A Novel Flucytosine-Resistant Yeast Species, Candida pseudoaaseri, Causes Disease in a Cancer Patient

Abstract

Some members of the genus Candida are among the most common human fungal pathogens and cause serious diseases especially in immunocompromised people. A yeast was isolated from a blood culture from an immunocompromised cancer patient who suffered from acute pneumonia. The growth characteristics of the yeast on CHROMagar Candida were similar to those of Candida tropicalis, whereas the API ID 32C system identified the yeast as Candida silvicola. On the basis of the nucleotide divergence in the D1/D2 domain of the 26S nuclear rRNA (nrRNA) gene, as well as the internal transcribed spacer (ITS) domain of the nrRNA gene region, a new species, Candida pseudoaaseri sp. nov. with type strain VK065094 (CBS 11170^T), which was found to be closely related to Candida aaseri, is proposed. While C. aaseri strains were susceptible to all tested antifungals, the new species is resistant to flucytosine and may also be distinguished from C. aaseri by its ability to assimilate l-rhamnose, whereas its colony morphology on CHROMagar Candida may be helpful for differentiation.

INTRODUCTION

Since the early 1980s, an increase in invasive fungal diseases (IFDs) in immunocompromised and critically ill hospitalized patients has been observed (35). IFDs are characterized by a high morbidity and mortality and have therefore emerged as an important public health problem (28, 35). The most common IFD is invasive candidiasis (IC). Since the 1990s, an increase in the numbers of non-Candida IFDs, such as aspergillosis, zygomycosis, and fusariosis, has been observed (28). The incidence of IC since 1995 in Europe increased from 1.9 cases up to 20 cases/100,000 population/year (28, 35). In the United States, a significantly higher incidence of cases of IC is evident as well (35).

Bloodstream infections and sepsis caused by Candida spp. are particularly important, as they are often associated with a higher mortality than bacterial sepsis (26, 45). Wisplinghoff et al. (45) found a 10% higher total crude mortality due to Candida species (39.2%) than due to bacteria (28.5%) by using retrospective analysis of pathogens that were isolated from monomicrobial nosocomial bloodstream infections. The most frequently isolated fungal pathogen is Candida albicans, which can be isolated in 50 to 70% of the cases. Other important species are Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei. However, the use of the triazole fluconazole since the 1990s led to an increase in the number of non-albicans Candida species in clinical specimens (from 25% non-albicans Candida species in 1990 to 45% in 1995). The most frequent non-albicans Candida species in the United States and Europe were C. glabrata (before 1990, 5 to 10%; after 1995, 15 to 20%) and C. parapsilosis (before 1990, 5%; after 1995, 20%), respectively (6, 8, 10, 28, 35). Even though this trend has not been observed in recent years, the huge influence of antifungal therapy and prophylaxis on the spectrum of pathogenic yeasts is obvious. The selection of natural and acquired species-specific resistances plays an important role. Both the identification to species level of Candida strains from clinical samples and the in vitro susceptibility tests have proven to be very important for the outcomes of the patients (22, 32, 35).

Numerous members of the genus Candida are presently included among the most common human fungal pathogens. Thirty species of this genus have already been shown to cause diseases in humans (1, 2, 13, 15, 29, 38, 39, 41, 42), whereas the substantial progress in medical research and the increasing number of immunocompromised patients will increase the number of medically important Candida species in the future. Furthermore, these numbers are expanding by the introduction of molecular methods that improve the identification of existing and novel Candida species and offer the possibility of distinguishing strains of closely related species that were previously believed to belong to the same species (1, 2, 13, 15, 29, 38, 39, 41).

Based on the nucleotide divergence in the partial nuclear rRNA (nrRNA) gene that includes the D1/D2 domain of the large-subunit nrDNA as well as the internal transcribed spacer (ITS) domain (ITS 1, ITS 2, and the intervening 5.8S nrRNA gene), a novel Candida species, isolated during the course of a microbiological diagnostic routine from different blood cultures from a cancer patient, is proposed and the effects that different antifungal drugs have on this species are discussed.

CASE REPORT

In June 2007, a 46-year-old man with a diagnosed metastasized stomach adenocarcinoma was hospitalized (Klinikum Niederlausitz, Lauchhammer, Germany) during the fifth cycle of chemotherapy with docetaxel, cisplatin, and 5-fluorouracil (TCF scheme), as he was suffering from high fever, prostration, exhaustion, and weight loss.

