholar 

  4. Brouwer AE, Rajanuwong A, Chierakul W, Griffin GE, Larsen RA, White NJ, Harrison TS (2004) Combination antifungal therapies for HIV-associated cryptococcal meningitis: a randomised trial. Lancet 363(9423):1764–1767

    Article  PubMed  CAS  Google Scholar 

  5. Varma A, Kwon-Chung KJ (2010) Heteroresistance of Cryptococcus gattii to fluconazole. Antimicrob Agents Chemother 54(6):2303–2311

    Article  PubMed  CAS  Google Scholar 

  6. Perkins A, Gomez-Lopez A, Mellado E, Rodriguez-Tudela JL, Cuenca-Estrella M (2005) Rates of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var. neoformans. J Antimicrob Chemother 56(6):1144–1147

    Article  PubMed  CAS  Google Scholar 

  7. Bii CC, Makimura K, Abe S, Taguchi H, Mugasia OM, Revathi G, Wamae NC, Kamiya S (2007) Antifungal drug susceptibility of Cryptococcus neoformans from clinical sources in Nairobi, Kenya. Mycoses 50(1):25–30

    Article  PubMed  CAS  Google Scholar 

  8. Sionov E, Chang YC, Garraffo HM, Kwon-Chung KJ (2009) Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence. Antimicrob Agents Chemother 53(7):2804–2815

    Article  PubMed  CAS  Google Scholar 

  9. Martinez LR, Mihu MR, Han G, Frases S, Cordero RJ, Casadevall A, Friedman AJ, Friedman JM, Nosanchuk JD (2010) The use of chitosan to damage Cryptococcus neoformans biofilms. Biomaterials 31(4):669–679

    Article  PubMed  CAS  Google Scholar 

  10. Steverding D, Evans P, Msika L, Riley B, Wallington J, Schelenz S (2012) In vitro antifungal activity of DNA topoisomerase inhibitors. Med Mycol 50(3):333–336

    Article  PubMed  CAS  Google Scholar 

  11. Verwer PE, Woodle MC, Boekhout T, Hagen F, Bakker-Woudenberg IA, van de Sande WW (2011) Cryptococcus and Trichosporon spp. are susceptible in vitro to branched histidine- and lysine-rich peptides (BHKPs). J Antimicrob Chemother 66(7):1649–1652

    Article  PubMed  CAS  Google Scholar 

  12. Martinez LR, Casadevall A (2007) Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light. Appl Environ Microbiol 73(14):4592–4601

    Article  PubMed  CAS  Google Scholar 

  13. Ramage G, Mowat E, Jones B, Williams C, Lopez-Ribot J (2009) Our current understanding of fungal biofilms. Crit Rev Microbiol 35(4):340–355

    Article  PubMed  CAS  Google Scholar 

  14. Martinez LR, Casadevall A (2006) Cryptococcus neoformans cells in biofilms are less susceptible than planktonic cells to antimicrobial molecules produced by the innate immune system. Infect Immun 74(11):6118–6123

    Article  PubMed  CAS  Google Scholar 

  15. Martinez LR, Casadevall A (2006) Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro. Antimicrob Agents Chemother 50(3):1021–1033

    Article  PubMed  CAS  Google Scholar 

  16. Robertson EJ, Wolf JM, Casadevall A (2012) EDTA inhibits biofilm formation, extracellular vesicular secretion, and shedding of the capsular polysaccharide glucuronoxylomannan by Cryptococcus neoformans. Appl Environ Microbiol 78(22):7977–7984

    Article  PubMed  CAS  Google Scholar 

  17. Walsh TJ, Schlegel R, Moody MM, Costerton JW, Salcman M (1986) Ventriculoatrial shunt infection due to Cryptococcus neoformans: an ultrastructural and quantitative microbiological study. Neurosurgery 18(3):373–375

    Article  PubMed  CAS  Google Scholar 

  18. Ingram CW, Haywood HB 3rd, Morris VM, Allen RL, Perfect JR (1993) Cryptococcal ventricular-peritoneal shunt infection: clinical and epidemiological evaluation of two closely associated cases. Infect Control Hosp Epidemiol 14(12):719–722

    Article  PubMed  CAS  Google Scholar 

  19. Banerjee U, Gupta K, Venugopal P (1997) A case of prosthetic valve endocarditis caused by Cryptococcus neoformans var. neoformans. J Med Vet Mycol 35(2):139–141

    Article  PubMed  CAS  Google Scholar 

  20. Johannsson B, Callaghan JJ (2009) Prosthetic hip infection due to Cryptococcus neoformans: case report. Diagn Microbiol Infect Dis 64(1):76–79

    Article  PubMed  Google Scholar 

  21. Cordeiro RA, Costa AK, Brilhante RS, de Lima RA, Castelo-Branco Dde S, Ribeiro JF, Monteiro AJ, Rocha FA, Sidrim JJ, Rocha MF (2011) PCR-REA as an important tool for the identification of Cryptococcus neoformans and Cryptococcus gattii from human and veterinary sources. Vet Microbiol 154(1–2):180–184

    Article  PubMed  CAS  Google Scholar 

  22. Enache-Angoulvant A, Chandenier J, Symoens F, Lacube P, Bolognini J, Douchet C, Poirot JL, Hennequin C (2007) Molecular identification of Cryptococcus neoformans serotypes. J Clin Microbiol 45(4):1261–1265

    Article  PubMed  CAS  Google Scholar 

  23. Meyer W, Castañeda A, Jackson S, Huynh M, Castañeda E; IberoAmerican Cryptococcal Study Group (2003) Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis 9(2):189–195

