A New European Species of the Opportunistic Pathogenic 2 Genus Saksenaea – S

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1 A New European Species of The Opportunistic Pathogenic 2 Genus Saksenaea – S. dorisiae Sp. Nov.

3 Roman Labudaa,c*, Andreas Bernreiterb,c, Doris Hochenauerb, Christoph Schüllerb,c, Alena 4 Kubátovád, Joseph Straussb,c and Martin Wagnera,c

a 5 Department for Farm Animals and Veterinary Public Health, Institute of Milk Hygiene, Milk Technology and 6 Food Science; Bioactive Microbial Metabolites group (BiMM), University of Veterinary Medicine Vienna, Konrad 7 Lorenz Strasse 24, 3430 Tulln a.d. Donau, Austria

8 bDepartment of Applied Genetics and Cell Biology, Fungal Genetics and Genomics Laboratory, University of 9 Natural Resources and Life Sciences, Vienna (BOKU); Konrad Lorenz Strasse 24, 3430 Tulln a.d. Donau, Austria

10 c Research Platform Bioactive Microbial Metabolites (BiMM), Konrad Lorenz Strasse 24, 3430 Tulln a.d. Donau, 11 Austria

12 d Charles University, Faculty of Science, Department of Botany, Culture Collection of Fungi (CCF), Benátská 2, 13 128 01 Prague 2, Czech Republic

14 15 *Corresponding author. Email: [email protected]

16 17

18 Abstract: A new species Saksenaea dorisiae (Mucoromycotina, Mucorales), recently isolated 19 from a water sample originating from a private well in a rural area of Serbia (Europe), is 20 described and illustrated. The new taxon is well supported by phylogenetic analysis of the 21 internal transcribed spacer region (ITS), domains D1 and D2 of the 28S rRNA gene (LSU), and 22 translation elongation factor-1α gene (TEF-1α), and it is resolved in a clade with S. 23 oblongispora and S. trapezispora. This fungus is characteristic by its moderately slow growth 24 at 15 and 37°C, sparse rhizoids, conical-shaped sporangia and short-cylindrical 25 sporangiospores. S. dorisiae is a member of the opportunistic pathogenic genus often 26 involved in severe human and animal mucormycoses encountered in tropical and subtropical 27 regions. Despite its sensitivity to several conventional antifungals (terbinafine and 28 ciclopirox), the fungus is potentially causing clinically challenging infections. This is the first 29 novel taxon of the genus Saksenaea described from the moderately continental climate area 30 of Europe.

31 Key words: Saksenaeaceae, mucormycosis, antifungal susceptibility, water quality

33 INTRODUCTION

34 The genus Saksenaea S.B. Saksena belongs to mucoralean microscopic fungi 35 (Mucoromycotina, Mucorales, Saksenaeaceae) comprising species often causing severe 36 human and animal cutaneous mucormycoses in both immunocompromised and 37 immunocompetent hosts (1, 2). The genus was described from a forest soil in India in 1953, 38 with Saksenaea vasiformis S.B. Saksena being a type and for more than 50 years a single- 39 species of the genus (nowadays considered as S. vasiformis species complex), reported in 40 soil, drift-wood, and grains (1). Interruption of skin integrity, such as needle sticks, insect 41 stings or spider bites, burn and accident wounds (up to 95% of cases) represent the most 42 common mode of infection by this fungus from contaminated soil or water (2-4). In addition 43 to these traumatic implantations infections through inhalation of spores and the use of 44 indwelling catheters have been reported (5). The species of the genus are responsible for 45 skin infections, characterized by rapid progression and invasion of neighboring tissues. 46 Infection of the Saksenaea fungi is diagnosed by angio-invasion leading to tissue necrosis 47 with cutaneous and subcutaneous involvement. Furthermore, rhino-orbito-cerebral and 48 disseminated infections have been reported (1, 2). Classic management of the infection site 49 usually includes a combination of broad, aggressive and repeated surgical debridement 50 (which may lead to amputation), and long–term systemic therapy with appropriate 51 antifungals, preferably liposomal amphotericin B and/or posaconazole (6-9). Antifungal 52 therapy alone seems to be inadequate to control infection (2).

53 Recent revisions by Alvarez et al. (1) and Crous et al. (10, 11) applying a polyphasic 54 approach including sequence analysis of three loci (ITS, LSU, EF-1α) revealed that a 55 monospecific genus Saksenaea is actually genetically heterogenous and encompasses more 56 species, namely S. vasiformis complex (with more putative cryptic species), S. 57 loutrophoriformis, S. erythrospora, S. oblongispora and S. trapezispora. So far these species 58 (except S. oblongispora) have been reported worldwide from human and animal clinical 59 cases. They were encountered in tropical and subtropical climates and have been reported 60 from Australia, Thailand, India and Sri Lanka, the Middle East, Tunisia, the USA, Central and 61 South America (1, 12). In Europe, there are only a few published cases available reporting S. 62 vasiformis infections, e.g. from Spain (3, 13-15), France (1), and Greece (2). Up to now, 63 approximately 45 cases of mostly cutaneous infections of Saksenaea, have been reported

64 around the world (2, 4, 5, 10, 11), although the actual number of clinical cases is possibly 65 underestimated (4). Despite its reproducibility in severe human infections, this genus 66 remains a poorly studied mucoralean genus, mainly due to the lack of sporulation on the 67 mycological culture media (e.g. sabourad agar, malt extract agar, corn-meal agar) routinely 68 used in clinical laboratories (1).

