Patient Characteristics. An 84-year-old man was treated for pneumonia a few months ago before his admission to the hospital. Moreover, he was also treated for diabetes, hyperthyroidism, atherosclerosis and kidney disorder. This patient was hospitalized because of severe keratitis of unknown aetiology. Cornel scrubs and cultures were taken for the microbiological analysis on different culture media. The negative results for bacteria and fungi were obtained after seven days of culture on the appropriate media. In spite of the empirical treatment with antibiotics and antifungal agents the corneal inflammation continued to be very severe. After nine days of local and systemic treatment we decided about corneal transplantation "on hot". The tissue of the cornea was taken to the microbiological examination. After the seven days of the culture on PDA and MA culture media, using standard laboratory methods no colonies were visible on the media. Despite on this we decided to carry on our analysis for a longer time.
The research material was kept in a physiological solution of 0,9% NaCl at room temperature. The mycological analysis of cornea pieces was carried on PDA and MA culture media, using standard laboratory techniques.
Two isolation methods were used: the first (I) - 0.5 mm cornea fragments were placed on solidified culture media in Petri dishes - 4 pieces per one dish; and the second (II) - suspension of the fungus hyphae that passed from the cornea to the physiological solution was transferred to sterile Petri dishes in a volume of 0.1 ml and 0.5 ml, and poured over a chilled liquid PDA (own method). The samples were then incubated in a thermostat at a temperature of 25 C. The visible colonies of fungus were transferred to slants with PDA medium and subsequently single spore cultures were prepared and allowed to grow and develop. The cultures were identified using classical and molecular methods.
The species name and author's initials were corrected in the current status of the Index Fungorum taxonomic species database in 2016 and according to elaboration.
Classical identification. Pure fungus cultures were placed as a small inoculum on agar media in Petri dishes, using three inocula per one Petri dish, which was then sealed with a parafilm to prevent drying. Five different culture media were used: Czapek-Dox, PDA, Sabouraud, YPD and a poor medium (5 g - glucose, 20 g - agar, 1 dm3 - distilled water). The cultures of fungus were carried out at a temperature of 25 C for one month. Considering the very slow growth of fungi, the temperature was raised to 30 C and the period of incubation was extended.
Macroscopic and microscopic observations and photographic documentation were performed from the start of visible colony growth, i.e. from day 17 of culture. Photographs of the morphological structures using a Scanning Electron Microscope (SEM) (Tescan Vega/LMU) were taken at the Central Laboratory of Agro-Ecological (CLA), the University of Life Sciences in Lublin.
Molecular identification. The research material used in this study consisted of 3 different isolates, obtained from the human cornea (Nos 1-3). The colonies prepared for DNA isolation were grown on PDA medium. Identification of the isolates to the species level was carried out using universal primers for rDNA genes: the ITS1-5.8S-ITS2 - (ITS1 and ITS4) region, 28S rDNA region - LSU - (LR5 and LROR) and for the beta-tubuline - gene (Bt2a and Bt2b) (Table I).
DNA isolation. Genomic DNA isolation was performed using the CTAB method by with some own modifications. Fungus isolates were grown at a temperature of 27 C for 45 days. The obtained mycelium was transferred to Eppendorf tubes of a 1.5 ml volume, and then frozen in liquid nitrogen. The frozen material was homogenized with a sterile pestle (Sigma-Aldrich) until mycelium homogeneity. Subsequently, 600 ml of CTAB lysis buffer was added to each tube and incubated at 65 C for 2 hours and then centrifuged (10 000 rpm for 10 minutes). In the next step, 1.0 ml phenol/chloroform/alcohol mixture in the volume ratio of 25:24:1 was added to the supernatant and centrifuged (10 000 rpm for 8 minutes). DNA was precipitated with 40 mul of sodium acetate (5 M) and 400 mul of isopropanol. The resulting precipitate was washed with 70% ethanol, and then centrifuged at 14 000 rpm for 15 minutes and dried. The supernatant was suspended in 50 mul of PCR water (Sigma-Aldrich), supplemented with RNase A, and then incubated for 25 hours at a temperature of 4 C, followed by one hour at 37 C, and 10 minutes at 65 C. DNA was run in 1.5% agarose gel electrophoresis and compared with GeneRuler 100 bp DNA Ladder Plus marker (Thermo Scientific). The DNA quantity was measured with a UV-Vis NanoDrop 2000c/2000 spectrophotometer (Thermo Scientific). Then, the DNA samples were diluted to a final concentration of 50 ng/mul and stored at 20 C for further analysis.
PCR amplification and sequence analysis. Three different primers were used for identification of Aspergillus spp. isolates: ITS1 and ITS4, LROR and LR5, and Bt2a and Bt2b (Table I). The amplification reaction volume was 25 mul and the reaction solution contained 20 ng/mul of genomic DNA, 1 x Buffer Tag (750 mM Tris HCl pH 8.8, 200 mM (NH4)2SO4, 0.1% Tween 20) (Thermo Scientific), 0.1 mM x MgCl2, dNTP mix 1 mM of each primer 0.2 muM and 1.0 U polymerase DNA (Fermentas). Amplification reaction was performed using a DNA Engine Dyad Thermal Cycler (Biorad). The PCR amplification reaction for ITS1 and ITS4 primer pair was carried out using the following program: initial denaturation for 5 min at 95 C, followed by 40 cycles consisting of 30 s at 95 C, 50 s at 57 C, 60 s at 72 C and final extension for 10 min at 72 C. PCR reaction programs for two primers pairs, the first, LROR and LR5, and the second, Bt2a and Bt2b, were subjected to minor changes, as follows: initial denaturation for 5 min at 94 C, then 36 cycles of annealing: 30 s at 94 C, 50 s at 56 C, 55 s at 72 C and final extension of the product at 72 C for 5 min. The obtained products were separated by electrophoresis using 1.5% agarose gel containing 7.5 mul of ethidium bromide in 1 x TBE buffer at 80 V for 1.5 hour in the presence of GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific). The PCR products were further sequenced using Applied Biosystems BigDay Terminator v. 3.1. kit (Life Technologies) and separated on a 3730xl DNA Analyser capillary sequencer. The nucleotide sequences obtained were compared with the nucleotide sequence deposited in GenBank at the National Centre for Biotechnology Information NCBI (http://www.ncbi.nlm.nih.gov/), using the Mega Blast program. The "local sequence alignment" method was used as a comparison criterion. The highest degree of sequence identity was used to determine the species of the isolates. The phylogenetic analysis of ITS sequences of our three isolates of A. penicillioides studied was carried out in several steps using the Maximum Likelihood method based on the Tamura-Nei model. Moreover, 13 other reference strains of four Aspergillus spp. i.e.: A. salinicola - strains EXF_10401, IBT_34266, KAS_6054, A. hordei - NRRL25826, NRRL25825, NRRL25830, A. clavato-phorus - DTO257-G5, NRRL_25874 and 5 strains of A. penicillioides (mentioned in Table II) were also included. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 16 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 455 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.