Morphological changes and induction of antifungal resistance in Aspergillus fumigatus due to different CO2 levels

Background and Purpose: Aspergillosis is one of the most common opportunistic fungal infections in immunocompromised and neutropenic patients. Aspergillus fumigatus (A. fumigatus) is the most common causative agent of this infection. Due to variable CO2 concentrations that pathogens are exposed to during the infection process and to understand the role of CO2, we examined the effects of various CO2 concentrations as one of the environmental factors on morphological changes and induction of antifungal resistance in A. fumigatus. Materials and Methods: A. fumigatus strains were cultured and incubated under 1%, 3%, 5%, and 12% CO2 atmospheres, each time for one, two, and four weeks. The control culture was maintained for one week without CO2 atmosphere. Morphological changes were investigated and antifungal susceptibility test was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. The results of different CO2 atmospheres were compared with that of the control sample. Results: We found that 1%, 3%, 5%, and 12% CO2 atmospheres were associated with morphological colony changes. Macroscopically, the colonies were shallow dark green, smooth, crisp to powdery with reduced growth; microscopic examination revealed the absence of conidiation. The induction of antifungal resistance in the susceptible strains to itraconazole, voriconazole, and amphotericin B increased after exposure to 12% CO2 atmosphere and four weeks of incubation. The MIC values for itraconazole, voriconazole, and amphotericin B were 16 g/ml, 1 g/ml, and 16 g/ml, respectively. These values for the control group were 0.125 g/ml, 0.125 g/ml, and 2 g/ml, respectively. Conclusion: Exposure to different CO2 atmospheres induced morphological changes in A. fumigatus, it seems to increase the MIC values, as well. In parallel, resistance to both itraconazole and voriconazole was also observed.


Introduction
spergillus species are widely distributed in the environment and have a wide range of manifestations from asymptomatic colonization or allergic reaction to invasive infection depending on host immunity [1,2]. Invasive aspergillosis is the most severe manifestation with high mortality rate in immunocompromised patients [3,4]. Amphotericin B (a member of the polyene antibiotics) and several azole-based medicines such as itraconazole, voriconazole, posaconazole, and isavuconazole are used for the treatment and prophylaxis of aspergillosis [5][6][7].
Besides, specific point mutations in the cyp51A gene in combination with tandem repeats in the cyp51A promoter region are indicated as major environmentally derived mutations among azoleresistant A. fumigatus isolates. Additionally, cyp51A- independent resistance mechanisms, such as the upregulation of drug efflux transporters, have been recognized [14,15]. Acquired resistance in A. fumigatus may be caused by long-term treatment and exposure of the fungal cells to azole fungicides used in agriculture. Along with these factors, environmental changes including the nutrient sources, humidity, temperature, and air pollution could potentially lead to physiological changes in microorganisms [16]. Atmospheric CO2 is considered an important agent for the biosphere homeostasis, which may cause changes in the physiological and morphological characteristics and the allergenic properties of saprophytic and pathogenic fungi. Such changes have been shown in Cryptococcus neoformans and Candida albicans during colonization and infection [17]. In addition, the effect of CO2 pressure on growth and aflatoxin production in Aspergillus flavus was previously reported [18][19][20][21]. Other studies revealed that increasing CO2 atmosphere in the culture medium could influence the pathogenicity of A. fumigatus [22,23]. Due to the effectiveness of CO2 pressure as one of the environmental factors, we examined the effects of different CO2 concentrations on morphology and induction of antifungal resistance in A. fumigatus.

A. fumigatus strain
The wild-type strain of A. fumigatus (ATCC 1028) was cultured on Potato Dextrose Agar (PDA; Merck, Germany) and incubated at 35°C for 5-7 days. The suspension was prepared according to Clinical And Laboratory Standard Institute (CLSI M38-A2) protocol [24]. Briefly, fungal spores were harvested from 3 to 7 day-old cultures (in logarithmic growth phase) and mixed with distilled water containing 0.05% Tween 40; the mixture was adjusted at the final concentration of 1×10 6 CFU/ml by spectrophotometer (530 nm) [24]. Afterwards, 200 μl of the obtained suspension was added to PDA culture medium plates [25]. The plates were incubated at 35°C for 1, 2, and 4 weeks under 1%, 3%, 5%, and 12% CO2 atmospheres separately.
CO2 atmosphere was provided by using a 20-L cell culture CO2 incubator (SLS, USA). The desired CO2 injection was controlled by the calibrated automatic control panel in this incubator. The control culture was prepared under the same conditions without CO2 pressure.