69 During our microbiological survey of water samples a fast growing mucoralean 70 nonsporulating fungus was recovered on a Pseudomonas isolation plate in a sample 71 originating from a private well in a rural area of Manastirica (Serbia) in October 2018. This 72 isolate was designated BiMM-F232 and further characterized in terms of morphology, 73 physiology, molecular phylogeny and antifungal susceptibility. Phylogenetically informative 74 sequences were obtained from three loci, i.e. internal transcribed spacer region including 75 5.8S rDNA (ITS), domains D1 and D2 of the 28S rRNA gene (LSU), and from the translation 76 elongation factor-1α locus (TEF1α). Overall, the resulting data revealed that this isolate 77 represents a novel species of the opportunistic pathogenic genus Saksenaea, and it is 78 described and illustrated here as Saksenaea dorisiae sp. nov.

80 MATERIALS AND METHODS

81 Sample collection and isolation of the fungus

82 A single sample of water from a private well in Manastirica-Petrovac (The Republic of Serbia, 83 Europe) was collected in October 2018. A 100 mL aliquot was aseptically filtered through a 84 filter paper disc (0.45 µm, 47 mm diameter, GN-6Metricel, Pall, Mexico) plated on 85 Pseudomonas Selective Agar amended with Glycerol (Carl Roth, Germany) and incubated for 86 3 days at 37 (±0,2°C) in the dark.

87 Cultivation of a strain, media and morphological analysis

88 For phenotypic determination, the strain was transferred (one agar block grown on CYA, ca 5 89 x 5 mm, in the middle) on Potato Dextrose Agar (PDA, Fluka), Malt Extract Agar (MEA, 90 Merck), Corn Meal Agar (Oxoid), Water Agar 1% (WA), Oatmeal Agar (OA), Synthetic 91 Nutrient-poor Agar (SNA), Czapek Agar (CZA), Czapek Yeast extract Agar (CYA) and Yeast 92 Extract Sucrose Agar (YES) as described by Samson et al. (16), and incubated for 5-30 days in 4

93 the dark at 25°C. Colony size (in mm), colony structure and characteristics were noted after 4 94 days, however the cultivation was prolonged up to 3 months in order to observe and record 95 changes in pigmentation of the colonies as well as to determine the onset of sporangia and 96 zygospore formation. For sporangial development, a recommended method of floating agar 97 blocks in yeast extract water according to Padhye et Ajello (17) was used in this study. In 98 order to determine the optimal and minimal/maximal temperatures for growth, the strain 99 was incubated on four different media (CZA, CYA, PDA, and MEA) at 12, 15, 20, 25, 30, 35, 100 37, 39, 40, and 42 °C (± 0.1-0.2°C). Colony diameters were measured on the 4th day of 101 cultivation. For comparative description of the macroscopic and microscopic characteristics, 102 CZA was used according to Alvarez et al. (1) and Crous et al., (10, 11).

103 The capability of three different vegetative form of the fungus to germinate or grow on more 104 extreme temperatures was assessed by incubation at 5±0.3°C during 1 to 50 days or at 105 40±0.1°C for 24 to 48 hours. For these tests the following procedure was applied: (a) spores 106 - 100 µL of spore suspension (4.0 x 105 CFU/mL physiologic solution) collected from CZA (6 107 days at 30°C) was applied on MEA plate (at 37°C) at defined time intervals (1, 2, 3 ,4, 7, 14, 108 21, 28, 42 and 50 d); (b) mycelial pellets-micro-colonies of ca 50-200 µm in diam. (~ 2.0 x 109 104/mL ) after 10-12 d incubation submerse YES5%, 140 rpm, 25°C, 100 µL of pellet 110 suspension applied on MEA plate (at 37°C); (c) mycelium - as a nonsporulating colony 111 growing on MEA plate after 4 d incubation (37°C). Both spores and pellets were exposed to 112 5°C in physiological or YES broth liquids, respectively, and then transferred (100 µl onto a 113 plate at 37°C). For exposure to 40°C, they were directly applied onto plates and after 24 and 114 48 hours transferred on a new MEA plate and further incubated at 37°C. Germination of 115 spores was determined in situ under low magnification (50 –100x). 116

117 A dried herbarium specimen of the holotype was deposited in the herbarium of the 118 Mycological Department, National Museum in Prague, Czech Republic (PRM); the ex-type 119 cultures were deposited in the Bioactive Microbial Metabolites (BiMM) Fungal Collection, 120 UFT- Tulln (AT) and in the Culture Collection of Fungi (CCF), Prague (CZ).

121 For determination of microscopic traits, CZA was used after 6-14 days. Sporangiophore 122 structures and sporangia formation were observed in situ under low magnification (50 – 123 100x). Details in sporangiophores, sporangia, sporangiospores and other microscopic 5

124 structures, such as width of hyphae, were observed in mounts with lactophenol blue (RAL 125 Diagnostics). Sporangiospores were measured in water to prevent their deformation. Except 126 on CZA, formation of sporangia was not observed, in any of the other media used after 8 127 days of cultivation. For these media incubation was prolonged for 3 months and the plates 128 were checked at 5-day intervals for the onset of sporangia production. The 129 photomicrographs were taken using a Motic BA 310 microscope with Motic Image Plus 3.0 130 software. Lactophenol blue was used as a mounting medium for microphotography. 131 Photographs of the colonies were taken with a Sony DSC-RX100.