Computer tomography (CT) indicated distinct basal inferior pneumonia in the right lobe and a smaller pneumonic infiltrate in the left lobe. Antibiotic treatment with ciprofloxacin (2× 400 mg/day) failed, and the results of additional tests on the blood cultures were compiled. Microbial isolates from blood cultures, taken on 25, 26, and 29 June 2007, presented only one morphological type of yeast cells. The clinical isolates were grown on CHROMagar Candida (BD Deutschland GmbH, Heidelberg, Germany) and had a colony morphology and color similar to those of C. tropicalis, whereas the additional classical methods used provided inconsistent results that did not confirm the initial diagnosis of C. tropicalis. Analysis using the API ID 32C system (bioMérieux, Nürtingen, Germany) identified the isolate as Candida silvicola (teleomorph, Pichia holstii), whereas the Vitek 2 analysis (ID-YST card; bioMérieux, Nürtingen, Germany) and Micronaut-Candida plate (Merlin Diagnostic GmbH, Bornheim-Hersel, Germany) provided no results. The Candida colonies did not produce the typical pseudohyphal structures on rice agar, which are characteristic for C. tropicalis. Therefore, one representative isolate, VK065094^T, was sent to the National Reference Laboratory for Systemic Mycoses in Göttingen, Germany, the National Konsiliar Laboratory for Dermatophytes in Berlin, Germany, and the CBS-KNAW Fungal Biodiversity Centre in Utrecht, The Netherlands, for further analyses.

Antifungal treatment with fluconazole (200 mg/day) for 20 days was successful, and the patient was released from the hospital. In order to find the source of infection, an interview was performed with the patient. It became clear that before his hospitalization, the patient was exposed to airborne dust containing fecal remains from pigeons while cleaning his attic without wearing a dust mask. At that time, his health condition was characterized by distinct leukopenia and anemia. However, a thorough sampling of the attic showed no sign of the Candida contaminant.

MATERIALS AND METHODS

Strains examined.

Strains were isolated from three cultures (six bottles) of blood collected from the patient on 26, 27, and 29 June 2007 and used in the primary morphological analyses. A representative isolate, VK065094^T, was used in further physiological, biochemical, and molecular analyses. The following isolates, representing different yeast species, were used in several of the analyses for comparative studies and quality controls: C. albicans ATCC 10231, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, C. tropicalis ATCC 4563, and Candida aaseri CBS 1913^T and CBS 2226.

Morphological, physiological, and biochemical characteristics.

Morphological, physiological, and biochemical characteristics were examined as described by Yarrow (46) and Barnett et al. (7), and the evaluations were done in duplicate. The incubation time for the fermentation reactions was 20 days, with readings done after 5, 9, 12, 14, 16, and 20 days, whereas the assimilation tests were done for 7 days. For cultivation and differentiation, the following media were used: Sabouraud glucose (2%) agar (SGA 2% Glc), Sabouraud glucose (2%) agar with chloramphenicol and gentamicin (SGA 2% Glc GM-C), Sabouraud glucose (2%) brain heart infusion glucose (2%) agar with chloramphenicol and gentamicin (SGA 2% Glc BHI GM-C), CHROMagar Candida, Sabouraud glucose (2%)-CHROMagar Candida biplate, malt-yeast-glucose-peptone broth (YM broth), malt-yeast-glucose-peptone agar (YMA), Columbia blood agar (CBA) (BD Diagnostic Systems, Heidelberg, Germany); Sabouraud glucose (2%) broth (SGB 2% Glc), Sabouraud glucose (4%) agar (SGA 4% Glc), potato dextrose agar (PDA), malt extract agar (MEA), Candida Ident agar, rice Tween agar (Heipha GmbH, Eppelheim, Germany); Sabouraud glucose (2%) agar Emmons (SGA 2% Glc Emmons), Brilliance Candida agar, Chromogen C. albicans agar (CALB agar) (Oxoid GmbH, Wesel, Germany); CandiSelect 4 agar (Bio-Rad Laboratories GmbH, Munich, Germany); CHROMagar Candida (Mast Diagnostic GmbH, Reinfeld, Germany); and chromID Candida (CAN2 agar) (bioMérieux, Nürtingen, Germany) (7, 15, 20, 33, 46). The cultures were, in general, incubated on these media aerobically at 37°C for 2 to 4 days.

