New Species of the Genus Curvularia: C. tamilnaduensis and C. coimbatorensis from Fungal Keratitis Cases in South India

Members of the genus Curvularia are melanin-producing dematiaceous fungi of increasing clinical importance as causal agents of both local and invasive infections. This study contributes to the taxonomical and clinical knowledge of this genus by describing two new Curvularia species based on isolates from corneal scrapings of South Indian fungal keratitis patients. The phylogeny of the genus was updated based on three phylogenetic markers: the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster as well as fragments of the glyceraldehyde-3-phosphate dehydrogenase (gpdh) and translation elongation factor 1-α (tef1α) genes. The maximum likelihood phylogenetic tree constructed from the alignment of the three concatenated loci revealed that the examined isolates are representing two new, yet undescribed, Curvularia species. Examination of colony and microscopic morphology revealed differences between the two species as well as between the new species and their close relatives. The new species were formally described as Curvularia tamilnaduensis N. Kiss & S. Kocsubé sp. nov. and Curvularia coimbatorensis N. Kiss & S. Kocsubé sp. nov. Antifungal susceptibility testing by the broth microdilution method of CLSI (Clinical & Laboratory Standards Institute) revealed that the type strain of C. coimbatorensis is less susceptible to a series of antifungals than the C. tamilnaduensis strains.


Introduction
The fungal genus Curvularia (Ascomycota, Pleosporales, Pleosporaceae) comprises of dematiaceous, melanin-producing molds with various lifestyles including saprophytism, plant endophytism [1], plant parasitism [2], and human pathogenicity [3].  Table 2 shows the strains and sequences involved in the phylogenetic analysis of the genus Curvularia, including four isolates derived from cases of fungal keratitis diagnosed and treated in the Aravind Eye Hospital, Coimbatore, Tamil Nadu, India. The tef1α dataset consisted of 902 characters of nucleotide alignment without binary characters. The gpdh dataset contained 684 characters with 601 characters of nucleotide alignment and 63 binary characters derived from indel coding. The length of the ITS alignment was 1193 characters long, containing 896 bp of nucleotide data and 297 binary characters.   On the phylograms obtained from each of the three loci, the four keratitis isolates of this study were resolved as two new species with over 80% of confidence values (data not shown), one of them represented by the single isolate SZMC 22225, while the other one by isolates SZMC 22226, SZMC 26758, and SZMC 26758. As the individual inferences were largely congruent, the three loci were concatenated and partitioned. The phylogenetic tree obtained from the concatenated dataset is shown in Figure 1. Maximum likelihood phylogeny of the genus Curvularia inferred from the concatenated internal transcribed spacer (ITS), translation elongation factor 1-α (tef1a), and glyceraldehyde-3-phosphate dehydrogenase (gpdh) sequences. The isolates examined in this study are shown as the new species Curvularia tamilnaduensis and Curvularia coimbatorensis (highlighted in color). Sequences of the reference Curvularia strains were collected from the GenBank Nucleotide database (Table 1). Bootstrap support values greater than 60% are shown above the branches. Bipolaris maydis CBS 136. 29    Vegetative hyphae septate, subhyaline to brown, branched, smooth walled, but often heavily asperulate, 2-3 µm in width. Colonies on PDA reaching approximately 6-7 cm in diameter after 7 d at 25 °C, surface lanose, aerial mycelium abundant, margin fimbriate, olivaceous green. Conidiophores erect, usually unbranched, in most cases uniformly brown, sometimes with paler tip, seminematous, septate, slightly flexuous, rarely geniculate towards the apex, up to 125 µm long, 2.5-4 µm wide. Conidiogenous cells integrated, terminal or intercalary, smooth, pale brown to brown, mono-or polytretic, proliferating sympodially. Chlamydospores present, subglobose, terminal and intercalary, 8-22 µm in diameter. Conidia ellipsoidal to clavate to obovoid, asymmetrical with paler basal and apical cells, usually curved at the third cell from the base which is darker than the other cells, (15-)20-23(-28) × (7-)8-10(-11) µm, (2-)3-distoseptate with non-protuberant, thickened, and darkened hila.

