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Biodiversity, pathogenicity and antifungal susceptibility of and relatives

Badali, H.

Publication date 2010

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Citation for published version (APA): Badali, H. (2010). Biodiversity, pathogenicity and antifungal susceptibility of Cladophialophora and relatives.

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Download date:24 Sep 2021 Chapter 4

Cladophialophora psammophila, a novel species of with a potential use in the bioremediation of volatile aromatic hydrocarbons

H. Badali 1, 2, 3, F.X. Prenafeta-Boldú 4, 5, J. Guarro6, C.H. Klaassen 7, J.F. Meis 7, G.S. de Hoog 1, 2, 8

1CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands;2 Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands; 3Department of Medical Mycology and Parasitology, School of Medicine/Molecular and Cell Biology Research Centre, Mazandaran University of Medical Sciences, Sari, Iran, 4GIRO Technological Centre, Mollet del Vallès, Spain; 5IRTA, Barcelona, Spain; 6Mycology Unit, Medical School, IISPV, Rovira i Virgili University, Reus, Spain; 7 Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands; and 8Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China

Submitted: (2010). 463 Chapter 4

Abstract Cladophialophora is a genus of asexual black yeast-like fungi with one-celled, hydrophobic conidia which is predicted to have teleomorphs in the ascomycete genus Capronia, a member of the order Chaetothyriales. Cladophialophora species are relatively frequently involved in human disease ranging from mild cutaneous lesions to cerebral abscesses. Although the natural niche outside humans is unknown for most opportunistic Cladophialophora species, the fungi concerned are rarely isolated from environmental samples such as dead plant material, rotten wood, or soil. The objective of the present paper is to describe a novel species of Cladophialophora which was isolated from soil polluted with benzene, toluene, ethylbenzene, and xylene (BTEX). It proved to be able to grow on toluene and other related alkylbenzenes as its sole carbon and energy source. This strain is of interest for the complete biodegradation of toluene and other related xenobiotics under growth limiting conditions, particularly in air biofilters, dry and/or acidic soil. A preliminary genetic analysis using MLST and AFLP showed that this was closely related to the pathogenic species C. bantiana, sharing a C. bantiana-specific intron in SSU rDNA. However, it was unable to grow at 40 °C and proved to be non-virulent in mice. The clear phylogenetic and ecophysiological delimitation of the species is fundamental to prevent biohazard in engineered bioremediation applications.

Key words: Cladophialophora, black yeasts, air biofilters, ITS rDNA, AFLP, in vitro antifungal activity, pathogenicity, neurotropism.

Introduction Cladophialophora is a genus of asexual black yeast-like fungi with one-celled, hydrophobic conidia arranged in long, branched, usually coherent chains and with nearly unpigmented conidial scars. The genus was initially e!rected to accommodate a species exhibitingPhialophora -like conidiogenous cells in addition to conidial chains (Borelli 1980), but thus far this feature is expressed in only a few strains of C. carrionii and thus has limited diagnostic value. Judging from multigene phylogeny (nucLSU, nucSSU, RPB1) Cladophialophora is predicted to have teleomorphs in the ascomycete genus Capronia, a member of the order Chaetothyriales in the family (Badali et al. 2008). Cladophialophora species are relatively frequently encountered in human disease, infections ranging from mild cutaneous lesions to cerebral abscesses (Li et al. 2009, de Hoog et al. 2007, Badali et al. 2009). At present the genus contains seven species proven to be involved in human disease (de Hoog et al. 2007, Badali et al. 2008). Among the most virulent species is the neurotrope Cladophialophora bantiana, which is potentially able to cause fatal infections in otherwise healthy individuals (Horré & de Hoog 1999, Kantarcioglu & de Hoog 1999). Cladophialophora carrionii and C. samoënsis are the main etiologic agents of the mutilating skin disorder . Also this type of chronic infection occurs primarily in immunocompetent individuals (Yeguëz- Rodriguez et al. 1992, de Hoog et al. 2007). The natural niche outside humans is unknown for most opportunistic Cladophialophora species. In contrast to frequently expressed opinions (Revankar 2007), listing black yeasts as common saprobes, the fungi concerned are rarely isolated from environmental samples such as dead plant material, rotten wood, or soil. A number of plant- associated Cladophialophora species has been described (Crous et al. 2007), but most of these are located at the phylogenetic base of the Chaetothyriales, remote from the human opportunists (Badali et al. 2008). For recovery of herpotrichiellaceous fungi, selective isolation methods are required, e.g., the use of high temperature (Sudhadham et al. 2008), a mouse vector (Gezuele et al. 1972), extraction