Macroscopic and microscopic examinations
Macroscopic and microscopic criteria were investigated during culture under various CO2 atmospheres. The color and texture features of the colony (i.e., flat, granular, downy to powdery, and radial grooves) were studied during the incubation time. By using Lactophenol Cotton Blue, the microscopic criteria were considered to assess the morphological changes [26,27]. All the cultures revealing morphological changes under CO2 atmospheres were inoculated in the Tryptic Soy Broth (TSB) medium (Merck, Germany) and stored at -20°C. After three weeks, the stocked cultures in TSB medium were sub-cultured in PDA medium and incubated at 35°C without CO2 atmosphere, and their morphological aspects were checked for reversibility evaluation.

Antifungal susceptibility test
The minimum inhibitory concentrations (MICs) of three antifungal agents were evaluated according to CLSI (M38-A2) protocol [24,28]. Itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer, NY, USA), and amphotericin B (Bristol-Myers-Squibb, Woerden, the Netherlands) were obtained as reagent-grade powders dissolved in dimethyl sulfoxide (DMSO) and were diluted in standard RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) buffered to pH 7.0 with 0.165 mol.L -1 morpholinepropanesulfonic acid buffer with L-glutamine without bicarbonate (MOPS, Sigma-Aldrich, St. Louis, MO, USA). The final concentrations of itraconazole, voriconazole, and amphotericin B ranged from 0.016 to 16 µg/ml. The plates were stored at -20°C prior to use [29]. All the cultured fungal cells under CO2 pressure were scraped after sporulation and the obtained spores were suspended in distilled water containing 0.05% Tween-40. The optical density (OD) of the supernatant was adjusted spectrophotometrically at 530 nm. The final concentrations of the stock inoculum suspensions were within the range of 0.5-4 × 10 4 CFU/ml. Microdilution plates were incubated at 35°C and examined visually for MIC determination within 48 h of incubation. The MICs for itraconazole, voriconazole, and amphotericin B were defined as the concentration that fully inhibits fungal growth. The strain Candida parapsilosis ATCC 22019 was used as the negative quality control. All the tests were performed in duplicate.
This study was approved by the Ethics Committee of Tehran University of Medical Sciences, Tehran, Iran (code: IR.TUMS.SPH.REC.1396.2289).

Results
We observed significant changes in the macroscopic and microscopic characteristics of A. fumigatus wild-type in different situations (CO2 atmosphere and incubation duration). The initial macro-and micro-colony changes occurred in the presence of low CO2 pressure (1%). However, the minimum of such changes were observed under 3% and 5% CO2 atmospheres after one, two, and four weeks, while maximum changes happened under 12% CO2 atmosphere after four weeks. The major macroscopic changes appeared in the culture media that included shallow colonies with dark green color, smooth and crisp to powdery appearance, and reduced growth. However, the major microscopic changes appeared in exposed fungal cells with short Curr Med Mycol, 2017, 3(3): 21-26  23 conidiophores, deformed vesicles, and declined conidia ( Figure 1). These results were obtained by comparison with the control colonies, which were blue-green, fast-growing, fluffy to powdery colonies with normal conidiophores, arranged vesicles, phialides, metulae, and smooth hyaline conidia ( Figure 1). The reversibility of morphological changes was reviewed through culture of the samples after four weeks in the absence of CO2 atmosphere.
The obtained results indicated that A. fumigatus could maintain the described changes even in the absence of CO2 atmosphere.
The MIC values for itraconazole, voriconazole, and amphotericin B under various CO2 atmospheres and incubation times are listed in Table 1. The MIC values after 48 h of incubation increased with elevated CO2 pressure and prolonged incubation time. Under 12% CO2 atmosphere and after four weeks of

