Three-Locus Identification, Genotyping and Antifungal Susceptibilities of Medically Important ...

27 Pages·2011·0.44 MB·English

JCM Accepts, published online ahead of print on 7 September 2011
J. Clin. Microbiol. doi:10.1128/JCM.00937-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Three-Locus Identification, Genotyping and Antifungal Susceptibilities of Medically
2 Important Trichosporon Species from China
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4 Li-Na Guo,1† Meng Xiao,1† Fanrong Kong,2† Sharon C-A. Chen,2 He Wang,1 Tania C. Sorrell,2
5 Wei Jiang,4 Hong-Tao Dou,1 Ruo-Yu Li,3 and Ying-Chun Xu1*
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6 †equal contribution to the work and are listed as joint first authors. w
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8 1Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy d

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9 of Medical Sciences, Beijing 100730, P. R. China m

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10 2Centre for Infectious Diseases and Microbiology, the University of Sydney, Westmead Hospital, ttp
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11 Westmead, NSW 2145, Australia jc
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12 3Peking University First Hospital, Research Center for Medical Mycology, Peking University, as
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13 Beijing 100034, P. R. China o
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14 4Department of Clinical Laboratory, First Affiliated Hospital of Chinese People's Liberation o
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15 Army General Hospital, Beijing 100048, China p
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17 *Correspondence to: 0
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18 Professor Ying-Chun Xu b
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19 Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy u
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20 of Medical Sciences, Beijing 100730, P. R. China. t
21 Phone: [phone]; Fax: [phone]
22 E-mail: [email protected]
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1 Key words: Trichosporon, species identification, genotypes, phylogenetic analysis, antifungal
2 susceptibility
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1 ABSTRACT
2 Three reference and 45 clinical isolates of Trichosporon were analysed by conventional
3 phenotypic and molecular methods to determine the species and genotypes of Trichosporon
4 isolates from China. Target loci for molecular methods included the internal transcribed spacer
5 (ITS) region, D1/D2 domain of the 26S rRNA gene and intergenic spacer 1 (IGS1) region.
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6 Identification of eight Trichosporon species was achieved of which T. asahii was the most w
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7 common. Of sequence-based molecular methods, the D1/D2 domain assigned 97.9% (47/48) of a
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8 isolates (seven species) correctly, whilst both ITS and IGS1 regions correctly identified all 48 d

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9 isolates. The commercial API 20C AUX and Vitek 2 Compact YST systems, respectively, m

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10 correctly identified 91.9% and 73% of isolates against species contained in the databases, and tp
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11 misidentified 63.6% and 36.4% isolates, respectively, of species that were unclaimed by the jc
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12 databases. The predominant genotype amongst T. asahii clinical isolates, genotype 4 (51.4%), is s
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13 rarely found in other countries. Voriconazole and itraconazole were the most active drugs in o
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14 vitro against all the Trichosporon species tested, whilst caspofungin and amphotericin B o
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15 demonstrated poor activity. p
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1 INTRODUCTION
2 Trichosporon species are emerging fungal pathogens capable of causing localized or systemic
3 mycoses. Disseminated infection typically occurs in patients with underlying hematological
4 malignancy, organ transplantation and those otherwise heavily immunocompromised (5, 9, 18).
5 Such infection is often lethal (mortality rates of 42-80%) (4, 13, 27). Furthermore, morbidity is
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6 significant with multiple sequelae including respiratory and renal failure, and disseminated w
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7 intravascular coagulation (5, 9, 30, 35). a
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8 With the advent of modern molecular identification techniques, the nomenclature within the d

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9 genus Trichosporon has been substantially revised. Whilst the major species previously m

