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Date ebook Published : 2012-12-11 Characterization of a New Clinical Yeast Species, Candida tunisiensis sp. nov., Isolated from a Strain Collection from Tunisian Hospitals Jamel Eddouzi, a,d Valérie Hofstetter, b Marizeth Groenewald, c Mohamed Manai, d Dominique Sanglard a Institute of Microbiology, University Hospital Lausanne and

Characterization of a New Clinical Yeast Species, Candida tunisiensis sp. nov., Isolated from a Strain Collection from Tunisian Hospitals Jamel Eddouzi, a,d Valérie Hofstetter, b Marizeth Groenewald, c Mohamed Manai, d Dominique Sanglard a Institute of Microbiology, University Hospital Lausanne and University Hospital Center, Lausanne, Switzerland a ; Agroscope Changins-Wädenswil, Plant Protection Field Crops and Grapevine/Viticulture and Enology, Nyon 1, Switzerland b ; CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands c ; Laboratory of Biochemistry and Molecular Biology, Faculty of Sciences of Tunis, Campus Universitaire El-Manar, Tunis, Tunisia d From a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ioniza- tion–time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and meta- bolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylo- genic analyses suggested that this isolate clustered at the base of the Dipodascaceae ( Saccharomycetales , Saccharomycetes , and Ascomycota ). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohy- phal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromo- somes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC  8  g/ml), voriconazole (MIC  0.5  g/ml), itraconazole (MIC  16  g/ml), and amphotericin B (MIC  4  g/ml) but still suscep- tible to posaconazole (MIC  0.008  g/ml) and caspofungin (MIC  0.5  g/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classi- fied based on phylogenetic approaches. T he incidence of fungal infection has significantly increased during the past 2 decades. These infections mainly occur in susceptible patients afflicted with hematological malignancies or undergoing organ or bone marrow transplantation ( 1–5). Patients and treatment have changed, as well as fungi, which are involved in infections. Indeed, species that were previously unknown to the medical community are now emerging ( 4, 6). Invasive fungal in- fections are associated with high mortality rates, often related to a delayed diagnosis (1, 3), and novel fungal species isolated in hu- man are being increasingly reported ( 6). Because fungal species show very different antifungal susceptibility profiles, it is impor- tant for clinicians to obtain accurate identifications in order to apply appropriate treatments (3, 7). Identification of yeast species is based on different criteria. Traditionally, fungi have been iden- and C. krusei ATCC 6258 were used as control strains. For some isolates, ID 32C metabolic galleries (bioMérieux, Marcy l’Etoile, France) were used as described by the manufacturer. Description of Candida tunisiensis J. Eddouzi, V. Hofstetter, M. Groenewald, M. Manai, D. Sanglard sp. nov. (JEY63). Candida tunisien- sis ( = sis, N.L. nom. fem. sing. adj., tunisiensis , referring to the region from which this yeast was isolated, Tunis, the capital of the Tunisia Republic). On YEPD agar, after 1 week of incubation at 25°C, the colonies are white in color, circular, rough, with convex elevation and invade agar. The cells are round to ovoid, single or in pairs, the budding is multipolar, and the cells are 3 to 4  mby5to6  m. Pseudohyphal growth is domi- nant, but no transition to a hyphal form is observed. The formation of ascospores is not observed. The physiological characteristics are presented in Table S1 in the supplemental material. The optimal growth tempera- ture is between 37 and 42°C. The type strain is JEY63 (CBS 12513), which was isolated from a 50-year-old male that suffered from oral thrush in a Tunisian hospital. The Mycobank accession number is 509092. Formation of chlamydospores. Yeasts were grown in complete YEPD medium (1% Bacto peptone [Difco]), 0.5% yeast extract [Difco], 2% glucose [Fluka]) at 30°C under constant shaking. The inoculum size was adjusted to 1.5  10 4 CFU/ml by measuring the absorbance in a spectro - photometer. A 50-  l of the third serial dilution was plated onto rice ex- tract agar (rice extract at 0.7 g/liter and agar at 14.3 g/liter [Fluka], sup- plemented with 1% Tween 80) under a sterile glass coverslip to maintain a semianaerobic condition and grown in the dark for 7 days at 25°C. The rice extract is the sole source of nutrient in the medium. The lack of nutrients together with the oxygen-deficient culture conditions creates an environment that induces the formation of specific morphological forms in particular chlamydospores in some yeast. The test was performed in triplicate. DNA isolation, amplification, and sequencing. Genomic DNA was prepared from yeast cultures after overnight incubation in YEPD medium at 30°C under constant shaking as previously described ( 17). Six DNA regions of the fungal strain JEY63 were amplified and sequenced: four