Isolation of cholesterol-dependent, multidrug-resistant Candida glabrata strains from blood cultures of a candidemia patient in Kuwait
BMC Infectious Diseases
Isolation of cholesterol-dependent, multidrug-resistant Candida glabrata strains from blood cultures of a candidemia patient in Kuwait
Ziauddin Khan 0
Suhail Ahmad 0
Leena Joseph 0
Khaled Al-Obaid 1
0 Department of Microbiology, Faculty of Medicine, Kuwait University , P. O. Box 24923, Safat 13110 , Kuwait
1 Microbiology Unit, Al-Amiri Hospital , Safat , Kuwait
Background: Candida glabrata has emerged as an important human pathogen associated with systemic and mucosal infections. Here, we describe isolation of two cholesterol-dependent Candida glabrata strains from a candidemia patient which failed to grow on the media devoid of a cholesterol source. Methods: Both the isolates were recovered from BACTEC Plus Aerobic/F blood culture bottles of a candidemic patient. Since these isolates failed to grow on Sabouraud dextrose agar, Mueller-Hinton agar and RPMI 1640 agar media, their definitive identification required PCR sequencing of the internally transcribed spacer (ITS)1 and ITS2 regions of rDNA and the D1/D2 region sequences within 26S rRNA gene. The cholesterol auxotrophy was determined by their ability to grow on media containing a cholesterol source. The minimum inhibitory concentrations (MICs) to antifungal agents were determined by Etest. Results: The identity of the isolates was confirmed by sequencing of the ITS1 and ITS2 regions of rDNA and the D1/D2 region sequences within 26S rRNA gene and also by matrix-assisted laser desorption and ionization-timeof-flight mass spectrometry with 99.9% confidence value. Both the isolates showed good growth only when media were supplemented with cholesterol, oxbile or blood. Additionally, these isolates were resistant to amphotericin B (MIC ≥32 μg/ml), fluconazole (MIC ≥256 μg/ml), voriconazole (MIC ≥32 μg/ml), itraconazole (MIC ≥32 μg/ml), and posaconazole (MIC ≥32 μg/ml), but susceptible to caspofungin (MIC range 0.064 to 0.19 μg/ml). Conclusion: This appears to be the first report on isolation of cholesterol-dependent strains of C. glabrata from a candidemia patient exhibiting resistance to azoles and amphotericin B. Further, the report demonstrates that induction of cholesterol/sterol auxotrophy is associated with resistance to antifungal drugs targeting ergosterol biosynthesis. These observations may have therapeutic implications for the treatment of infections caused by such C. glabrata strains.
Candida glabrata; Drug resistance; Cholesterol auxotrophy; Candidemia
Candida glabrata has emerged as the second most
important yeast associated with mucosal and systemic
infections in critically ill patients in some tertiary care hospitals
in North America [
]. The species is intrinsically less
susceptible to azoles and it is generally believed that extensive
topical and systemic use of these drugs might have
contributed to its rising incidence as a human pathogen.
There are multiple mechanisms that impart resistance
against azoles. These include alterations in ERG11 gene
that encodes for 14- α-methyl sterol demethylase in
ergosterol biosynthesis and/or upregulation of efflux pumps
encoded by CgCDR1 and CgCDR2 genes [
Additionally, alterations in ERG3 gene encoding Δ5,6 sterol
desaturase have been noted in strains carrying mutations in ERG11
to allow survival under aerobic conditions [
]. Here, we
describe the isolation of two C. glabrata strains from
bloodstream of a candidemia patient which required exogenous
cholesterol/sterol for growth in media that are routinely
used in clinical mycology laboratories.
Isolation of C. glabrata strains
The isolates, Kw1018/12 and Kw1154/12, were obtained
9 days apart from two blood samples at Al-Amiri hospital,
Kuwait. Initially, the isolates grew slowly in BACTEC
Plus blood culture bottles with detection time of 54 and
75 hours, respectively and were tentatively identified by
Vitek2 yeast identification system as C. glabrata. The
isolates were received at Mycology Reference Laboratory,
Faculty of Medicine, Kuwait University for further
identification and antifungal susceptibility testing. When the
isolates were subcultured on Sabouraud dextrose agar (SDA),
they failed to grow. Similarly, no growth was obtained on
RPMI medium with 2% glucose and Mueller-Hinton agar
(MHA). Surprisingly, the isolates grew well on blood agar
and chocolate agar. Addition of oxbile (2%), cholesterol
(20 μg/ml) or sheep blood (5%) to SDA, MHA or RPMI
supported good growth of the isolates.
Molecular characterization of the isolates
Further species-specific identification of the cultured
isolates was carried out by PCR sequencing of the internally
transcribed spacer (ITS)1 and ITS2 regions of rDNA
and the D1/D2 region sequences within 26S rRNA gene.
