Candida haemulonii was originally described from a
strain obtained from the gut of a blue-striped grunt
(Haemulon sciurus) in 1962 (17). Lavarde et al. (6)
reported the first clinical isolation of this microorganism
from the blood of a patient in 1984. Subsequently, this
fungus has been isolated from the nails and toes of
patients in the United States (3). Clinical isolates iden-
tified as C. haemulonii can be divided into types I and II
based on their isoenzyme and protein profiles, and this
division was supported by a DNA-DNA hybridization
study by Lehmann et al. (7). Type I includes the type
strain of this microorganism. Although the type II
strain should be treated as a new species, the new taxon
remains to be established. At present, the name “C.
haemulonii types I and II” is used in the field of yeast
taxonomy. During a taxonomic investigation of clinical
isolates from Thai patients, we found a strain that was
resistant to amphotericin B and azoles in a patient’s
blood. The clinical isolate was phylogenetically similar
to C. haemulonii, which is also resistant to ampho-
tericin B. In this paper, we propose a new species, C.
pseudohaemulonii, for the isolate.
Strain DMST 17134 was isolated from the blood of a
Thai patient at Mae Sot Hospital. The Candida haemu-
lonii type I and II strains were purchased from the Cen-
traalbureau voor Schimmelcultures and were studied for
comparison (Table 1).
Fungal DNA was extracted using the method of
Makimura et al. (8). The internal transcribed spacer
(ITS) and D1/D2 regions of the 26S rDNA of the rRNA
gene were amplified using the primer pairs pITS-F (5'
GTCGTAACAAGGTTAACCTGCGG) and pITS-R (5'
TCCTCCGCTTATTGATATGC) (13) and NL1 (5'
GCATATCAATAAGCGGAGGAAAAG) and NL4 (5'
GGTCCGTGTTTCAAGACGG) (5), respectively. The
PCR products were sequenced using the same four
primers. The sequences were aligned using Clustal W
software (15). For the neighbor-joining analysis (12),
the distances between sequences were calculated using
Kimura’s two-parameter model (4). A bootstrap analy-
sis was conducted with 100 replications (1). Figure 1
shows the tree constructed using the D1/D2 26S rDNA
Candida pseudohaemulonii Sp. Nov.,
an Amphotericin B- and Azole-Resistant Yeast
Species, Isolated from the Blood of a Patient from
Takashi Sugita*, 1, Masako Takashima2, Natteewan Poonwan3, and Nanthawan Mekha3
1Department of Microbiology, Meiji Pharmaceutical University, Kiyose, Tokyo 204–8588, Japan, 2Microbe Division, Japan
Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351–0198, Japan, and 3Mycology Section,
National Institute of Health, Department of Medical Sciences, Nonthaburi, 11000 Thailand
Received February 28, 2006; in revised form, March 9, 2006. Accepted March 13, 2006
Abstract: Candida haemulonii (types I and II) is rarely isolated from clinical specimens. We isolated a
strain that is phylogenetically close to C. haemulonii from the blood of a Thai patient, and named it C.
pseudohaemulonii sp. nov. (CBS 10099T?JCM 12453T?DMST 17134T). The new species and C. haemu-
lonii types I and II were resistant to amphotericin B and azole agents but were susceptible to a 1,3-?-D-glu-
can synthetase inhibitor, micafungin, and 5-flucytosine. The species were easily distinguished using an
ID32 yeast identification kit. The taxonomic description of C. pseudohaemulonii sp. nov. is presented.
Key words: Candida haemulonii, New species, Amphotericin B, Azole
Microbiol. Immunol., 50(6), 469–473, 2006
Abbreviations: CBS, Centraalbureau voor Schimmelcultures,
Utrecht, The Netherlands; DDBJ, DNA Data Bank of Japan;
DMST, National Institute of Health, Department of Medical Sci-
ences, Nonthaburi, Thailand; ITS, internal transcribed spacer;
JCM, Japan Collection of Microorganisms, RIKEN, Japan;
NRRL, Agricultural Research Service Culture Collection,
National Center for Agricultural Utilization Research, Peoria, Ill.,
*Address correspondence to Dr. Takashi Sugita, Department
of Microbiology, Meiji Pharmaceutical University, 2–522–1
Noshio, Kiyose, Tokyo 204–8588, Japan. Fax: ?81–424–95–8762.
T. SUGITA ET AL
Table 1. Strains examined
Species StrainSource Locality
DDBJ accession number
D1/D2 26S rDNA
C. haemulonii type I
Ulcer on toe
Ulcer on foot
Rhode Island, U.S.A.