64 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons via mineral oil (Vicente et al. 2008) or enrichment on volatile aromatic hydrocarbons (Zhao et al. 2010). The success of the latter method, enabling isolation of black yeasts where previously direct plating had failed, has supported the hypothesis that herpotrichiellaceous black yeasts are potent degraders of aromatic hydrocarbons. Previously, the alkylbenzene enrichment has been applied in the biofiltration of air polluted with volatile aromatic compounds (Kennes & Veiga 2004, Cox et al. 1993, Weber et al. 1995). The objective of the present paper is to describe a novel species of Cladophialophora, based on the strain ATCC MYA-2335 (CBS 110553) which was isolated from soil polluted with benzene, toluene, ethylbenzene, and xylene (collectively known as BTEX). It proved to be able to grow on toluene and other related alkylbenzenes as its sole carbon and energy source (Prenafeta-Boldú et al. 2001). This strain is of interest for the complete biodegradation of toluene and other related xenobiotics under growth limiting conditions, particularly in air biofilters, dry and/or acidic soil (Prenafeta-Boldú et al. 2004, Prenafeta-Boldú et al. 2008). A preliminary genetic analysis showed that this fungus was closely related, perhaps even conspecific, to the pathogenic speciesC. bantiana (Prenafeta-Boldú et al. 2006). A clear phylogenetic and ecophysiological delimitation of the species is therefore fundamental to prevent biohazard in engineered bioremediation applications. 4 Materials and Methods

Fungal strain The isolate used in this study was obtained fromBS-KNAW theC Fungal Biodiversity Centre, Utrecht, The Netherlands (Table 1). Stock cultures were maintained on slants of 2 % malt-extract agar (MEA, Difco) and oatmeal agar (OA, Difco) and incubated at 24 °C for two weeks (Gams et al. 2007). The studied strain (CBS 110553) had been isolated from soil in a former gasoline station polluted with BTEX hydrocarbons, which was being treated by means of bioventing (Bennekom, The Netherlands). Soil texture was homogeneously sandy, and a sample was taken from about 2 m deep into the unsaturated zone of the contamination plume. About 10 g of soil were enriched in a solid state-like cultivation batch system that used perlite humidified with a mineral nutrient medium as carrier material (Prenafeta-Boldú et al. 2001). Toluene was supplied via the gas phase as the only carbon and energy source and the incubation was performed at 30 ºC under a relatively low water activity (0.9). Intense dematiaceous growth was observed on the top perlite layer after 5 weeks of incubation. The predominant fungal strain was isolated and purified by re-suspending colonized perlite granules and plating serial dilutions on mineral medium agar plates incubated under a toluene atmosphere.

Morphological and physiological characterization The isolate was cultured on 2 % MEA and OA and incubated at 24 °C in the dark fortwo weeks (Gams et al. 2007). Provisional identification was based on macroscopic and microscopic morphology. Microscopic observations were made with slide culture techniques using potato dextrose agar (PDA, Difco) or OA because these media readily induce sporulation and suppress growth of aerial hyphae (de Hoog et al. 2000). Mounts of two-week-old slide cultures were made in lactic acid or lactophenol cotton blue and light micrographs were taken using a Nikon Eclipse 80i microscope with a Nikon digital sight DS-Fi1 camera. Cardinal growth temperatures were determined by incubating MEA culture plates in the dark for 2 weeks at temperatures ranging from 21 to 40 °C at intervals of 3 °C (Crous et al. 1996). Experiments consisted of three simultaneous replicates for each isolate; the entire procedure was repeated once.