Genomic DNA from the isolates was prepared and the ITS
region of rDNA and D1/D2 regions of 26S rRNA gene were
amplified and sequenced as described previously [
PCR Sequencing of ERG11 and ERG3 genes
The complete ERG11 gene (including 5′ and 3′
untranslated regions) was amplified by PCR using rTth DNA
polymerase (Applied Biosystems, Foster City, CA, USA),
ERG11F (5′-TCCACCTCGAACCCGTATA-3′) and ERG11R
(5′-TCCATGTTGATATTCACGATGACT-3′) primers and
by following the amplification and cycling conditions
described previously [
]. The 1923 bp amplicons were
sequenced by using Big-Dye terminator cycle sequencing
kits (Applied Biosystems) and automated DNA sequencer
3130xl using ERG11FS1 (5′-GAACCCGTATACTCATC
TCGTA-3′), ERG11FS2 (5′-GGTGATATCTTCTCTTTC
ATGCTA-3′), ERG11FS3 (5′-GACGTGAGAAGAACGA
TATCCA-3′), ERG11FS4 (5′-GTTACACTCACTTGCAA
GAAGAA-3′), ERG11RS1 (5′-CACGATGACTTACTAT
TAGGCTAA-3′), ERG11RS2 (5′-CGAAACCGTAATCA
ACTTCGTCA-3′), ERG11RS3 (5′-ATCAAGACACCAA
TCAATAGGTT-3′), or ERG11RS4 (5′-AGTAAGCAGCT
TCAGCGGAAACA-3′) as sequencing primer [
complete ERG3 gene (including 5′ and 3′ untranslated
regions) was also amplified by PCR using rTth DNA
polymerase, ERG3F (5′-AGAGATGAGGCCTGGAAG
AAGA-3′) and ERG3R (5′-AAATATGAGAACCCAGG
TCAGCA-3′) primers and by following the amplification
and cycling conditions described previously [
1647 bp amplicons were sequenced by using ERG3FS1
(5′-CTTGTGCAAGGCCTTGTAGACA-3′), or ERG3RS4
sequencing primer [
]. The complete ERG11 and ERG3 gene
sequences were assembled and were compared with the
corresponding sequences from reference C. glabrata
strain 2001-L5 by using the program Clustal Omega
Since both bloodstream C. glabrata strains failed to grow
on RPMI medium, minimum inhibitory concentrations
(MICs) were determined by Etest on RPMI medium
containing 2% glucose and supplemented with cholesterol
(20 μg/ml) and Mueller-Hinton agar (MHA) medium
supplemented with cholesterol (20 μg/ml) or 5% sheep blood.
For comparison, C. glabrata strain (ATCC 90030) and a
recent blood culture isolate (Kw2273/13) were used as
controls. The Etest method was performed using Etest
strips (bioMérieux, Marcy-ĺEtoile, France) for fluconazole,
itraconazole, voriconazole, caspofungin and amphotericin
B. A standardized inoculum suspension of each isolate
equivalent to a 0.5 McFarland standard was prepared.
Plates were inoculated uniformly with cotton swab and
allowed to dry before Etest strips were applied. MICs were
determined after 48 h of incubation at 35°C. Azole MICs
were read as the lowest concentrations producing an
80% reduction of growth. The isolates were considered
resistant following the breakpoints described recently
for Candida spp. [
Since these investigations were part of the routine
diagnostic service offered by Mycology Reference Laboratory,
no patient consent was required.
The isolates showed moderate to good growth on blood
agar, chocolate agar and routine culture media
supplemented with oxbile (2%), cholesterol (20 μg/ml) or sheep
The complete ITS and D1/D2 region sequences of isolates
Kw1018/12 and Kw1154/12 were assembled and the
corresponding sequences from the two isolates were identical.
In BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST/
Blast.cgi?), the ITS and D1/D2 region sequences of isolates
Kw1018/12 and Kw1154/12 (EMBL accession nos. HE
993756/7 and HE 998775/6, respectively) showed only 7
and 1 nucleotide differences with the corresponding
sequences from reference C. glabrata strain (CBS 138). The
ITS region sequence is also available from another
reference C. glabrata strain (ATCC 90030) and showed only 1
nucleotide difference with the corresponding sequences
from isolates Kw1018/12 and Kw1154/12. Thus, the ITS
and/or D1/D2 region sequences of both the isolates
exhibited >99% sequence identity with the corresponding
sequences from the reference C. glabrata strains confirming
their identity as C. glabrata. The species-specific identity
of the isolates was also confirmed by matrix-assisted laser
desorption and ionization–time-of-flight mass
spectrometry (MALDI–TOF MS; bioMeriuex) with 99.9%
The ERG11 gene sequences of our isolates (EMBL
accession number HF952117) showed four synonymous
mutations within the coding region compared to the sequence
from reference C. glabrata strain 2001-L5 [
]. Similarly, the
ERG3 gene sequences of our isolates (EMBL accession
number HF952118) showed three synonymous mutations
within the coding region compared to the sequence from
reference C. glabrata strain 2001-L5. No non-synonymous
mutations were detected and no promoter mutations were
apparent in both ERG11 and ERG3 genes.