Discussion
A. fumigatus is one of the most important fungal pathogens and the main causative agent of invasive aspergillosis. This species is affected by environmental conditions such as CO2 [23,30]. Studies showed that CO2 could significantly affect fungal physiology and morphology [31]. Previous studies on the effects of CO2 atmosphere on pathogenesis and metabolism of C. albicans and Cryptococcus neoformans revealed the induction of invasion to blood in C. albicans cells under 5% CO2 atmosphere. Besides, filamentation and pseudohyphal formation were reported to be induced in such cells [25,32,33]. Capsular polysaccharide synthesis of C. neoformans was stimulated after exposure to 5% CO2 atmosphere [23,[34][35][36]. Another study indicated the occurrence of arthroconidium formation, a resistance factor in T. rubrum, after 10 days of incubation on Sabouraud Dextrose Agar medium at 37°C under 10% CO2 atmosphere [37]. Some investigations have considered the effect of CO2 on fungal growth and found a general inhibitory effect under CO2 atmospheres [31]. Coelho et al. reported that high CO2 pressure could have detrimental effects on growth and metabolism of yeasts, and therefore, could contribute to the inactivation of Saccharomyces cerevisiae cells [38,39]. Due to its antimicrobial activity, CO2 can be particularly used for storing food commodities through the inactivation of the microorganisms [40]. Changed CO2 atmosphere, carbonic anhydrases (CAs), and C: N ratio have been reported to affect growth by influencing the production of proteins and the content of fungal spores [41,42]. The effects of CO2 on morphology, growth, and citric acid production in A. niger were also reported by McIntyre and McNeil [31].
Recent investigations indicated that A. fumigatus and A. flavus spores grown under varying CO2 atmosphere showed higher allergenicity [22,32]. In line with the previous investigations, we appraised the effect of CO2 on morphological changes in A. fumigatus. The obtained results indicated that CO2 atmosphere could enhance some morphological (macroscopic and microscopic) changes such as sporulation, vesicle deformation, and the development of Chlamydia spores in A. fumigatus strains. The majority of the mentioned changes were also indicated to occur under 12% CO2 atmosphere and after four weeks of incubation. Previous reports revealed the capability of pathogenic Aspergillus species in growth under CO2 atmosphere; they also showed that this capacity might affect the pathogenicity and antifungal activity of these species [20]. Our findings may help with deeper understanding of this process. Besides, variation in CO2 atmosphere in environments where A. fumigatus is able to grow reveals the importance of effective factor and CO2 absorb regulator genes in these fungi.
Our results manifested that MICs of itraconazole, voriconazole, and amphotericin B against A. fumigatus increased near high CO2 environments. However, the mechanism of resistance induction by CO2 remains inconspicuous. Some medicines and environmental factors have been used for the induction of drug resistance in A. fumigatus [43]. In some studies, different types of resistance mechanisms, including changes in the target lanosterol-14-demethylase and induction of high-capacity drug efflux pumps, have been shown to promote drug resistance following UVinduced mutations [44][45][46]. In the study of Escribano et al., the MICs of itraconazole, voriconazole, and posaconazole against A. fumigatus cells were determined before and after exposure of the fungal cells to itraconazole. The obtained results indicated that the final MICs were substantially higher than the initial ones [47]. Faria-Ramos et al. investigated crossresistance in A. fumigatus to clinical azoles after the exposure of fungal cells to prochloraz (an agronomical azole) and found that resistance to prochloraz increased after the initial exposure [48]. In addition, prochloraz exposure caused some morphological changes in fungal colony such as changing the appearance to white-colored colonies, losing the typical pigmentation, and absence of conidiation [48]. Our results also indicated the macroscopic and microscopic morphological changes in A. fumigatus due to CO2 pressure. However, these morphological changes were not similar to the alterations described by Faria-Ramos et al.
The results of these studies indicate that A. fumigatus can be resistant to antifungal compounds due to antifungal agents in medicine, fungicides used in agriculture, and environmental factors. The results of this study also indicated that high concentrations of CO2 could be considered as an environmental factor affecting the occurrence of drug resistance in fungi.

Conclusion
Different levels of CO2 exposure induced morphological changes in A. fumigatus, an evident increase in MIC values, and the development of crossresistance to itraconazole and voriconazole. Our next step is assessing the underlying molecular resistance mechanisms in these induced resistant strains, as well as in isolates with naturally high MIC values in different levels of CO2 and evaluating resistance to antifungal drugs without any prior in vitro induction.

Conflicts of interest
None declared.

Financial disclosure
No pecuniary interest related to the material of this writing has been declared.