65 Chapter 4 Geography USA USA, Kentucky Carolina USA, North Chiba Japan, Netherlands The - Virginia USA, America South Netherlands Brazil Brazil del Queguay Grande Isla Uruguay, Wageningen Netherlands, Curitiba Paraná, Brazil, Curitiba Paraná, Brazil, del Queguay Grande Isla Uruguay, Apeldoorn Netherlands, Kaiserslautern Germany, Atlanta USA, Georgia, Colombo Parana, Brazil, Sarandi Parana, Brazil, Brazil UK Cayman Islands USA, Grand Curitiba Paraná, Brazil, Germany Source brain abscess Human, brain abscess Human, brain abscess Human, source unknown Human, soil Gasoline-polluted Cat, subcutaneous lesion phaeohyphomycosis Human, chromoblastomycosis Male, aspiration-biopsy Lymphnode, Soil chromoblastomycosis, Male, tree Dead biofilter Toluene toe lesion, child Interdigital cover/soil vegetable Litter, cut tree Trunk, soil Gasolin-station inoculated with soil Biofilter subcutaneous phaeohyphomycosis Male, stem romanzoffianum, Syagrum vegetable cover Decaying eumycetoma Male, phaeohyphomycosis Cutaneous disseminated infection Human, cover/soil vegetable Litter, abscess, male Tracheal GenBank number GenBank EF1, nucSSU ITS, EU103989, EU140585, AY554284 -,-, - -,-, - -,-, - -, AY150798 AY857517, EU103995, - , EU140584 EU103996, -, EU140583 -, - AY366914, - AY366925,-, - AY366927,-, - AY366926,-, EU103983, EU140601, - EU140603, - AY857507, EU103984, EU140602, - EU140600, - AY857508, FJ385270,-, - FJ385274, EU137261, - FJ385271, EU137260, - EU137318, EU137257, - FJ385269 , EU137259, - FJ385272, EU137258, - -, - -, - EU103985, EU140595, - FJ385275,-, - EU103986, EU140593, - Other reference Other 10958, CDC B-1940 (T) ATCC dH 12086 dH12082 IFM 41438 MYA-2335 (T) ATCC 4991 CDC B-3634; UAMH CDC B-3875; NCMH 2247 (T) 18658; dH 15659 ATCC dH 15691 dH 11602 dH 11613 dH 12333; IHM 1727 200384 ATCC dH 12939 dH 11591 dH 12335 dH15250 dH 11474 CDCB-5680; dH 10680 (T) dH 11588 dH 11601 dH 18473 - 56280; dH15405 (T) ATCC ISO 13F (T) CBS no. 173.52 110013 110009 644.96 110553 640.96 979.96 271.37 289.93 102238 102248 109628 114326 118724 102230 109630 110551 109797 834.96 102227 102237 122636 123977 147.84 118720 306.94 Taxon name Taxon bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora psammophila Cladophialophora emmonsii Cladophialophora emmonsii Cladophialophora pedrosoi Fonsecaea monophora Fonsecaea monophora Fonsecaea monophora Fonsecaea saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora devriesii Cladophialophora devriesii Cladophialophora arxii Cladophialophora Table. 1. Isolation data of examined strains. 1. Isolation Table.

66 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

Molecular characterization Mycelia were grown on 2 % (MEA) plates for 2 weeks at 24 °C. A sterile blade was used to scrape off the mycelium from the surface of the plate and transferred to a 2 ml Eppendorf tube containing 400 µL TEX-buffer (Tris 1.2 % w/v, Na-EDTA 0.38 % w/v, pH 9.0) and glass beads (Sigma G9143). The fungal materials were homogenized by Mobio vortex for 5−10 min. Subsequently Geography Chiba Yachimata, Japan, Shiroi Japan, Jicaltepec Mexico, Netherlands The 120 µL SDS 10 % and 10 μl proteinase K were added and incubated for 30 min at 65 °C, the mixture was vortexed for 3 min. After addition of 120 μl of 5 M NaCl and 1/10 vol CTAB 10 % (cetyltrimethylammonium bromide) buffer, the material was incubated for 60 min at 55 °C. Subsequently 700 μl SEVAG (24:1, chloroform: isoamylalcohol) was slowly mixed, incubated for 30 min on ice water and centrifuged for 10 min at 14 000 r.p.m. The supernatant was transferred to a new tube with 225

μl of 5 M NH4-acetate, mixed carefully by inverting, incubated 4 Source wood Rotting wood Decaying eumycetoma Male, contaminant Culture for 30 min on ice water, and centrifuged again for 10 min at 14 000 r.p.m. The supernatant was transferred to another tube with 0.55 vol isopropanol and centrifuged for 5 min at 14 000 r.p.m. Finally, the pellet was washed with 1000 μl ice cold 70 % ethanol. After drying at room temperature, it was re-suspended in 100 μl TE buffer (Tris 0.12 % w/v, Na-EDTA 0.04 % w/v) plus 1.5 μl RNAse 20 U/mL and incubated for 15–30 min at 37 °C. DNA extracts were stored at −20°C until use (Gerrits van den Ende et al. 1999). GenBank number GenBank EF1, nucSSU ITS, EU103988, EU140599 EU140598, - AY251087, FJ385276,-, - EU137293, EU137235, - Three genes were amplified for phylogenetic characterization of the new species: the small subunit of the nuclear ribosomal RNA gene (nucSSU), the large subunit of the nuclear ribosomal RNA gene (nucLSU), and the largest subunit genes of RNA polymerase I (RPB1). For multilocus sequencing typing (MLST), the internal transcribed spacer region (ITS rDNA), the translation elongation factor 1 alpha (EF1-α), and the partial beta tubulin (TUB) genes were amplified. The primers used for