Both our auxotrophic C. glabrata strains were
resistant to amphotericin B (MIC ≥32 μg/ml), fluconazole
(MIC ≥256 μg/ml), voriconazole (MIC ≥32 μg/ml),
itraconazole (MIC ≥32 μg/ml), and posaconazole (MIC ≥32 μg/ml),
but appeared susceptible to caspofungin (MIC range 0.064
to 0.19 μg/ml) on all the media used (Figure 1, Table 1)
]. In contrast, the MICs of control strain of C. glabrata
(ATCC 90030) as well as of a recent blood culture isolate
(Kw2273/13) were within susceptible range (Table 1).
A literature review revealed that some strains of C. glabrata
require exogenous supply of cholesterol for their growth
]. Both our blood culture isolates grew on blood
agar and chocolate agar, but exhibited growth on SDA,
RPMI agar or MHA only when supplemented with
oxbile (2%), cholesterol (20 μg/ml) or sheep blood (5%).
Isolation of cholesterol-requiring C. glabrata strains
from candidemia patients may not be difficult since
patient’s blood can serve as a source of cholesterol.
However, C. glabrata strains requiring exogenous cholesterol
for growth have also been recovered from non-blood
specimens and were found to be resistant to antifungal
drugs that act on ergosterol biosynthesis [
patients infected with such strains may pose significant
diagnostic and therapeutic challenges because of lack of
growth on routine culture media, but will exhibit in vivo
growth due to their ability to maintain integrity of cell
membrane by utilizing exogenous cholesterol available
in blood/tissue milieu. Consequently, such strains can
persist at the site of infection in spite of adequate therapy
with azoles or amphotericin B. Although
cholesteroldependent strains of C. glabrata are rarely encountered, it
is possible that isolation of some such strains might be
missed if specimens are cultured only on routine media
(devoid of a cholesterol source). Here, it is pertinent to
mention that C. glabrata is prone to developing reduced
susceptibility/cross-resistance to multiple antifungal
agents without cholesterol/sterol auxotrophy [
The development of cholesterol auxotrophy seemingly
does not affect susceptibility to caspofungin or other
echinocandins, which is the treatment of choice for C.
glabrata infections .
The patient from whom the two isolates (Kw1018/12
and Kw1154/12) were recovered was initially treated
with liposomal amphotericin B (AmBisome, 5 mg/kg)
for three weeks and later with caspofungin (70 mg loading
dose, followed with 50 mg daily dose) for 7 days. However,
the patient died of multi-organ failure and septic shock. It
is probable that the appropriate therapy with caspofungin
was delayed in our patient until C. glabrata was isolated
and its resistance against amphotericin B and azoles was
The major mechanisms known to mediate resistance to
azoles in C. glabrata involve upregulation and/or other
mutations in ERG11 and efflux pumps. However,
resistance to azoles in our isolates was not due to mutations in
ERG11 gene and non-synonymous mutations were also
not detected in the ERG3 gene. Although the nucleotide
sequences of both genes varied slightly, no promoter
mutations were detected and the encoded protein sequences
(EMBL accession numbers HF952117 and HF952118)
were also identical to reference C. glabrata strain 2001-L5
]. Upregulation of efflux pumps was not studied in the
present investigation. Thus, the molecular basis of
resistance to azoles in our isolates remained unidentified. It is
probable that upregulation of efflux pumps or mutation(s)
in some other genes that have been recently shown to
confer resistance to antifungal drugs in C. glabrata
are involved in our isolates [
]. Occurrence of
such strains poses a major threat for proper
management of such patients, particularly when resistance to
echinocandins among C. glabrata strains is also
emerging rapidly .
Two cholesterol-dependent C. glabrata strains isolated
from the blood of a candidemic patient are described.
The report demonstrates that induction of cholesterol/
sterol auxotrophy in C. glabrata may impart resistance
to antifungal drugs targeting ergosterol biosynthesis.
The observations may have therapeutic implications.
Early therapy with echinocandins may be optimal to
overcome the problem of triazole/amphotericin B
resistance in such isolates. A limitation of our study is that
the molecular basis of resistance to azoles in our isolates
remained unidentified as only two genes involved in
ergosterol biosynthesis were studied while efflux pumps
that can also contribute towards resistance to azoles
were not investigated. To our knowledge, this is the first
report on the isolation of cholesterol-dependent strains
of C. glabrata from a candidemia patient exhibiting
resistance to azoles and amphotericin B.
AP: Amphotericin B; FL: Fluconazole; VO: Voriconazole; IT: Itraconazole;
POS: Posaconazole; CS: Caspofungin.
The authors declare that they have no competing interests.
ZUK and SA drafted the manuscript. LJ performed antifungal susceptibility
and molecular identification studies. KA collected the clinical data. All
authors read and approved the final version of the manuscript.
The authors are thankful to Ms. Sandhya Vayalil and Rachel Chandy for
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