Population structure and molecular genetic characterization of clinical Candida tropicalis isolates from a tertiary-care hospital in Kuwait reveal infections with unique strains
Population structure and molecular genetic characterization of clinical Candida tropicalis isolates from a tertiary-care hospital in Kuwait reveal infections with unique strains
Khaled Al-Obaid 0 1
Mohammad Asadzadeh 1
Suhail Ahmad 1
Ziauddin Khan 1
☯ These authors contributed equally to this work. 1
0 Microbiology, Department of Medical Laboratories, Al-Amiri Hospital , Sharq , Kuwait , 2 Department of Microbiology, Faculty of Medicine, Kuwait University , Safat , Kuwait
1 Editor: Vishnu Chaturvedi, Wadsworth Center , UNITED STATES
Candida tropicalis is a frequently isolated yeast species causing bloodstream, urinary tract and other infections particularly in patients admitted to intensive care units (ICUs) and those requiring prolonged urinary catheterization (UC) or receiving broad-spectrum antibiotics (BSA). This study investigated clinical characteristics and genetic relatedness among C. tropicalis strains isolated from patients at Al-Amiri Hospital in Kuwait. C. tropicalis strains (n = 63) isolated from blood, genito-urinary, respiratory (RT) and digestive (GIT) tracts and wound sites from 54 patients were used. All isolates were phenotypically identified and tested against six antifungal drugs by using Vitek 2 system. Molecular identification was performed by PCR amplification of rDNA. Fingerprinting was achieved by 6-loci-based multilocus sequence typing (MLST) and data were analyzed by BioNumerics software for phylogenetic relationships. Patients mean age was >65 years and >20% patients were hospitalized in ICUs. Most patients had underlying conditions that included UC, BSA, diabetes and RT/GIT abnormalities. Most candiduria cases had UC, ureteric stent or suprapubic catheters. All isolates were identified as C. tropicalis by Vitek 2 and by species-specific PCR. Sixty-two isolates were susceptible to all tested antifungal drugs. MLST identified 59 diploid sequence types (DSTs) including 54 newly-identified DSTs. C. tropicalis isolates from multiple sites of same patient usually belonged to different DSTs. Interestingly, 56 of 57 isolates from 48 patients belonged to unique genotypes. Only six isolates from six patients belonged to three DSTs (clusters), however, C. tropicalis strains in each cluster were isolated >3 months apart. Our data show diverse origins of C. tropicalis infections in Kuwait as most isolates were unique strains. There was no obvious correlation between cluster isolates with time of isolation and/or hospital ward of their origin. This study presents the first MLST analysis of C. tropicalis isolates from Middle East and may be useful for studying genetic relationships among global C. tropicalis strains.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The study was funded by Kuwait
University Research Sector grant MI02/15 and
core facility grant SRUL02/13.
Competing interests: The authors have declared
that no competing interests exist.
Candida spp. are important constituent of human microbial flora of skin, mucous membranes
of mouth and vagina and gastrointestinal tract. They are also opportunistic human pathogens.
The infections range in severity from mild superficial infections to life-threatening
bloodstream and invasive infections inflicting considerable morbidity and mortality, particularly in
immunocompromised and hospitalized patients [
]. Candida spp. are now the fourth most
common cause of all bloodstream infections and the third most common cause of bloodstream
infections in patients in the intensive care unit (ICU) in many tertiary-care hospitals across
the world with an attributable mortality of 15±35% in adults and 10±15% in neonates [3±5].