132

133 Antifungal susceptibility testing

134 A 50 µL aliquot of sporangiospore suspension (4.0 x 105 CFU/mL) of Saksenaea dorisii strain 135 BiMM-F232 (collected from CZA grown at 30°C for 14 days into physiological solution) was 136 evenly distributed on RPMI-1640 (Sigma) medium plates. In addition to the spores, a 50 µL 137 aliquot of suspension of mycelial pellets (micro-colonies sized roughly 50-200 µm in 138 diameter collected from YES 5% liquid medium grown at 25°C, 150 rpm after 10 days) was 139 inoculated at a density of 2.0 x 104/mL in the same way on the RPMI plates. Antifungal 140 susceptibility testing was performed by applying the antifungal discs with 10 and 15 µg 141 amphotericin B, 10 µg griseofulvin, 5 µg caspofungin, 1 µg flucytosin, 100 IU nystatin, 10 µg 142 econazole, 25 and 100 µg fluconazole, 1 µg voriconazole, 10 and 15 µg ketoconazole, 5 µg 143 posaconazole, 10 µg miconazole, 8 and 50 µg itraconazole, and 50 µg clotrimazole 144 (Antifungal Discs, Liofilchem Diagnostici, Via Scozia, Italy), 50 µg ciclopirox and 30 µg 145 terbinafine (Neo-Sensitabs, Rosco Diagnostica, Taastrup, Denmark) directly on the plates. 146 MIC values were measured by applying MIC test strip with amphotericin B, flucytosin, 147 caspofungin, ketoconazole, posaconazole, itraconazole, and voriconazole with a 148 concentration range of 0.002-32 µg, and fluconazole with a concentration range of 0.016- 149 256 µg (Antifungal Discs, Liofilchem Diagnostici, Via Scozia, Italy). A single MIC test strip was 150 applied per plate, while 4-6 discs per a plate and incubated for 3-7 days at 37°C.

151 DNA extraction, PCR amplification and sequencing

152 DNA was extracted using a standard cetyltrimethyl ammonium bromide (CTAB) procedure, 153 as described previously (18). Polymerase chain reaction (PCR) analysis was performed by 154 amplification of the internal transcribed spacer (ITS) region with primers ITS1-F (19) and ITS4 6

155 (20). Partial translation elongation factor (TEF-1α) gene was amplified with primers 983F (21) 156 and 2218R (22). The D1/D2 domains of the large-subunit (28S) rRNA gene were 157 amplified and sequenced using the primer pair ITS1/TW14 (20, 23). All reactions were 158 performed in an Eppendorf Gradient MasterCycler (Eppendorf, Hamburg). Conditions for 159 amplification of ITS and D1/D2 domains: 95°C for 5 min; 35 cycles of 95°C for 30 s, 54°C for 160 30 s, 72°C for 90 s and finally 5 min at 72°C. Touchdown amplification of TEF was performed 161 as follows: 95°C for 5 min; 9 cycles of 30 s at 95°C, 30 s at 66°C (−1°C every cycle) and 1 min 162 at 72°C, followed by 30 cycles of 30 s at 95°C, 30 s at 56°C and 1 min at 72°C and a final 163 elongation step of 7 min at 72°C.

164 The PCR products were sequenced with the same primers used for the PCR amplifications 165 (Microsynth AG, Balgach, Switzerland). All sequences obtained in this study were deposited 166 in GenBank. For information on fungal strains used in this study see Table 1. The list provides 167 GenBank accession numbers to ITS, TEF-1α and D1/D2 domains of 28S rRNA gene (LSU) 168 sequences for all accepted species in the genus Saksenaea.

169

170 Phylogenetic analysis

171 For phylogenetic analysis, sequences were aligned with MUSCLE algorithm (24) implemented 172 in SEAVIEW 4.6 (25) software. Phylogenetic trees were constructed using Neighbor Joining 173 (NJ) method in SEAVIEW and genetic distances were computed with the Kimura‐2‐parameter 174 (K2P) model. Bootstrap analyses were performed in NJ with 1000 bootstrap replicates. 175 Apophysomyces elegans CBS 476.78T was selected as outgroup for phylogenetic evaluation.

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182 7

183 RESULTS

184 Taxonomy

185 Saksenaea dorisiae R. Labuda, A. Bernreiter, C. Schüller, J. Strauss & M. Wagner sp. nov. 186 Figs 1, 2 and 3

187 MycoBank MB 830072

188 Etymology: Latin, dorisiae = named after Doris Hochenauer, who isolated the fungus.

189

190 Culture characteristics (Fig. 1) - Colonies reaching 25-30 mm in diam. after 4 d of incubation 191 at 37°C on CZA, whitish, with very scare (cobweb-like) aerial mycelium, with colorless 192 reverse. Colonies at 37°C on MEA, CYA, PDA, CMA and SAB more abundant floccose, 193 mycelium moderately slow growing (30-65 mm) after 4 days, with colorless reverse, 194 remaining sterile (without sporulation).

195 The optimum temperature for growth on CZA was between 20 and 35 °C (45-60 mm diam.), 196 reduced growth was observed at 15°C (15-20 mm diam.) and 37°C (25-30 mm diam.). 197 Minimum growth was observed at 12°C (3-4 mm diam.), and the maximum temperature for 198 growth was 39°C (0.5-1 mm diam.). The fungus did not grow at 40°C on any of the media 199 used (CZA, CYA, MEA and PDA). An overall growth on CYA, MEA and PDA was approximately 200 20-25 % faster after 4 days of incubation at the optimum temperatures compared to CZA 201 (Fig. 4). Germination and growing ability of the spores, mycelial pellets and mycelium, after 202 exposure of 5°C and 40°C is listed in Table 3 and 4.