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10 encompassed Trichosporon beigelii (10, 32, 33), today, at least 13 Trichosporon species have tp
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11 been reported as human pathogens. These include T. asahii, T. asteroides, T. coremiiforme, T. jc
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12 cutaneum, T. dermatis, T. domesticum, T. faecale, T. inkin, T. japonicum, T. jirovecii, T. loubieri, s
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13 T. montevideense, and T. mucoides (5, 25). Yet the identification of Trichosporon and o
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14 closely-related yeasts to species level still largely relies on phenotypic-based methods which are o
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15 insensitive and time consuming (20). Further, none of the available commercial phenotypic p
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16 identification systems include all new taxonomic categories, or species, in their databases. 11
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17 Specifically, the API 20C AUX (bioMérieux, Marcy l’Etoile, France) as well as the Vitek 2 0
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18 Compact YST (bioMérieux) systems are only able to identify three Trichosporon species: T. b
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19 asahii, T. inkin and T. mucoides, which may lead to erroneous species assignment (6, 11, 17, 23, u
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20 24). Species identification is important for epidemiological purposes and to better define t
21 species-specific clinical associations (5, 11). In addition, certain Trichosporon species may be
22 more resistant to antifungal drugs (2, 5, 25).
23 To enable accurate species identification, a number of molecular methods have been
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1 developed of which DNA sequencing of the internal transcribed spacer (ITS) region, D1/D2
2 domain of the 26S subunit of the rRNA gene region, and of the intergenic spacer 1 (IGS1) region
3 are the most frequently used (5, 7, 12, 19, 25, 26, 28-31). The IGS1 gene region has been
4 particularly useful in phylogenetic studies and in delineating intraspecies variation. These data
5 are fundamental to better understanding both species distribution and genotype differences that
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6 have been reported according to geographic region, such as that observed with T. asahii (5, 12, w
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7 19, 26, 28, 29). a
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8 In contrast to data from South and North America and Japan, studies examining the species d

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9 distribution, phylogenetic diversity and antifungal susceptibilities of Trichosporon pathogens in m

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10 China are few. In the present study, we studied a collection of 45 clinical Trichosporon isolates tp
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11 obtained from three hospitals in China along with three well-characterized reference strains. jc
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12 Species identification of all isolates was undertaken by sequence analysis of the ITS region, s
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13 D1/D2 domain and IGS1 region and compared with that obtained by the API 20C AUX and o
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14 Vitek 2 Compact YST methods. The antifungal susceptibility testing of Trichosopron isolates to o
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15 five antifungal agents was also performed. p
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16 11
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17 MATERIALS AND METHODS 0
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18 Trichosporon strains and DNA extraction. b
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19 Forty-eight Trichosporon isolates were studied including (i) three reference strains namely T. u
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20 asahii CBS 2479 (PUMCH strain ID no. PUMCHBY15), T. cutaneum ATCC 28592 (PUMCH t
21 strain ID no. PUMCHBY28) and T. laibachii CGMCC 2.1963 (PUMCH strain ID no.
22 PUMCHMC31) (see Table 1), and (ii) 45 clinical isolates collected from three hospitals (Peking
23 Union Medical College Hospital, Peking University First Hospital and the First Affiliated
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1 Hospital of Chinese People's Liberation Army General Hospital) in Beijing, China. Of the 45
2 clinical isolates, 22 strains were isolated from sputum, 12 from tissue or body fluid, three from
3 urine, two isolates from blood and six from unknown clinical sources (Table 1). All isolates were
4 initially identified by the API 20C AUX and Vitek 2 Compact YST according to the
5 manufacturer’s instructions (Table 1), and then subjected to DNA sequencing (see below). DNA
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6 was extracted from pure cultures of each of the isolates as described previously (16) and stored at w
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7 -20oC before use. loa
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9 PCR and DNA sequencing. m

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10 Amplification of the ITS region, D1/D2 domain and IGS1 region were performed as previously tp
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11 described with the primer pairs ITS1/ITS4, F63/R635 and 26SF/5SR, respectively (7, 29). A jc
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12 total of 8 µl of amplified PCR products were visualized by 2% agarose gel after staining with s
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13 SYBR safe DNA gel stain (Invitrogen, Mt. Waverley, VIC, Australia). The PCR products were o
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14 then sequenced by TSINGKE Co. Ltd. (Beijing, P. R. China) with the DNA analyser ABI o
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15 3730XL system (Applied Biosystems, Foster City, CA) in both directions using the PCR p
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16 amplification primers. 11
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18 Sequencing-based species identification and phylogenetic analysis. b
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19 All DNA sequence chromatograms were checked manually to ensure high quality sequences. For u
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20 species identification, sequences of the ITS region, D1/D2 domain and IGS1 region obtained in t
21 this study were queried against sequences of type strains for all Trichosporon species in the
22 Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center database using
23 pairwise sequence alignment with the BioloMICSNet software
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1 (http://www.cbs.knaw.nl/collections/BioloMICSSequences.aspx). The sequences of each target
2 gene region and concatenated sequences of the ITS region and D1/D2 domain were then aligned
3 using Clustal W software (36), and adjusted manually to form the consensus sequences for all 48
4 isolates. Phylogenetic trees were computed with MEGA version 4 (Molecular Evolutionary
5 Genetic Analysis software, v4.0.2; http://www.megasoftware.net) using maximum-parsimony
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6 analysis (34), with all positions containing gaps and missing data eliminated from the data set. w
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7 ITS region, D1/D2 domain and IGS1 region sequences from whole-genome sequence of a
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8 Cryptococcus neoformans var. neoformans strain B-3501A (15) were used as outgroups to d