Antifungal Susceptibilities of Curvularia Strains Isolated from Fungal Keratitis
The minimum inhibitory concentrations (MIC) of nine antifungal agents towards C. coimbatorensis SZMC 22225, C. tamilnaduensis SZMC 22226, SZMC 26758, and SZMC 26759, as well as the type strains of C. australiensis (CBS 172.57), C. hawaiiensis (CBS 173.57), and C. spicifera (CBS 274.52) are shown in Table 3. The MIC of natamycin was 2 µg mL −1 for both new species and all other strains tested, while substantial differences between them could be observed in the case of clotrimazole, econazole, miconazole, and terbinafine, with the type strain of C. coimbatorensis having 4, 8, 4, and 4-8 times higher values, respectively. Among the tested isolates, the type strain of C. spicifera proved to be the less susceptible to clotrimazole, econazole, fluconazole, ketoconazole, and miconazole. Notable strain-to-strain variations between the C. tamilnaduensis strains could be observed only in the case of itraconazole and ketoconazole with detected MIC ranges of 0.03-0.25 and 0.06-0.25, respectively.

Discussion
The phylogenetic tree obtained from the concatenated dataset of three loci presents an update about the phylogeny of Curvularia, which is mostly in agreement with the recently published phylogenies of this genus (Figure 1). C. ischaemi formed a clade with C. coicis, which is in contradiction with the results of Tan et al. [8] and Tibpromma et al. [27], where C. ischaemi formed a sister clade to C. gladioli, but in agreement with the phylogram obtained by Madrid et al. [7] and Manamgoda et al. [28]. Our analysis placed C. perotidis as a sister clade to C. australiensis, however, other studies [7,8,27,29] suggested that this species is closer to C. spicifera. The placement of C. variabilis was also different from previously published articles [8,29]. According to the analyses of Tan et al. [8] and Marin-Felix et al. [29], C. variabilis forms a clade with C. hawaiiensis, C. nodosa, C. dactyloctenicola, and C. beasleyi, however, in this study we found C. variabilis as a sister clade of C. tsudae and C. mebaldsii. The same authors found C. tripogonis, C. pseudorobusta, C. robusta, C. alcornii, C. protuberata, and C. inaequalis as members of two distinct monophyletic clades, while our results indicate that these species are closely related and paraphyletic, however, none of the topologies have strong statistical supports. The observed slight differences between the previous inferences and our analyses did not Vegetative hyphae septate, subhyaline to brown, branched, smooth walled, but often heavily asperulate, 2-3 µm in width. Colonies on PDA reaching approximately 6-7 cm in diameter after 7 days at 25 • C, surface lanose, aerial mycelium abundant, margin fimbriate, olivaceous green. Conidiophores erect, usually unbranched, in most cases uniformly brown, sometimes with paler tip, seminematous, septate, slightly flexuous, rarely geniculate towards the apex, up to 125 µm long, 2.5-4 µm wide. Conidiogenous cells integrated, terminal or intercalary, smooth, pale brown to brown, mono-or polytretic, proliferating sympodially. Chlamydospores present, subglobose, terminal and intercalary, 8-22 µm in diameter. Conidia ellipsoidal to clavate to obovoid, asymmetrical with paler basal and apical cells, usually curved at the third cell from the base which is darker than the other cells, (15-)20-23(-28) × (7-)8-10(-11) µm, (2-)3-distoseptate with non-protuberant, thickened, and darkened hila.

Antifungal Susceptibilities of Curvularia Strains Isolated from Fungal Keratitis
The minimum inhibitory concentrations (MIC) of nine antifungal agents towards C. coimbatorensis SZMC 22225, C. tamilnaduensis SZMC 22226, SZMC 26758, and SZMC 26759, as well as the type strains of C. australiensis (CBS 172.57), C. hawaiiensis (CBS 173.57), and C. spicifera (CBS 274.52) are shown in Table 3. The MIC of natamycin was 2 µg mL −1 for both new species and all other strains tested, while substantial differences between them could be observed in the case of clotrimazole, econazole, miconazole, and terbinafine, with the type strain of C. coimbatorensis having 4, 8, 4, and 4-8 times higher values, respectively. Among the tested isolates, the type strain of C. spicifera proved to be the less susceptible to clotrimazole, econazole, fluconazole, ketoconazole, and miconazole. Notable strain-to-strain variations between the C. tamilnaduensis strains could be observed only in the case of itraconazole and ketoconazole with detected MIC ranges of 0.03-0.25 and 0.06-0.25, respectively.

