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Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light

  • Microbial Genetics, Genomics and Molecular Biology
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Abstract

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.

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References

  • Ahearn, D.G., Simmons, R.B., Zhang, S., Stulting, R.D., Crow, S.A.Jr., Schwam, B.L., and Pierce, G.E. 2007. Attachment to and penetration of conventional and silicone hydrogel contact lenses by Fusarium solani and Ulocladium sp. in vitro. Cornea 26, 831–839.

    Article  PubMed  Google Scholar 

  • Al-Fattani, M.A. and Douglas, L.J. 2006. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J. Med. Microbiol. 55, 999–1008.

    Article  CAS  PubMed  Google Scholar 

  • Al-Hatmi, A.M., Hagen, F., Menken, S.B., Meis, J.F., and de Hoog, G.S. 2016. Global molecular epidemiology and genetic diversity of Fusarium, a significant emerging group of human opportunists from 1958 to 2015. Emerg. Microbes Infect. 5, e124.

    Article  PubMed  PubMed Central  Google Scholar 

  • Baillie, G.S. and Douglas, L.J. 2000. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J. Antimicrob. Chemother. 46, 397–403.

    Article  CAS  PubMed  Google Scholar 

  • Beauvais, A. and Latgé, J.P. 2018. Special issue: Fungal cell wall. J. Fungi (Basel) 4, E91.

    Article  Google Scholar 

  • Beauvais, A., Schmidt, C., Guadagnini, S., Roux, P., Perret, E., Henry, C., Paris, S., Mallet, A., Prévost, M.C., and Latgé, J.P. 2007. An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus. Cell. Microbiol. 9, 1588–1600.

    Article  CAS  PubMed  Google Scholar 

  • Bhoomibunchoo, C., Ratanapakorn, T., Sinawat, S., Sanguansak, T., Moontawee, K., and Yospaiboon, Y. 2013. Infectious endophthalmitis: review of 420 cases. Clin. Ophthalmol. 7, 247–252.

    PubMed  PubMed Central  Google Scholar 

  • Bispo, P.J., Haas, W., and Gilmore, M.S. 2015. Biofilms in infections of the eye. Pathogens 4, 111–136.

    Article  PubMed  PubMed Central  Google Scholar 

  • Bizerra, F.C., Nakamura, C.V., de Poersch, C., Estivalet Svidzinski, T.I., Borsato Quesada, R.M., Goldenberg, S., Krieger, M.A., and Yamada-Ogatta, S.F. 2008. Characteristics of biofilm formation by Candida tropicalis and antifungal resistance. FEMS Yeast Res. 8, 442–450.

    Article  CAS  PubMed  Google Scholar 

  • Borghi, E., Borgo, F., and Morace, G. 2016. Fungal biofilms: Update on resistance. Adv. Exp. Med. Biol. 931, 37–47.

    Article  PubMed  Google Scholar 

  • Bozzola, J.J. and Russell, L.D. 1999. Specimen preparation for scanning electron microscopy, pp. 48–70. In Electron microscopy. 2nd ed. Jones and Bartlett Press, New York, N.Y., USA.

    Google Scholar 

  • Chandra, J., Kuhn, D.M., Mukherjee, P.K., Hoyer, L.L., McCormick, T., and Ghannoum, M.A. 2001. Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance. J. Bacteriol. 183, 5385–5394.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Choi, K.S., Yoon, S.C., Rim, T.H., Han, S.J., Kim, E.D., and Seo, K.Y. 2014. Effect of voriconazole and ultraviolet—A combination therapy compared to voriconazole single treatment on Fusarium solani fungal keratitis. J. Ocul. Pharmacol. Ther. 30, 381–386.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M., and 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.

  • Coleman, J.J. 2016. The Fusarium solani species complex: ubiquitous pathogens of agricultural importance. Mol. Plant Pathol. 17, 146–158.

    Article  PubMed  Google Scholar 

  • Costa-Orlandi, C.B., Sardi, J.C., Santos, C.T., Fusco-Almeida, A.M., and Mendes-Giannini, M.J. 2014. In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms. Biofouling 30, 719–727.

    Article  CAS  PubMed  Google Scholar 

  • Dabas, Y., Bakhshi, S., and Xess, I. 2016. Fatal cases of bloodstream infection by Fusarium solani and review of published literature. Mycopathologia 181, 291–296.

    Article  PubMed  Google Scholar 

  • Fanning, S. and Mitchell, A.P. 2012. Fungal biofilms. PLoS Pathog. 8, e1002585.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Finkel, J.S. and Mitchell, A.P. 2011. Genetic control of Candida albicans biofilm development. Nat. Rev. Microbiol. 9, 109–118.