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9 generate the maximum-parsimony trees. The IGS1 region was further targeted to determine m

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10 genotypes of T. asahii isolates as described previously (26, 28, 29). tp
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11 jc
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12 Antifungal susceptibility testing. s
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13 Antifungal susceptibility testing was performed according to the Clinical and Laboratory o
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14 Standards Institute (CLSI) document M27-A3 and M27-S3 broth microdilution method (21, 22). o
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15 The following antifungal drugs were tested at the indicated concentration ranges: AMB, 0.032 to p
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16 16 µg/ml (North China Pharmaceutical Group Corporation, Shijiazhuang, China); caspofungin 11
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17 (CAS), 0.032 to 16 µg/ml (Merck Research Laboratories, Rahway, New Jersey, USA); 0
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18 fluconazole (FLC), 0.125 to 64 µg/ml (Pfizer Incorporated, New York, USA); itraconazole b
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19 (ITC), 0.032 to 16 µg/ml (National Institute for Food and Drug Control, Beijing, China); and u
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20 voriconazole (VRC), 0.032 to 16 µg/ml (Pfizer Incorporated). The microdilution plates were t
21 incubated at 35°C except for T. cutaneum ATCC 28592 (strain PUMCHBY28) where plates
22 were incubated at 28oC as described previously (14). All plates were observed for visible growth
23 at 48 h. Microdilution wells were scored with the aid of a reading mirror and the growth in each
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1 well compared with that of the growth control (drug-free) well (21). Candida parapsilosis ATCC
2 22019 and Candida krusei ATCC 6258 were used as the quality control strains.
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4 Nucleotide sequence accession numbers.
5 New data for sequences within the ITS region, D1/D2 domain and IGS1 region generated in this
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6 study have been deposited in the GenBank database with accession numbers w
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7 EU863532-EU863579 (for ITS region), EU882088-EU882103 (for D1/D2 domain) and a
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8 JF302977-JF303024 (for IGS1 region). d

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9 m

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10 RESULTS tp
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11 Sequencing-based identification of Trichosporon species. jc
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12 All 48 Trichosporon isolates were identified by querying their unknown ITS, D1/D2 and IGS1 s
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13 sequences against sequences of Trichosporon type strains in the CBS Fungal Biodiversity Center o
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14 database, and results were summarized in Supplementary Table S1. o
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15 The three loci correctly identified the reference strains to species level (Table 1, p
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16 Supplementary Table S1). Amongst 45 clinical isolates, six species were identified. 11
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17 Trichosporon asahii was the most common species (35 isolates, 77.8%), followed by T. 0
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18 domesticum (four isolates, 8.9%), T. japonicum and T. jirovecii (two isolates each, 4.4%), T. b
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19 dermatis and T. inkin (one isolate each, 2.2%) (Table 1, Supplementary Table S1). Sequencing of u
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20 both the ITS and IGS1 regions successfully identified all 45 clinical isolates. D1/D2 domain t
21 sequences assigned 44 of 45 (97.8%) strains to species level correctly, with the exception of
22 strain PUMCHBY24, which was identified as T. mucoides but as T. dermatis by ITS and IGS1
23 sequencing; T. dermatis was assigned as the correct species for this strain (Supplementary Table
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1 S1).
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3 Comparison of phenotypic-based and molecular identification.
4 Identification results of 48 Trichosporon isolates tested with the Vitek 2 Compact YST and API
5 20C AUX are summarized in Tables 1 and 2. As the databases of both the API 20C AUX and
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6 Vitek 2 Compact YST systems only contain three Trichosporon species (T. asahii, T. inkin and T. w
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7 mucoides), isolates in the present study were further subgrouped as ‘species claimed by a
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8 databases’ (37 isolates, two species) and ‘species unclaimed by databases’ (11 isolates, six d