In order to test the growth at different temperatures, VK065094^T was cultured on YMA and growth under anaerobic, CO₂, and microaerophilic conditions was tested using the GENbag anaer, GENbag CO₂, and GENbag microaer (bioMérieux, Nürtingen, Germany) methods, respectively. Biochemical characteristics were also investigated using the API ID 32C, ID YST card (Vitek 2) (bioMérieux, Nürtingen, Germany), and Micronaut Candida (Merlin Diagnostic GmbH, Bornheim-Hersel, Germany) systems in accordance with the manufacturers' instructions.

Ascospore production of VK065094^T was examined using four different media: YMA, MEA, acetate agar, and Gorodkowa agar, with incubation at 25°C for 6 weeks.

Preliminary molecular identification.

DNA extractions were done with a Nexttec genomic DNA isolation kit for yeast from Biozym (Oldendorf, Germany). The protocol of the manufacturer was slightly modified with respect to centrifugation during the lysis step, which was omitted. The PCR analysis of the ITS region was performed with the primer pair LSU266 and V9G (14). Sequence analysis was commissioned at SMB GmbH (Berlin, Germany) using the internal primers ITS 4 and ITS 5 (44) and the general protocol that is described by this company. The ITS sequence of VK065094^T was submitted to a BLASTN search using the GenBank, EMBL, DDBJ, and PDB (http://blast.ncbi.nlm.nih.gov/) databases as well as the freely available CBS yeast sequence database (www.cbs.knaw.nl/). VK065094^T was also sent to CBS to confirm the results of the initial identification and to conduct phylogenetic analyses using the ITS and D1/D2 regions of VK065094^T and additional closely related species.

Phylogenetic analyses.

DNA was extracted from cultures grown on YMA for 3 days using the FastDNA kit (Bio101, Carlsbad, CA). Primers V9G (14) and LR5 (43) were used to amplify the D1/D2 and ITS regions as described by Knutsen et al. (25). The amplicons were sequenced in both directions using the primers LR0R (43) and LR5 for the D1/D2 domain, whereas the primers V9G and ITS 4 (44) were used for the ITS domain. The BigDye terminator version 3.1 cycle sequencing kit (Applied Biosystems) was used according to the manufacturer's recommendations, and the products were analyzed on an ABI Prism 3730XL DNA sequencer (PerkinElmer). A consensus sequence was computed from the forward and reverse sequences with SeqMan from the Lasergene package (DNASTAR).

All sequences of VK065094^T were searched against sequences in GenBank, EMBL, DDBJ, and PDB (http://blast.ncbi.nlm.nih.gov/) as well as against available sequences of strains present in the CBS yeast sequence database (www.cbs.knaw.nl/yeast/) to find sequences of closely related species that were then used in the phylogenetic analyses. All newly produced sequences as well as published sequences of closely related species were assembled and aligned using MAFFT (version 6) (24). The sequence data were analyzed using Phylogenetic Analysis Using Parsimony (PAUP) version 4.0b10 (Sinauer Associates, Sunderland, MA), and the resulting trees were printed as described by Groenewald et al. (18). Maximum parsimony analyses were performed using the heuristic search option with 1,000 random taxon additions, and the robustness of the trees was evaluated by 1,000 bootstrap replicates. Other measures calculated included tree length, consistency index, retention index, and rescaled consistency index (TL, CI, RI, and RCI, respectively). Neighbor-joining analyses using different substitution models (HKY85, Kimura 2-parameter, and uncorrected “P”) were also performed. All analyses were done where gaps were treated as fifth characters (“new state”). The sequences generated during this study were deposited in GenBank.

MALDI-TOF MS analyses.

VK065094^T was tested by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the SARAMIS software and database (bioMèrieux, formerly AnagnosTec). The spectrum analysis was performed after 24 h of cultivation on Sabouraud glucose (4%) agar (SGA 4% Glc) at 37°C using the direct smear method, and CHCA (alpha-cyano-4-hydroxy-cinnamic acid) was used as the matrix. All analyses were done in duplicate. Mass analyses were performed in the linear mode with positive-ion delayed extraction (acceleration voltage, 20 kV) on an AXIMA Confidence (Shimadzu Europe, Duisburg, Germany) mass spectrometer equipped with a nitrogen laser (k = 337 nm). Spectra were accumulated from 500 automatically acquired laser pulse cycles. All spectra were processed by the Biotech Launchpad software (Shimadzu Europe, Duisburg, Germany) with baseline correction, peak filtering, and smoothing. The resulting peak lists were exported to the SARAMIS software package. The spectra were compared with the SuperSpectra and reference spectrum databases without matching. Using the SARAMIS functions for comparison and cluster analysis shows the confirmation of a new species and no significant similarities with spectra in the existing SARAMIS database (version 2010).

