Identification and characterization of Daldinia eschscholtzii isolated from skin scrapings, nails, and blood

Background Daldinia eschscholtzii is a filamentous wood-inhabiting endophyte commonly found in woody plants. Here, we report the identification and characterization of nine D. eschscholtzii isolates from skin scrapings, nail clippings, and blood. Methods The nine isolates were identified based on colony morphology, light microscopy, and internal transcribed spacer (ITS)-based phylogeny. In vitro antifungal susceptibility of the fungal isolates was evaluated by the Etest to determine the minimum inhibitory concentration (MIC). Results The nine isolates examined were confirmed as D. eschscholtzii. They exhibited typical features of Daldinia sp. on Sabouraud Dextrose Agar, with white felty colonies and black-gray coloration on the reverse side. Septate hyphae, branching conidiophore with conidiogenous cells budding from its terminus, and nodulisporium-like conidiophores were observed under the microscope. Phylogenetic analysis revealed that the nine isolates were clustered within the D. eschscholtzii species complex. All the isolates exhibited low MICs against azole agents (voriconazole, posaconazole, itraconazole, and ketoconazole), as well as amphotericin B, with MIC of less than 1 µg/ml. Discussion Early and definitive identification of D. eschscholtzii is vital to reducing misuse of antimicrobial agents. Detailed morphological and molecular characterization as well as antifungal profiling of D. eschscholtzii provide the basis for future studies on its biology, pathogenicity, and medicinal potential.


INTRODUCTION
Members of the genus Daldinia are pyrenomycetes, which are characterized by internal horizontally zonated stromata that develop conspicuously on woody plants (Stadler et al., 2014). Daldinia spp. are initial colonizers as evident from the early appearance of stromata following stress or damage to the woody host plant. Initial colonization is a trait of Daldinia spp. owing to their habit as endophytes (Stadler et al., 2014). As an early colonizer, they remain dormant in the host without triggering symptoms before wood decay. Formation of stromata on the woody host plant is triggered by dehydration that may be caused by climatic stress, fire, or lightning (Johannesson, Laessøe & Stenlid, 2000;Srutka, Pazoutova & Kolarik, 2007). At this stage, Daldinia spp. becomes wood-decaying in its habit, and produces anamorphic structures under favorable conditions of humidity and temperature to colonize the substrate further (Stadler et al., 2014).
D. eschscholtzii is a wood-inhabiting endophyte or wood-decaying fungus that is widespread in warm tropical climate (Stadler et al., 2014). It is characterized by colonies that are white to smoky gray with a slight olivaceous-tone, and by conidiogenous structures with a nodulisporium-like branching pattern (Ju, Rogers & Martin, 1997;Stadler et al., 2014). D. eschscholtzii grows preferentially on dead or decaying wood substrates, and is commonly isolated from dead woody plants such as dicotyledonous crop plants, trees, and occasionally, marine algae (Karnchanatat et al., 2007;Tarman et al., 2012;Zhang et al., 2008).
Compelling data in the last decade has demonstrated the presence of a wide array of secondary metabolites in this fungus, such as 1,1 -binaphthalene-4,4 -5,5 -tetrol (BNT) (a polyketide derived from 1,8-dihydroxynaphthalene biosynthesis), cytochalasins (metabolites of mixed polyketide/NRPS origin), concentricols (terpenoids derived from the acetate-mevanolate pathway), dalesconol A and B (polyketides), and helicascolide C (polyketides) (Fang et al., 2012;Stadler et al., 2001a;Stadler et al., 2001b;Tarman et al., 2012;Zhang et al., 2008;Zhang et al., 2011). Some of these secondary metabolites are precursors of biologically active medicinal compounds. Dalesconol A and B have immunosuppressive activity (Zhang et al., 2008;Zhang et al., 2011) while helicascolide C exhibits antifungal activity against the phytopathogenic fungus Cladosporium cucumerinum (Tarman et al., 2012). In a previous study, genome analysis of D. eschscholtzii clinical isolates showed that our isolates UM 1400 and UM 1020 are potentially rich in secondary metabolites (Chan et al., 2015). The presence of the gene encoding lovastatin nonaketide synthase suggests that these isolates can synthesize the drug lovastatin that is used to induce a hypocholesterolemic effect (Chan et al., 2015). D. eschscholtzii had not been reported as a human pathogen until we isolated this species from skin scrapings and the blood of patients with suspected fungal infections (Chan et al., 2015;Ng et al., 2012;Yew et al., 2014). To the best of our knowledge, all previous isolations of D. eschscholtzii from humans were by our group (Chan et al., 2015;Ng et al., 2012;Yew et al., 2014). Nevertheless, the clinical evidence of infection caused by this fungus remains unclear. In this study, we identified a total of nine D. eschscholtzii clinical isolates, including the aforementioned isolates in the past five years. Here, we present a detailed morphological, molecular, phenotypic characterization, and antifungal susceptibility profile of D. eschscholtzii. These data may serve as a reference for the mycological research community for rapid detection of D. eschscholtzii.

