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Fluconazole was used to test the susceptibility of Candida albicans isolated from different clinical samples, and to detect mutations in ERG11 gene, and their relationship to fluconazole resistance. Forty-eight isolates of Candida albicans were tested for susceptibility using the disc diffusion method (M-44). ERG11 genes of six isolates were amplified (four resistant, two susceptible) and sequenced. The sequenced genes were analyzed to detect the mutations. Out of 48 isolates of Candida albicans, 4 (8%) were resistant to fluconazole. Sixteen-point mutations were detected included 13 silent mutations, and three missense mutations. The mutations of A945C (E266D) and G1609A (V488I) were found only in susceptible Candida albicans isolates, while the mutation of G1456A (V437I) was detected only in resistant Candida albicans isolates. Candida albicans had a high susceptibility against fluconazole. The amino acid substitutions of E266D and V488I have no role in fluconazole resistance, while the substitution of V437I may have a role in developing resistance against fluconazole. Multiple point mutations in ERG11 gene may develop resistance to fluconazole.
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MJBMB, 2020, 1, 57 - 61
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MUTATIONS IN ERGOSTEROL 11 GENE OF FLUCONAZOL RESISTANT Candida albicans
ISOLATED FROM DIFFERENT CLINICAL SAMPLES
Dhiey A. Al-Aameri1 and Bareq N. Al-Nuaimi2*
1Department of Biology, University of Baghdad, Baghdad, Iraq
2Department of Biology, Madenat Alelem University College, Baghdad, Iraq
*Corresponding Author: bariqnihad.bn@gmail.com
History
Abstract
Received: 13th October 2019
Accepted: 4th March 2020
Fluconazole was used to test the susceptibility of Candida albicans isolated from different
clinical samples, and to detect mutations in ERG11 gene, and their relationship to fluconazole
resistance. Forty-eight isolates of Candida albicans were tested for susceptibility using the disc
diffusion method (M-44). ERG11 genes of six isolates were amplified (four resistant, two
susceptible) and sequenced. The sequenced genes were analyzed to detect the mutations. Out of
48 isolates of Candida albicans, 4 (8%) were resistant to fluconazole. Sixteen-point mutations
were detected included 13 silent mutations, and three missense mutations. The mutations of
A945C (E266D) and G1609A (V488I) were found only in susceptible Candida albicans isolates,
while the mutation of G1456A (V437I) was detected only in resistant Candida albicans isolates.
Candida albicans had a high susceptibility against fluconazole. The amino acid substitutions of
E266D and V488I have no role in fluconazole resistance, while the substitution of V437I may
have a role in developing resistance against fluconazole. Multiple point mutations in ERG11 gene
may develop resistance to fluconazole.
Keywords:
Fluconazole, mutations, resistance,
susceptibility
INTRODUCTION
Candida albicans is the most common human opportunistic
fungal pathogen among other Candida species [1]. Candida
spp. infects different anatomical sites of the human body. It
resides on the mucosa of the gastrointestinal tract, vagina,
mouth, and esophagus. [2][3] Candidiasis is classified as
superficial in cutaneous and mucosal infections of healthy
individuals, and invasive or systemic in deep and widespread
infections of immunocompromised individuals. [4] Candida
albicans is the most prevalent species developing
candidiasis. About 50% of world wild candidiasis infections
are caused by C. albicans.
[5] Immunosuppression and azole long-term therapy
are the main factors for developing azole resistance. Because
of its high oral bioavailability, fluconazole is prescribed to
treat AIDS patients with oropharyngeal candidiasis. Most of
HIV-patients have fluconazole-resistant C. albicans in their
oral cavities. [6] While azole-resistant C. albicans is less
common in patients with vaginal candidiasis and
Candidemia. These patients have azole resistance incidence
about 0-5% because they receive a shorter course of azole
therapy [7]. The mechanism of Azole resistance can be either
qualitative or quantitative changes in the target 14 α-
demethylase enzyme.
Qualitative changes include reducing the affinity
between the antifungal and the target enzyme. Quantitative
changes include the accumulation of the enzyme inside the
cell. Modifications in ergosterol of Candida cell wall lead to
impaired uptake of the drug, or decreased accumulation of
drug inside the cell [8] [9]. Different molecular mechanisms
contribute to cell wall modifications. These mechanisms
include alteration in ERG11 gene encoding of the 14 α-
demethylase enzyme, and overexpression of genes encoding
for membrane transport proteins [10].