ribosomal gene regions (the small subunit [18S], the large subunit [26S], the 5.8S and flanking internal transcribed spacers 1 and 2 [ITS1-5.8S- ITS2], part of the mitochondrial small subunit [12S]), two protein-coding gene regions (part of the second largest subunit of the RNA polymerase II [subunit B150, regions 6 and 7; RPB 2] and part of translation elongation factor 1  [ TEF1  ]). The primers used for amplification and/or sequenc- ing are listed in Table 1. Amplification of nuclear ribosomal genes was performed in the presence of 200  M concentrations of each deoxy- nucleoside triphosphate, 250 nM concentrations of each primer, 1.5 mM MgCl 2 , 2.6 U of Expand high-fidelity PCR System (Roche, Switzerland), and 5 ng of total genomic DNA. After enzyme activation at 94°C for 5 min, the reactions were subjected to 35 thermal cycles of 94 and 54°C for 60 s for each temperature, 72°C for 4 min, and a final elongation at 72°C for 10 min. Different regions of 12S, RPB2 , and TEF1  were amplified and se- quenced under the conditions and with the primers described by Zoller et al. (18), Matheny et al. (19), and Morehouse et al. (20), respectively. Se- quencing was performed using reagents and conditions of the BigDye Terminator v1.1 cycle sequencing kit and on an ABI Prism 3130 XL DNA analyzer (Perkin-Elmer/Applied Biosystems, Foster City, CA). Final se- quences were assembled and edited using the software package Se- quencher 3.0 (Gene Codes Corp., USA). The GenBank accession numbers for ribosomal rRNA, mitSSU, and TEF1 are reported in Table S1 in the supplemental material. The GenBank accession number for 2118, C. dubliniensis ATCC 2119, C. dubliniensis CBS 7987, C. glabrata ATCC 90030, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, C. parap- silosis ATCC 90018, C. tropicalis ATCC 750, and Cryptococcus neoformans ATCC 90112) as a validation method. Each sample was tested in duplicate to ensure reproducibility of the spectra. A characterization score cutoff value was attributed to each sample and was interpreted as recommended by the manufacturer (scores of  1.7 indicate unreliable identification, scores from 1.7 to 1.999 indicate identification to the genus level; scores of 2 to 2.299 indicate probable species identification, and scores of  2.3 indicate highly probable species identification). Susceptibility testing. MIC values were determined by broth microdi- lution using the reference procedure of the Antifungal Susceptibility Test- ing Subcommittee of the European Committee for Antimicrobial Suscep- tibility Testing (EUCAST) for the testing of fermentative yeasts ( 28) with slight modification (5). The strains were cultivated overnight in YEPD at 30°C. Tests were performed in flat-bottom microdilution plates with RPMI 1640 medium supplemented with 2% of glucose, glutamine, and phenol red as pH indicator, but without bicarbonate. The inoculum sizes were adjusted to 0.5  10 5 to 2.5  10 5 CFU/ml by measuring the absor - bance in a spectrophotometer. Drug dilutions were made for each in the corresponding solvents with concentration ranges for fluconazole (128 to 0.0625 mg/liter) and for itraconazole, voriconazole, posaconazole, am- photericin B, and caspofungin (16 to 0.0078 mg/liter). C. albicans ATCC 90028, C. glabrata ATCC 90030, and C. tropicalis ATCC 750 were included as control isolates. MIC values were determined with a spectrophotome- ter (at 530 nm) after 24 h of incubation as the lowest concentration of drug resulting in  50% (MIC 50 ) and  90% (MIC 90 ) growth inhibition, re - spectively. Flow cytometry. The estimation of the nuclear DNA content in yeast was achieved by flow cytometry with propidium iodide as fluorochrome staining. Yeast cells of three strains ( C. albicans SC5314, C. glabrata CBS 138, and JEY63) were grown in YEPD broth overnight to the stationary phase of growth. Then, 10-ml samples of each culture were harvested, and the cells were fixed in 70% ethanol for 12 h at 4°C. The samples were washed once with 5 ml of 50 mM sodium citrate (pH 7.5) and resus- pended in 1 ml of 50 mM sodium citrate. The cell concentration was diluted to 1.5  10 7 cells/ml. To each sample, 25  l of RNase A (10 mg/ml) was added. After1hofincubation at 50°C, 100  l of proteinase K (10 mg/ml) was added. The incubation was continued for an additional hour at 50°C, after 1 ml of 50 mM sodium citrate containing 16  g of pro- pidium iodide/ml was added. The samples were then incubated overnight BLAST of ribosomal data. In order to determine the species of the 10 remaining isolates, we undertook the sequencing of their D1/D2 and ITS1/ITS2 domains from the large subunit rDNA. This method resulted in the presumptive identification of 9 isolates to the following species ( Table 2): Hanseniaspora opuntiae (3 isolates) Candida palmioleophila (2 isolates), Kodamea ohmeri (2 isolates), Debaryomyces hansenii (1 isolate), and Pichia caribbica (1 isolate). Only strain JEY63, for which the MALDI-TOF spec- trum was unique (see Fig. S1 in the supplemental material) and not present in the MALDI-TOF instrument database, remained unknown after these