Ethical statement
The isolates used in this study were obtained from an archived fungal collection. No patient information is disclosed except for specimen type. As such, this study is exempt from ethical approval by the UMMC Medical Ethics Committee. Notes. a Inviable isolates, the morphological study and unique DNA signature evaluation for these two isolates were excluded in this study while the antifungal susceptibility Etest MIC readings were adopted from previous study (Yew et al., 2014).

Fungal isolates
UM 230, UM 1020, UM 1094, UM 1104, UM 1134, UM 1216, UM 1217, UM 1218, and UM 1400 were isolated from skin scrapings, nail clippings, and blood of patients with suspected fungal infection in the Mycology diagnostic laboratory, UMMC, Kuala Lumpur, Malaysia ( Table 1). The isolates were processed according to the laboratory's standard operating procedures (SOP) (Yew et al., 2014) with direct wet mount microscopy followed by culture on Sabouraud Dextrose Agar (SDA; Oxoid Ltd., Basingstoke, UK) for incubation at 30 • C for seven days. The isolates were archived at 4 • C in SDA slants and maintained by periodic subculturing on SDA slants at 30 • C. UM 1020 and UM 230 isolates were not included in the morphological study as both isolates were no longer viable at the point of analysis.

Morphological study
Morphological and colony features such as color, texture, and topography of the isolates were examined on SDA, potato dextrose agar (PDA; Difco Laboratories, Detroit, MI), and V8 juice agar (V8; HiMedia Laboratories, Mumbai, India). The isolates were incubated at 30 • C with alternate-day examination for fungal growth. Slide cultures of the fungi on SDA, PDA, and V8 agar were performed as previously described (Kuan et al., 2015). After a 7-day incubation at 30 • C, the fungal slide cultures were stained with lactophenol cotton blue stain and examined under the light microscope (Leica DM3000 Led, Germany).

DNA extraction
DNA extraction was carried out as previously described (Yew et al., 2014). The pure cultures on SDA were harvested by scraping the mycelia from the agar surface and transferred to phosphate buffer saline (PBS, pH 7.4). The mycelial suspension was then transferred into a 15 ml centrifuge tube containing washed glass beads and then vortexed for five minutes. Subsequently, a total of 200 µl of lysate was subjected to DNA extraction using ZR Fungal/Bacterial DNA MiniPrep TM (Zymo Research, USA) according to the manufacturer's protocol.

PCR amplification and DNA sequencing
The ITS1-5.8S-ITS2 region was PCR amplified from the isolates' genomic DNA in a 25 µl reaction consisting of 10× PCR buffer, 10 µM each of ITS1 (5 -TCCGTAGGTGAACCT GCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) primers (White et al., 1990), 25 mM MgC1 2 , 2 mM deoxynucleoside triphosphate, 2.5 unit of HotStarTaq DNA polymerase, and 10 µg of each genomic DNA. The PCR was performed for 30 cycles at 94 • C for 30 s, 58 • C for 30 s, and 72 • C for 60 s. The PCR products were then purified using Expin TM PCR SV (GeneAll, Korea), and confirmed by Sanger sequencing (First Base Laboratories Kuala Lumpur, Malaysia). TraceTuner version 3.0.6 (Denisov, Arehart & Curtin, 2004) was used for base and quality calling of the sequenced ITS. The lowquality called bases (Phred value < 20) of both ends of the sequences were then trimmed by running Lucy version 1.20 (Chou & Holmes, 2001) and the included zapping.awk script. The processed ITS sequences were searched against the NCBI non-redundant (nr) nucleotide database using the nucleotide BLAST program.