C. haemulonii type IIAB118790a)
T, type strain.
Fig. 1. Molecular phylogenetic trees constructed using the D1/D2 26S rDNA sequences of Candida pseudohaemulonii and
related Candida species including pathogenic species. The DDBJ/GenBank/EMBL accession numbers are indicated in paren-
theses. The numbers indicate the confidence level from 100 replicate bootstrap samplings (frequencies below 50% are not indi-
cated). Knuc, Kimura’s parameter (Kimura, 1980).
sequences of strain DMST 17134, and phylogenetically
closely related species including pathogenic Candida
species. Strain DMST 17134 formed a cluster with the
two types of C. haemulonii with 100% bootstrap sup-
port. The ITS 1 and 2 regions in the two types of C.
haemulonii and DMST 17134 were only 66 to 70 bp
and 70 to 81 bp long, respectively. The DNA sequence
similarities in ITS 1 and 2 between C. haemulonii type
II and DMST 17134 were 79.1 and 92.5%, respectively.
The divergence between the two types of C. haemulonii
and our blood isolate was sufficient to resolve them as
individual species (10, 13). Therefore, we named the
new isolate C. pseudohaemulonii.
Most of the morphological, biochemical, and physio-
logical characteristics were examined using the meth-
ods described by Yarrow (18). An ID32 yeast identifi-
cation kit (bioMérieux SA, Lyon, France) was also used
for practical identification, according to the manufac-
turer’s instructions. Table 2 shows the characteristics
that distinguish C. pseudohaemulonii from the two
types of C. haemulonii, using the ID32 yeast identifica-
tion kit. They were easily distinguished by their pattern
of utilization of five compounds: trehalose, glycerol, L-
rhamnose, melezitose, and esculine.
In vitro testing of susceptibility to amphotericin B,
5-flucytosine (5-FC), fluconazole, itraconazole, micona-
zole, and micafungin was performed using a Yeast-like
fungi DP, EIKEN kit (Eiken Chemical, Tokyo), accord-
ing to the manufacturer’s instructions. The microplates
were incubated at 35 C for 48 hr. The susceptibilities of
the three species to six antifungal drugs were similar.
They were all susceptible to micafungin and 5-FC, and
they were resistant to the three azole agents (flucona-
zole, itraconazole, and miconazole) and amphotericin B
Candida pseudohaemulonii was resistant to both
amphotericin B and the azole agents. Amphotericin B
exerts its antifungal effect through cell membrane
destabilization resulting from its selective binding to
ergosterol. Despite more than 40 years of clinical use,
resistance to amphotericin B is very rare. Only C. lusi-
taniae, C. haemulonii, and Trichosporon asahii have
reduced sensitivity to this drug (2, 9, 11, 16). Although
C. pseudohaemulonii and the two types of C. haemu-
lonii are resistant to both amphotericin B and azole
agents, they are very susceptible to 5-FC and micafun-
gin. Unfortunately, we could not obtain clinical infor-
mation on the patient from whom the strain was isolat-
ed, and the patient’s outcome was unknown. Neverthe-
less, based on the results of our in vitro drug suscepti-
bility testing, 5-FC and micafungin should be effective
for treating infection with this species. Candida
pseudohaemulonii and the two types of C. haemulonii
show the same spectrum against antifungal agents and
Table 2. Differential characteristics of C. pseudohaemulonii and C. haemulonii type I and II strains using ID32 yeast identification kit
C. haemulonii type I
C. haemulonii type II
?, positive; ?, negative; V, variable.
Table 3. In vitro susceptibility testing against six antifungal agents
MIC (µg/ml) of
C. haemulonii type I
C. haemulonii type II
AMPB, amphotericin B; FCZ, fluconazole; ICZ, itraconazole; MCZ, miconazole; MFG, micafungin; 5-FC, 5-flucy-
are very close phylogenetically. As the numbers of
immunocompromised patients have increased, many
more fungal species have been isolated from clinical
specimens. We previously isolated several Pseudozy-
ma, which are usually isolated from plants, from the
blood of Thai patients (14). In the future, fungi that are
generally regarded as non-pathogenic saprophytes or
new species will likely be isolated from clinical speci-
Taxonomically, it is desirable that a description of a
new species be based on more than one isolate. Howev-
er, as our new species was resistant to amphotericin B
and azole agents, the microorganism appears to be very
significant clinically. Therefore, we propose a new
species based on a single isolate.
Latin description of Candida pseudohaemulonii
Sugita, Takashima, Poonwan et Mekha sp. nov.
