Exophiala species in household environments and their antifungal resistance profile

The black fungus Exophiala causes a wide range of infections from superficial to subcutaneous, but also invasive fungal infections in immunocompromised patients as well as healthy individuals. In addition, Exophiala, is a common colonizer of the air ways of patients with cystic fibrosis. However, the source of infection and mode of transmission is still unclear. The aim of this study was to investigate the presence of Exophiala in samples collected from Swedish indoor environments. We found that the Exophiala species were commonly found in dishwashers and that Exophiala dermatitidis was the most common Exophiala species, being identified in 70% (26 out of the 37) of samples. Almost all E. dermatitidis isolates had the ability to grow at 42 °C (P = 0.0002) and were catalase positive. Voriconazole and posaconazole exhibited the lowest MICs, while caspofungin and anidulafungin lack the antifungal activities in vitro. Future studies are needed to illuminate the transmission mode of the fungi.

followed by bathrooms (Table 1).E. dermatitidis was predominantly found in samples from dishwashers, representing 12 out of 13 isolates from these samples.

The physiological activity of Exophiala species
Almost all of the E. dermatitidis isolates, but only two of the non-dermatitidis Exophiala isolates (E.spinifera and E. mesophila), had the ability to grow at 42 °C (P = 0.0002) (Table 3).All isolates, from both E. dermatitidis and non-dermatitidis, were catalase positive, and the majority of isolates were urease positive.Esculin hydrolase activity was found only in 8 non-dermatitidis Exophiala isolates (80).None of the Exophiala isolates showed oxidase activity.

The susceptibility of Exophiala to anti-fungal drugs
The minmum inhibitory concentration (MIC) ranges, including the MIC 50 values of antifungal agents to E. dermatitidis, are shown in Fig. 1.Voriconazole and posaconazole exhibited the lowest MICs, followed by itraconazole and amphotericin.Caspofungin and anidulafungin had very broad range of MICs to E. dermatitidis (Fig. 1).Caspofungin and anidulafungin did not show any antifungal activity regarding the non-dermatitidis Exophiala isolates (Table 4).A higher MIC for fluconazole tested in this study were found for non-dermatitidis Exophiala (> 256 µg/ml) compared with E. dermatitidis (32 µg/ml).

Dishwasher Bathroom Coffee machine Faucet opening Washing machine
Exophiala alcalophila 1

Discussion
We found that almost 10% of the samples collected from indoor environments were positive for black fungus, being E. dermatitidis the dominant species.The black fungi E. dermatitidis has been found to be a frequent colonizer of patients with cystic fibrosis 4 , causing airway infection/colonization in this patient group 6 .Little is known about the natural habitat of Exophiala or possible infection routes.Various studies reported the presence of E. dermatitidis in tropical rainforests, steam baths, bathrooms, kitchen sinks and dishwashers [9][10][11][12] .However, the presence of Exophiala in the human population of the Nordic countries has not been previously investigated.
In the present study we analyzed 388 indoors samples of which 10% were positive for black fungus.The fungus was more frequently isolated from dishwashers, being present in 17% of the dishwashers analyzed.This finding agrees with a previous study by Dogen et al. 10 who showed that E. dermatitidis was the most common fungi isolated from dishwashers in Turkish household, being detected in 24 out of 158 (15%) dishwashers analyzed in their study.Earlier studies showed that the highest frequencies of E. dermatitidis in indoor environments are found in steam baths and dishwashers, where high temperatures, water and changing pH combine 7 .In our hands, only one sample collected from a washing machine was positive, which correlates with a previous study in which the number of E. dermatitidis was very low or absent in washing machines 10,13 .All E. dermatitidis isolated in this study had catalase activity.Catalases are antioxidant enzymes that are found in aerobic microorganisms and protect the cells from oxidative damages.Catalase activities have previously been reported for Candida albicans, Cryptococcus neoformans and Aspergillus species.Catalase production may be a virulence factor of Candida albicans and Cryptococcus neoformans 14 .Sav et al. reported catalase activity in all environmental and clinical E. dermatitidis 15 .
E. dermatitidis is a pathogenic fungus, which can cause neutropenic infections in humans.E. dermatitidis poses enormous ecological plasticity, which might play a role in fungus distribution and enables the transition of the fungus from the environment to a human host 16 .This thermotolerant fungus has been found in high temperature man-made habitats such as sauna facilities and dishwashers 9,11 .The majority of E. dermatitidis isolates in this study were thermotolerant and are able to grow at 42 °C, while only two non-dermatitidis Exophiala species were able to grow at this temperature.
The antifungal susceptibility testing exhibited that voriconazole and posaconazole followed by amphotericin and itraconazole, were the most effective antifungals against E. dermatitidis, which are in concordance with previous published studies 4,17 .Echinocandines (anidulafungin and caspofungin) and 5-flucytosine had no effect on Exophiala species, in vitro.
In conclusion, Exophiala species are found in humid common indoor environments and E. dermatitidis is the most common species.This thermotolerant fungus was frequently found in dishwashers.Its ability to produces various enzymes and grow at higher temperature may play an important role in its pathogenicity.The fungus also exhibits reduced susceptibility to several antifungal agents.Future studies are needed to clarify the infection's sources and mode of transmission.