Nearly 90% of invasive Candida infections are caused by only four species or species complexes
which include Candida albicans, Candida glabrata, Candida parapsilosis, and Candida
tropicalis [3±6]. Candida tropicalis is the second most frequently isolated Candida from bloodstream
in many countries and is also the leading cause of nosocomial fungemia and invasive fungal
infections in patients with hematologic and other malignancies in several countries [5±10]. C.
tropicalis infections are more common in older patients admitted to ICUs and those requiring
prolonged urinary catheterization (UC) or receiving broad-spectrum antibiotics (BSA) [
Antifungal drug susceptibility testing of Candida spp. isolates have also shown that the
frequency of fluconazole-resistant (including isolates that show intermediate susceptibility) C.
tropicalis strains is high (~10%) and is comparable to that of C. glabrata isolates in some Asian
6, 12, 13
Candiduria, Candida colonization or infection of the urinary tract, is also common among
hospitalized patients with predisposing diseases such as diabetes mellitus and abnormalities of
the kidney and urinary tract (urinary tract obstruction, surgery or instrumentation), especially
those in the ICU as they acquire several common risk factors such as urinary drainage devices,
parenteral nutrition, mechanical ventilation and broad-spectrum antibacterial therapy [
]. Candida spp. are third most common organisms isolated from urine samples of
hospitalized patients and age older than 65 years, female sex, diabetes mellitus and urinary tract
abnormalities are independent risk factors for developing candiduria [11, 14±16]. In many tertiary
care facilities, nearly 10% of all cultures from urine samples yield Candida spp. isolates with C.
tropicalis as the second or third most common Candida species causing candiduria [11, 15±
19]. Treatment of candiduria in patients with symptoms of urinary tract infection (UTI) is
beneficial and mortality rates are higher in hospitalized patients who have candiduria
compared to similar patients without candiduria [
7, 11, 14, 20
The origin of nosocomial Candida infections could be endogenous strains brought into the
hospital environment by the patients themselves or exogenous strains transmitted to the
patients from biomedical devices, contaminated infusates, hospital surroundings and health
care workers [
]. Two molecular typing techniques, capable of discriminating closely
related but non-identical isolates, multilocus sequence typing (MLST) based on
single-nucleotide polymorphisms within housekeeping genes with discriminatory power of 0.99 and
multilocus microsatellite typing (MLMT) based on microsatellite length polymorphism with
discriminatory power of 0.97 have mostly been used in recent years for determining genetic
relatedness among clinical C. tropicalis isolates [23±25]. Of these, MLST is a standardized
scheme based on six conserved housekeeping genes and has a publicly accessible and curated
online C. tropicalis MLST database (http://pubmlst.org/ctropicalis/) for worldwide
comparisons. Previous studies based on MLMT and MLST have shown that most C. tropicalis
infections develop as a consequence of endogenous colonization with this species, however,
outbreaks of C. tropicalis candiduria due to poor management of medical waste (urine) in
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ICUs have also been described [11, 26±29]. However, such studies have not been carried out
from any of the Middle Eastern countries.
This study performed molecular genetic characterization of C. tropicalis strains isolated at a
major tertiary-care hospital in Kuwait by MLST to ascertain nosocomial clusters/common
source of infection among patients and compared MLST data with data obtained previously
from a global collection of isolates to better understand the population structure of C.
Materials and methods
Reference strains and clinical isolates of C. tropicalis
Reference strains of C. tropicalis (ATCC 750), Candida viswanathii (CBS 1924), C. albicans
(ATCC 90028), Candida dubliniensis (CD36), C. parapsilosis (ATCC 22019), Candida
orthopsilosis (ATCC 96139), Candida metapsilosis (ATCC 96143), Lodderomyces elongisporus (CBS
2605), C. glabrata (ATCC 15545), Candida nivariensis (CBS 9983), Candida bracarensis (CBS
10154), Candida krusei (ATCC 6258) and Candida haemulonii (CBS 5149) were used.
Fiftyseven C. tropicalis isolates obtained from 48 consecutive patients at Al-Amiri, a major
tertiarycare hospital in Kuwait, during an 8-month period (March 2015 to October, 2015) were used.