203 Micromorphology (Fig. 2) - Hyphae mostly coenocytic (non-septate), branched, hyaline, 204 smooth and thin walled, up to 22 µm wide. Sporangiophores erect, generally arising singly, 205 unbranched, brown, , 75-130 long (mostly 85-100 µm), 6-12 µm wide (mostly 7-10 µm), 206 slightly to distinctively verrucose (asperulate) covered with bacilliform protuberances, 207 terminating into hemispherical columellae (15-35 µm wide), and with sparse, dichotomously 208 branched rhizoids (root-like structure). Sporangia terminal, multi-spored, hyaline, flask- 209 shaped (vasiform), slightly to distinctively verrucose (asperulate), 70-190 µm long (mostly 210 90-160 µm), at maturity with a conical venter up to 55 µm wide (mostly 40-50 µm) and 211 gradually narrowing into a long neck (70-100 µm x 6-10 µm) with a rounded apex (closed

212 with a mucilaginous plug) in young sporangia (Fig. 2 F, G and H), truncated and opened at 213 maturity. Sporangia observed after 5 days on CZA most abundantly at 30°C (up to 30-50 per 214 plate), less so at 25 and 35°C (up to 10 per plate), while none at 20 and 37 °C. The sporangia 215 were formed at the center of the colony nearby or at agar block used for inoculation. 216 Sporangiospores during development and at maturity (4-8 days) mainly short-cylindrical 217 (capsulate) with rounded ends, a few also more or less trapezoidal in lateral view, smooth, 218 thin walled, hyaline, (4.5-)5.0-5.5(-6.0) x (2.0-)2.5-3.0(-3.5) µm (mean = 5.1±0.4 x 2.8±0.3 219 µm, n = 70). Zygospores not observed. 220 221 The main distinguishing phenotypic characteristics of the new species compared with the 222 other taxa of the genus Saksenaea are listed in the Table 2.

223

224 Holotype: Serbia, Manastirica (Petrovac) isolated from a private, 65 m deep well - water 225 sample (Code DOO33) 08.10. 2018, isolated by Doris Hochenauer; PRM 951593 [dried 226 culture].

227 Ex-type strain: BiMM-F232 = CCF 6174.

228 DNA sequences: GenBank MK559697 (ITS), GenBank MK569515 (TEF-1α), GenBank 229 MK570305 (LSU). 230

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239 9

240 Based on a search of NCBI's GenBank nucleotide database, the closest hits using the ITS 241 sequence are S. trapezispora (UTHSC DI 15-1; Genbank: NR_147690; Identities = 601/644 242 (93%), gaps 23/644 (3%)) and S. oblongispora (CBS 133.90; Genbank: NR_137569; Identities 243 = 595/652 (91%), gaps 35/652 (5%)). By using TEF-1α sequence the closest hits are S. 244 oblongispora (CBS 133.90; Genbank: HM776687; Identities = 473/476 (99 %), no gaps) and S. 245 trapezispora (UTHSC DI 15-1; Genbank: LT607408; Identities = 467/476 (98%), no gaps)). A 246 region of the 28S rRNA, containing D1 and D2 regions (LSU) shows highest similarities with S. 247 trapezispora (GenBank NG_060019; identities = 704/720 (98%), gaps 1/720(0%)) and S. 248 oblongispora (GenBank NG_057868; identities = 702/719 (98%), no gaps). The phylogenetic 249 trees built for each ITS (Fig. 5) and LSU (Fig. 6) as well as by combining ITS, LSU and TEF (Fig. 250 7), indicate that the isolate represents a new species, being closest to S. trapezispora. 251

252 In vitro antifungal susceptibility testing. The MIC Test Strips with 8 antibiotics used for the 253 strain BiMM-F232 (Saksenaea dorisiae) sensitivity testing showed that the fungus is sensitive 254 towards ketoconazole, posaconazole and itraconazole at rather high concentrations, i.e. 3.0, 255 2.0 and 4.0 µg/mL, respectively. The fungus showed resistance towards caspofungin, 256 flucytosine, and voriconazol (MIC > 32 µg/mL) as well as fluconazole (> 256 µg/mL). The MIC 257 value for amphotericin B was unclear as no clear inhibition zone was formed (Fig. 8). 258 Additional application of antibiotic discs (15 antibiotics) with definedconcentrations revealed 259 sensitivity of the strain also towards terbinafine (25 mm zone at 30 µg), ciclopirox (25 mm 260 zone at 50 µg), econazole (15 mm zone at 10 µg) and clotrimazole (25 mm zone at 50 µg). 261 The antibiotic discs containing amphotericin B (with 10 and 20 µg), griseofulvin (10 µg), 262 flucytosin (1 µg), fluconazole (20 and 100 µg), miconazole (10 µg), and voriconazole (1 µg) 263 showed very limited (up to 8mm) or no activity at all (Table 5). It has been observed, 264 however, that all antifungals strips and/or discs, except terbinafine and ciclopirox, were 265 overgrown after incubation for 5 days (37°C). The same type of multi-resistance towards all 266 antifungals (except terbinafine) was observed also when mycelial pellets of the fungus were 267 used for antifungal susceptibility test during this study. 268 269 270 271 272 10

