Halotolerance (ionic NaCl) and chaotolerance (ionic MgCl2) of the human pathogen Wallemia mellicola isolated, for the first time, from indoor air in Poland

We isolated the human pathogen Wallemia mellicola from the class Wallemiomycetes for the first time in Poland. The fungus was isolated from indoor dust in Cracow. The strain belonged to the genus Wallemia, as confirmed by molecular methods (i.e., ITS1-5.8S-ITS2 nrDNA sequencing). We compared the halotolerance and chaotolerance of W. mellicola to other those of halotolerant fungi (Talaromyces diversus and Aureobasidium pullulans) isolated from an anchialine cave. The W. mellicola strain tolerated up to 20% NaCl and up to 15% MgCl2. As the concentration of NaCl in the culture medium increased, the colony diameter of W. mellicola decreased slightly. Dose–response curves for the two reference fungi T. diversus and A. pullulans revealed much lower tolerance of these fungi to increasing concentrations of NaCl. Our results indicated that W. mellicola is an advanced kosmotrope that is distinctly adapted to saline environments.


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Species in the W. sebi complex produce toxic metabolites, i.e., walleminol A, walleminone [7,8], azasteroid [9], and wallimidione [10], which may cause subcutaneous infections referred to as phaeohyphomycosis [11]. Some reports have suggested that species in this complex could be the causal agents for lung disease in farmers in Scandinavia, France, and China and allergic activity [12]. These slow-growing fungi are easily overgrown by fast-growing hyphomycetes on agar plates and this could explain why they are rarely isolated by traditional cultivation techniques. Real-time PCR appears to be the most sensitive technique for detecting the W. sebi complex [13].
We isolated several microfungal strains from 90 flats in Cracow old town. The strains were temporarily identified as Wallemia sp. and were found in a single locality. Fungi from the genus Wallemia have not been reported in Poland. We examined the taxonomic and physiological status of the isolated strain.
Some culture features, including micromorphological and ecophysiological characters of the Wallemia strain enabled the correct identification of the species. The size of the cerebriform colony on YMA, relatively large conidia, growth at 34°C, degree of halotolerance, and growth on YMA with 13% MgCl 2 [14] indicated that the strain is W. mellicola Jančič, Nguyen, Seifert & Gunde-Cimerman.
To better describe the taxonomic position of the investigated strain, we performed ecophysiological tests [3], such as halotolerance (ionic NaCl) and chaotolerance (ionic MgCl 2 ) assays. We compared the results for W. mellicola to those for two other moderately halotolerant (Chlebicki and Jakus, unpublished data) species isolated from anchialine caves, Aureobasidium pullulans and Talaromyces diversus. Growth in highly saline environments requires specific adaptations [3]. We examined decreases in the size of the strain colony with increasing salinity to estimate halotolerance. Additionally, we used molecular methods to confirm the strain identification.