    Article  PubMed  Google Scholar 

  24. Hanafy A, Uno J, Mitani H, Kang Y, Mikami Y (2007) In vitro antifungal activities of sulfa drugs against clinical isolates of Aspergillus and Cryptococcus species. Jpn J Med Mycol 48(1):47–50

    Article  CAS  Google Scholar 

  25. Clinical and Laboratory Standards Institute (CLSI) (2008) Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard—third edition. CLSI document M27-A3. CLSI, Wayne, PA

  26. Cordeiro Rde A, Astete-Medrano DJ, Marques FJ, Andrade HT, Perdigão Neto LV, Tavares JL, de Lima RA, Patoilo KK, Monteiro AJ, Brilhante RS, Rocha MF, de Camargo ZP, Sidrim JJ (2011) Cotrimoxazole enhances the in vitro susceptibility of Coccidioides posadasii to antifungals. Mem Inst Oswaldo Cruz 106(8):1045–1048

    Article  CAS  Google Scholar 

  27. Costa AK, Sidrim JJ, Cordeiro RA, Brilhante RS, Monteiro AJ, Rocha MF (2010) Urban pigeons (Columba livia) as a potential source of pathogenic yeasts: a focus on antifungal susceptibility of Cryptococcus strains in Northeast Brazil. Mycopathologia 169(3):207–213

    Article  PubMed  Google Scholar 

  28. Moran GP, Pinjon E, Coleman DC, Sullivan DJ (2007) Analysis of drug resistance in pathogenic fungi. In: Kavanagh K (ed) Medical mycology: cellular and molecular techniques. Wiley, Hoboken, pp 93–113

    Google Scholar 

  29. Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72(2):157–165

    Article  PubMed  CAS  Google Scholar 

  30. Navarro-Martínez MD, Cabezas-Herrera J, Rodríguez-López JN (2006) Antifolates as antimycotics? Connection between the folic acid cycle and the ergosterol biosynthesis pathway in Candida albicans. Int J Antimicrob Agents 28(6):560–567

    Article  PubMed  Google Scholar 

  31. Hahn RC, Morato Conceição YT, Santos NL, Ferreira JF, Hamdan JS (2003) Disseminated paracoccidioidomycosis: correlation between clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole. Mycoses 46(8):342–347

    Article  PubMed  Google Scholar 

  32. Brilhante RS, Fechine MA, Cordeiro Rde A, Rocha MF, Ribeiro JF, Monteiro AJ, de Lima RA, Mesquita JR, de Camargo ZP, Sidrim JJ (2010) In vitro effect of sulfamethoxazole–trimethoprim against Histoplasma capsulatum var. capsulatum. Antimicrob Agents Chemother 54(9):3978–3979

    Article  PubMed  CAS  Google Scholar 

  33. Lim PL, Zhou J, Ditangco RA, Law MG, Sirisanthana T, Kumarasamy N, Chen YM, Phanuphak P, Lee CK, Saphonn V, Oka S, Zhang F, Choi JY, Pujari S, Kamarulzaman A, Li PC, Merati TP, Yunihastuti E, Messerschmidt L, Sungkanuparph S; TREAT Asia HIV Observational Database (2012) Failure to prescribe pneumocystis prophylaxis is associated with increased mortality, even in the cART era: results from the Treat Asia HIV observational database. J Int AIDS Soc 15:1

    Article  PubMed  CAS  Google Scholar 

  34. Pfaller MA, Boyken L, Hollis RJ, Messer SA, Tendolkar S, Diekema DJ (2005) In vitro susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species to itraconazole: global survey of 9,359 isolates tested by clinical and laboratory standards institute broth microdilution methods. J Clin Microbiol 43(8):3807–3810

    Article  PubMed  CAS  Google Scholar 

Download references

Funding declaration

This work was supported by grants from the National Council for Scientific and Technological Development (CNPq; Brazil; Processes 302574/2009-3 and 562296/2010-7). We would like to thank the Mycology Laboratory of the Evandro Chagas Clinical Research Institute in Rio de Janeiro (IPEC/FIOCRUZ) for providing the reference strains from its cryptococcal culture collection.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Pathology and Legal Medicine, School of Medicine, Postgraduate Program in Medical Microbiology and Specialized Medical Mycology Center, Federal University of Ceará, Fortaleza, CE, Brazil

    R. de Aguiar Cordeiro, C. I. Mourão, M. F. G. Rocha, F. J. de Farias Marques, C. E. C. Teixeira, D. F. de Oliveira Miranda, L. V. P. Neto, R. S. N. Brilhante, T. de Jesus Pinheiro Gomes Bandeira & J. J. C. Sidrim

  2. Department of Clinical Medicine, School of Medicine, Post-Graduation Program in Medicine Science, Federal University of Ceará, Fortaleza, CE, Brazil

    R. de Aguiar Cordeiro, C. I. Mourão, R. S. N. Brilhante & J. J. C. Sidrim

  3. School of Veterinary Medicine, Postgraduate Program in Veterinary Science, State University of Ceará, Fortaleza, CE, Brazil

    M. F. G. Rocha

Authors
  1. R. de Aguiar Cordeiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. I. Mourão
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. F. G. Rocha
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. J. de Farias Marques
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. E. C. Teixeira
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D. F. de Oliveira Miranda
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. L. V. P. Neto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. R. S. N. Brilhante
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. T. de Jesus Pinheiro Gomes Bandeira
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J. J. C. Sidrim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. de Aguiar Cordeiro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Aguiar Cordeiro, R., Mourão, C.I., Rocha, M.F.G. et al. Antifolates inhibit Cryptococcus biofilms and enhance susceptibility of planktonic cells to amphotericin B. Eur J Clin Microbiol Infect Dis 32, 557–564 (2013). https://doi.org/10.1007/s10096-012-1774-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-012-1774-8

Keywords

Search

Navigation