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9 species) for analysis (Table 2). m

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10 Compared with results obtained by our three-locus sequencing scheme, the API 20C AUX tp
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11 system correctly identified 34 of 37 (91.9%) isolates amongst species claimed by databases, with jc
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12 the remaining three isolates unidentified (with their biochemical profiles yielding either ‘low s
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13 discrimination’ or ‘no identification’) and no isolates misidentified; while amongst species o
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14 unclaimed by databases, four of 11 isolates were correctly shown as unidentified but seven were o
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15 misidentified (Tables 1 and 2). The ability of the Vitek 2 Compact YST system to identify p
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16 Trichosporon spp. amongst species claimed by databases was even lower - only 27 T. asahii 11
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17 isolates (75.0% of 36 T. asahii isolates studied) were correctly identified; however, amongst 0
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18 species unclaimed by databases, seven of 11 isolates were correctly shown as unidentified but b
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19 four isolates were misidentified (Tables 1 and 2). u
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21 Phylogenetic analysis of IGS1 region and genotyping.
22 Phylogenetic analysis was performed based on the results of sequence analysis of the ITS region
23 (595-bp), D1/D2 domain (663-bp) and IGS1 region (1,110-bp) as well as using concatenated
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1 sequences of the ITS region and D1/D2 domain (Table 3). Based on parsimony informative
2 characters (PIC) per bp (Table 3), the IGS1 region was more phylogenetically informative than
3 either the ITS region or D1/D2 domain. Maximum parsimony trees (MPT) were generated for
4 the IGS 1 region (Figure 1, left MPT) and ITS region plus D1/D2 domain (Figure 1, right MPT)
5 rooted by the outgroup C. neoformans var. neoformans strain B-3501A.
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6 Amongst the 48 isolates, the IGS1 region recognized 13 sequence types/genotypes (Table 3). w
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7 Genotypes of T. asahii isolates were assigned as described by Sugita et al. and Rodriguez-Tudela a
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8 et al. (26, 28). Trichosporon asahii reference strain PUMCHBY15/CBS 2479 was correctly d

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9 classified as genotype 1 (5). The remaining 35 clinical T. asahii isolates belonged to four m

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10 genotypes, namely genotype 1 (9 isolates, 25.7%), genotype 3 (7 isolates, 20.0%), genotype 4 tp
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11 (18 isolates, 51.4%) and genotype 6 (1 isolate, 2.9%) (Table 1 and Figure 1). Four T. domesticum jc
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12 isolates studied had a single IGS1 sequence type, while two sequence types were found for both s
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13 T. japonicum and two T. jirovecii strains (Table 1, Supplementary Table S1). o
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14 o
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15 Antifungal susceptibility testing. p
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16 The susceptibilities of 48 isolates representing eight species (based on the molecular 11
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17 identification result) to five antifungal agents are shown in Table 1. MIC ranges (and geometric 0
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18 mean [GM]) MICs were 0.125 to 32 µg/ml (1.09 µg/ml) for AMB, 1 to 16 µg/ml (9.15 µg/ml) for b
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19 CAS, 0.125 to 8 µg/ml (3.97 µg/ml) for FLC, 0.125 to 2 µg/ml (0.66 µg/ml) for ITC and 0.032 to u
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20 1 µg/ml (0.12 µg/ml) for VRC (Table 1). CAS MICs for all Trichosporon isolates tested were t
21 consistently high (>= 2 µg/ml for all strains tested except one T. asahii isolate CAS MIC = 1
22 µg/ml). Two T. japonicum isolates exhibited AMB MICs of >= 4 µg/ml, whilst all T. asahii
23 isolates had AMB MICs of <=1 µg/ml (GM MIC 0.91 µg/ml). With regard to the azoles, FLC
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