Phylogenetic relationships matter - antifungal susceptibility among clinically relevant yeasts ...

43 Pages·2013·0.93 MB·English

AAC Accepts, published online ahead of print on 23 December 2013
Antimicrob. Agents Chemother. doi:10.1128/AAC.01799-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 Title: Phylogenetic relationships matter - antifungal susceptibility among clinically
2 relevant yeasts
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4 Running title: Azole-susceptibility according to phylogeny
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6 A.F. Schmalreck1, M. Lackner2,#, K. Becker3, W. Fegeler3, V. Czaika4, H. Ulmer5, C. w
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7 Lass-Flörl2 loa
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9 1 MBS, Microbiology, Sauerbruchstraße 10, 81377 Munich, Germany m

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10 2Division of Hygiene and Medical Microbiology, Innsbruck Medical University, 6020 tt
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11 Innsbruck, Austria a
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12 3 Institute of Medical Microbiology, University Hospital, Domagkstraße 10, 48149 as
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13 Münster, Germany o
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14 4 Department of Dermatology, Medical University Charité, Hindenburgdamm 30, 12203 o
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15 Berlin, Germany p
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16 5 Department of Medical Statistics, Informatics, and Health Economy, Innsbruck Medical il 1
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17 University, Austria. 0
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19 # Corresponding author: y
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20 Mag. Dr. rer. nat. Michaela Lackner e
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21 Schöpfstrasse 41/II, 6020 Innsbruck, Austria. Phone: [phone]. Fax: + 43
22 512 9003 73700. Email: [email protected]
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24 Key words: taxonomy, ascomycetes, basidiomycetes, nomenclature, azole,
25 susceptibility
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27 Abstract
28 The objective of this study was twofold: to evaluate whether phylogenetically closely
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29 related yeasts share common antifungal susceptibility profiles (ASP), and whether these w
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30 ASP can be predicted from phylogeny. To address this question, 9,627 yeast strains a
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31 were collected and tested for their antifungal susceptibility. Isolates were re-identified d

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32 considering recent changes in taxonomy and nomenclature. A phylogenetic code m

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33 (PHYLO-code) based on multilocus sequence analyses (large subunit rRNA, small tt
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34 subunit rRNA, translation elongation factor-1α, RNA polymerase II subunit 1 and 2), and a
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35 the classification of cellular neutral sugar composition of the coenzyme Q and 18S rDNA as
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36 was created to group related yeasts into PHYLO-groups. ASP per PHYLO-group was o
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37 performed for fluconazole, itraconazole, and voriconazole. The majority (95%) of yeast o
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38 strains were ascomycetes. After re-classification a total of 23 genera and 54 species p
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39 were identified, resulting in an increase of 64% of genera and decrease of 5% of species 1
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40 compared with initial identification. These taxa were assigned to 17 distinct PHYLO- 0
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41 groups (Ascomycota, n=13; Basidiomycota, n=4). ASP for azoles was similar among b
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42 members of the same, and different between the various PHYLO-groups. u
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43 Yeast phylogeny may be an additional tool to significantly enhance the assessment of t
44 MIC values and to predict antifungal susceptibility, thereby more rapidly initiating
45 appropriate patient management.
46
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47 Introduction
48 The taxonomy of fungi has been under significant discussion over the last years, in
49 particular due to the molecular/sequence data that have become available. Fungi are
50 classified primarily based on the structures associated with sexual reproduction, which
51 tend to be evolutionarily conserved. However, many fungi reproduce only asexually, and
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52 cannot be assigned to a classification based on sexual characters; some produce both w
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53 asexual (anamorphs/synanamorphs) and sexual (teleomorph) states. These problematic a
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54 species are often members of the Ascomycota, but may also belong to the d

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55 Basidiomycota. Even among fungi that reproduce both sexually and asexually, often m