Discussion
The phylogenetic tree obtained from the concatenated dataset of three loci presents an update about the phylogeny of Curvularia, which is mostly in agreement with the recently published phylogenies of this genus (Figure 1). C. ischaemi formed a clade with C. coicis, which is in contradiction with the results of Tan et al. [8] and Tibpromma et al. [27], where C. ischaemi formed a sister clade to C. gladioli, but in agreement with the phylogram obtained by Madrid et al. [7] and Manamgoda et al. [28]. Our analysis placed C. perotidis as a sister clade to C. australiensis, however, other studies [7,8,27,29] suggested that this species is closer to C. spicifera. The placement of C. variabilis was also different from previously published articles [8,29]. According to the analyses of Tan et al. [8] and Marin-Felix et al. [29], C. variabilis forms a clade with C. hawaiiensis, C. nodosa, C. dactyloctenicola, and C. beasleyi, however, in this study we found C. variabilis as a sister clade of C. tsudae and C. mebaldsii. The same authors found C. tripogonis, C. pseudorobusta, C. robusta, C. alcornii, C. protuberata, and C. inaequalis as members of two distinct monophyletic clades, while our results indicate that these species are closely related and paraphyletic, however, none of the topologies have strong statistical supports. The observed slight differences between the previous inferences and our analyses did not affect the validity of any of the previously described species, and some of them might be the result of the slightly broader taxon sampling.
One of the newly described species, C. coimbatorensis is only known from the type specimen isolated from corneal ulcer. Phylogenetic analysis based on three loci placed C. coimbatorensis as a sister clade to the other newly described species C. tamilnaduensis. The two species are closely related, but can be distinguished by tef1a, gpdh, and ITS sequences, with percentage identities of 99%, 98%, and 99%, respectively. C. petersonii [8] is also closely related and can be distinguished by all three loci (98% in tef1a, 93% in gpdh and 96% in ITS). C. coimbatorensis differs from C. tamilnaduensis in colony morphology, the lack of chlamydospores, and the size of conidia. C. petersonii is very similar in colony morphology, however, has significantly shorter (up to 110 µm) and only slightly geniculate conidiophores bearing narrower (5-)5.5-6(-7) conidia [8]. C. coimbatorensis has longer conidiophores.
The phylogenetic analysis based on three loci placed the other newly described species, C. tamilnaduensis as a sister clade to the recently described species C. petersonii. C. tamilnaduensis can be reliably distinguished from the ex-type of C. petersonii by tef1a, gpdh and ITS sequences with percentage identities of 99%, 95%, and 96%, respectively. The two species also differ by morphology, as C. petersonii has not been reported to produce chlamydospores and has different conidial dimensions (17-19 × 5.5-6) [8]. C. americana [7] and C. verruculosa [30] are also related species with considerable amount of genetic distances and none of these species have been reported before to have chlamydopores.
The antifungal susceptibilities of the examined strains of C. coimbatorensis and C. tamilnaduensis to amphotericin B, clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, natamycin, and terbinafine were within the MIC ranges reported for other clinically relevant Curvularia species in the study of Guarro et al. [11] and the review of Krizsán et al. [3]. The type strain of C. coimbatorensis proved to be less susceptible than the strains of C. tamilnaduensis to all antifungals except for natamycin. For itraconazole and ketoconazole our results are in agreement with the study of Guarro et al. [11], who reported that amphotericin B, itraconazole, miconazole and ketoconazole are highly effective against a series of Curvularia species known from fungal keratitis (C. brachyspora, C. clavata, C. geniculata, C. lunata, C. pallescens, C. senegalensis, and C. verruculosa).

