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Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole.

Faria-Ramos I, Farinha S, Neves-Maia J, Tavares PR, Miranda IM, Estevinho LM, Pina-Vaz C, Rodrigues AG - BMC Microbiol. (2014)

Bottom Line: The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure.PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation.PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.

View Article: PubMed Central - HTML - PubMed

Affiliation: Microbiology Department, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal. agr@med.up.pt.

ABSTRACT

Background: The purpose of this study was to unveil whether azole antifungals used in agriculture, similar to the clinical azoles used in humans, can evoke resistance among relevant human pathogens like Aspergillus fumigatus, an ubiquitous agent in nature. Additionally, cross-resistance with clinical azoles was investigated. Antifungal susceptibility testing of environmental and clinical isolates of A. fumigatus was performed according to the CLSI M38-A2 protocol. In vitro induction assays were conducted involving daily incubation of susceptible A. fumigatus isolates, at 35°C and 180 rpm, in fresh GYEP broth medium supplemented with Prochloraz (PCZ), a potent agricultural antifungal, for a period of 30 days. Minimal inhibitory concentrations (MIC) of PCZ and clinical azoles were monitored every ten days. In order to assess the stability of the developed MIC, the strains were afterwards sub-cultured for an additional 30 days in the absence of antifungal. Along the in vitro induction process, microscopic and macroscopic cultural observations were registered.

Results: MIC of PCZ increased 256 times after the initial exposure; cross-resistance to all tested clinical azoles was observed. The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure. PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation.

Conclusions: PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.

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Photomicrographs of Aspergillus fumigatus colonies using the cellotape flag technique preparation with lactophenol cotton blue staining. Microscopic morphological changes in the development of conidiation of A. fumigatus following exposure to subinhibitory concentration of PCZ. A. Initial morphological aspect (control). B. After fifteen days. C. After thirty days.
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Figure 2: Photomicrographs of Aspergillus fumigatus colonies using the cellotape flag technique preparation with lactophenol cotton blue staining. Microscopic morphological changes in the development of conidiation of A. fumigatus following exposure to subinhibitory concentration of PCZ. A. Initial morphological aspect (control). B. After fifteen days. C. After thirty days.

Mentions: There are several studies that have characterized azole resistance in A. fumigatus, and most recently some addressed the possible cross-resistance between environmental and medical azoles [8-11]. Our study demonstrated the time frame between the introduction of a widely used agricultural antifungal and the emergence of cross-resistance to medical triazoles. During the induction assay, we found that besides the emergence of cross-resistance, PCZ exposure caused marked morphological colony changes, both macroscopically and microscopically. Macroscopic modification of the pigmentation of A. fumigatus colonies, changing from the original green colour to white (Figure 1A, B and C) was remarkable at the beginning of the assay. With the increase of MIC values of PCZ the colonies became scarcer, smaller and totally white (Figure 1C). Microscopic examination showed a progressive absence of conidiation: the original strain (Figure 1A) showed normal microscopic features regarding conidiation (Figure 2A) while almost white colonies (Figure 1B) showed nearly complete absence of conidiation (Figure 2B). The totally white mycelia (Figure 1C) corresponded solely to hyphae and immature little conidiophore structures without conidia (Figure 2C). These changes in pigmentation and in conidiation as a consequence of exposure to azoles have already been reported. Varanasi and colleagues speculate that azoles may bind to a phytochrome-like regulatory molecule inhibiting the initiation and subsequent development of conidiophores in Aspergillus species [13]. Such mechanism of action could also explain the different levels of inhibition displayed by other tested azoles and why echinocandins and polyenes did not show this effect [13]. Notably, such morphological changes may be responsible for laboratorial diagnostic misidentification of the fungal genus/species [14]. The high MIC values for PCZ that were achieved in vitro maintained stable following removal of the selective pressure of the drug. For VRC, the MIC value decreased only after 30 days of incubation without the selective pressure, changing the susceptibility phenotype from resistant to intermediate. For POS, the developed MIC value also decreased but not enough to change the phenotype of resistance. Regarding ITZ, for both LMF11 and LMN60, it was observed the complete reversibility of the resistant phenotype in the absence of PCZ, ie, the MIC reverted to the initial value (susceptible). However, strain LMF05 had, since day zero, ITZ MIC of 2 mg/L, which falls in resistant category. In all the isolates conidiation reappeared together with the typical green colour of mature colonies following the removal of PCZ.


Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole.