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56 only one method of reproduction can be observed at a specific point in time or under tt
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57 specific conditions (1-6). a
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58 The term “holomorph” has been assigned to fungi with teleomorphic sporulation together as
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59 with all their sporulating or vegetative anamorphs. This dual naming system can be o
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60 confusing. o
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61 Former binomial nomenclature was according to the International Code of Botanical p
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62 Nomenclature (ICBN) - current is International Code of Nomenclature for Algae, Fungi 1
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63 and Plants (ICNAFP). (7-8). Since 1st January 2013, only one name is allowed to be 20
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64 assigned to all newly described fungi, and the system permitting to use of separate b
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65 names for anamorphs and teleomorphs was repealed (6). In practice, the terms u
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66 anamorph and teleomorph shall be dropped in favour of the terms asexual and sexual t
67 stage, and all legitimate names proposed for a species, regardless of which reproduction
68 stage they are typified by, can serve as the correct name for that species (6, 9, 10). The
69 usage of molecular tools has led to recognition of many novel polymorphic and cryptic
70 species (two or more distinct species classified as a single) and species complexes, e.g.
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71 in C. parapsilosis, C. guilliermondii, C. glabrata, and Aspergillus niger (11-18). To avoid
72 uncertainties caused by frequent renaming and reassigning of human-pathogenic fungi,
73 a plea was published by de Hoog et al. (19) that aim to uncouple molecular phylogeny
74 from nomenclature. The authors state that taxonomic novelties should only be
75 transferred into nomenclatural changes when there is clinical relevance (e.g., different
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76 antifungal susceptibility patterns and/or virulence) is connected to it. w
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77 New molecular genetic identification techniques and the increased number of a
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78 immunocompromised and severe ill patients lead to the emergence of uncommon, rare, d

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79 and novel species of opportunistic human and animal pathogens (20-21). The mortality m

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80 rate due to yeast infections is still high (22) and may therefore reinforce the need for tt
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81 antifungal susceptibility testing in order to guide antifungal treatment. However a
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82 antifungal susceptibility testing takes time (>3 days), needs the fungus to be cultured, as
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83 implies technical experience and is not easily available in routine laboratories. In such o
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84 case, allocation to PHYLO-groups may provide sufficient information for appropriate o
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85 selection of antifungal drugs. p
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86 The goal of this study was to evaluate whether phylogenetically closely related yeasts 1
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87 share common antifungal susceptibility patterns and whether sensitivity of yeast 0
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88 pathogens to antifungals can be predicted from phylogeny. Thus, a large collection of b
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89 clinically relevant yeasts from both the Ascomycota and Basidiomycota, which had u
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90 previously been collected in the framework of German/Austrian multicentre studies from t
91 1998 to 2009, were re-examined. All strains investigated were studied for their
92 phylogentic relationship, were reassigned to valid species names according to recent
93 taxonomic changes (23-48), and were evaluated for their antifungal susceptibility
94 profiles.
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96 Material and Methods
97 Yeast strains and antifungal susceptibility testing. A total of 15,606 clinical yeast
98 isolates (Table 1) were collected by 39 participating study centres, running four
99 multicenter studies between November 1997 and January 2009. The participating
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100 centres were distributed over throughout Germany and Austria. To avoid the bias of w
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101 redundant data, multiple isolates obtained from an individual patient were eliminated. a
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102 The final collection re-analysed included 9,627 strains that were tested against the d

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103 antifungal agents fluconazole (FLC), itraconazole (ITC), and voriconazole (VRC) using a m