273 DISCUSSION 274 Phenotypically, the new species, Saksenaea dorisiae, is characteristic and differs from the 275 other taxa in the genus Saksenaea S.B. Saksena (Mucorales, Saksenaeaceae) by the 276 combination of the following features: (1) no growth at 40°C and slow to moderate growth 277 at 15 and 37°C (15-20 mm and 25-30 mm, respectively), (2) conical sporangial venter, (3) 278 morphology of sporangiospores (short-cylindrical, av. = 5.0 x 3.0 µm), and (4) sparse rhizoids. 279 Especially the shape of sporangia and sporangiospores are very distinctive, rendering 280 Saksenaea dorisiae to be easily discernable from the other hitherto known species of the 281 genus. Based on ITS (Fig. 5) and LSU (Fig. 6) S. dorisiae is resolved phylogenetically in a 282 cluster with S. oblongispora and S. trapezispora. Furthermore, phylogenetic reconstruction 283 applying a combination of ITS, LSU and TEF-1α sequences also resulted in clustering of the 284 new species with these two species, being closest to S. trapezispora (Fig. 7). All three species 285 do not grow at 42°C in contrast to species in sister clades growing at this temperature, i.e. S. 286 vasiformis complex, S. erythrospora and S. loutrophoriformis (1, 11). At the time of this 287 study, the genus Saksenaea contains 6 species, including the new taxon. Each of them is 288 characteristic by a particular combination of morphological traits (mainly morphology of 289 sporangia and sporangiospores) and on this basis also easily distinguished from S. dorisiae on 290 these phenotypic bases. The conical-shaped sporangia, sparse rhizoids as well as capsulate 291 sporangiospores formed by the new species are very distinctive, morphologically separating 292 this fungus from all the others in the Saksenaea genus. Since it is obvious that formation of 293 sporangia is necessary for phenotypical identification of isolates into the genus and/or 294 species level, incubation on CZA and 30°C are highly recommended conditions to reach 295 sporulation. All species of Saksenaea are known for their rapid growth on traditional 296 microbiological media (e.g. SAB, PDA, MEA, WA etc.) but without sporulation. To stimulate 297 sporulation, Padhye et Ajello (17) suggested a simple technique, i.e. floating agar blocks in 298 water with yeast extract, but we could not recapitulate this method with S. dorisiae. Out of 299 9 media used, only CZA was effective in stimulating sporangia development within 4-5 days, 300 at 30°C. This medium has been used for morphological description of all known species of 301 Saksenaea (1, 10, 11).

302 Compared to the phylogenetically close S. trapezispora (10), the new species grows 303 substantially slower on CZA at 15°C and 37°C after 4 days (15-20 mm vs 35-45 mm, and 25- 304 30 mm vs 35-40 mm, respectively). Compared to S. dorisiae, S. trapezispora has longer 11

305 sporangiophores (up to 230 µm vs 130 µm), spherical instead of conical sporangia, profuse 306 rhizoids versus sparse ones, and substantially larger trapezoid sporangiospores (av. = 7 x 3.5 307 µm) versus capsulate ones (av. = 5 x 3 µm). In S. trapezispora, growth was similar to S. 308 dorisiae on CZA at its optimal temperature (30-35°C) after 4 days of incubation. The other 309 species in the genus grow very fast on this medium, their mycelium totally covering the 9 cm 310 Petri dish after 4 days incubation at their optimal temperatures.

311 Apart of the growth characteristic, shape and size of sporangiospores are important 312 distinguishing morphological characters within the genus (1), also emphasized by Crous et 313 al., (10, 11). In S. vasiformis complex and S. lautrophoriformis sporangiospores are long 314 cylindrical (to bacilliform) up to 7 x 3.5 µm large, in S. erythrospora biconcave (erythrocyte- 315 like) up to 5.5 x 3 µm large, in S. oblongispora (ellipsoidal to oblong) up to 6.5 x 4.5 µm large, 316 and in S. trapezispora (trapezoidal) up to 8 x 4 µm large. The new species forms mostly short 317 cylindrical (capsulate) sporangiospores, up to 6 x 3 µm large. The phylogenetically closest 318 species, S. oblongispora (1), S. trapezispora (10), and S. dorisiae, are represented by only a 319 single strain. Strain S. oblongispora CBS 133.90T has been isolated from a forest soil in Brazil, 320 while a strain S. trapezispora CBS 141687T from knee wound of a soldier in Texas, USA.

321 Mucormycoses caused by Saksenaea are difficult to treat. We analyzed sensitivity to 322 antifungal drugs, and found that S. dorisiae overgrew all antifungals strips and/or discs used, 323 with exception of terbinafine and ciclopirox. Moreover, the fungus overgrew the inhibition 324 zone after prolonged incubation 5 days at 37°C. Out of 15 antifungals, terbinafine showed 325 stable activity towards the mycelial form (growth from mycelial pellets) of the fungus even 326 after prolonged incubation. It is interesting to note that amphotericin B, a drug that is being 327 the first choice for treatment of mucormycoses (2) including those caused by the Saksenaea 328 species (e.g. (6-9), showed only very weak activity against S. dorisiae. From the azoles used 329 in the study, ketoconazole, posaconazole and itraconazole showed weak activity at rather 330 high minimum effective concentrations, i.e. 3.0, 2.0, 4.0 µg/mL, respectively. Azoles, such as 331 posaconazole, have been proposed as second-line agents against mucormycosis or as an 332 alternative to amphotericin B in situations when high toxicity of amphotericin B has been 333 detected (2). As indicated here by in vitro tests, in case of potential infections caused by S. 334 dorisiae, traditional antibiotic therapy applying amphotericin B and posaconazole might not 335 be the optimal treatment strategy. Similarly, in his review on Saksenaea, Dannaoui (27)

336 stated that these fungi seem to be less susceptible to amphotericin B than other 337 Zygomycetes. Noteworthy, we found that the fungus is not able to grow at 40°C and is 338 rapidly inactivated at this temperature. After 1 day exposure to 40°C there was no growth or 339 spore germination observed and further cultivation under optimal temperatures 340 demonstrated that the fungus was not surviving this heat treatment. This physiological 341 limitation of the fungus could potentially be used as a subsidiary treatment strategy, along 342 with the suggested terbinafine and/or ciclopirox – therapy.