ERG11 gene (previously ERG16 and CYP51A1)
encodes the enzyme lanosterol demethylase in ergosterol
biosynthesis pathway. This gene contains 1851 bp of
nucleotides. The transcription start codon is at 148-150 bp
and the stop codon is at 1732-1734 bp. ERG11 reference
sequence in gene bank is deposited under the no. of X13296
[11]. Molecular alteration in ERG11 gene is either point
mutations in the encoding region, which lead to decreased
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MJBMB, 2020, 1, 57 - 61
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affinity between the enzyme and azoles, or overexpression
of ERG11 gene that increases the production of the enzyme,
making it difficult for the drug to inhibit the enzyme [10].
ERG11 point mutations lead to native amino acid
replacement in the product protein (ERG11P). By analyzing
the sequence of C. albicans resistant isolates, point
mutations in ERG11 were identified. These mutations not
distributed randomly. They were clustered in a hot spot
region of the protein's structure, therefore, reducing the
affinity of the ERG11P protein to the drug [12].
MATERIALS AND METHODS
Samples Collection
Samples collected from forty-eight (48) patients of
candidiasis. They were isolated from sputum, vagina, mouth,
and urine and according to ethical considerations.
Fluconazole Susceptibility Test
Susceptibility of C. albicans isolates to fluconazole was
measured using M-44 disc diffusion method, which is a
global guideline recommended by CLSI for disk diffusion
testing of Candida sp. Mueller Hinton agar (2% glucose +
0.5 methylene blue dye), and Fluconazole 25 µg disc were
used to perform the test.
Reading of Results of Disc Diffusion
After 24 hours of incubation the diameter of the inhibition
zone was measured in millimeters using a metric ruler.
According to the measured diameters, the results translated
as sensitive (S), susceptible dose-dependent (S-DD), and
resistance (R), and they were compared with the standard
inhibition zone in (Table 1).
Table 1: Zone diameter and corresponding minimal inhibitory
concentrations (MIC) breakpoints for Candida spp. (NCCLS,
2004)
agent
Disk
content
Zone diameter
(mm)
Equivalent MIC
breakpoints
(µg/mL)
Fluconazole 25 µg
R S-DD S R S-DD
S
4
15-18
19
64
16-32
8
DNA Extraction
DNA extraction was performed using Quick-DNATM
Fungal/Bacterial Miniprep Kit from zymoresearch. Quick-
DNATM Fungal/Bacterial Miniprep Kit is designated for
simple, rapid isolation of DNA from tough to lyse fungi
including Aspergillus fumigatus, Candida albicans,
Neurospora crassa, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and from mycelium and Gram
(+) and () bacteria. The procedure is easy and can be
completed in 15 minutes. The fungal and bacterial samples
were added to a ZR Bashing BeadsTM Lysis tube (0.1mm &
0.5 mm) and rapidly and efficiently lysed by bead beating
without using organic denatures or proteinases. The DNA
was isolated and purified using Zymo-spinTM technology
and ideal for downstream molecular-based applications.
Amplifying and Sequencing of ERG11 Gene
ERG11 gene of six isolates (four resistant, two susceptible)
was amplified and sequenced. Three pairs of primers were
designated by referring to Xu et al. (2008) (Table2).
Polymerase chain reaction (PCR) was carried out in 25 µl
reaction containing 12.5 µl of green master mix, 1µl of 10
pmol/µl of each primer, and 2µl of the template DNA. The
volume was topped up to 25 µl by adding 8.5 µl of nuclease-
free water. PCR condition was as follows: 95 ºC for 5 min,
95 ºC for 30 sec, 56 ºC for 30 sec, 72 ºC for 30 sec, for 33
cycles, and 72 ºC for 7 min. The PCR products were
sequenced using ABI3730XL automated DNA sequencer.
RESULTS AND DISCUSSION
Fluconazole susceptibility
Out of 48 C. albicans isolates only 8% was resistant to
fluconazole. The four isolates (89, 90, 82, and 20) were
isolated from vaginal swab, and sputum. About 10.42% was
susceptible to dose-dependent, and 81.25% was susceptible
to fluconazole (Table3).
Amplifying and mutations of ERG11 gene
The gene amplified was classified into three sections with a
length of 488, 454, and 462 bps, respectively. The three
sections were analyzed by comparing them with the
referenced ERG11 sequence in the gene bank under the
accession number of X123296 using BioEdit multiple
alignment. The amplified product was extended from 322 to
1664 bps of the gene. Based on the results of gene sequence
analysis, thirteen silent mutations, and three missense
mutations were detected in the ERG11 gene (Table 4). The
amplified sequences are under the accession number of
MH468713-MH468727 in NCBI gene bank.