Antifungal susceptibility.

Antifungal susceptibility tests were performed using broth dilution testing in accordance with the guidelines of Clinical and Laboratory Standards Institute (CLSI) document M27-A3 (12) and the commercially available systems Etest, AST-YS01 card (Vitek 2) (bioMérieux, Nürtingen, Germany), and Micronaut-AM (Merlin Diagnostic GmbH, Bornheim-Hersel, Germany) according to the manufacturers' instructions. For the Etest method, 90-mm agar plates containing RPMI 1640 medium supplemented with 2% glucose and buffered with MOPS (morpholinepropanesulfonic acid) to pH 7.0 (bioMérieux, Nürtingen, Germany) were used. Etests were read at 24 h and 48 h. The MICs were rounded up to the next even log₂ concentration. The Micronaut-AM test was deduced from the CLSI M27-A2 broth microdilution method. The susceptibility tests using Micronaut-AM plates were based on the rehydration of antimycotics by the addition of a standardized yeast suspension in RPMI 1640 broth. Growth of the yeasts is indicated by a color change from blue to pink, mediated by the aspartate transaminase (AST) indicator that was added to the test medium. The addition of methylene blue solution facilitates the reading of antimycograms of yeast with trailing effects. After 22 to 24 h of incubation at 37°C, the result is read photometrically with the plate reader and evaluated with Micronaut software. For all media, C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were included as quality control strains and the CLSI, Etest, Vitek 2, and Micronaut tests were repeated six, two, four, and six times, respectively, for each strain tested against each of the antifungal agents.

MycoBank accession number.

The description of Candida pseudoaaseri is available online (http://www.MycoBank.org/) under MycoBank accession number MB 560230.

Nucleotide sequence accession numbers.

The D1/D2 sequences obtained in this study for C. pseudoaaseri CBS 11170^T have been deposited in GenBank under the accession number JN241689, and the ITS sequences of C. pseudoaaseri CBS 11170, C. aaseri CBS 6421, and C. aaseri CBS 1913^T have been deposited in GenBank under the accession numbers JN241686 to JN241688, respectively.

RESULTS

Phenotypic characteristics.

In Table 1 all phenotypic characteristics of VK065094^T tested are listed next to those observed for the closely related C. aaseri strains CBS 1913^T and CBS 2226 and C. albicans ATCC 10231. As the phenotypic characteristics of C. aaseri CBS 6421 (originally described as Candida butyri Nakase) were previously found to be comparable to those of other C. aaseri strains (31), this species was not included in this part of the study. The macromorphology of VK065094^T, cultured on different media, was significantly different from that of the C. aaseri strains. While regular, semishiny colonies with an even margin were formed by VK065094^T, irregular partly folded colonies were produced by C. aaseri (Fig. 1).

Table 1.

Key physiological and morphological features of Candida pseudoaaseri VK065094^T, C. aaseri CBS 1913^T, C. aaseri CBS 2226, and C. albicans ATCC 10231^a

Characteristic	Reaction of strain:
	C. pseudoaaseri VK065094T	C. aaseri CBS 1913T	C. aaseri CBS 2226	C. albicans ATCC 10231
Morphology
Germ tubes	−	−	−	+
Pseudohyphae	+	+	+	+
Chlamydospores	−	−	−	+
Ascospores	−	−	−	−
Growth at 37°C
YMA	+	+	+	+
MEA	+	+	+	+
SGA 2% Glc	+	+	+	+
SGA 2% Glc GM-C	−	−	−	+
Growth on chromatogenic agar
Brilliance Candida agar (Oxoid)	+ (blue)	+ (blue)	+ (blue)	+ (green)
CALB agar (Oxoid)	−	−	−	+ (blue)
CAN2 agar (bioMérieux)	−	−	−	+ (blue)
CandiSelect 4 agar (Bio-Rad)	−	−	−	+ (pink)
Candida Ident agar (Heipha)	−	−	−	+ (green)
CHROMagar Candida (BD)	−	−	−	+ (green)
CHROMagar Candida biplate (BD)	+ (blue)	+ (blue)	+ (blue)	+ (green)
CHROMagar Candida (Mast)	+ (blue)	+ (blue)	+ (blue)	+ (green)
Growth on YMA
37°C	+	+	+	+
40°C	−	−	−	+
Fermentationb
d-Glucose	+ (s)	−	+ (s)	+
d-Galactose	+ (s)	−	+ (s)	+
Maltose	−	−	−	+
α,α-Trehalose	−	−	−	+
Cellobiose	+ (s)	−	+ (s)	−
Assimilationb
l-Sorbose	−	−	d	−
d-Glucosamine	+	−	+	+
N-Acetyl-glucosamine	+	−	+	+
d-Ribose	+	+	+	−
l-Arabinose	+	+	+	−
l-Rhamnose	+	−	−	−
Sucrose	+	−	+	+
Methyl-α-d-glucoside	+	+	−	+
Cellobiose	+	+	+	−
Melezitrose	+	−	d	−
Glycerol	+	+	+	d
Erythritol	+	+	+	−
d-Glucitol	+	−	+	+
d-Mannitol	+	+	+	+
2-Keto-d-gluconate	−	−	−	+
d-Gluconate	+	−	+	+
d-Galacturonate	−	−	−	d
dl-Lactate	−	−	−	+
Nitrate	−	−	−	−
Other characteristics
Esculin hydrolysis	+	+	+	−
Susceptibility to cycloheximide (0.01%)	−	−	−	+
Urease activity (37°C, 4 h)	−	−	−	−