343 Even though S. dorisiae has been isolated from a single water sample of a private well 344 located in a rural area of the Republic of Serbia (village Manastirica), there is no clear 345 evidence on its connection to water as a primary habitat. Most likely, the fungus originated 346 from surrounding soil resulting in pollution of ground water in the well. In fact, soil is 347 supposed to be the primary habitat of the fungus and a known source for infection typically 348 seen after skin rupture and subsequent contact with contaminated soil (3, 26) or 349 contaminated water (2, 4). Although, the presence of the Saksenaea fungi (as S. vasiformis) 350 associated with soil environments has been rarely reported, the fungus has been found in 351 different parts of the world, suggesting a wide distribution (27). Species of this genus have 352 been isolated from a clay loam soil in Tamil Nadu state (India), soil from forest tree 353 nurseries in Georgia (the USA), soil in Barro Colorado Island (Panama), in turtle nest sand on 354 the Nancite beach in Costa Rica, soil of banana-producing areas in Cortes province 355 (Honduras), soil of groundnut fields in Israel, soil sample in I-lan prefecture (Taiwan), 356 pineapple field soil in Okinawa (Japan), and also in Africa from intertidal driftwoods Mitsiua 357 (Ethiopia). Infection associated with water or water environment of human and animals 358 (dolphins and a killer whale) have also been reported already (3, 4, 28). 359 The results of bacteriological investigation (unpublished data) of the water sample revealed 360 a high level contamination by enterococci and other fecal bacterial species indicating that 361 the water of this well was of poor microbiological quality not suitable for consumption. It is 362 noteworthy that the fungus was isolated from an area (village Manastirica, the Republic of 363 Serbia) characterized by cold winters typical for moderately continental climate. 364 Interestingly, we found that the white mycelium of the fungus and mycelial pellets (micro- 365 colonies in a liquid medium) did not survive at 5°C for more than 2 and 7 days, respectively. 366 However, spores in a suspension in water were viable at low temperature for up to 50 days.

367 Thus, especially during cold seasonal periods, spores are vital for propagation and survival of 368 this fungus. 369 Up to date, all known Saksenaea species, were initially described from areas outside of 370 Europe, namely S. vasiformis (India: NRRL 2443T), S. lautrophoriformis (USA: UTHSC 08-379T 371 and India: M-1012/15), S. erythrospora (USA: UTHSC 08-3606T and Middle East: UTHSC 06- 372 576), S. oblongispora (Brazil: CBS 133.90T), and S. trapezispora (USA: CBS 141687T). To the 373 best of our knowledge, reports dealing with Saksenaea fungi from Europe have been 374 associated only with S. vasiformis and areimited to the Mediterranean climate area of Spain 375 (3, 13-15) and/or Greece (2), and. Hence, S. dorisiae represents a first described species of 376 this genus originating from a moderately continental climate area in Europe. 377

378 ACKNOWLEDGMENTS

379 The authors thank Stephan Bruck (AQA GmbH, Vienna, Austria) for providing data on the 380 sample origin. The Bioactive Microbial Metabolites research platform (BiMM) is supported 381 by grants K3-G-2/026-2013 and COMBIS/ LS16005 funded by the Lower Austria Science and 382 Education Fund (NfB).

383

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454 22. Rehner SA, Buckley E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha 455 sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. 456 Mycologia 97:84-98. 457 23. Mori Y, Sato Y, Takamatsu S. 2000. Evolutionary analysis of the powdery mildew fungi using 458 nucleotide sequences of the nuclear ribosomal DNA. Mycologia 92:74-93. 459 24. Edgar RC. 2004. MUSCLE: a multiple sequence alignment method with reduced time and 460 space complexity. BMC Bioinformatics 5:113. 461 25. Gouy M, Guindon S, Gascuel O. 2010. SeaView version 4: A multiplatform graphical user 462 interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221-4. 463 26. Hospenthal DR, Chung KK, Lairet K, Thompson EH, Guarro J, Renz EM, Sutton DA. 2011. 464 Saksenaea erythrospora infection following combat trauma. J Clin Microbiol 49:3707-9. 465 27. Dannaoui E. 2011. Saksenaea., p 791-798. In Dongyou L (ed), Molecular detection of human 466 fungal pathogens. CRC Press, Taylor & Francis Group, USA. 467 28. Robeck TR, Dalton LM. 2002. Saksenaea vasiformis and Apophysomyces elegans zygomycotic 468 infections in bottlenose dolphins (Tursiops truncatus), a killer whale (Orcinus orca), and 469 pacific white-sided dolphins (Lagenorhynchus obliquidens). J Zoo Wildl Med 33:356-66.

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485 TABLE 1 Table 1 List of strains included in the study

GenBank accession no.