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Table2: Primers and expected PCR products for ERG11 amplification
Primer Strand Sequence
Expected PCR
product
ERG11sec1
Forward
-TTAGTGTTTTATTGGATTCCTTGGTT-
259-777 bp
Reverse
-TCTCATTTCATCACCAAATAAAGATC-
ERG11sec2
Forward
-ACCAGAAATTACTATTTTCACTGCTTCA-
723-1204 bp
Reverse
-AAGTCAAATCATTCAAATCACCACCT-
ERG11sec3
Forward
-AGGTGGTGATTTGAATGATTTGACTT-
1179-1667 bp
Reverse
-GAACTATAATCAGGGTCAGGCACTTT-
Table 3: Distribution of Candida albicans in samples and their fluconazole susceptibility
Site of isolation (%)
Fluconazole susceptibility
(%)
Fungus
Vaginal
swab
Sputum
Oral
swab
Urine
S
S-DD
R
Candida
albicans
18 (37.50)
17 (35.42)
8 (16.67)
5 (10.42)
39
(81.25)
5 (10.42)
4
(8.33)
*S: sensitive, R: resistant, S-DD: susceptible dose-dependent
Amplifying and Mutations of ERG11 Gene
The gene amplified was classified into three sections with a
length of 488, 454, and 462 bps, respectively. The three
sections were analyzed by comparing them with the
referenced ERG11 sequence in the gene bank under the
accession number of X123296 using BioEdit multiple
alignment. The amplified product was extended from 322 to
1664 bps of the gene. Based on the results of gene sequence
analysis, thirteen silent mutations, and three missense
mutations were detected in the ERG11 gene (Table 4). The
amplified sequences are under the accession number of
MH468713-MH468727 in NCBI gene bank.
Disc diffusion is a qualitative test, but CLSI
provided a correlation between MIC values in broth dilution
methods and zone inhibition measurements of disc diffusion.
It is suitable for the susceptibility testing of water-soluble
antifungals such as fluconazole. [13] Candida albicans in the
area of the study showed a high susceptibility to fluconazole.
The isolates were from patients with no immunosuppression
or patients with HIV, the latest usually show a lower
susceptibility to fluconazole, due to long-term antifungal
therapy. [11]
The amplified product in our study was extended
from 332 to 1664 bp of the gene, since the hot spot regions
of ERG11P located at 105-165, 266-287, and 405-488 [14].
The isolates (3, 10) were isolated from different patients, but
sharing the same mutations with nine silent (T462C, C558T,
C805T, T1143C, A1173G, C1257T, T1350C, C1443T, and
T1449G) including two missense mutations (A945C, and
G1609A), and four silent mutations (A945C, G1609A,
C1443T, and T1449C) detected only in these susceptible
isolates (Table 5). These data can be an evidence that these
two isolates are belonging to the same strain. The point
mutations are allelic variation between different strains, not
necessarily associated with resistance [15].
The missense mutations (A945C) and (G1609A)
led to the substitution of Glutamic acid by Aspartic acid in
position of 266 (E266D), and Valine by Isoleucine in
position of 488 (V488I), respectively found together in two
fluconazole susceptible isolates (48, 70) with no other
missense mutations in both of them. The same mutations in
C. albicans isolates presented by Maebashi et al ) .2003 .( The
substitution of E266D located in G-helix of an enzyme
product, which covers a part of the active site, but probably
does not contribute to fluconazole resistance, because it
detected in both resistant and susceptible C. albicans isolates
[11][15]. Golabek et al. (2014) reported that D266E
decreasing gene expression. The amino acid encoded by the
mutation of G1609A with the substitution of V488I is at a
position far from the active site [16]. Maebashi et al. (2003)
had detected this mutation in resistant and susceptible
isolates, so it may not confirm fluconazole resistance. The
substitution of V437I was found in three resistant isolates
(89, 90, and 82). The substitution of V437I located in the
third portion of the hot spot region indicating V437I may
have a role in raising resistance to fluconazole. V437I also
reported in resistant C. albicans by Wang et al. (2015).
The silent mutations of T462C, C558T, C805T,
A1173G, C1257T, and T1350C were found in resistant and
susceptible isolates. While C1443T and T1449C were found
only in susceptible isolates. These two mutations were also
reported by Taraskina et al. (2016), but in resistant isolates.
