Research Article: Effect of amphotericin B and voriconazole on the outgrowth of conidia of Aspergillus fumigatus followed by time-lapse microscopy

Date Published: April 3, 2019

Publisher: Springer Berlin Heidelberg

Author(s): Laszlo Talas, Zsuzsa M. Szigeti, Gaspar Banfalvi, Gabor Szeman-Nagy.


Studies of morphological measurements from the outgrowth of cells to a network of hyphae have been extended from Candida albicans (Nagy et al. in Appl Microbiol Biotechnol 98(11):5185–5194. 10.1007/s00253-014-5696-5, 2014) to invasive conidiospores of Aspergillus fumigatus upon treatment with antifungal agents. The understanding of mycelial processes is important to optimize industrial processes such as fermentation and contributes to the fight against pathogenic fungi. This brief study combines TLS with digital image analysis. The TLS system was adapted to get information related to the adherence and growth dynamics of filamentous fungi. This approach was used earlier to distinguish among subphases of bacterial and fungal infections of mammal cells by detecting Mycoplasma infection in cell cultures causing serious damages in cell cultures. We describe changes in adherence, germination of spores, and hyphal growth of A. fumigatus, taking place in the absence and presence of amphotericin B (AMB) and voriconazole (VRC). These growth parameters were measured by TLS in CO2 incubator under physiological Photomicrography by TLS and extended for a longer period of time up to several weeks combined with image analysis represents a comfortable and reliable means to characterize the growth dynamism of A. fumigatus. The most important observation of medical importance related to the pathomechanism of VRC was that it did not adhere to conidiospores, i.e. that it did not contribute to the attachment of spores to the growth surface, and did not prevent germination but delayed hypha protrusion and elongation. In contrast AMB adhered to conidia, inhibited germination, hypha elongation and branching. It was concluded that AMB was efficient against the therapy of growth but not against the prevention of fungal infection.

Partial Text

Filamentous fungi play an important role in the so-called ‘white biotechnology’, by producing antibiotics and enzymes. ‘White biotechnology’ uses living cells to synthesize products that are biodegradable, require less energy and create less waste during their production (Frazzetto 2003) and in a more general term during the implementation of biotechnology in the industrial sphere (Heux et al. 2015). Some of the filamentous fungi are human pathogens, including different Aspergillus species. The human pathogen Aspergillus fumigatus can cause among others invasive pulmonary aspergillosis, aspergilloma, immunoglobulin-mediated allergic rhinitis, asthma, hypersensitive pneumonitis, chronic necrotizing pneumonia, allergic bronchopulmonary aspergillosis in immunocompromised individuals (Chaudhary and Marr 2011; Simon-Nobbe et al. 2008). The route of infection of Aspergillus conidia is mostly by inhalation to the alveoli of lung due to their small size (2–3 µm) and subjected to conidium-host interactions by exposure to alveolar macrophages. Aspergillus fumigatus can invade the host through alternative routes causing other serious biomaterial-related biofilm infections, through catheters, joint replacements, cardiac pacemakers, heart valves and breast augmentation implants (Escande et al. 2011; Jeloka et al. 2011; Muldoon et al. 2017). A. fumigatus can form biofilms on biomaterials and on other medical instruments: sensors, gloves, incubators and blankets (Gallais et al. 2017; Groll et al. 1998; Müller et al. 2011; Müller 2014; Perzigian and Faix 1993; Singer et al. 1998; Stock et al. 2010). In the past decades the number of immunocompromised patients increased due to the escalated use of biomaterials in medicine, which led to the elevated number of invasive pulmonary aspergillosis (Blankenship and Mitchell 2006; Calton et al. 2014; Munoz et al. 2014).

Parameters that distinguish between the stages of infection of conidiospores and the time of sporal outgrowth serve as important data to be utilized in the antifungal therapy against filamentous fungi such as Aspergillus, Fusarium and yeast cells e.g. Candida or Cryptococcus species. Images of conidiosporal development of A. fumigatus obtained by TLS were subjected to digital image analysis to understand fermentation processes related to apical filament growth. In this work the conidial and hyphal morphology were analysed to understand the mechanism of action of the best known antifungal agents. Methods based on image analysis were originally applied to Candida albicans (Nagy et al. 2014) to avoid the use of expensive commercial software. These methods were extended to the measurement of outgrowth of conidiospores of A. fumigatus. After preparation of conidia and young hyphae as well as their separation it is recommended to analyze all forms of fungi from conidium to the growth of branching hyphae.




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