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104 microdilution assay (DIN 58940) (49-50). This method provides reproducible, tt
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105 respectively similar results to EUCAST procedure (50-53). DIN-MIC distributions were in a
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106 agreement with EUCAST MICs and data obtained were applied for EUCAST breakpoint as
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107 setting (54). In addition, control limits of the MIC target values of control strain o
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108 C. parapsilosis ATCC 22019 (American Type Culture Collection) are similar for FLC, o
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109 ITC, and VRC for DIN and EUCAST (50, 55). Also, C. krusei (CL-CNM CL 3403) and C. p
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110 albicans (CL-CNM F 8555), both EUCAST quality control strains displayed similar MIC 1
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111 ranges according to DIN (50, 52). In 2002, DIN standards were replaced by EUCAST, 0
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112 the major difference is based on the fact that DIN methodology recommended the usage b
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113 of HR-medium instead of RPMI 1640. At study time, the individual test centres applying u
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114 routine standard laboratory procedures performed initial species-identification and t
115 species-confirmation was performed using Fourier-Transform Infrared-Spectroscopy
116 (FT-IR). FT-IR was reported to be a rapid, reliable and cost-saving tool to identify and
117 differentiate bacteria, dermatophytes and yeasts (56-64). For FT-IR based identification
118 a IFS 28/B spectrophotometer (Bruker GmbH, Germany) was used; resulting spectra
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119 were transformed and evaluated using opus software from Bruker GmbH, as previously
120 described by Tietz et al. (60). As reference served a comprehensive, validated in-house
121 database consisting of at least one reference strain per yeast species (including also all
122 known rare clinical relevant yeasts) gained form international collections, as follows:
123 American Type Culture Collection (ATCC; USA; n =31), Collection of Biomedical Fungi
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124 and Yeasts (BCCM/MUCL; Belgium; n = 64), Collection of (Agro) Industrial Fungi and w
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125 Yeasts (BCCM/MUCL; Belgium; n = 49), Centraal Bureau voor Schimmelcultures (CBS; a
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126 The Netherlands; n = 12), German Collection of Microorgansisms and Cell Cultures d

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127 (DSMZ; Germany; n = 19), National Collection of Pathogenic Fungi (NCPF; UK; n = 18), m

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128 and National Collection of Type Cultures (NCTC; UK; n = 9). In addition, the database tt
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129 contains spectra of 3,264 clinical yeasts. Additionally, difficult to differentiate isolates a
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130 were identified by an in-house, pan-fungal PCR by a qualified reference laboratory as
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131 selected by the multicentre study coordinators. Internal transcribed spacer region (ITS) o
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132 was amplified using the primers ITS 1 (5’-TCC GTA GGT GAA CCT GCG G) and ITS 2 o
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133 (5’-GCTGCG TTC TTC ATC GAT GC) (Metabion, Germany) as previously described by p
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134 White et al. (65). DNA was extracted from pure cultures using DNeasy Plant Mini Kit 1
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135 (Qiagen) according to the manufactures’ instructions. PCR was performed on a 0
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136 TProfessioal standard thermoycyler (Biometra), as previously described by (66). PCR b
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137 products were purified using ExoSAP-IT (Affymetrix) enzymatic clean up according to u
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138 manufactors’ instructions. For DNA sequencing ITS 1 primer and the BigDye Terminator t
139 (version 3.1) cycle sequencing kit in the ABI PRISM 3100 genetic analyzer (Applied
140 Biosystems). ITS sequences were identified using NCBI BLAST® sequence analyses
141 search function (http://blast.ncbi.nlm.nih.gov/Blast). Together with their origin
142 (clinic/ward), the type of specimens investigated is given in Table S1.
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143 State of the art taxonomy. All fungi were re-evaluated according to the “one fungus,
144 one name” principle, and new current valid species names are derived from Index
145 Fungorum (www.species fungorum.org), Mycobank (www.mycobank.org), and from the
146 database of the Fungal Biodiversity Centre (www.cbs.knaw.nl).
147 PHYLO-code and PHYLO-group. An easy computable artificial phylogenetic code
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148 (PHYLO-code) was developed to group all yeast isolates according to their elsewhere w
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149 described phylogenetic relationship. The PHYLO-code of ascomycetous yeasts takes a
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150 into account: a) the 12 different clusters of Saccharomycetales obtained by multilocus d