^aAll strains were negative in their ability to ferment sucrose, lactose, and raffinose; positive in their ability to assimilate d-glucose, d-galactose, d-xylose, maltose, α,α-trehalose, xylitol, citrate, acetate, and palatinose; negative in their ability to assimilate melibiose, lactose, raffinose, myo-inositol, d-glucuronate, and levulinate. +, positive; −, negative; d, positive or negative; s, slow or delayed.

^bOnly variations observed in a specific character tested for the four strains are listed.

Fig. 1.

Colony morphology of Candida pseudoaaseri VK065094^T (a and d), C. aaseri strains CBS 1913^T (b and e) and CBS 2226 (c and f) on YMA (a to c) and CHROMagar Candida (biplate; BD) (d to f) grown for 3 days at 37°C. Bars a to c and d to f, 2 mm.

Good growth was observed for VK065094^T on YMA at 37°C under aerobic, microaerophilic, and capnophilic conditions but not under anaerobic conditions. The growth behavior of VK065094^T on different selective culture media and under various growth conditions (e.g., temperature, atmosphere, etc.) was identical to that of CBS 1913^T and CBS 2226. VK065094^T and CBS 1913^T, as well as CBS 2226, were positive in their ability to grow on YMA, MEA, PDA, SGA 2% Glc, SGA 4% Glc, SGA 2% Glc Emmons, CHROMagar Candida (BD and Mast), and Brilliance Candida agar at 37°C. These strains were not able to grow on SGA with antibiotic additions and also not able to grow on most chromatogenic agars (Table 1). Similar fermentation and assimilation profiles were also produced by VK065094^T, CBS 1913^T, and CBS 2226, except that only VK065094^T assimilated l-rhamnose (Table 1). When VK065094^T was cultured on different media (CBA, SGA 2% Glc Emmons, and SGA 2% Glc), the API ID 32C profiles were also reproducible (code, 5555771315).

Molecular analyses.

No D1/D2 and ITS sequences were found in the public sequence databases that were similar to those of VK065094^T. The species closest to the studied strain is C. aaseri (Fig. 2). The D1/D2 sequences (531 bp) of VK065094^T (GenBank accession no. JN241689) and that of C. aaseri CBS 1913^T (U45802) differ by three substitutions and two gaps from one another. However, many variations were found between the ITS sequences (594 bp) of VK065094^T (GenBank accession no. JN241686) and that of CBS 1913^T (GenBank accession no. JN241688), as these sequences differ by 26 nucleotide substitutions and 15 gaps.

Fig. 2.

The first of two equal most parsimonious trees obtained from a heuristic search with 1,000 random taxon additions of the combined D1/D2 and ITS sequence alignment (TL, 310 steps; CI, 0.777; RI, 0.625; RCI, 0.486). The scale bar shows 10 changes, and bootstrap support values (>49%) from 1,000 replicates are shown as percentages at the nodes. Thickened lines indicate the branches present in the strict consensus tree. The new species, Candida pseudoaaseri, is indicated in bold.

