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

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.

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™ (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 MgC1₂, 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™ 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 low-quality 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 spread 500 µl fungal suspension evenly on a RPMI plate. Etest strips were placed on plates that had been dried for at least 10 min at room temperature. The MICs were determined after 72 h of incubation at 30 °C. Both on-scale and off-scale MICs were included in the data. For the calculation of geometric mean (GM), the high off-scale MICs (>32 and >256 µg/ml) were rounded up to the next intermediate Etest dilution (48 and 384 µg/ml), while the low off-scale MICs (<0.002 and <0.016 µg/ml) remained unchanged.

Nucleotide sequence accession numbers

The ITS nucleotide sequences of UM 1094, UM 1104, UM 1134, UM 1216, UM 1217, and UM 1218 were deposited in the GenBank database with the accession numbers KT936494, KT936495, KT936496, KT936497, KT936498, and KT936499, respectively (Table 1).

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 group (Group I), D. concentrica group (Group II), D. vernicosa/loculata group (Group III), D. childiae group (Group IV), and D. petriniae group (Group V). In this study, all the isolates were clustered within the D. eschscholtzii group, forming a cluster with the reference isolates of D. eschscholtzii.

In vitro antifungal susceptibility test

The MICs of the nine isolates tested are shown in Table 4. The antifungal susceptibility profiles obtained were isolate-dependent. In general, VRC, PSC, ITC, KTC, and AMB displayed very low MICs to D. eschscholtzii, with all the isolates exhibiting MICs of ≤1 µg/ml. ANID also showed low MICs to (≤1 µg/ml) the different isolates, with the exception of UM 1134 and UM 1218 (>32 µg/ml). Similarly, low MIC values (≤1 µg/ml) were obtained for CAS, with two thirds of the isolates eliciting MICs of ≤1 µg/ml, and the remaining eliciting MICs of >1 µg/ml. Overall, PSC exhibited the highest in vitro anti-fungal activity (GM MIC 0.016 µg/ml) against all isolates, followed by VRC (GM MIC 0.021 µg/ml), KTC (GM MIC 0.027 µg/ml), AMB (GM MIC 0.031 µg/ml), ANID (GM MIC 0.062 µg/ml), ITC (GM MIC 0.089 µg/ml), and CAS (GM MIC 0.481 µg/ml). Relatively high MICs against D. eschscholtzii (88.89% with MIC >1 µg/ml; GM MIC value of 3.530 µg/ml) were found for FLC, indicating potential resistance of D. eschscholtzii to this drug.