    Article  CAS  PubMed  Google Scholar 

  • Fox, E.P., Bui, C.K., Nett, J.E., Hartooni, N., Mui, M.C., Andes, D.R., Nobile, C.J., and Johnson, A.D. 2015. An expanded regulatory network temporally controls Candida albicans biofilm formation. Mol. Microbiol. 96, 1226–1239.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fredheim, E.G., Klingenberg, C., Rohde, H., Frankenberger, S., Gaustad, P., Flaegstad, T., and Sollid, J.E. 2009. Biofilm formation by Staphylococcus haemolyticus. J. Clin. Microbiol. 47, 1172–1180.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Godoy, P., Cano, J., Gené, J., Guarro, J., Höfling-Lima, A.L., and Lopes Colombo, A. 2004. Genotyping of 44 isolates of Fusarium solani, the main agent of fungal keratitis in Brazil. J. Clin. Microbiol. 42, 4494–4497.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • González-Ramírez, A.I., Ramírez-Granillo, A., Medina-Canales, M.G., Rodríguez-Tovar, A.V., and Martínez-Rivera, M.A. 2016. Analysis and description of the stages of Aspergillus fumigatus biofilm formation using scanning electron microscopy. BMC Microbiol. 16, 243.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guevara-Suarez, M., Cano-Lira, J.F., de García, M.C., Sopo, L., De Bedout, C., Cano, L.E., García, A.M., Motta, A., Amézquita, A., Cárdenas, M., et al. 2016. Genotyping of Fusarium isolates from onychomycoses in Colombia: Detection of two new species within the Fusarium solani species complex and in vitro antifungal susceptibility testing. Mycopathologia 18, 165–174.

    Article  CAS  Google Scholar 

  • Gutiérrez-Correa, M., Ludeña, Y., Ramage, G., and Villena, G.K. 2012. Recent advances on filamentous fungal biofilms for industrial uses. Appl. Biochem. Biotechnol. 167, 1235–1253.

    Article  CAS  PubMed  Google Scholar 

  • Gutiérrez-Correa, M. and Villena, G.K. 2003. Surface adhesion fermentation: a new fermentation category. Rev. Peru. Biol. 10, 113–124.

    Google Scholar 

  • Harding, M.W., Marques, L.L., Howard, R.J., and Olson, M.E. 2009. Can filamentous fungi form biofilms? Trends Microbiol. 17, 475–480.

    Article  CAS  PubMed  Google Scholar 

  • Hughes, K.A., Lawley, B., and Newsham, K.K. 2003. Solar UV-B radiation inhibits the growth of Antarctic terrestrial fungi. Appl. Environ. Microbiol. 69, 1488–1491.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Imamura, Y., Chandra, J., Mukherjee, P.K., Lattif, A.A., Szczotka-Flynn, L.B., Pearlman, E., Lass, J.H., O’Donnell, K., and Ghannoum, M.A. 2008. Fusarium and Candida albicans biofilms on soft contact lenses: model development, influence of lens type, and susceptibility to lens care solutions. Antimicrob. Agents Chemother. 52, 171–182.

    Article  CAS  PubMed  Google Scholar 

  • Kaur, S. and Singh, S. 2014. Biofilm formation by Aspergillus fumigatus. Med. Mycol. 52, 2–9.

    CAS  PubMed  Google Scholar 

  • Kuhn, D.M., Chandra, J., Mukherjee, P.K., and Ghannoum, M.A. 2002. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect. Immun. 70, 878–888.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kuthan, M., Devaux, F., Janderová, B., Slaninová, I., Jacq, C., and Palková, Z. 2003. Domestication of wild Saccharomyces cerevisiae is accompanied by changes in gene expression and colony morphology. Mol. Microbiol. 47, 745–754.

    Article  CAS  PubMed  Google Scholar 

  • Legrand, C., Anaissie, E., Hashem, R., Nelson, P., Bodey, G.P., and Ro, J. 1991. Experimental fusarial hyalohyphomycosis in a murine model. J. Infect. Dis. 164, 944–948.

    Article  CAS  PubMed  Google Scholar 

  • Lohse, M.B., Gulati, M., Johnson, A.D., and Nobile, C.J. 2018. Development and regulation of single- and multi-species Candida albicans biofilms. Nat. Rev. Microbiol. 16, 19–31.

    Article  CAS  PubMed  Google Scholar 

  • Lopez-Ribot, J.L. 2005. Candida albicans biofilms: More than filamentation. Curr. Biol. 15, 453–455.

    Article  CAS  Google Scholar 

  • Martínez, L.R. and Casadevall, A. 2006. Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro. Antimicrob. Agents Chemother. 50, 1021–1033.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mayayo, E., Pujol, I., and Guarro, J. 1999. Experimental pathogenicity of four opportunist Fusarium species in a murine model. J. Med. Microbiol. 48, 363–366.