These mutations have no role in fluconazole-resistant but
may have if they combine with specific mutations or other
mechanisms [12]. Four silent mutations T696C, T1404C,
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Table 4: Mutations detected in Candida albicans isolates showing the nucleotide changes and resulting amino acid substitutions in the
protein.
Mutations
(Nucleotide
changes)
Mutations in codons Type of mutations ERG11p changes/positions
(amino acid substitution)
T462C
TTT→TTC
Silent mutation
F105
C558T
TCC→TCT
Silent mutation
S137
T696C
CAT→CAC
Silent mutation
H183
C805T
CTA→TTA
Silent mutation
L220
A945C
GAA→GAC
Missense
mutation
E266D
T1143C
GTT→GTC
Silent mutation
V332
A1173G
AAA→AAG
Silent mutation
L340
C1257T
CTC→CTT
Silent mutation
K342
T1350C
TAT→TAC
Silent mutation
Y401
T1404C
CCT→CCC
Silent mutation
P419
C1443T
GCC→GCT
Silent mutation
A432
T1449C
GCT→GCC
Silent mutation
A434
G1456A
GTT→ATT
Missense
mutation
V437I
A1587G
TTA→TTG
Silent mutation
L480
G1609A
GTT→ATT
Missense
mutation
V488I
T1617C
AAT→AAC
Silent mutation
N490
Table 5: Distribution of nucleotide changes and the resulted amino acid substitutions of each isolate.
ERG11p
Samples
48 (S) 70 (S) 89 (R) 90 (R) 82 (R) 20 (R)
F105
T462C
T462C
T462C
T462C
T462C
T462C
S137
C558T
C558T
C558T
C558T
C558T
C558T
H183
---
---
T696C
T696C
---
T696C
L220
C805T
C805T
C805T
C805T
C805T
C805T
E266D
A945C
A945C
---
---
---
---
V332
T1143C
T1143C
T1143C
T1143C
T1143C
---
L340
A1173G
A1173G
---
---
---
A1173G
K342
C1257T
C1257T
C1257T
C1257T
C1257T
C1257T
Y401
T1350C
T1350C
T1350C
---
---
---
P419
---
---
T1404C
---
---
T1404C
A432
C1443T
C1443T
---
---
---
---
A434
T1449C
T1449C
---
---
---
---
V437I
---
---
G1456A
G1456A
G1456A
---
L480
---
---
A1587G
A1587G
A1587G
A1587G
V488I
G1609A
G1609A
---
---
---
---
N490
---
---
T1617C
T1617C
T1617C
T1617C
*S= sensitive, R= resistant.
MJBMB, 2020, 1, 57 - 61
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C1587G, and T1617C were detected only in resistant
isolates. T696C was reported by Taraskina et al. (2016) in
resistant C. albicans, while T1404C, C1587G, and T1617C
were detected by Strzelczyk et al. (2013) in both sensitive
and susceptible isolates. Isolate no. 20, which was FLC
resistant, contained nine-point mutations, and no amino acid
substitution. These silent mutations that present only in
resistant isolates may be associated with FLC resistance by
effecting gene expression or by combining with other
mutations. Multiple mutations in ERG11 can result in
decreasing azole susceptibility, since one mutation is not
enough to raise the resistance. Some mutations do not have
an effect until they combine with one or more mutation(s).
[17][12].
Resistance to fluconazole evolves when the target
enzyme changed either by point mutations in ERG11 gene or
increasing gene expression. ERG11 genes characterized by a
genetic polymorphism, more than 140 missense mutations
have been reported. The presence of single point mutation in
the gene is not enough to change the affinity between
fluconazole and the target enzyme, whereas the presence of
multiple mutations could raise the resistance of C. albicans
[12]. Other mechanisms like upregulation of active efflux
transporter gene, alterations in other genes that responsible
for ergosterol biosynthesis pathway polymorphism, and
biofilm formation also contribute to fluconazole resistance
[18].
ACKNOWLEDGMENT
The authors would like to thank Dr. Batool, Dr. Imran, Dr. Ghusoon
Ali and Dr. Nemat Jamel for scientific support. Special thanks also
to Dr. Nasr Noori Al Anbari for the statistical work and the staff of
teaching laboratories of medical city teaching hospital.
CONFLICT OF INTEREST
The article including the tables is original. It was written by the
stated authors and was not published elsewhere. This manuscript
was also not submitted to, nor under review at another journal or
other publishing venue. The authors have no affiliation with any
organization with a direct or indirect financial interest in the subject
matter discussed in the manuscript.
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