The combined D1/D2 and ITS sequence alignment containing nine strains, including the outgroup sequences, had a total length of 1,036 characters, of which 835 were constant, 81 were phylogenetically uninformative, and 102 were informative. Parsimony analysis resulted in two equal most parsimonious trees, the first of which is shown in Fig. 2. The two trees obtained do not differ with regard to the position of VK065094^T within the tree or its most closely related species. It is clear from the sequence and phylogenetic analyses that this novel species is part of the Yamadazyma complex, with C. aaseri as the most closely related species. Candida pseudoaaseri and C. aaseri are clustered together with a 98% bootstrap support (Fig. 2).

MALDI-TOF MS analyses.

The MALDI-TOF MS fingerprint analyses (23) are shown in Fig. 3 and confirm that VK065094^T represents a yeast species that is not yet included in the currently available database. The used version of the SARAMIS database (version 2010) contains 85 different species of the order Saccharomycetales, including the genera Candida, Clavispora, Debaryomyces, Geotrichum, Kluyveromyces, Issatchenkia, Lodderomyces, Metschnikowia, Pichia, Saccharomyces, Stephanoascus, Yarrowia, and others. Figure 4 is a presentation of the SARAMIS cluster analysis of VK065094^T and additional reference strains that shows no significant similarities with any other yeast species in the existing SARAMIS database.

Fig. 3.

MALDI-TOF mass spectra of Candida pseudoaaseri VK065094^T (a), C. aaseri CBS 2226 (b), and C. albicans DSM 11225 (c).

Fig. 4.

SARAMIS cluster analysis of reference strains C. aaseri CBS 2226, C. aaseri CBS 1913^T, C. albicans DSM 11225, C. glabrata DSM 70164, and the new C. pseudoaaseri VK065094^T. The cluster displays duplicate measurements and relative identities based on the identical mass signals.

Antifungal susceptibilities.

The in vitro susceptibilities of VK065094^T and the C. aaseri strains to the antifungal agents amphotericin B, flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and micafungin are listed in Table 2. The results for the single antifungal agents were comparable, independent of the system that was used (CLSI reference method, Etest, Vitek 2, or Micronaut). It is known that the Etest correlates well with the CLSI reference method for determining the susceptibility of Candida isolates (3, 4, 5, 11, 34) and that the results from the YST card of Vitek 2 have a good correlation with the results from the standard method (9, 11, 36, 37). In contrast to both C. aaseri strains, VK065094^T is resistant to flucytosine. For all other tested antifungal agents, the MIC or breakpoint values were comparable among the strains tested.

Table 2.

In vitro susceptibility testing of antifungal agents on Candida pseudoaaseri VK065094^T, C. aaseri CBS 1913^T, and C. aaseri CBS 2226^a

Antifungal agent and strain	MIC (μg/ml)
	CLSI (48 h)	Etest (48 h)	Vitek 2	Micronaut
Amphotericin B
VK065094T	0.25–0.5	0.25	≤0.25–0.5	0.25–0.5
CBS 1913T	0.063–0.125	0.032	≤0.25	0.125–0.25
CBS 2226	0.031–0.063	0.064	≤0.25	0.125–0.25
Flucytosine
VK065094T	≥16	≥32	≥64	≥64
CBS 1913T	≤0.031	0.016	≤1	≤0.063
CBS 2226	≤0.031	0.016	≤1	≤0.063
Ketoconazole
VK065094T	NT	0.064	NT	0.031–0.125
CBS 1913T	NT	0.008	NT	0.016–0.031
CBS 2226	NT	0.008	NT	0.016–0.031
Fluconazole
VK065094T	0.25–0.5	0.5	≤1	0.25–0.5
CBS 1913T	0.25	0.25	≤1	0.25
CBS 2226	0.125	0.25	≤1	0.25
Itraconazole
VK065094T	0.063–0.125	0.5	NT	0.125–0.5
CBS 1913T	0.031–0.063	0.032	NT	≤0.031–0.063
CBS 2226	0.016	0.032	NT	≤0.031–0.063
Voriconazole
VK065094T	0.031–0.063	0.016	≤0.12	0.016–0.063
CBS 1913T	0.031	0.008	≤0.12	≤0.016
CBS 2226	≤0.008	0.008	≤0.12	≤0.016
Posaconazole
VK065094T	0.063–0.125	0.064	NT	0.063
CBS 1913T	0.016–0.063	0.016	NT	≤0.016
CBS 2226	≤0.008	0.016	NT	≤0.016
Caspofungin
VK065094T	0.5	0.5–1	NT	0.125–0.5
CBS 1913T	0.25–0.5	0.25	NT	0.125–0.5
CBS 2226	0.125–0.5	0.25	NT	0.125–0.25
Anidulafungin
VK065094T	0.125	0.25–0.5	NT	0.25–1
CBS 1913T	0.125–0.25	0.5	NT	0.25–0.5
CBS 2226	0.125–0.25	0.5	NT	0.25–0.5
Micafungin
VK065094T	0.5–1	1	NT	0.5–1
CBS 1913T	0.5–1	0.5	NT	0.5–1
CBS 2226	0.5	0.5	NT	0.5–1

^aResults were compared with the standard dilution testing (CLSI) and three commercial test systems, Etest, Vitek 2, and Micronaut. The CLSI, Etest, Vitek 2, and Micronaut tests were repeated six, two, four, and six times, respectively. NT, not tested.