steps, even including ITS1 or ITS2, which are considered the best loci for fungal barcodes (38). The GenBank accession numbers of JEY63 ribosomal se- quences (18S, ITS1-5.8S-ITS2, and 26S) are reported in Table S1 in the supplemental material. The BLAST top scores of the 18S and 26S ribosomal regions were the most similar to members of the Saccharomycetes ( Ascomycota ) but with BLAST top scores that were too low to confirm JEY63’s affiliation to this fungal class TABLE 2 Species assignment of isolates not identified by MALDI-TOF MS analysis Collection Pairwise identity (%) of regions to existing database Species assignment D1/D2 a region ITS1/ITS2 region JEY63 86.9 70.0 JEY182 100 98.7 Kodamaea ohmeri JEY234 100 99.7 Kodamaea ohmeri JEY258 100 99.5 Hanseniaspora opuntiae JEY267 100 99.8 Pichia caribbica JEY269 100 99.5 Hanseniaspora opuntiae JEY270 100 99.8 Hanseniaspora opuntiae JEY379 100 99.8 Candida palmioleophila JEY380 100 99.8 Candida palmioleophila JEY420 100 99.8 Debaryomyces hansenii a That is, domains D1 and D2 of the large subunit rDNA (LSU rDNA). FIG 1 Germ tube formation by C. albicans SC5314 and JEY63 after incubation of 10 6 cells/ml at 35°C in 50% calf serum. (A and B) C. albicans SC 5314 incubated for 1.5 and 3 h, respectively; (C and D): JEY63 incubated for 1.5 and 3 h, respectively. Bar, 10  m. Eddouzi et al. 34 Journal of Clinical Microbiology on March 27, 2019 by guest (BLAST top scores for 18S: Blastobotrys adeninivorans and several other Candida species, 95% sequence similarity; BLAST top scores for 26S: Sugiyamaella smithiae , Nakazawaea holstii , Spen- cermartinsiella europaea , Pichia xylosa , Scheffersomyces stipitis , and several other Candida species, 91% sequence similarity). Clinical data from strain JEY63. JEY63 was isolated from a 50-year-old man suffering from a pemphigus vulgaris disease. He was admitted to the University hospital of Habib Thameur in Tunis between February and May 2009. The diagnosis of pemphi- gus vulgaris was confirmed by histology and direct immunofluo- rescence. The patient showed extended mucocutaneous vesicles with clear or blood-stained contents. The treatment was con- trolled by corticosteroids (1.5 mg/kg/day). In March 2010, the patient was again admitted, and he was marked by the recurrence of extensive bullo-erosive lesions with involvement of the genital area. The second period of hospitalization lasted 4 months (11 March to 7 July 2010). During this period, three oral swabs were obtained from the patient each month. In these specimens, the same type of yeast was isolated, and JEY63 was the last sample. The patient also developed bacterial infections ( Staphylococcus spp.). Therefore, he often received antibacterial agents such as bristo- pen, ciprofloxacin, and fucidin. During this period, no docu- mented antifungal therapy was given. Phenotypic and physiological characteristics. Microscopic observations showed the formation of germ tubes in C. albicans SC5314 in calf serum at 35°C, while only pseudohyphae were de- tected in JEY63 (Fig. 1A and C). When the incubation was contin- ued for 3 h, the extension of gem tubes slowly elongated in C. albicans SC5314, but the dominant morphology was the develop- ment of pseudohyphae in JEY63 ( Fig. 1B and D). The new strain did also produce pseudohyphae after overnight incubation in YEPD medium at 30°C under constant shaking ( Fig. 2). Chlamy- dospore formation on rice extract agar plate was observed as ex- pected in C. albicans SC5314; however, only pseudohyphae were observed in JEY63 (Fig. 3). The results from physiological charac- terization of JEY63 are shown in Table S2 in the supplemental material. According to the metabolic database available at the CBS Fungal Biodiversity Centre, the profile does not correspond to any deposited fungal isolate. One striking feature of this yeast species is its ability to grow at high temperature (42°C). It is also positive for glucose fermentation but does not exhibit a high metabolic capac- ity in metabolizing diverse sugars. Phylogenetic analyses. The GenBank accession numbers of JEY63 sequences (mitSSU and TEF1 ) are reported in Table S1 in the supplemental material. Since we were not able to determine the placement of JEY63 in the classification of Ascomycota using classical methods, including MALDI-TOF or BLAST, we first in- ferred the phylogenetic placement of JEY63 within the Ascomy- cota , adding this taxon to the alignment of Schoch et al. (21) and running MP analyses. This 6-locus/435-taxon alignment included 26,665 characters. After the exclusion of ambiguously aligned re- gions, 2,811 of the 5,128 characters that were kept for analyses were parsimony informative. Phylogenetic analyses of the 6-locus/ 435-taxon data set suggested with significant support (MPbs  74%) that JEY63 was nested within the Saccharomycetes and re- solved as a sister to Yarrowia lipolytica , though without support FIG 2 Morphology of yeast and pseudohyphal forms of JEY63 after overnight incubation in YEPD medium at 30°C with constant shaking. Bar, 10

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