    Article  CAS  PubMed  Google Scholar 

  • Mitchell, K.F., Zarnowski, R., and Andes, D.R. 2016. The extracellular matrix of fungal biofilms. Adv. Exp. Med. Biol. 931, 21–35.

    Article  PubMed  Google Scholar 

  • Mlouka, M.A.B., Cousseau, T., and Martino, P.D. 2016. Application of fluorescently labelled lectins for the study of polysaccharides in biofilms with a focus on biofouling of nanofiltration membranes. AIMS Mol. Sci. 3, 338–356.

    Article  CAS  Google Scholar 

  • Mowat, E., Butcher, J., Lang, S., Williams, C., and 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, 1205–1212.

    Article  CAS  PubMed  Google Scholar 

  • Mowat, E., Lang, S., Williams, C., McCulloch, E., Jones, B., and Ramage, G. 2008. Phase-dependent antifungal activity against Aspergillus fumigatus developing multicellular filamentous biofilms. J. Antimicrob. Chemother. 62, 1281–1284.

    Article  CAS  PubMed  Google Scholar 

  • Mukherjee, P.K., Chandra, J., Yu, C., Sun, Y., Pearlman, E., and Ghannoum, M.A. 2012. Characterization of Fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis. Invest. Ophthalmol. Vis. Sci. 53, 4450–4457.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nett, J.E. and Andes, D.R. 2015. Fungal biofilms: In vivo models for discovery of anti-biofilm drugs. Microbiol. Spectr. 3, E30.

    Article  PubMed Central  Google Scholar 

  • Orlandi-Sardi, J.C., Pitangui, N.S., Gullo, F.P., Fusco-Almeida, A.M., and Mendes-Giannini, M.J.S. 2016. Fungal biofilms: Formation resistance and pathogenicity, pp. 291–314. In Razzaghi-Abyaneh, M., Shams-Ghahfarokhi, M., and Rai, M. (eds.), Medical mycology: Current trends and future prospects. Taylor & Francis Group LLC, CRC Press, New York, N.Y., USA.

    Google Scholar 

  • Peeters, E., Nelis, H.J., and Coenye, T. 2008. Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J. Microbiol. Methods 72, 157–165.

    Article  CAS  PubMed  Google Scholar 

  • Peiqian, L., Xiaoming, P., Huifang, S., Jingxin, Z., Ning, H., and Birun, L. 2014. Biofilm formation by Fusarium oxysporum f. sp. cucumerinum and susceptibility to environmental stress. FEMS Microbiol. Lett. 350, 138–145.

    Article  CAS  PubMed  Google Scholar 

  • Pierce, C.G., Srinivasan, A., Ramasubramanian, A.K., and López-Ribot, J.L. 2015. From biology to drug development: New approaches to combat the threat of fungal biofilms. Microbiol. Spectr. 3, 1–18.

    Article  CAS  Google Scholar 

  • Pierce, C.G., Vila, T., Romo, J.A., Montelongo-Jauregui, D., Wall, G., Ramasubramanian, A., and Lopez-Ribot, J.L. 2017. The Candida albicans biofilm matrix: Composition, structure and function. J. Fungi (Basel) 3, 14.

    Article  CAS  Google Scholar 

  • Pitangui, N.S., Sardi, J.C., Silva, J.F., Benaducci, T., Moraes da Silva, R.A., Rodríguez-Arellanes, G., Taylor, M.L., Mendes-Giannini, M.J., and Fusco-Almeida, A.M. 2012. Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface. Biofouling 28, 711–718.

    Article  CAS  PubMed  Google Scholar 

  • Rajendran, R., Williams, C., Lappin, D.F., Millington, O., Martins, M., and Ramage, G. 2013. Extracellular DNA release acts as an antifungal resistance mechanism in mature Aspergillus fumigatus biofilms. Eukaryot. Cell 12, 420–429.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Ramage, G., Rajendran, R., Gutierrez-Correa, M., Jones, B., and Williams, C. 2011. Aspergillus biofilms: clinical and industrial significance. FEMS Microbiol. Lett. 324, 89–97.

    Article  CAS  PubMed  Google Scholar 

  • Ramage, G., Rajendran, R., Sherry, L., and Williams, C. 2012. Fungal biofilm resistance. Int. J. Microbiol. 2012, 528521.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ramírez Granillo, A., Canales Medina, M.G., Espíndola Sánchez, M.E., Martínez Rivera, M.A., de Lucio, V.M., and Rodriguez Tovar, A.V. 2015. Antibiosis interaction of Staphylococcus aureus on Aspergillus fumigatus assessed in vitro by mixed biofilm formation. BMC Microbiol. 15, 33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sardi, J.D.C.O., Pitangui, N.D.S., Rodríguez-Arellanes, G., Taylor, M.L., Fusco-Almeida, A.M., and Mendes-Giannini, M.J.S. 2014. Highlights in pathogenic fungal biofilms. Rev. Iberoam. Micol. 31, 22–29.