TAXONOMY

Based on the results obtained from the various analyses done during this study, we conclude that a novel species, closely related to C. aaseri, was isolated from a blood sample from a cancer patient and is proposed here as a new species.

Latin diagnosis of Candida pseudoaaseri Pfüller, Gräser, Erhard, M. Groenew. sp. nov.

In medio liquido YM post 3 dies 25°C, cellulae vegetativae ovoideae, ellipsoidae aut cylindricae, 1.4 to 4 × 2.2 to 6 μm, singulae, binae, brevibus catenis connexae. Post 1 mensem 25°C velum fragile at sedimentum formantur. In agaro YM post 1 mensem 25°C cultura est butyrosa, cremea, plana aut integro margine. Post 3 dies 25°C in PDA aut agaro Oryzae cum Tween, pseudohyphae et blastoconidia formantur, sed chlamydosporae nullae. Ascosporae non fiunt. Glucosum, galactosum, cellobiosum et fermentatur (lente). d-Glucosum, d-galactosum, d-glucosaminum, N-acetyl-d-glucosaminum, d-ribosum, d-xylosum, d-arabinosum, l-rhamnosum, sucrosum, maltosum, trehalosum, palatinosum, α-methyl-d-glucosidum, cellobiosum, melezitosum, glycerolum, erythritolum, xylitolum, d-glucitolum, d-mannitolum, d-gluconas, acetum, acidum citricum, assimilantur at non l-sorbosum, melibiosum, lactosum, raffinosum, inositolum, 2-keto-d-gluconas, d-glucuronas d-galacturonicum, dl-acidum-lacticum, levulinas, natrium niticum. Non crescit in medio 0.01% cycloheximido addito. Ureum non hydrolysatur. Crescit in 37°C, non crescit in 40°C. Typus: VK065094^T (CBS 11170^T) exsiccata et vivus in collectione culturarum Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum praeservantur.

Description of Candida pseudoaaseri Pfüller, Gräser, Erhard, M. Groenew. sp. nov. (MycoBank accession number MB 560230).

In YM broth, at 25°C for 3 days, the vegetative cells are ovoidal, ellipsoidal, tear shaped, and cylindrical (1.4 to 4 by 2.2 to 6 μm), single, in pairs, in short chains, or in groups with multilateral budding (Fig. 5a and b). After 1 month of incubation in YM broth at 25°C, a fragile film of mold and sediment are present. On YMA, after 1 month at 25°C, the streak culture is creamy, semishiny, and flat with an uneven center and entire margin. At the margin, a single submerse mycelium is developed (Fig. 5d). Germ tubes are not formed. On slide culture on rice Tween agar and potato dextrose agar, after 3 days at 25°C, pseudohyphae with blastoconidia are produced (Fig. 5c), and no chlamydospores are formed. Ascospores are not detected on sporulation media. Fermentation and growth on various carbon and nitrogen compounds are presented in Table 1. Esculin can be hydrolyzed; susceptibility to 0.01% cycloheximide; urease activity is negative. Growth is positive at 37°C but negative at 40°C. The type strain VK065094 (CBS 11170^T) was isolated in Lauchhammer, Germany, in 2007 from a blood culture from a human presenting with pneumonia after chemotherapy for adenocarcinoma of the stomach.

Fig. 5.

Candida pseudoaaseri VK065094^T. (a and b) Cells grown in YM broth after 3 days at 25°C. Bars, 20 μm (a) and 10 μm (b). (c) Pseudohyphae on rice Tween agar after 3 days at 25°C. Bar, 20 μm. (d) Colony morphology on YMA after 1 month at 25°C. Bar, 5 mm.

Etymology: pseudoaaseri, the epithet is chosen because of the close phylogenetic relationship to C. aaseri.