Faria-Ramos I, Farinha S, Neves-Maia J, Tavares PR, Miranda IM, Estevinho LM, Pina-Vaz C, Rodrigues AG - BMC Microbiol. (2014)

Photomicrographs of Aspergillus fumigatus colonies using the cellotape flag technique preparation with lactophenol cotton blue staining. Microscopic morphological changes in the development of conidiation of A. fumigatus following exposure to subinhibitory concentration of PCZ. A. Initial morphological aspect (control). B. After fifteen days. C. After thirty days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4061453&req=5

Figure 2: Photomicrographs of Aspergillus fumigatus colonies using the cellotape flag technique preparation with lactophenol cotton blue staining. Microscopic morphological changes in the development of conidiation of A. fumigatus following exposure to subinhibitory concentration of PCZ. A. Initial morphological aspect (control). B. After fifteen days. C. After thirty days.
Mentions: There are several studies that have characterized azole resistance in A. fumigatus, and most recently some addressed the possible cross-resistance between environmental and medical azoles [8-11]. Our study demonstrated the time frame between the introduction of a widely used agricultural antifungal and the emergence of cross-resistance to medical triazoles. During the induction assay, we found that besides the emergence of cross-resistance, PCZ exposure caused marked morphological colony changes, both macroscopically and microscopically. Macroscopic modification of the pigmentation of A. fumigatus colonies, changing from the original green colour to white (Figure 1A, B and C) was remarkable at the beginning of the assay. With the increase of MIC values of PCZ the colonies became scarcer, smaller and totally white (Figure 1C). Microscopic examination showed a progressive absence of conidiation: the original strain (Figure 1A) showed normal microscopic features regarding conidiation (Figure 2A) while almost white colonies (Figure 1B) showed nearly complete absence of conidiation (Figure 2B). The totally white mycelia (Figure 1C) corresponded solely to hyphae and immature little conidiophore structures without conidia (Figure 2C). These changes in pigmentation and in conidiation as a consequence of exposure to azoles have already been reported. Varanasi and colleagues speculate that azoles may bind to a phytochrome-like regulatory molecule inhibiting the initiation and subsequent development of conidiophores in Aspergillus species [13]. Such mechanism of action could also explain the different levels of inhibition displayed by other tested azoles and why echinocandins and polyenes did not show this effect [13]. Notably, such morphological changes may be responsible for laboratorial diagnostic misidentification of the fungal genus/species [14]. The high MIC values for PCZ that were achieved in vitro maintained stable following removal of the selective pressure of the drug. For VRC, the MIC value decreased only after 30 days of incubation without the selective pressure, changing the susceptibility phenotype from resistant to intermediate. For POS, the developed MIC value also decreased but not enough to change the phenotype of resistance. Regarding ITZ, for both LMF11 and LMN60, it was observed the complete reversibility of the resistant phenotype in the absence of PCZ, ie, the MIC reverted to the initial value (susceptible). However, strain LMF05 had, since day zero, ITZ MIC of 2 mg/L, which falls in resistant category. In all the isolates conidiation reappeared together with the typical green colour of mature colonies following the removal of PCZ.

Bottom Line: The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure.PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation.PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.

View Article: PubMed Central - HTML - PubMed

Affiliation: Microbiology Department, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal. agr@med.up.pt.

ABSTRACT

Background: The purpose of this study was to unveil whether azole antifungals used in agriculture, similar to the clinical azoles used in humans, can evoke resistance among relevant human pathogens like Aspergillus fumigatus, an ubiquitous agent in nature. Additionally, cross-resistance with clinical azoles was investigated. Antifungal susceptibility testing of environmental and clinical isolates of A. fumigatus was performed according to the CLSI M38-A2 protocol. In vitro induction assays were conducted involving daily incubation of susceptible A. fumigatus isolates, at 35°C and 180 rpm, in fresh GYEP broth medium supplemented with Prochloraz (PCZ), a potent agricultural antifungal, for a period of 30 days. Minimal inhibitory concentrations (MIC) of PCZ and clinical azoles were monitored every ten days. In order to assess the stability of the developed MIC, the strains were afterwards sub-cultured for an additional 30 days in the absence of antifungal. Along the in vitro induction process, microscopic and macroscopic cultural observations were registered.

Results: MIC of PCZ increased 256 times after the initial exposure; cross-resistance to all tested clinical azoles was observed. The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure. PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation.

Conclusions: PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.

Show MeSH
Related in: MedlinePlus