ITS EF-1alpha D1/D2 domains Straina Source of 28S rRNA gene (LSU) Saksenaea dorisiae Water from a private well, MK559697 MK569515 MK570305 BiMM-F232T Serbia Saksenaea vasiformis Cutaneous lesion, Tarragona, FR687326 HM776689 HM776678 FMR 10131 Spain Saksenaea vasiformis Skin lesions, France FR687325 HM776688 HM776677 CNRMA F9-83 Saksenaea vasiformis Soil, India FR687327 HM776690 AF113483 NRRL 2443T Saksenaea vasiformis Human tissue, USA FR687329 HM776692 HM776681 UTHSC 09-528 Saksenaea vasiformis Human tissue, USA FR687332 HM776695 HM776684 UTHSC R-2974 Saksenaea vasiformis Craniofacial tissue and brain, FR687322 HM776685 HM776674 ATCC 28740 USA Saksenaea loutrophoriformis Palate necrotic tissue, India LT796164 LT796166 LT796165 M-1012/15 Saksenaea loutrophoriformis Eye, USA FR687330 HM776693 HM776682 UTHSC 08-379T Saksenaea erythrospora Bovine fetus, USA NR 149333 HM776691 NG 059935 UTHSC 08-3606T Saksenaea erythrospora Blood, Middle East FR687331 HJN206536M776694 HM776683 UTHSC 06-576 Saksenaea oblongispora Forest soil, Brasil JN206283 HM776687 NG 057868 CBS 133.90T Saksenaea trapezispora Knee wound, USA NR 147690 LT607408 NG 060019 UTHSC DI 15-1 Apophysomyces elegans Soil, India NR 149336 AF157231 JN206536 CBS 476.78T 486 a FMR, Facultat de Medicina in Ciències de la Salut, Reus, Spain; CNRMA, Centre National de Référence 487 Mycologie et Antifongiques, Paris, France; NRRL, ARS Culture Collection (= the NRRL Collection), Peoria, IL, USA; 488 UTHSC, Fungus Testing Laboratory, University of Texas Health Sciences Center, San Antonio, TX, USA; ATCC, 489 American Type Culture Collection, Manasas, VA, USA; M-1012/15 = FMR 14516; CBS, Centraalbureau voor 490 Schimmelcultures, Utrecht, Netherlands; BiMM, Bioactive Microbial Metabolites Unit, UFT-Tulln, Austria; T, 491 type strain.

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496 TABLE 2 Comparison of the main phenotypic characteristics of the species in the genus Saksenaea. Species Growth Growth at Sporangiophore Shape of Spore size Spore shape References at 37°C 42°C length mature (µm) on CZA sporangia- (µm) venters ** after 4d (mm)

S. vasiformis complex >85* + 65-100 spherical 5-7 x 2-3 long 1 cylindrical

S. loutrophoriformis >85 + 50-75 spherical 3.5-7x2-3.5 long 11 cylindrical

S. erythrospora >85 + 100-150 spherical 5-5.5x2.5-3 ellipsoid- 1 biconcave

S. oblongispora >85 - 80- 100 spherical 5-6.5x3-4.5 oblong 1

S. trapezispora 35-40 - 150-230 spherical 5.5-8x3.5-4 trapezoid 10

S. dorisiae sp. nov. 25-30 - 75-130 µm conical 4.5-6 x2.5-3 Short This study cylindrical - capsulate

497 *Filling the Petri dish (diameter, 9 cm); ** shape of sporangia listed are based on available drawings and 498 illustrations in the referred studies

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514 TABLE 3 Effect of low-temperature (5±0.2°C) on viability of Saksenaea dorisiae (BiMM-F232) Fungus 1 d 2 d 3 d 4d 7 d 14d 21d 28d 42d 50d

Spores* +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

Pellets ** +++ +++ +++ +++ + - -

Mycellium*** +++ ++ - -

515 Growth: +++ = maximum (very good); ++ = good; + = poor; - = none; * spores (4.0 x 105 CFU/mL physiologic 516 solution) collected from CZA; 100 µL applied on plate (MEA, 37°C); **micro-pellets of ca 50-200 µm in diam. ~ 517 2.0 x 104/mL ) after 10-12 d incubation submerse YES5%, 140 rpm, 25°C; 100 µL applied on plate (MEA, 37°C); 518 ***mycelium without sporulation; colony growing on MEA plate after 4 d incubation (37°C) exposed to low 519 temperature 5±0.5°C °C 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 19

563 TABLE 4 Effect of high-temperature (40 ±0.1°C) on viability of Saksenaea dorisiae (BiMM-F232) Fungus 40°C (24h) 40°C (48h)

Spores* - -

Pellets** - -

Mycelium*** + -

564 Growth: + = poor; - = none; * spores (4.0 x 105 CFU/mL physiologic solution) collected from CZA (noticed in 565 material and method); 100 µL applied on plate (MEA, 37°C); **micro-pellets of ca 50-200 µm in diam. ~ 2.0 x 566 104/mL ) after 10-12 d incubation submerse YES5%, 140 rpm, 25°C; 100 µL applied on plate (MEA, 37°C); 567 ***mycelium without sporulation; colony growing on MEA plate after 4 d incubation (37°C) exposed to high 568 temperature (40 ±0.5°C)

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592 TABLE 5 In vitro antifungal susceptibility of Saksenaea dorisiae (BiMM-F232) towards 15 antifungal discs after 593 3 days of incubation at 37°C. Antimycotic Antifungal Disc Spores Mycelial pellets

(concentration µg/disc) Resistant/Sensitive (mm zone) Resistant/Sensitive (mm zone)

Amphotericin B 20 R/S (9)* R Terbinafine 30 S (25) S (30) Ciclopirox 50 S (25) R Caspofungin 5 R R Griseofulvin 10 R R Nystatin 100 IU R/S (6) * R Flucytosin 1 R R Fluconazole 100 R R Econazole 10 S (15) * R Clotrimazole 50 S (25) * R Itraconazole 8 S (15) * R Ketoconazole 15 R/S (8) * R Miconazole 10 R/S (4) * R Voriconazole 1 R R Posaconazole 5 S (15) * R 594 R –resistant, S- sensitive. Responses with an asterisk * indicate re-growing of the fungus into the inhibition 595 zones after prolonged incubation (5 days) at 37°C; Spores (4.0 x 105 CFU/mL), Mycelial pellets in size of 50-200 596 µm in diam. (2.0 x 104/mL)

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624 FIG 1 Saksenaea dorisiae (BiMM-F232). Colonies on CZA and MEA (4 days old) at 35 (bottom) and 37°C (top)

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638 FIG 2 Saksenaea dorisiae (BiMM-F232). (A, B, C, D and E) sporangiophores with sporangia (on CZA, 6 days old). 639 (F, G and H) young sporangia with rounded neck (closed). (I) detail of asperulate sporangiophore on CZA (6 days 640 old). (J, K and L) sporangiospores and details of sporangial neck (on CZA, 6 days old). Scale bars = 500 µm (A), 50 641 µm (B-G), 10 µm (H-L).