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151 sequence analyses of the genes large subunit rRNA [LSU], small subunit rRNA [SSU], m

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152 translation elongation factor-1α [EF-1α], RNA polymerase II subunit 1 and 2 [RPB1 and tt
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153 RPB2] by Kurtzman et al. (23,24) and Suh et al. (25), b) the ubiquinone classification a
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154 system which consider the cellular neutral sugar composition of the coenzyme Q (46, as
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155 47), c)18S rDNA analysis resulting in six clades (A-F) as described by Tsui et al. (31), .o
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156 and based on nuclear sequences of the largest subunit and second largest subunit of o
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157 the RNA polymerase II gene, actin, the second subunit of the mitochondrial cytochrome p
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158 oxidase gene and the D1/D2 LSU rRNA gene. 1
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159 Thus, the basis for the PHYLO-code results from the presence of the main clusters of 0
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160 Kurtzman et al. (23) (A1-A9), the coenzyme-Q (Q6-Q9), and the clades (A-F) given by b
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161 Tsui et al. (31). As Exophiala dermatitidis strains belong to the ascomycetous yeast of u
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162 the order Chaetothyriales - family Herpotrichiellaceae, and therefore are not covered by t
163 the PHYLO-codes A1-A9, the artificial PHYLO-code AH standing for Ascomycota and
164 Herpotrichiellaceae was assigned (Table 1 and Figure 1). In addition, for further
165 differentiation between galactose-containing and galactose-deficient species (59) the
166 PHYLO-group A9Q9G had to be introduced.
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167 The PHYLO-code of basidiomycetous yeasts was based on the ribosomal DNA
168 sequence data (18 S rDNA, SSU, D1/D2 of the LSU, internal transcribed spacer region
169 [ITS]) described by Haase et al. (67), Suh and Sugiyama (68), and Scorzetti et al. (69). It
170 has been designated as B1-B4, embedding the same ubiquinone (Co-Q) system (46-47)
171 as used for the ascomycetous yeasts (Q8-Q10). The list with the current valid species
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172 names and their assigned PHYLO-codes is provided in Table 1 and Figure 1. w
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174 Results d

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175 Species classification. Aside from the study centre differentiations, identification of all m

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176 yeasts by FT-IR was made in one study centre where this established method was tt
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177 performed routinely (70-71), relying on a data base of almost 5000 yeast species, a
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178 including 495 strains from ATCC (American Type Culture Collection), DSM (Deutsche as
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179 Sammlung für Mikroorganismen = German collection of microorganisms), NCTC (British o
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180 National Collection of Type Cultures), and BCCM (Belgian Culture Collection of o
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181 Microorganisms). The initial identification (by study centres) displayed 14 different p
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182 genera with 56 different species, from the latter, 31 species had been reported as 1
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183 different Candida varieties. The application of the “one fungus one name” principle and 0
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184 the reassignment of the new nomenclature resulted in an increase of 23 genera (64%) b
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185 and in a reduction to 54 species (5%). Overall, 9110 (95%) fungi were categorized as u
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186 ascomycetous yeasts and 517 (5%) as basidiomycetous yeasts. According to the t
187 reassessment, only 16 from the initial 31 Candida species could be truly assigned to the
188 genus Candida (decrease of 48%), while the other 15 previously reported species had to
189 be reassigned in 11 distinct genera. In summary, a total of 14 new genus names were
190 adopted (Table 1). Despite the fact that multiple species had to be reassigned to other
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191 genera than Candida, the genus Candida represents 70% out of all 9,627 clinical
192 isolates. Candida albicans remains the predominant yeast found in clinical specimens
193 with a rate of 46%. Among the five most prevalent yeasts are C. glabrata (16.8%), C.
194 tropicalis (8.4%), Issatchenkia orientalis (7.4%), and C. parapsilosis (5.4%) (Table 1).
195 The specimens and the source (clinic/ward) from which the clinical isolates were derived
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196 are given in Table 2. In this context, it should be mentioned that for the study centres w
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197 and the collaborative in vitro testing it was not mandatory to report the source area of the a
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198 isolated species. Therefore, much information (specimen-type, source) was not d