In liquido ‘YM,’ post dies 3 ad 25 C, cellulae glo-
bosae, ovoideae, aut ellipsoideae (2–7.5)?(2–15) µm,
singulae, binae, aut in fasciculis. Sedimentum for-
matur. Post unum mensem ad 17 C, pellicula fragilis et
imperfecta, insulae, et sedimentum formantur. In agaro
‘YM,’ post unum mensem ad 17 C, cultura flavido-alba
aut albo-armeniaca, seminitida, laevigata, mollis, margo
glabra. In lamina vitrea in ‘CMA,’ post dies 14 ad 17 C,
pseudomycelium formantur. Glucosum, fermentantur.
Glucosum, galactosum, L-sorbosum (lente), saccharo-
sum, maltosum, trehalosum, raffinosum, melezitosum,
amylum solubile, D-xylosum (lente), L-arabinosum
(variabile), D-ribosum (lente), L-rhamnosum, D-glu-
cosaminum, N-acetyl-D-glucosaminum, ethanolum
(lente), glycerolum, ribitolum, galactitolum, D-manni-
tolum, D-glucitolum, α-methyl-D-glucosidum (vari-
abile), glucono-δ-lactonum, acidum D-gluconicum,
acidum 2-ketogluconicum, acidum succinicum, acidum
citricum, hexadecanum, xylitolum, et L-arabinitolum
(lente) assimilantur, autem cellobiosum, lactosum,
melibiosum, inulinum, D-arabinosum, methanolum,
erythritolum, salicinum, acidum 5-ketogluconicum,
acidum DL-lacticum, inositolum, acidum saccharicum,
1,2-propanediolum, 2,3-butanediolum, acidum D-glu-
curonicum et acidum D-galacturonicum non assimilan-
tur. Ethylaminum, L-lysinum et cadaverinum assimilan-
tur, autem natrium nitrosum et kalium nitricum non
assimilantur. Ad crescentiam biotinum necessarium
est. Cultura crescens ad 37 C sed non ad 40 C. Materia
amyloidea iodophila non formatur. Ureum non
hydrolysatur. Ubiquinonum majus: Q-9. Proportio
molaris guanini?cytosini in acido deoxyribonucleico:
47 mol%. Cummutatio colori per diazonium caeruleum
Typus CBS 10099Tisolatus ex homine, in collectione
zymotica Centraalbureau voor Schimmelcultures,
Utrecht, the Netherlands.
Description of Candida pseudohaemulonii Sugita,
Takashima, Poonwan et Mekha sp. nov.
In YM broth, after 3 days at 25 C, the vegetative
cells are globose, ovoid, or ellipsoid, (2–7.5)?(2–15)
µm, single, in pairs, or in groups. Sediment is formed.
After 1 month at 17 C, an almost complete fragile ring,
islets, and sediment are present. On YM agar, after 1
month at 17 C, the streak culture is yellowish white to
orange-white, smooth, semi-shiny, soft, and has an
entire margin. On slide culture on cornmeal agar, after
14 days at 17 C, pseudomycelia with blastoconidia are
produced (Fig. 2). Glucose is fermented. Assimilation
of carbon compounds: glucose, galactose, L-sorbose
(latent), sucrose, maltose, trehalose, raffinose, melezi-
tose, soluble starch, D-xylose (latent), L-arabinose (vari-
able), D-ribose, L-rhamnose, D-glucosamine, N-acetyl-
glucosamine, ethanol (latent), glycerol, ribitol, galactitol,
D-mannitol, D-glucitol, α-methyl-D-glucoside (variable),
glucono-δ-lactone, D-gluconate, 2-ketogluconic acid,
succinic acid, citric acid, hexadecane, xylitol, and L-ara-
binitol (latent). No growth occurs on cellobiose, lac-
tose, melibiose, inulin, D-arabinose, methanol, erythritol,
salicin, 5-ketogluconic acid, DL-acetic acid, inositol,
saccharate, propane-1,2-diol, butane-2,3-diol, D-glu-
curonic acid, or D-galacturonic acid. Assimilation of
nitrogen compounds: ethylamine hydrochloride, L-
lysine, and cadaverine; negative for potassium nitrate or
sodium nitrite. Biotin required for growth. Growth is
positive at 37 C, but negative at 40 C. Starch-like com-
pounds are not produced. Urease activity and diazoni-
um blue B reaction are negative. The major ubiquinone
is Q9. Mol% G?C is 47%.
The type strain (CBS 10099T?JCM 12453T?
DMST 17134T) was isolated from a human.
T. SUGITA ET AL
Fig. 2. Pseudomycelia of Candida pseudohaemulonii CBS
10099T. Scale bar indicates 10 µm.
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