Dishwashers are a major source of human opportunistic yeast-like fungi in indoor environments in Mersin, Turkey

Medical Mycology July 2013, 51, 493–498 Dishwashers are a major source of human opportunistic yeast-like fungi in indoor environments in Mersin,Turkey The natural habitat of opportunistic fungal pathogens is outside of the host; therefore, it is crucial to understand their ecology and routes of transmission. In this study, we investigated the presence of black and filamentous fungi in moist indoor environments in the city of Mersin in subtropical Turkey. In total, 177 private dwellings were screened and 893 samples obtained using cotton swabs and moistened with physiological saline from dishwashers, washing machines, refrigerators, bath-tubs, bathroom walls, and shower heads. These were then inoculated onto malt extract agar supplemented with chloramphenicol, followed by incubation at 37°C. Thirty samples (3.4%) were positive for fungi, which were then identified by sequencing the rDNA internal transcribed spacer region. Exophiala dermatitidis was the most common species (23), followed by E. phaeomuriformis (three), Magnusiomyces capitatus (two), and Candida parapsilosis (two). Genotype A of E. dermatitidis (14) was more prevalent than genotypes B (eight) and C (one) and E. phaeomuriformis was also represented by two genotypes. Our findings suggest that dishwashers are a major indoor niche for thermophilic black yeasts. The occurrence of the opportunistic filamentous fungus M. capitatus in dishwashers is consistent with a recent report. Keywords Chaetothyriales, Exophiala dermatitidis, Exophiala phaeomuriformis, extremophile, filamentous fungi Introduction The black yeast genus Exophiala in the ascomycete order Chaetothyriales contains numerous species that are involved in human infection [1]. To date, 18 of 29 known Exophiala species have been proven or suggested to cause cutaneous, subcutaneous, or systemic infections [2,3]. The severity of human infections due to black fungi differs according to the species, with E. dermatitidis being among the more virulent species within the genus [2,4–6]. It has Received 25 July 2012; Received in final revised form 14 September 2012; Accepted 5 October 2012. Correspondence: Macit Ilkit, Division of Mycology, Department of Microbiology, Faculty of Medicine, University of Çukurova, Adana, 01330, Turkey. Tel.: ⫹90 532 286 0099; Fax: ⫹90 322 457 3072; E-mail: © 2013 ISHAM been reported to be responsible for, among other clinical conditions, relatively uncommon, but fatal cerebral infections in immunocompetent adults in East and South-East Asia [5,7,8]. In addition, it may be responsible for asymptomatic colonization of the lungs in patients with cystic fibrosis (CF). In the latter patient population, the prevalence of E. dermatitidis ranges from 5% in Germany [4] to 19% in Sweden [6]. In addition, E. dermatitidis was identified in the intestinal tract in 0.5% of the European population [9]. Uijthof et al. [10] and Sudhadham et al. [11] demonstrated that E. dermatitidis is distributed worldwide, but the route of human infections due to black fungi is still not known. Thus, screening for black fungi in indoor environments would enhance our understanding of potential health risks [12–14]. Sudhadham et al. [15] noted the presence DOI: 10.3109/13693786.2012.738313 AYLİ N DÖĞEN*, ENGİ N KAPLAN†, ZEHRA ÖKSÜZ*, MEHMET SAMİ SERİ N*, MACİ T ILKİ T‡ & G. SYBREN DE HOOG§ *Department of Pharmaceutical Microbiology, Faculty of Pharmacy, and †Advanced Technology Education, Research, and Application Center, Mersin University, Mersin, ‡Division of Mycology, Department of Microbiology, Faculty of Medicine, University of Çukurova, Adana,Turkey, and §CBS-KNAW Fungal Biodiversity Centre, Utrecht, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam,The Netherlands, Peking University Health Science Center, Research Center for Medical Mycology, Beijing, and Sun Yat-sen Hospital, Sun Yat-sen University, Guangzhou, P. R. China 494 Döğen et al. Materials and methods Sampling area The average monthly water temperature in February and March in Mersin is 14°C (annual average: 17.4°C). In addition, the average rainfall in these 2 months is 95 mm (kg/m2) and 70 mm (kg/m2), respectively. Water is supplied to Mersin’s city center by the 124 km (77 mile)-long Berdan Dam which has an average flow rate of 42 m3/s, higher than those of short rivers in its vicinity. The drainage basin of the dam covers an area of 1,592 km2; the river flows into the Mediterranean Sea at 36.47° N and 34.50° E. Sample collection From February to March 2012, a total of 893 samples were collected from dishwashers (153), washing machines (178), refrigerators (182), shower heads (173), bathtubs (171), and bathroom walls (36) from 177 private dwellings in Mersin, Turkey. The samples were obtained using sterile cotton swabs moistened with sterile physiological saline. Specifically, the inside of the invaginations of the rubber seals located on the folding doors of dishwashers were sampled, as described by Zalar et al. [14]. Samples were transported in sterile tubes and inoculated onto malt extract agar (MEA; Oxoid, Hampshire, UK) culture plates containing 100 μg/ml chloramphenicol (Sigma, Steinheim, Germany). The plates were incubated at 37°C and monitored daily for up to 15 days for evidence of growth. DNA extraction, PCR, and molecular analysis DNA extraction and PCR amplification were performed in accord with the protocol described by Turin et al. [19]. rDNA regions spanning the internal transcribed spacer (ITS) 1 were amplified using the universal fungal primers ITS1 and ITS4 on an ABI PRISM 3130XL genetic analyzer at Refgen Biotechnologies (Ankara, Turkey). CAP contig assembly software, included in the BioEdit Sequence Alignment Editor 7.0.9.0 software package, was used to edit the sequences [20]. Assembled DNA sequences were examined using the BLAST (nucleotide-nucleotide) software from the National Center of Biotechnology Information (NCBI) database. Genotyping Exophiala isolates Sequences were aligned with the ClustalW program [21] and compared by neighbor-joining phylogenetic tree analyses using MEGA version 5.05 (www.megasoftware.net). Genotype indications of E. dermatitidis were taken from Sudhadham et al. [11], and those for E. phaeomuriformis from Uijthof et al. [10]. Genotypes were submitted to and verified in a local database at CBS (www.cbs.knaw.nl), containing 390 comparable sequences of these two species, for research purposes. Physiology The Exophiala strains were tested for several growth characteristics. First, thermotolerance studies were performed in 96-well microtiter plates; each well contained 300 μl malt extract broth (MEB) into which 10 μl of a cell suspension was inoculated, after which plates were incubated at 5°C, 10°C, 40°C, 42°C, 45°C, and 47°C and assessed for growth daily for 2 weeks. Visual and spectrophotometric readings were performed at 450 nm (Thermo Scientific MultiScan Go reader, 1510-012, Vankaa, F (...truncated)