Additionally, six isolates collected eight months later from six patients were also used for
comparison purposes. Most (54 of 63, 86%) isolates originated from genito-urinary and respiratory
(RT)/gastro-intestinal (GIT) tract specimens including urine (n = 38), vaginal swab (n = 1),
sputum (n = 1), endotracheal aspirate (n = 2), oral/throat swab (n = 4), tracheostomy (n = 1),
percutaneous endoscopic gastrostomy (n = 4), stool/anal swab (n = 2) and abdominal fluid
(n = 1). The remaining nine isolates were obtained from bloodstream (n = 5) and skin wound
swabs (n = 4). Thus, a total of 63 C. tropicalis isolates from 54 patients were used. The source,
date of isolation and underlying conditions among patients yielding C. tropicalis isolates are
provided in S1 Table. The clinical specimens were collected after obtaining verbal consent
from patients as part of routine patient care for the isolation of fungal pathogens and the data
were analyzed anonymously. The consent procedure and the study were approved by the
Ethics Committee of the Faculty of Medicine, Health Sciences Center, Kuwait University
(Approval no. VDR/EC/2336 dated 2-6-2015). The phenotypic identity of the isolates was
initially based on assimilation profiles on commercial Vitek 2 yeast identification system
(bioMeÂrieux, Marcy-l'EÂtoile, France) which was used according to manufacturer's instructions.
The isolates were sub-cultured on Sabouraud dextrose agar medium for molecular
identification and MLST analyses.
DNA extraction and species-specific identification of C. tropicalis isolates
Genomic DNA was extracted from 1 ml of cell suspension in Sabouraud dextrose broth by
using Gentra Puregene Yeast DNA extraction kit (Qiagen, Hilden, Germany) according to kit
instructions or by the rapid method using Chelex-100 as described previously [
identification was performed by PCR amplification of internally transcribed spacer (ITS)
region of ribosomal DNA (rDNA) by designing and using two C. tropicalis-specific primers;
CTROPF (5’-TTTATTTACAGTCAAACTTGAT-3’) and CTROPR (5’-TTAAATTCTTTCAA
ACAAACC-3’). PCR amplification and detection of amplicons by agarose gel electrophoresis
was carried out as described previously [
] except that CTROPF and CTROPR primers
were used. DNA sequencing of the entire ITS region (including ITS-1-5.8S rRNA-ITS-2) of
rDNA was also performed, as described previously [
], for five selected isolates to confirm
the results of species-specific PCR amplification. Basic local alignment search tool (BLAST)
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were carried out and >99% sequence identity with reference strain of C. tropicalis (ATCC 750)
were used for species identification. The ITS region sequence data have been submitted to
EMBL/GenBank databases under accession numbers LT837794 to LT837798.
Antifungal drug susceptibility testing
The susceptibility of C. tropicalis isolates against six antifungal drugs; amphotericin B,
5-flucytosine, fluconazole, voriconazole, caspofungin and micafungin was determined by using Vitek
2 AST system according to the manufacturer's instructions (bioMeÂrieux). The results were
interpreted according to the revised interpretive susceptibility breakpoints as recommended
by Clinical Laboratory Standards Institute (CLSI) [
]. Quality control was ensured by testing
C. albicans ATCC 90028, C. parapsilosis ATCC 22019 and C. tropicalis ATCC 750, as
recommended by CLSI.
Fingerprinting of C. tropicalis isolates by MLST
All 63 C. tropicalis isolates were analyzed by using the MLST scheme based on PCR
amplification and DNA sequencing of six housekeeping gene (ICL1, MDR1, SAPT2, SAPT4, XYR1 and
ZWF1a) fragments as described by Tavanti et al. [
]. However, we used new sets of primers
for PCR amplification of each gene target and internal primers (to avoid any interference by
primer-dimer artifacts) for more efficient sequencing of PCR amplicons [
]. The sequences
and other features of the primers used for MLST are listed in S2 Table.