    Article  Google Scholar 

  • Schlafer, S. and Meyer, R.L. 2017. Confocal microscopy imaging of the biofilm matrix. J. Microbiol. Methods 138, 50–59.

    Article  PubMed  Google Scholar 

  • Shopova, I., Bruns, S., Thywissen, A., Kniemeyer, O., Brakhage, A.A., and Hillmann, F. 2013. Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus. Front. Microbiol. 4, 141.

    Article  PubMed  PubMed Central  Google Scholar 

  • Simões, L.C., Simões, M., and Lima, N. 2015. Kinetics of biofilm formation by drinking water isolated Penicillium expansum. Biofouling 31, 349–362.

    Article  CAS  PubMed  Google Scholar 

  • Singh, R., Shivaprakash, M.R., and Chakrabarti, A. 2011. Biofilm formation by zygomycetes: quantification, structure, and matrix composition. Microbiology 157, 2611–2618.

    Article  CAS  PubMed  Google Scholar 

  • Šišić, A., Baćanović-Šišić, J., Al-Hatmi, A.M.S., Karlovsky, P., Ahmed, S.A., Maier, W., de Hoog, G.S., and Finckh, M.R. 2018. The ‘forma specialis’ issue in Fusarium: A case study in Fusarium solani f. sp. pisi. Sci. Rep. 8, 1252.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Srinivasan, M. 2004. Fungal keratitis. Curr. Opin. Ophthalmol. 15, 321–327.

    Article  CAS  PubMed  Google Scholar 

  • Sun, Y., Chandra, J., Mukherjee, P., Szczotka-Flynn, L., Ghannoum, M.A., and Pearlman, E. 2010. A murine model of contact lens-associated Fusarium keratitis. Invest. Ophthalmol. Vis. Sci. 51, 1511–1516.

    Article  PubMed  PubMed Central  Google Scholar 

  • Trafny, E.A., Lewandowski, R., Zawistowska-Marciniak, I., and Stepinska, M. 2013. Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids. World J. Microbiol. Biotechnol. 29, 1635–1643.

    Article  CAS  PubMed  Google Scholar 

  • Vázquez-Nin, G. and Echeverría, O. 2000. Introducción a la microscopia electrónica aplicada a las ciencias biológicas. México: Fondo de Cultura Económica. Mexico City, Mexico.

    Google Scholar 

  • Villena, G.K., Fujikawa, T., Tsuyumu, S., and Gutiérrez-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, 1920–1926.

    Article  CAS  PubMed  Google Scholar 

  • Virag, A. and Harris, S.D. 2006. The Spitzenkörper: a molecular perspective. Mycol. Res. 110, 4–13.

    Article  CAS  PubMed  Google Scholar 

  • Wu, T.G., Keasler, V.V., Mitchell, B.M., and Wilhelmus, K.R. 2004. Immunosuppression affects the severity of experimental Fusarium solani keratitis. J. Infect. Dis. 190, 192–198.

    Article  PubMed  Google Scholar 

  • Zhang, S., Ahearn, D.G., Stulting, R.D., Schwam, B.L., Simmons, R.B., Pierce, G.E., and Crow, S.A.Jr. 2007. Differences among strains of the Fusarium oxysporum-F. solani complexes in their penetration of hydrogel contact lenses and subsequent susceptibility to multipurpose contact lens disinfection solutions. Cornea 26, 1249–1254.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, N., O’Donnell, K., Sutton, D.A., Nalim, F.A., Summerbell, R.C., Padhye, A.A., and Geiser, D.M. 2006. Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment. J. Clin. Microbiol. 44, 2186–2190.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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  1. Medical Micology Laboratory, Microbiology Department National School of Biological Sciences, National Polytechnique Institute (IPN), C.P. 11340, Ciudad de México, México

    Itzel Margarita Córdova-Alcántara, Diana Laura Venegas-Cortés, María Ángeles Martínez-Rivera & Aida Verónica Rodriguez-Tovar

  2. Research and Development Department, Probiomed S.A. de C.V. Cruce de Carreteras Acatzingo-Zumpahuacan S/N, C.P. 52400, Tenancingo, Estado de México, México

    Néstor Octavio Pérez

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  1. Itzel Margarita Córdova-Alcántara
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Córdova-Alcántara, I.M., Venegas-Cortés, D.L., Martínez-Rivera, M.Á. et al. Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light. J Microbiol. 57, 485–497 (2019). https://doi.org/10.1007/s12275-019-8637-2

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