DISCUSSION

A very important aspect that has to be kept in mind in disease control in humans and animals is species- or even strain-specific resistance to presently used antifungal drugs. The selection of natural and acquired species-specific resistances to antifungal drugs plays an important role and should be studied in detail during the emergence of an unknown disease-causing species. This is also true in common disease-causing fungal species, as the use of new antifungal drugs like echinocandins cannot prevent the development of new resistance mechanisms in the future. It has been demonstrated in the past, as well as during this study, that both the correct identification to the species level of fungal strains isolated from clinical samples and the in vitro susceptibility tests are very important for the outcomes of the patients (22, 32, 35).

Members of the genus Candida are presently among the most common human fungal pathogens, and as the number of immunocompromised patients increases in the future, the number of Candida species, causing disease, will increase as well. This trend can already be seen in the fact that 30 Candida species have already been found to cause human diseases. From the current study and others (1, 2, 13, 15, 29, 38, 39, 41), it is clear that these numbers are expanding drastically by the use of molecular methods that improve the identification of existing and novel Candida species.

From the molecular analyses, it is clear that the novel species described during this study, C. pseudoaaseri, is part of the Yamadazyma complex, with C. aaseri, a species that has previously been isolated from humans, as the closest relative. The variations found in the ITS sequences of VK065094^T and C. aaseri were more sufficient to distinguish them from one another than the sequences of the commonly used D1/D2 region. Groenewald et al. (19) also indicated that the ITS region is a good marker to use in species delimitation within this complex. Additional species that are part of the Yamadazyma complex were isolated from diverse habitats such as water, soil, and plants, and others have also been associated with insects (19, 40).

A formerly known species, C. butyri (type strain CBS 6421), has D1/D2 and ITS sequences similar to those of C. aaseri (CBS 1913^T) and was described previously as conspecific; C. butyri is now listed as an obligate synonym of C. aaseri by several authors (16, 27). The type strains of C. aaseri (CBS 1913^T) and C. butyri (CBS 6421^T) were isolated from the sputum of a Norwegian patient without clinical relevance and from butter, respectively (30). In contrast to its closest relatives, VK065094^T was the etiological agent of the candidemia of an immunocompromised cancer patient. The isolation of VK065094^T from a human sample increases the number of medically important species present in the Yamadazyma complex. It is also very likely that some of the closely related environmental species, such as C. aaseri and the formerly known C. butyri, may also be found in human samples as opportunists awaiting the proper change in environmental conditions to become pathogens, e.g., in immunocompromised hosts.

As the physiological profiles of VK065094^T and the two C. aaseri strains are similar, except in their ability to assimilate l-rhamnose, it is important to have a rapid system that can correctly distinguish among closely related species that does not rely only on tests for physiological characteristics, such as the API ID 32C, ID YST card (Vitek 2), and Micronaut Candida systems. Inclusion of novel human pathogens, such as C. pseudoaaseri, in currently used versions of databases that are frequently used for species identification in clinical diagnostic laboratories, e.g., the MALDI-TOF system, is extremely important for rapid identification of a potential pathogen. It is quite interesting, but also problematic, that VK065094^T, CBS 1913^T, and CBS 2226 were unable to grow on SGA with antibiotic additions and also unable to grow on most chromatogenic agars. Many of these media are used in routine primary isolation of yeasts in a clinical laboratory, and thus, the examined Candida strains of this case study cannot be detected using these media. Candida pseudoaaseri (VK065094^T) has low MICs for all tested antifungal agents except flucytosine. The high susceptibility of C. pseudoaaseri to fluconazole explains the good recovery of the patient in this study after 20 days of treatment with this antifungal drug. The clear resistance to flucytosine (CLSI MIC ≥ 16 μg/ml) differentiated C. pseudoaaseri from both C. aaseri strains. The antifungal resistance is most probably intrinsic, since strain VK065094^T was isolated before the use of antifungal agents and flucytosine was not used during the course of treatment. For other clinically important yeasts, e.g., C. albicans and C. tropicalis, resistances to flucytosine were also described in the literature (17, 21). These resistances are determined not only genetically; they also cause differences in phenotypical and epidemiological characteristics of susceptible and nonsusceptible yeast populations, respectively. Desnos-Ollivier et al. (17) have shown that a flucytosine-nonsusceptible C. tropicalis subgroup isolated from blood cultures has a higher incidence in patients with malignancies and is associated with lower mortality related to the infection. Currently, flucytosine has only a marginal therapeutic impact; however, the identification of the resistance is important for differentiation of Candida species, as demonstrated in this study.