Phylogenetic analysis
Unique ITS nucleotide sequences from the isolates, together with an additional 72 reference sequences for the ITS region of Daldinia spp., were compiled for ITS-based phylogenetic analysis (Table 2). Two Hypoxylon fragiforme sequences were used as outgroup strains in the analysis (Table 2). Multiple sequence alignments of all ITS sequences were performed using M-Coffee (Moretti et al., 2007). The alignments were then trimmed using trimAl version 1.4.rev10 to remove the alignment regions with ≥50% gaps (Capella-Gutierrez, Silla-Martinez & Gabaldon, 2009). The trimmed alignments were subsequently used for phylogenetic analysis conducted using MrBayes version 3.2.1 (Huelsenbeck & Ronquist, 2001). Bayesian Markov chain Monte Carlo (MCMC) analysis was initiated by sampling across the entire general time reversible (GTR) model space. A total of 1,500,000 generations were run with a sampling frequency of 100, and diagnostics were calculated for every 1,000 generations. The first 2,500 trees were discarded with a burn-in setting of 25%. Convergence was assessed with a standard deviation of split frequencies below 0.01, no noticeable trend in the generation versus log probability of the data plot, and a potential scale reduction factor (PSRF) close to 1.0 for all parameters (Ronquist, Huelsenbeck & Teslenko, 2011).

In vitro antifungal susceptibility test
The Etest (bioMérieux, France) was performed according to the manufacturer's instructions to determine the MICs of anidulafungin (ANID), amphotericin B (AMB), caspofungin (CAS), fluconazole (FLC), itraconazole (ITC), ketoconazole (KTC), posaconazole (PSC), and voriconazole (VRC). The concentration gradient of ANID, AMB, CAS, ITC, KTC, PSC, and VRC ranged from 0.002 to 32 µg/ml, while that of FLC ranged from 0.016 to 256 µg/ml. The test was performed on RPMI 1640 medium containing 2% glucose and MOPS. Each culture growing on SDA was harvested, suspended in sterile saline solution, and adjusted to a turbidity of a 0.5 McFarland standard. A sterile cotton swab was used to

Morphological study
All seven isolates grew rapidly on SDA. Initially, white hyphae grew from the inoculated site and formed a felty azonate mycelium. With aging, it turned to smoky gray color with a slight olivaceous tone as the mycelium became fully differentiated (Fig. 1). The smoky gray coloration was indicative of sporulation. All isolates had a black-gray coloration on the reverse side. The cultural characteristics of all fungal isolates that grew on SDA were similar to those that growing on PDA plates. However, their growth rates on PDA were different. The strains UM 1134, UM 1216, and UM 1218 grew faster (five days to reach the periphery of the 9 cm plate) than UM 1400, UM 1094, UM 1104, and UM 1217 (seven days) (Fig. 2). On V8 agar, all the colonies reached the edge of plate after a 5-day incubation; they had a felty to fluffy texture appearance (Fig. 3). The surfaces of the colony changed from white to gray or black after 5 days of incubation. The reverse side of V8 agar plate was initially colorless, and became slightly black after culture for five days. UM 1134 showed denser mycelial growth on SDA, PDA, and V8 agar as compared to the other isolates (Figs. 1D, 2D and 3D). Light microscope analysis revealed that all isolates that grew on SDA, PDA, and V8 agar had similar morphology. The septate hyphae could be hyaline thin-walled or melanized thick-walled (Fig. 4A). The thick-walled hyphae showed black exudates on their surfaces (Fig. 4B). Septate conidiophores were irregularly branched into mononematous, dichotomous or trichotomous structures with one to three conidiogenous cells originating from the terminus (Figs. 4C-4E). The conidiophores were hyaline and black with occasional pigmented exudates. The conidiogenous cells were hyaline and cylindrical. On the apex of conidiogenous cells, conidia were produced holoblastically in a sympodial sequence (Fig. 4E). The conidia were hyaline and ellipsoid with an attenuated base as shown in Fig. 4F. Table 3 summarizes the morphological features of this fungal species.