Other reference Other 5022 IFM 4701; UAMH 52853; IMI 298056 ATCC dH 18909 (T) dH 15898 amplification and sequencing were described previously (Badali et al. 2008, Gueidan et al. 2008). PCR reactions were performed on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) in 50 μL volumes containing 25 ng of template

CBS no. 987.96 556.83 122637 454.82 DNA, 5 μl reaction buffer (0.1 M Tris-HCl, pH 8.0, 0.5 M

KCl, 15 mM MgCl2, 0.1 % gelatine, 1 % Triton X-100), 0.2 mM of each dNTP and 2.0 U Taq DNA polymerase (ITK Diagnostics, Leiden, The Netherlands). Amplification of those genes were performed with cycles of 2 min at 94 °C for primary denaturation, followed by 35 cycles at 94 °C (45 s), 52 °C (30 s) and 72 °C (120 s), with a final 7 min extension step at 72 °C. Annealing temperatures used to amplify SSU, LSU, RPB1, ITS, EF1-α and TUB genes were 55 °C, 52 °C, 52 °C, 52 °C, Taxon name Taxon minourae Cladophialophora minourae Cladophialophora mycetomatis Cladophialophora mycetomatis Cladophialophora Atlanta, U.S.A.; DCU = Prevention, and Disease Control Netherlands; CDC = Centers for The Utrecht, Centre, Biodiversity Fungal U.S.A.; CBS = CBS-KNAW Collection, Manassas, Culture Type = American ATCC Abbreviations: of University = The UAMH Chiba, Japan; Chiba University, Toxicoses, and Microbial Fungi Center for Pathogenic collection; IFM = Research working dH = G.S. de Hoog Chiba, Japan; School of Medicine, Department of Dermatology, strain. Type T = Canada; Edmonton, Collection and Herbarium, Alberta Microfungus Table 1 (continued) Table 50 °C and 58 °C, respectively. Amplicons were purified using

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Cladophialophora bantiana CBS 173.52 100 Cladophialophora bantiana CBS 110013

85 Cladophialophora bantiana CBS 110009 Cladophialophora bantiana CBS 644.96 98 Cladophialophora psammophila CBS 110553 100 Cladophialophora emmonsii CBS 640.96 Cladophialophora emmonsii CBS 979.96 Fonsecaea pedrosoi CBS 271.37 100 100 Fonsecaea monophora CBS 289.93 Fonsecaea monophora CBS 102238 Fonsecaea monophora CBS102248 Cladophialophora saturnica CBS 109628 100 Cladophialophora saturnica CBS 114326 Cladophialophora saturnica CBS 118724 99 Cladophialophora saturnica CBS 102230 Cladophialophora saturnica CBS 109630 x10 100 86 Cladophialophora immunda CBS102237 Cladophialophora immunda CBS 122636 72 Cladophialophora immunda CBS 834.96 82 Cladophialophora immunda CBS 109797 Cladophialophora immunda CBS 110551 Cladophialophora immunda CBS 123977 Cladophialophora immunda CBS 102227 96 88 Cladophialophora devriesii CBS 147.84 Cladophialophora devriesii CBS 118720 76 Cladophialophora immunda dH18474 Fig. 1. Phylogenetic reconstruction