The PCR amplification and cycling conditions for the six gene fragments were same as
described previously [
] except that gene-specific amplification primers (listed in S2 Table)
were used for each gene. The amplicons were purified and subjected to bi-directional
sequencing as described previously [
] except that gene-specific primers (listed in S2 Table) were
used as sequencing primers. Each sequence chromatogram was reviewed for heterozygous
(two equally strong and overlayed fluorescence peaks) nucleotide positions. The allelic profile
(allele number) for each gene and allele combinations (diploid sequence type, DST) for the
six loci for each isolate were assigned or new allele and new DST numbers were provided by
the curator, Prof. Frank C. Odds of the C. tropicalis MLST database (http://pubmlst.org/
ctropicalis/). The phylogenetic relationship of C. tropicalis isolates was also determined. Based
on the allele number for the six loci for each isolate, a dendrogram was constructed with the
clustering method, using unweighted pair group method with arithmetic averages (UPGMA)
settings of BioNumerics v7.5 software (Applied Maths, Saint-Martens-Latem, Belgium). The
isolates belonged to the same DST when they contained the same alleles for all six loci. A
cluster was defined as a group of two or more patients infected with a C. tropicalis strain
belonging to the same DST. The MLST data were further analyzed for clonal clusters by using
the eBURST package. The genetic relationship between the 63 isolates from Kuwait with 804
isolates in the central data library of C. tropicalis MLST database as of January 2017 was also
studied by using the minimum spanning tree of the BioNumerics software. The minimum
spanning tree predicts putative relationships among the isolates and records the isolates as
more closely related when five of six loci are identical.
Characteristics of patients yielding C. tropicalis isolates
We performed molecular genetic characterization and fingerprinting of 63 C. tropicalis isolates
obtained from 54 patients. One isolate each was tested from 47 patients, two isolates each were
obtained from five patients and three isolates each were tested from two patients. Multiple
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isolates from the same patient were from different anatomic sites. The majority (38 of 63, 60%)
of the isolates were obtained from urine samples from patients with candiduria while only five
isolates were obtained from bloodstream of candidemia patients.
The demographic details of the 54 patients yielding 63 C. tropicalis isolates were as follows.
Most (48, 89%) patients were Kuwaiti nationals while six patients were non-Kuwaiti expatriate
residents. The country of origin of non-Kuwaiti patients included Egypt (n = 2), Iran (n = 2),
Bahrain (n = 1) and Pakistan (n = 1). The age range of the patients varied from 11 years to 97
years old. Nearly two-thirds (35 of 54, 65%) of the patients were >65 years of age and the ratio
of male to female was nearly equal (28 males and 26 females). Four C. tropicalis isolates were
obtained from four patients who were attending the clinic or out-patient department while the
remaining 59 isolates were cultured from 50 hospitalized patients including 12 (22%) patients
from an ICU, six patients from the emergency room, three patients from the dialysis unit and
29 patients from different wards in the hospital. Various underlying conditions for Candida
infections were present in 49 of 54 patients (no information was available for four patients
who attended the clinic or out-patient department and one patient who was admitted to the
emergency room) and are summarized in Table 1. The most common underlying conditions
were the presence of a urinary catheter, kidney disease and/or urinary tract infection in 29
patients, treatment with broad-spectrum antibiotics in 29 patients and diabetes mellitus in 24
patients. Other factors included prostate hyperplasia and/or cancer in 12 patients and
gastrointestinal bleeding, liver disease and/or abdominal surgery in 10 patients. The most common
underlying condition among patients with candiduria included the presence of a urinary
catheter, urinary obstruction (e.g. benign prostatic hyperplasia) and/or kidney disease and older
(>65 years) age (Table 1).
Phenotypic and molecular identification of C. tropicalis isolates
Each isolate was subjected to identification by using Vitek 2 yeast identification system which
identified all 63 isolates as C. tropicalis. For genotypic identification, the genomic DNA
prepared from each isolate was subjected to PCR amplification of rDNA by using species-specific
CTROPF and CTROPR primers. The species-specificity of the primers for C. tropicalis DNA
was indicated by BLAST searches and was confirmed by lack of PCR amplification of rDNA
with genomic DNA from common Candida species other than C. tropicalis, as expected. Only
genomic DNA from reference C. tropicalis strain (ATCC 750) yielded an expected amplicon of
259 bp (S1 Fig). The genomic DNA from all 63 isolates yielded an amplicon of ~259 bp
confirming the identification of all isolates as C. tropicalis. The DNA sequencing of the entire ITS
region (including ITS-1-5.8S rRNA-ITS-2) of five selected isolates was also performed and
No. of patients
>65 years old
5 / 16
showed variation at only one or two nucleotide positions (<1% difference) with the
corresponding sequence from reference C. tropicalis strain (ATCC 750) further confirming the
identification of all five isolates as C. tropicalis. The sequencing data also identified three ITS
haplotypes among five C. tropicalis isolates (S2 Fig).