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199 disclosed, and had to be marked as “not available” (n.a.). m

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200 Susceptibility data for the five most frequent Candida species and non-Candida species tt
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201 are given in Table 3. Among the basidiomycetous yeasts most commonly encountered a
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202 were Cryptococcus neoformans (3.3%), Malassezia furfur (0.6%), and Trichosporon as
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203 asahii (0.5%) (Table 1). Isolation frequencies of all individual species together with their o
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204 valid names, synonyms, and the assigned PHYLO-codes are given in Table 1. o
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205 PHYLO-groups. Applying the phylogenetic relationship of all distinct entities as p
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206 published by Kurtzman and Robnett and Suh (23-25), their five main clusters designated 1
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207 as A1, A2, A5, A6, and A9 covered all 9,059 yeast isolates associated to the 0
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208 Saccharomycetales (phylum Ascomycota: subphylum Saccharomycotina: class b
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209 Saccharomycetes: order Saccharomycetales). u
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210 As all species in PHYLO-group A9Q9 (yeasts sharing this PHYLO-code) contain only t
211 Co-Q9 as major ubiquinone. The artificial subgroup G of A9Q9 does not refer to a
212 different CoQ-system, however, had to be introduced to distinguish between galactose-
213 containing and galactose-deficient species (59). By further discrimination, all 9,110
214 ascomycetous yeasts could be assigned to a total of 13 PHYLO-codes, respectively
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215 PHYLO-groups (Figure 1 and Table 1). The 517 basidiomycetous yeasts were assigned
216 to four different PHYLO-groups referred to as PHYLO-code B1-B4 (Figure 1 and Table
217 1). In total, all 9,627 clinical isolates were phylogenetically separated into 17 PHYLO-
218 groups. C. albicans forms together with C. africana, C. dubliniensis, C.
219 membranifaciens, C. parapsilosis, and C. tropicalis the largest PHYLO-group (A1Q9A)
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220 covering 5,841 strains (Table 1 and 3). The smallest PHYLO-group within the w
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221 Ascomycota is A1Q9C consisting only of H. uvarum (n = 3). A complete summary of all a
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222 species and their association to the different PHYLO-groups is given in Figure 1 and d

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223 Tables 1 and 3. m

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224 Antifungal susceptibility. According to the reassessment, the minimum inhibitory tt
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225 concentrations (MICs) of the five most frequent Candida and non-Candida species are a
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226 given as cumulative percentages in Table 3, indicating MIC50 and MIC90 values by as
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227 shadowed numbers. Four of the five listed Candida species in Table 3 belong to the o
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228 PHYLO-group A1Q9A, whereas C. glabrata is affiliated to PHYLO-group A1Q6F. In o
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229 addition, Kluyveromyces marxianus and Saccharomyces cerevisiae belong to this group. p
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230 The two PHYLO-groups A1Q9A and A1Q6F are covering 72% of all strains, 1
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231 representing the two main clusters with major importance for the clinical setting. The 0
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232 MIC-values for the PHYLO-groups are given in the supplementary Tables S2 and S3. b
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233 Similar to the different species the phylogenetic groups vary in their antifungal u
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234 susceptibility profiles. Remarkable is that strains within an individual PHYLO-group t
235 share a similar susceptibility profile. Furthermore, it turned out that the PHYLO-group
236 which encompass the majority of yeasts (A1Q9A) has distinct lower MIC values for all
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237 tested azoles (FLC MIC = 0.5 mg/L, ITC MIC = 0.031 mg/L, and VRC MIC = 0.031
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238 mg/L) than the second largest group A1Q6F (FLC MIC = 4.0 mg/L, ITC MIC = 0.25
50 50
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