100 Cladophialophora immunda dH 18476 obtained from a ML analysis of three Cladophialophora immunda dH 18478 combined loci (ITS rDNA, EF1-α and Cladophialophora immunda CBS 122255 TUB) using RAxML. Bootstrap support Cladophialophora immunda dH 18477 values were estimated based on 500 Cladophialophora immunda CBS 122257 replicates, and are shown above the CBS 306.94 100 Cladophialophora arxii 100 Cladophialophora minourae CBS 987.96 branches (values ≥ 70 %). New species Cladophialophora minourae CBS 556.83 are highlighted using with grey boxes. Cladophialophora mycetomatis CBS 122637 The tree was rooted with two strains Cladophialophora mycetomatis CBS 454.82 of Cladophialophora mycetomatis (CBS 0.01 substitutions/site 454.82 and CBS 122637). QIAquick PCR purification Kit (Cat. No 28104, U.K.). Sequencing was performed as follows: 95 °C for 1 min, followed by 30 cycles consisting of 95 °C for 10 s, 50 °C for 5 s and 60 °C for 2 min. Reactions were purified with Sephadex G-50 fine (GE Healthcare Bio-Sciences, Uppsala, Sweden) and sequencing was done on an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, U.S.A.). Sequence data obtained in this study were adjusted using the SeqMan of Lasergene software (DNAStar Inc., Madison, Wisconsin, U.S.A.). Alignments of DNA sequences were done manually for each gene using MacClade 4.08 (Maddison et al. 2003) with the help of amino acid sequences for protein coding loci. Ambiguous regions and introns were excluded from the alignments. The program RAxML-VI-HPC v.7.0.0 (Stamatakis et al. 2008), as implemented on the Cipres portal v. 1.10, was used for the tree search and the bootstrap analysis (GTRMIX model of molecular evolution and 500 bootstrap replicates). Bootstrap values equal or greater than 70 % were considered significant (Hillis et al. 1993). Data were analysed as Clade II (Badali et al. 2008). TheBantiana -clade comprised 36 taxa. Phylogenetic reconstructions and bootstrap values were first obtained for each locus separately using RAxML (as described above). The congruence between loci was assessed using a 70 % reciprocal bootstrap criterion (Mason-Gamer et al. 1996). The loci were then combined and analysed using RAxML (as described above). The phylogenetic trees were edited using Tree View v. 1.6.6. AFLP analysis was performed as described previously (Badali et al. 2010).

68 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

Fig. 2. Amplified fragments length polymorphism (AFLP) profile of Cladophialophora psammophila (CBS 110553) in relation to that of reference strains of Cladophialophora bantiana (CBS 173.52) and Cladophialophora minourae (CBS 556.83).

4

Fig. 3. Survival rates of OF-1 mice infected intravenously with 1x107 CFU/animal. T = type strain.

Table. 2. Minimum inhibitory (azoles) and effective (echinocandins) concentrations in mg/L of different antifungal drugs against Cladophialophora psammophila (CBS 110553) and other Cladophialophora reference strains

Name CBS no AmB FLU ITC VOR POS ISA CAS ANI

C. bantiana 173.52 (T) 0.25 16 0.016 0.125 0.016 0.031 1 0.125

C. bantiana 110009 1 16 0.031 0.25 0.031 0.031 8 0.125

C. bantiana 110013 0.5 32 0.063 1 0.063 0.5 2 0.063

C. psammophila 110553 (T) 0.25 32 0.016 0.25 0.016 0.125 2 0.031

C. minourae 555.83 (T) 1 32 0.063 0.25 0.031 0.25 1 0.063

Virulence testing The virulence of the new species was compared with that of three reference strains of Cladophialophora bantiana and a strictly non-pathogenic strain of C. minourae using a murine model, which was previously described by Mariné et al. (2009) determining the mortality rates of the infected animals. Briefly, six-week-old OF-1 male mice (ten per group) (Charles River, Criffa SA, Barcelona, Spain) weighing 28-30 g were used. Animals were housed five per cage in standard boxes with corncob redding and free access to food and water. Conditions were approved by the Animal Welfare committee of the Medical School of the University Rovira i Virgili. The isolates tested were Cladophialophora sp. CBS 110553, C. bantiana (CBS 110013, CBS 110009 and CBS 173.52 as ex-type strain), and C. minourae (CBS 556.83). They were stored on corn meal agar (CMA, 30 g corn and 15 g agar per litre) slants covered with paraffin oil, and prior to the study they were cultured on PDA for 5–8 days at 30 ºC. Inocula were prepared by flooding the surface of the agar plate with sterile saline, scraping the sporulating mycelium with a culture loop, and

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Fig. 4. Cladophialophora psammophila (CBS 110553). A–B. Culture on MEA and PDA) produced velvety to hairy, powdery and olivaceous-black with a wide well-defined margin. C. Hyphal coil. D–G. Conidiophores laterally or terminally on undifferentiated hyphae, ellipsoidal to fusiform or sub-cylindrical. F. One-celled and long conidia, produced inlong, strongly coherent chains. Scale bars = 10 μm.