Material and methods
The investigated strain of Wallemia was isolated from the indoor dust of an old flat located in Cracow, Poland (Targowa Street). Air for mycological studies was collected using the Eco Mas 100 sampler (Microbial Air Sampler MBV Switzerland, distributed by Merck Eurolab, Darmstadt, Germany, V4, 100 liters) and Petri dishes contained PDA (potato dextrose agar). After sampling, the plates were stored at room temperature (ca. 22°C) in the dark. After 10 days, small colonies were transferred to the following media: PDA, MEA (malt extract agar), and MEA supplemented with 5% NaCl. Strains of A. pullulans and T. diversus used for comparison were isolated from anchialine caves located in Croatia (Chlebicki and Jakus, unpublished data). Halotolerance (ionic NaCl) and chaotolerance (ionic MgCl 2 ) were determined on YMA (yeast malt agar; Sigma-Aldrich, St. Louis, MO, USA). Conidia obtained from the cultivated strain were suspended in 0.9% NaCl and 0.05% agar and were point-inoculated in triplicate on each plate onto 9-cm Petri dishes with YMA medium supplemented with ionic MgCl 2 at 4%, 9%, 11%, 13%, 15%, and 17% and ionic NaCl at 10%, 12%, 14%, 16%, 18%, 20%, 24%, and 28%. Cultures in six replicates for each combination (in two series) were incubated in the dark at room temperature for 7, 14, and 28 days. Colony diameters were measured after 14 days. The fungal colonies were deposited in the fungal collection of the Department of Mycology, W. Szafer Institute of Botany in Cracow.
DNA was extracted using a modification of the CTAB protocol described by Särkinen et al. [15] and Healey et al. [16]. Amplification of the ITS region was performed by touchdown polymerase chain reaction (PCR). Amplification was performed in a 15-μL reaction volume containing 1× REDTaq PCR Reaction Buffer (Sigma-Aldrich), 0.2 mM dNTPs at an equimolar ratio, 0.01 mg/mL bovine serum albumin, 0.05 units/ μL REDTaq DNA Polymerase (Sigma-Aldrich), and 0.2 μM each of the ITS1-F [17] and ITS4 primers [18]. Stock DNA (1 μL) was added to each reaction as a template. The PCR protocol was as follows: 3 min at 94°C, 10 cycles of 30 s at 94°C, 30 s at 60°C, reducing the annealing temperature by 1°C every successive cycle, and 1 min at 72°C, 25 cycles of 30 s at 94°C, 30 s at 50°C, and 1 min at 72°C, followed by 7 min at 72°C. The reaction was held at 4°C until further processing. PCR products were purified using ExoSAP-IT PCR Product Cleanup (Affymetrix, Santa Clara, CA, USA). Sequencing reactions (3 min at 96°C and 30 cycles of 10 s at 96°C, 5 s at 50°C, and 2 min at 60°C) were performed using the primers mentioned above and the BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, MA, USA) together with BDX64 Sequencing Enhancement Buffer (Nimagen, Lagelandseweg, Netherlands), according to the manufacturer's protocol (Nimagen) for a 32× dilution. Labelled fragments were separated on the POP-7 polymer, using an ABI Prism 3130 automated DNA sequencer.
The morphological characters of the living fungi were examined in water and cotton blue in lactophenol by light microscopy (Nikon SMZ 1500, Nikon Eclipse 800; Nikon, Tokyo, Japan) equipped with a digital camera for microphotography documentation. For scanning electron microscope (SEM) studies, the mycelium was fixed in 3% buffered glutaraldehyde (pH 7), washed two times in buffer for 10 min and subsequently dehydrated in ethanol and acetone. The prepared mycelium was coated with gold and images were obtained using a LEO 1430 VP microscope (SEM; Zeiss, Oberkochen, Germany) with a working distance of ca. 10 mm. The nomenclature for fungal species followed Index Fungorum. Description. Colonies were initially punctiform after spreading on the agar, grew on MEA without additional solutes, purplish brown and brown in reverse, cerebriform (Fig. 1), surface was velvety, irregular in shape, powdery, with margins darker than the colony (Fig. 1B). Conidia were initially cubic, then spherical, verruculose, (2.5)2.9-3.5 × 1.8-2.2 µm (Fig. 2) We used a DNA sequence analysis to confirm the correct strain identification. The ITS1-5.8S-ITS2 sequences of our strain PKA-35 were 583 bp. In Blast searches, two strains (GenBank accession numbers KJ409882.1 and AY302532.1) had 100% query coverage with our strain. Wallemia mellicola strain DAOM 242695 = KJ409882.1 [2] was chosen as the leading sequence. There were no differences between DAOM 242695 and PKA-35 = KX977321 sequences. Therefore, based on culture features, micromorphological data, as well as the ITS sequence, we may conclude that the investigated strain belongs to the species W. mellicola. It is worth noting that W. mellicola, isolated and cultured from an old flat in Cracow, has not been previously recorded in Poland. It is also the first species of Wallemiomycetes noted in Poland.

Results and discussion
The tested strain of W. mellicola grew at up to 20% NaCl and up to 15% MgCl 2 (Fig. 3, Fig. 4). According to Zajc et al. [19], the type of salt added to media affects the growth of strains. Extensive studies have identified fungi that thrive on media with NaCl. In contrast, few strains are able to growth in the presence of MgCl 2 [19].
Jančič et al. [14] noted a halotolerance of 4-24% NaCl and chaotolerance of 4-13% MgCl 2 for W. mellicola. The most closely related W. canadensis exhibits no growth at 34°C on MEA and no growth on YMA with 13% MgCl 2 [14]. With increasing concentrations of NaCl in the culture medium, the colony diameter of our W. mellicola strain decreased only slightly (Fig. 4). The growth curves obtained for T. diversus and A. pullulans indicated much greater declines than that for W. mellicola (Tab. 1). These results suggest that W. mellicola is more adapted to saline    environments than the other two fungal species. The examined strains of A. pullulans and T. diversus were isolated from anchialine caves (Chlebicki and Jakus, unpublished data). Its distinct adaptation to a saline environment suggests that the strain is a longstanding and evolutionary advanced kosmotrope.