Sampling
From January to September 2016, a total of 388 samples from 89 households located in Region Västra Götaland, Gothenburg, were collected.The samples were collected from faucet opening (n = 83), coffee machines (n = 76), dishwashers (n = 70), washing machines (n = 75), and bathrooms (floor and walls, n = 84) using sterile cotton swabs.The samples were cultivated on Sabouraud dextrose agar plates and erythritol-chloramphenicol (ECA) agar plates.All plates were incubated for up to 3 weeks at 30 °C.The plates were checked for growth twice a week.

Isolation and identification of Exophiala species
Brownish colonies were cultivated on Sabouraud agar plates for confirmation of purity and maintenance of the isolates.Pigmented fungi were identified to the species level using morphological criteria, and molecular identification as previously described 4 .www.nature.com/scientificreports/

Physiological activity
DNase.The extracellular DNase activity was analyzed by using a DNase test agar plate as previously described.
The microorganism suspension was cultured on DNase medium and incubated at 30 °C for 7 days.The plates were then flooded with 1N hydrochloric acid.After 5 min, the excess fluid was removed and clear zones around the colonies were measured 18 .Staphylococcus aureus CCUG 17,621 was used as control.
Esculin activity.The ability of fungi to hydrolyse esculin (a coumarin glucoside) into glucose is measured by incubation of a fungal colony a the medium (3 ml) composed of esculin 1 mg/ml and ferric citrate 0.5 mg/ml in peptone water.The fungal suspension was incubated at 37 °C for 48 h.The production of a black coloured component in medium is considered as a positive reaction 19 .www.nature.com/scientificreports/Catalase activity.A colony of fungal isolate was transferred on to a glass microscope slide and mixed with 20 µl of hydrogen peroxidase at room temperature.The formation of air bubbles was considered as a positive catalase activity.Staphylococcus aureus CCUG 17,621 was used as a control.
Oxidase.Peroxidase (20 µl) was added to a glass microscope slide.A small portion of a fungal colony was removed from the agar surface and mixed with oxidase substrate.The formation of a color change to deep blue or purple within 10 s were considered as a positive reaction.Staphylococcus aureus CCUG 17621 was used as a control.
Urease.The fungal isolates were cultured on Urease agar medium and incubated for three days at 25 °C.The fungal isolates were considered urease positive when a pinkish discoloration in medium was observed.
Thermotolerance.The fungal tolerance to higher temperatures was studied by incubation of the fungal isolates on Sabouarud agar plates at 42 °C for three days.

Antifungal susceptibility testing
Antifungal susceptibilities were determined by Etest (bioMerieux, Marcy-l'Étoile, France) as previously described 4 .The concentrations of the antifungal agents on Etest strips ranged from 0.002 to 256 μg/mL for fluconazole and from 0.002 to 32 μg/mL for itraconazole, voriconazole, posaconazole, caspofungin, amphotericin B, and flucytosine.Briefly, fungal colonies were suspended in 0.85% saline solution, and the turbidity was adjusted to 0.5 on the McFarland scale (1 × 10 6 to 5 × 10 6 CFU/mL).The suspensions were further diluted 1:5 in 0.85% saline and flooded on agar plates consisting of RPMI 1640 medium supplemented with 2% glucose and buffered with morpholinopropanesulfonic acid.Excess fluid was removed, and the plates were allowed to dry at room temperature before the Etest strips were applied and incubated for 72 h at 37 °C.The MICs of antifungal agents were read as the lowest concentration at which the border of the elliptical inhibition zone intercepted the scale of the strips.The MIC 50 results are the concentrations of each antifungal agent necessary to inhibit the growth of 50% of the fungal isolates.

Statistical analysis
The microbiological data was analyzed by Fisher's exact test using GraphPad Prism, version 9 (GraphPad, San Diego, CA, USA).

FluconazoleFigure 1 .
Figure 1.Minimum inhibitory concentration (MIC) of eight antifungal agents to environmental Exophiala dermatitidis.The MIC 50 represents the antifungal concentration that inhibited the growth of 50% of the Exophiala isolates.

Table 1 .
Number of isolated Exophiala spp from various areas in a household.

Table 2 .
Non-dermatitidis Exophiala species isolated from areas in a household.

Table 4 .
Minimum inhibitory concentration (MIC) of eight antifungal agents to environmental nondermatitidis Exophiala species.