70 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

4

Fig. 5. Line drawing of microscopic morphology of Cladophialophora psammophila (CBS 110553). Scale bar = 10 µm.

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Fig. 6. Colony diameters at different temperatures ranging from 27 °C to above 40 °C, measured after two weeks on 2 % MEA were calculated for Cladophialophora bantiana (CBS 173.52) and Cladophialophora psammophila (CBS 110553). drowning up the resultant suspension with a sterile Pasteur pipette. Suspensions were filtered once through sterile gauze to remove hyphae. Conidial suspensions were adjusted after counting with a haemocytometer. The viability of these inocula was verified by plating dilutions of the suspension on PDA plates. Infection was established intravenously with 0.2 ml of inoculum via the lateral tail vein. Inocula administered were 2 × 107 conidia/animal. Preliminary experiments with several reference strains of C. bantiana demonstrated that this concentration produced infections with subsequent 100 % mortality within 20 days of inoculation (data not shown).

Antifungal susceptibility In vitro antifungal susceptibilities of different drugs against CBS 110553 and reference strains (C. bantiana and C. minourae which are a neurotropic pathogen and a saprobe, respectively) were performed by microbroth dilution as described in the Clinical and Laboratory Standards Institute (CLSI) document M38-A2. Minimum inhibitory concentrations (MIC) for (AmB, Bristol-Myers Squib, Woerden, The Netherlands); fluconazole (FLU, Pfizer Central Research Sandwich, UK); (ITC, Janssen Research Foundation, Beerse, Belgium); voriconazole (VOR, Pfizer); posaconazole (POS, Schering-Plough, Kenilworth, USA); isavuconazole (ISA, Basilea, Basel, Switzerland), and minimum effective concentrations (MEC) for caspofungin (CAS, Merck Sharp & Dohme, Haarlem, The Netherlands) and anidulafungin (ANI, Pfizer) were determined. As per the CLSI guidelines, stock solutions of the drugs were prepared in the appropriate solvent. The drugs were diluted in the standard RPMI-1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) with L-glutamine without bicarbonate to yield two times their concentrations were as follows: amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin 0.016–16 mg/ml; fluconazole 0.063–64 mg/ ml; isavuconazole 0.002–2 mg/ml and anidulafungin 0.008–8 mg/ ml. Plates were stored at –70 °C until they were used. Methods for sporulation and preparation of conidial suspensions were those of Badali et al. (2010). Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were used as quality controls.

72 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

Results Using the phylogenetic markers including nucSSU, nucLSU and RPB1, Cladophialophora species are predicted to belong to at least four different lineages. Most of the human-opportunistic species belong to two well supported clades within the Herpotrichiellaceae (Badali et al. 2008). Strain CBS 110553 was nested in lineage one corresponding to the family Herpotrichiellaceae (Tree not shown) that contained the pathogenic Cladophialophora species (C. carrionii, C. boppii, C. bantiana, C. arxii, C. immunda, C. devriesii, C. saturnica, C. emmonsii C. mycetomatis) as well as the pathogenic genus Fonsecaea (F. pedrosoi and F. monophora) and Phialophora (P. verrucosa and P. mericana). Phylogenetic reconstructions were first performed for each gene separately (ITS: 495 characters, EF1-α: 133 characters, TUB: 355 characters). Topological conflicts were detected only within the species C. saturnica, the incongruence involving only below species-level relationships, and therefore – in agreement with the genealogical recognition species concept (Taylor et al. 2000) – the conflicts were ignored and the three loci were combined. In this multilocus analysis, Cladophialophora mycetomatis, an agent of true mycetoma was taken as an outgroup (Fig. 1). This analysis showed that CBS 110553, originating from hydrocarbon polluted soil was phylogenetically Cladophialophora distinct from other species of , and a sister taxon (85 % bootstrap support) of the 4 pathogenic species C. bantiana. Hence, it is described below as a novel species of Cladophialophora. The AFLP profile of C. psammophila (CBS 110553) was compared with strains of C. bantiana, yielding similar data as obtained by sequencing analysis in that banding patterns were invariable within C. bantiana and deviated considerably from the CBS 110553 profile: no major bands were shared by the two entities (Fig. 2). Survival rates of mice experimentally infected with strains of Cladophialophora are shown in Fig. 3. All animals infected with three different strains of C. bantiana died between days 12 and 17; differences between strains were insignificant. Animals infected with C. psammophila and C. minourae survived and remained alive until the end of the experiment. Table 2 summarizes the MIC values of eight antifungal drugs against three highly neurotropic isolates (C. bantiana) and two non-pathogenic species (CBS 110553 and C. minourae). There was a uniform pattern of low MICs for itraconazole, posaconazole and isavuconazole. The highest MICs were for fluconazole followed by amphotericin B, the echinocandins and voriconazole.