Curvularia Strains, Culture Conditions, and Morphological Examination
The Curvularia strains involved in this study derived from corneal scrapings from fungal corneal ulcers of keratitis patients attending the Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, India. All cases were initially screened by experienced ophthalmologists, and the corneal scrapings were collected following the clinical diagnosis of fungal keratitis. The samples were initially processed microbiologically for the isolation of the causative agents as described earlier [31]. The corneal scrapings of all patients were subjected to Gram stain, Giemsa stain, and 10% KOH wet mount. Culture methods involved direct inoculation of specimens onto 5% sheep blood agar, chocolate agar, non-nutrient agar, potato dextrose agar, thioglycolate broth, and brain-heart infusion broth. The microbial cultures were considered positive only if the growth of the same organism was demonstrated on two or more solid media, or there was confluent growth at the site of inoculation on one solid medium with consistent direct microscopic findings. The isolates were deposited in the Szeged Microbiology Collection (SZMC, Szeged, Hungary) under the accession numbers SZMC 22225, SZMC 22226, SZMC 26758, and SZMC 26759. Colony morphology of the isolates was examined on PDA (BioLab, Budapest, Hungary) medium after 7 days of incubation at 25 • C under normal day/night light conditions. Micromorphological characters were examined with a Leica DMI 4000B (Leica, Wetzlar, Germany) microscope equipped with a Leica DFC 295 camera. Microscopic features were examined in lactic acid (100% v/v) on glass slides. Conidiophores were studied in the same mounting fluid with the transparent tape method. Conidiophores and conidia were measured using the software ImageJ v2.52a (National Institute of Mental Health, Bethesda, MD, USA). Size ranges of the conidia were derived from 50 measurements. Lengths and widths are given as (minimum value) mean size minus SD-mean size plus SD (maximum value).

DNA Extraction, Amplification, Sequencing, and Phylogenetic Analysis
Genomic DNA was isolated from the examined Curvularia strains SZMC 22225, SZMC 22226, SZMC 26758, and SZMC 26759 with the Masterpure™ Yeast DNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer's instructions. Fragments of tef1a and gpdh were amplified as described previously [5,32,33]. The ITS region of the ribosomal RNA gene cluster was amplified according to White et al. [34]. Sequencing of the amplicons was carried out on a 3500 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) by the sequencing service of the Biological Research Centre, Szeged, Hungary. Resulting sequences were deposited in the GenBank Nucleotide database (www.ncbi.nlm.nih.gov) under the accession numbers shown in Table 2.
Sequences of the four clinical isolates were aligned with publicly available sequences of 108 previously described Curvularia species, as well as Bipolaris maydis as the outgroup (Table 2). Phylogenetic analyses were conducted using three loci (tef1α, gpdh and ITS). Sequences of all three loci were aligned with the phylogeny-aware sequence alignment tool Canopy v0.1.4 using RAxML as tree estimator and PRANK [35] with the -F option as the aligner with 10 iterations and seed decomposition strategy. Alignments of the three loci were concatenated and partitioned by region. The tef1α sequences formed one partition while in the case of gpdh sequences the dataset was partitioned to exons and introns. The ITS dataset was divided to rDNA and ITS1-ITS2 regions. Alignments of gpdh and ITS datasets contained high number of indels with important phylogenetic signal, therefore gaps were coded as absence/presence characters by SequenceMatrix v1.8 [36] using the simple indel coding algorithm [37]. The two indel matrices were concatenated and added as a single partition to the dataset. Maximum likelihood analysis was performed using RAxML-NG v0.9.0 [38] under the GTR model with gamma-distributed rate heterogeneity using empirical base frequencies. As indel-based datasets do not contain constant sites, the ascertainment bias correction described by Lewis [39] was used for this partition. Statistical support of the best ML tree was obtained with 1000 thorough bootstrap replicates.

Conclusions
The present study demonstrates, that although the phylogeny of the genus Curvularia is resolved and well established, further expansion can be expected both in the list of described Curvularia species and in the known spectrum of clinically relevant members of the genus. The collection of further keratitis isolates from the genus Curvularia and gaining data about their antifungal susceptibilities are therefore tasks of increasing importance. Furthermore, comparing the infectivity of various Curvularia species causing keratitis-including the recently described ones-in animal keratitis models would be an intriguing topic for future research.