Molecular study
All isolates were identified as D. eschscholtzii following the BLASTn searches. The ITS-based phylogenetic tree in this study comprised members from the genus Daldinia (Fig. 5), and was divided into groups as described by Stadler et al. (2014), namely the D. eschscholtzii

In vitro antifungal susceptibility test
The MICs of the nine isolates tested are shown in

DISCUSSION
Daldinia eschscholtzii is a filamentous fungus commonly found as an endophyte or a wood-decaying fungus in woody plants (Karnchanatat et al., 2007;Karnchanatat et al., 2008;Stadler et al., 2014). Although we previously reported isolations of this organism  (Yew et al., 2014). c GM, geometric mean. d MIC categories: Category A: ≤1 µg/ml, Category B: >1-32 µg/ml or >1-256 µg/ml, Category C: >32 µg/ml or >256 µg/ml. from humans, it is unclear whether it is the cause of an actual infection, or if it merely exists as a harmless colonizer living in the nail plate or skin surface damaged by trauma or other diseases. In this study, we obtained nine D. eschscholtzii isolates from blood specimens, skin scrapings, and nail clippings. While the clinical significance of D. eschscholtzii remains in question, repeated isolation of this fungal species from humans recently suggests that it is not a mere environmental contaminant in patients. Chan et al. (2015) report that the genomes of D. eschscholtzii harbor several stress adaptation mechanisms for their survival in human hosts. Hence, it would not be surprising for the species to have undergone rapid evolution to select for fitness attributes as well as virulence factors related to pathogenicity in humans. Filamentous fungi are routinely identified by colony morphology and microscopy. The former would not precisely identify D. eschscholtzii owing to their natural variation among the isolates and tendency towards media-dependency, as evident from their macroscopic appearances. Identification to species level based on morphological examination alone would be difficult as many species of Daldinia are morphologically very similar (Ju, Rogers & Martin, 1997;Stadler et al., 2014), and hence considerable expertise and experience are required of the examiner in this regard.
The ITS region of the nuclear rDNA can be used to examine species level relationship in fungi due to its higher degree of variation. Thus, PCR-based ITS sequence analysis has been widely used to identify D. eschscholtzii (Chan et al., 2015;Hu et al., 2014;Tarman et al., 2012;Yuyama et al., 2013). Despite recent studies reporting limitations of the ITS region in distinguishing between the species complexes of Daldinia spp., this region has the broadest taxa covered in Daldinia (Stadler et al., 2014). The phylogenetic analysis showed that our clinical isolates and reference environmental isolates of D. eschscholtzii formed a cluster. However, further studies based on protein coding genes are needed to segregate members of the D. eschscholtzii species complex reliably.
The Etest is a simple, reliable, and reproducible assay that has been shown to correlate with the Clinical and Laboratory Standards Institute (CLSI) method in antifungal susceptibility testing of filamentous fungi (Espinel-Ingroff, 2001;Szekely, Johnson & Warnock, 1999). In line with this, we applied the Etest to study the antifungal profiles of our isolates. The results showed that D. eschscholtzii elicited low MICs in all the antifungal agents tested, except for FLC. Among these antifungals, VRC, PSC, ITC, KTC, and AMB were the more active in vitro, with all isolates inhibited by concentrations of less than 1 µg/ml. Since there is no available information on antifungal susceptibility profiles for D. eschscholtzii, this work will contribute towards establishing an optimal antifungal precautionary treatment for this fungus.

CONCLUSIONS
In this paper, we report the isolation of D. eschscholtzii from superficial sites in humans, predominantly skin and nails. If these fungi are confirmed to be of clinical importance, the in vitro antifungal activities determined here might be useful in clinical practice. The characterization of these fungi is important to understand the basic fungal biology of