642

643 FIG 3 Line drawing of micromorphology of Saksenaea dorisiae (BiMM-F232). (A, B and C) sporangiophores, 644 sporangia and sporangiospores on CZA (6-8 days old). (A) sporangiophore with sporangium and mature 645 sporangiospores. (B) sporangiophores with sporangia (in situ). (C) sporangiospores. Scale bars = 50 µm (A-B), = 646 5 µm (C).

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Temperature dependent growth of Saksenaea dorisiae BiMM-F232 on different media (plates) 90 CZA 80 70 MEA

60 50 CYA 40 PDA

Growth (mm) 30 20 10 0 10 12 15 20 25 30 35 37 39 40 Temperature (°C) 658 659 FIG 4 Temperature dependent growth of Saksenaea dorisiae (BiMM-F232) and its maximal colony extension 660 (in mm) on Czapek agar (CZA), Malt extract agar (MEA), Czapek yeast extract agar (CYA) and Potato dextrose 661 agar (PDA)

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0. 01

T A.elegans CBS 476. 78

T S.v as iformis N RRL 2443

5 3 S. vasif ormis UTHSC R -2974

2 7 S.v as if orm is CN RMA F9-83

8 0 3 0 S. vasiformis FMR 10131 8 0 S. vasiform is UTH SC 09-528

S.vasif ormis AT CC 28740 5 0 5 4 S.loutrophorif ormis M -1012/ 15

1 00 T S.loutrophoriformis UT HSC 08-379

T S.eryt hros pora U THSC 08-3606

1 00 S. ery throspora UTHSC 06-576

T S.t rapezispora UT HSC DI 15-1

T 1 00 S.oblongispora C BS 133.90

4 4 T 676 S.dorisiae BiMM-F 232 677

678 FIG 5 Neighbor joining tree constructed with sequences of the ITS1-5.8S-ITS2 of rDNA. The new taxon S. 679 dorisiae is compared with species from the same genus. Numbers at nodes indicate bootstrap values 680 (expressed as percentages of 1,000 replications). Apophysomyces elegans was used as outgroup. Scale bar 681 indicates 0.01 substitutions per nucleotide position. S. = Saksenaea, A. = Apophysomyces. T, type strain.

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0 .01

T A.elegans CBS477. 78

S.v as if ormis ATCC 28740

T 8 1 S.vasiformis NRRL2443

S_vasif ormis HM776678.1 8 9 8 1 S. vasiform is CNRM A/ F9-83

9 0 5 4 S.vasiformis UTHSC09-528 6 3 S.vasiformis U THSCR-2974

S.lout rophoriformis M1012/ 15 1 00

T S.lout rophoriformis UTH SC 08-379

3 1 T S.eryt hrospora UT HSC 08-3606

1 00 S.erythrospora U THSC06-576

T S. trapezispora U THSC D I_15-1 5 5

T S. oblongis pora C BS 133.90 10 0 T 695 S. doris iae BiMM -F232 696 FIG 6. Neighbor joining tree based on a partial sequence the 28S rDNA including D1, D2 domains (LSU) for the 697 new taxon S. dorisiae is compared with species from the same genus. Numbers at nodes indicate bootstrap 698 values (expressed as percentages of 1,000 replications). Apophysomyces elegans was used as outgroup. Scale 699 bar indicates 0.01 substitutions per nucleotide position. S. = Saksenaea, A. = Apophysomyces. T, type strain.

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0. 01

T A.elegans CBS 476. 78

S.vasiformis ATC C 28740

T 8 9 S.v asiformis NRR L 2443

S. vasiform is UTH SC 09-528 7 5 8 7 S.v as iformis U THSC R-2974

9 7 6 8 S.vasif orm is FM R 10131

7 3 S.v as if orm is CN RMA F9-83

S.loutrophoriformis M-1012/15

1 00 T S.loutrophoriformis UT HSC 08-379

T S. ery throspora UTH SC 08-3606 1 00

S. ery throspora UTH SC 06-576

T 5 5 S. oblongis pora CBS 133.90

T 1 00 S.t rapezispora UT HSC DI 15-1

7 2 T 714 S. dorisiae BiMM -F 232 715 FIG 7. Neighbor joining tree based on a concatenated set of 3 sequences (ITS, LSU and TEF-1α) for the new 716 taxon S. dorisiae is compared with species from the same genus Saksenaea. Numbers at nodes indicate 717 bootstrap values (expressed as percentages of 1,000 replications). Apophysomyces elegans was used as 718 outgroup. Scale bar indicates 0.01 substitutions per nucleotide position. S. = Saksenaea, A. = Apophysomyces. T, 719 type strain.

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746 FIG 8. In vitro antifungal susceptibility of Saksenaea dorisiae BiMM-F232 towards selected antifungals after 3 747 days incubation at 37°C. MIC Test strips – (a) posaconazole (POS), (b) itraconazole (ITC), (c) ketoconazole (KE), 748 (d) caspofungin (CAS), (e) flucytosine (FC), (f) fluconazole (FLU), (g) voriconazole (VO), (h) amphotericin B 749 (AMB), (i) antifungal discs - left top to left bottom (ketoconazole KCA 15 µg, itraconazole ITC 8 and 15 µg); - 750 right top to right bottom (clotrimazole CLO 50 µm, fluconazole FLU 25 and 100 µm); all plates were overgrown 751 by the fungus after prolonged incubation (5 days) at 37°C. 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 29