Cladophialophora psammophila Badali, Prenafeta-Boldú, Guarro & de Hoog, sp. nov. MycoBank MB 515525. Figs 4, 5

Coloniae fere lente crescentes, ad 30 mm diam post 14 dies 33 °C, velutinae vel lanosae, conidiis maturis pulverulentae, olivaceo-nigrae, margine obscuriore circumdatae. Hyphae fertiles erectae, cylindricae, sursum ramosae, 2.0−2.5 µm latae. Conidia unicellularia, in catenis longis persistentibus, raro ramosis connexa, ex hyphis indistinctis orientia, pallide olivacea, levia, tenuitunicata, ellipsoidea vel fusiformia vel subcylindrica, 6.5−10.0 × 2.0−3.5 μm. Teleomorphosis ignota.

Description based on CBS 110553 at 27 °C after 2 wk in darkness.

Macroscopy on PDA: Colonies moderately expanding, with a daily growth rate of 2.1 mm (1.5 mm at 37 °C). Colonies velvety to hairy, powdery when sporulating, olivaceous-black with a wide well-defined margin which is darker than the colony centre; reverse olivaceous-black (Figs 4A−B). Microscopy on MEA: Fertile hyphae septate, ascending to erect, smooth- and thin-walled, hyaline to pale olivaceous, guttulate, branched, 2−3 μm wide, forming hyphal strands and hyphal coils.

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Conidiophores laterally or terminally on undifferentiated hyphae, pale olivaceous, smooth- and thin-walled, ellipsoidal, fusiform or sub-cylindrical, 6.5−10.0 × 2.0−3.5 μm, erect. Conidia one- celled, produced in long, strongly coherent, poorly branched chains (Figs 4C-H, 5). Teleomorph unknown.

Physiology: Cardinal temperatures: optimal development at 27−30 °C, with growth abilities between 9−37 °C. No growth at 40 °C (Fig. 6).

Specimen examined: The Netherlands, Bennekom, isolated from sandy soil in a former gasoline station polluted with BTEX hydrocarbons (benzene, toluene, ethylbenzene, and xylene), CBS H-20384 (Holotype) = CBS 110553 (ex-type culture).

Discussion The new species C. psammophila morphologically resembles C. bantiana, which is a virulent neurotropic agent of fatal cerebral phaeohyphomycosis in humans, causing granolumatous abscesses in the brain and having a maximum growth temperature of 42 °C. The multiple sequences analyzed in this study deviate sufficiently from those of C. psammophila to be sure that the two species can be considered to represent separate though highly related species. Members of C. bantiana are unique by consistently containing a distinctive 558-bp intron beginning at position 1768 in the small subunit of the ribosomal DNA gene (Gerrits van den Ende et al. 1999). Interestingly, this intron is also present in C. psammophila, underlining the close kinship of the two species. Cladophialophora bantiana has a worldwide distribution, but until now it could be isolated from the environment only by using a mammal vector (Conti-Diaz et al. 1977, Dixon et al. 1980). The natural niche of this organism remains unknown and it is presumably introduced via inhalation as the main infection route. In contrast, C. psammophila was isolated by hydrocarbon enrichment from a location where hydrocarbon pollution was prevalent. It does not grow at temperatures above 37 °C. Pathogenicity tests on a murine model indicated that C. psammophila is non-virulent, whereas C. bantiana kills mice within two weeks. Both Cladophialophora species, although at short phylogenetic distance, thus exhibit striking ecological differences. The ecophysiological divergence between C. bantiana and the newly described C. psammophila is puzzling. Future genome comparisons will serve to reveal the essential virulence factors involved in the pathogenicity of the Bio-Safety Level-3 species C. bantiana. Phylogenetically the genus Cladophialophora – morphologically consistently characterised by poorly or profusely branched chains of dry, rather strongly coherent, moderately melanized conidia – is polyphyletic within the order Chaetothyriales (Badali et al. 2008). In part the genus is closely related to members of genera that are consistently associated with human infection, such as Exophiala, Fonsecaea and Rhinocladiella (lineage 1 in combined phylogenetic reconstructions (nucSSU, nucLSU and RPB1) of Chaetothyriales published by of Badali et al. 2008). This part of the tree comprises a number of Capronia teleomorphs and may therefore be regarded to represent the family Herpotrichiellaceae. This family also encompasses several species that have been related to the metabolism of monoaromatic hydrocarbons and other related aromatic substrates, both in polluted and natural environments. Consequently, these organisms are of interest in biotechnological applications for the bioremediation of hydrocarbon pollution. The distantly related Cladophialophora species have been described as host-specific plant-associates (Crous et al. 2007) and are unlikely to belong to the Chaetothyriales. No association to aromatic substrates has ever been reported in this latter group. Enrichment on toluene as the sole sources of carbon and energy was a key factor for the isolation

74 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons of the strain CBS 110553. The physiological and metabolic characterization of axenic cultures demonstrated that assimilation of aromatic hydrocarbons was limited to certain alkylbenzenes, such us toluene and ethylbenzene (Prenafeta-Boldú et al. 2001). This biodegradation pattern was confirmed in biodegradation assays of complex BTEX mixtures simulating gasoline pollution, in which only toluene and ethylbenzene were mineralized, while the xylene isomers were co- metabolized and benzene remained undegraded throughout the experiment (Prenafeta Boldú et al. 2002). Alkylbenzene degradation proceeded through the initial oxidation of the alkyl side chain, rather than the aromatic ring (Prenafeta Boldú et al. 2001). Growth of C. psammophila in liquid cultures under agitation was predominatly yeast-like. Furthermore, enzymological studies on the related Cladophialophora saturnica CBS 114326, initially misidentified as a sphaerospermum, showed the involvement of a cytochrome P450 monooxygenase in the initial oxidation of the alkyl group (Luykx et al. 2003). The exceptional metabolic and physiological capacities of CBS 110553 have been investigated in relation to the biofiltration of waste gases polluted with volatile aromatic hydrocarbons and for the bioremediation of BTEX pollution in soil microcosms (Prenafeta-Boldu et al. 2004 and 2008). The striking close phylogenetic proximity of strain CBS 110553 to the notorious human pathogen C. bantiana has put its applicability in bioremediation into question due to potential 4 biohazard (Prenafeta-Boldu et al. 2006). Cladophialophora bantiana has regularly been recognized in human disease by its high mortality rate despite the application of combined surgery and antifungal therapy, and has therefore been considered as one of the most dangerous pathogenic fungi known to date (de Hoog et al. 2003). The majority of reported CNS infections caused byC. bantiana were found to be brain abscesses with no predisposing factors or immunodeficiency (Horré & de Hoog 1999). The pathogenesis may be haematogenous spread from an initial, presumably subclinical, focus which may be pulmonary, subcutaneous or intestinal (Li et al. 2009). It remains unclear why this fungus preferentially causes CNS disease in immunocompetent individuals. It has been hypothesized that, in addition to other virulence factors that are common in herpotrichiellaceous fungi, such as thermotolerance, dimorphic yeast-like growth, wall melanization, etc., the tendency to infect central nervous tissue in mammals is also related to the capacity of metabolizing aromatic compounds, which is also conspicuous in the Herpotrichiellaceae (Prenafeta-Boldu et al. 2006). Being primary neurotropic, a characteristic from just a few species in the Herpotrichiellaceae, these fungi must also possess other, as yet undiscovered attributes conferring infective capacity towards the brain. An association between metabolism of aromatic hydrocarbons and opportunistic pathogenicity has also been observed in Cladophialophora immunda and C. saturnica, as well as in the related genus Exophiala, particularly E. oligosperma and E. xenobiotica. Representatives have been reported from opportunistic human infections and from hydrocarbon polluted sites (Badali et al. 2008, de Hoog et al. 2003, Vicente et al. 2008). However, infection patterns of these species tend to be coincidental, and are mostly limited to skin and nails. Cladophialophora psammophila thus far has never been reported from clinical cases, and virulence test on the strain CBS 110553 indicated that the new species is, in principle, non-pathogenic.

Acknowledgments This study and the work of H. Badali have been funded by a research grant (No. 13081) from the Ministry of Health and Medical Education of the Islamic Republic of Iran and the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. The authors thank Walter Gams for his comments on the manuscript and for providing the Latin descriptions.

75 Chapter 4

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