Phenotypic antifungal drug susceptibility testing of C. tropicalis isolates
The antifungal drug susceptibility testing (AST) of all 63 C. tropicalis isolates was performed
against six (amphotericin B, 5-flucytosine, fluconazole, voriconazole, caspofungin and
micafungin) antifungal drugs by using the Vitek 2 system and the results of AST are presented in
S3 Table. All isolates were uniformly susceptible to amphotericin B, fluconazole, voriconazole,
caspofungin and micafungin. The minimum inhibitory concentration (MIC) range for
amphotericin B varied between 0.25 mg/L to 0.5 mg/L only. The MIC values for fluconazole,
voriconazole, caspofungin and micafungin showed no variation among the isolates (S3 Table).
Only one isolate (Kw45-15) with MIC value of 16 mg/L was resistant to 5-flucytosine while the
remaining 62 isolates were susceptible to 5-flucytosine.
Genetic diversity of C. tropicalis isolates in Kuwait by MLST
The genetic diversity and population structure of C. tropicalis isolates in Kuwait was studied by
MLST. All six gene fragments were successfully amplified, sequenced by using the
corresponding gene-specific forward and reverse primers (S2 Table) and the DNA sequences were used to
obtain the allelic profiles for each gene fragment and allele combinations to obtain DSTs from
the C. tropicalis MLST database (http://pubmlst.org/ctropicalis/). A total of 88 cases of
heterozygosity were detected in the six sequenced fragments and 144 polymorphic sites were
identified, including 26 in ICL1, 36 in MDR1, 40 in SAPT2, 19 in SAPT4, 12 in XYR1 and 11 in
ZWF1a. The highest typing efficiency (1.83 genotypes per polymorphism) was obtained with
XYR1 while the lowest typing efficiency (0.27 genotypes per polymorphism) was obtained with
SAPT2. Twenty-two new alleles were identified among C. tropicalis isolates from Kuwait
during searches of the C. tropicalis MLST database (http://pubmlst.org/ctropicalis/).
The polymorphic alleles combined to form 59 DSTs among 63 C. tropicalis isolates from 54
patients. Of the 59 DSTs, only 5 DSTs (DST94, DST99, DST114, DST168 and DST238) for 6
isolates (DST238 was shared among two isolates) were present in the C. tropicalis MLST
database while 54 DSTs (DST677 to DST731) for 57 isolates (DST678, DST681 and DST688 were
shared among two isolates each) were new and were added to the C. tropicalis MLST database.
Of the 54 new DSTs, 34 DSTs were generated by rearrangement of previously known alleles
while the remaining 20 DSTs were formed due to the presence of a new allele for 1±3 genes.
The allelic profile and the final DST for each isolate are shown in S1 Table.
The fingerprinting data further showed that multiple isolates from the same patient but
recovered from different anatomic sites were genotypically different as they belonged to
different DSTs. The seven urine C. tropicalis isolates obtained from seven candiduria patients were
genotypically different than the isolates cultured from other clinical specimens of the same
patient (Table 2). Six of seven candiduria patients were >65 years old while one patient was 60
years old and six of seven were male patients. Furthermore, bloodstream isolate from one
patient belonged to a different DST than the isolates from urine and endotracheal aspirate
from the same patient. Only two non-urine isolates from one patient and originating from the
GIT (oral swab and anal swab) belonged to the same DST (Table 2). Interestingly, the two
isolates from one patient (patient no. 18) differed at all six loci, two isolates from two patients
(patient no. 9 and 14) differed at five loci, two isolates from one patient (patient no. 25)
differed at four loci and two isolates from another patient (patient no. 30) differed at three loci.
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aET aspirate, endotracheal aspirate.
bMLST-based DST, multi locus sequence type-based diploid sequence type; new DSTs detected in this study are shown in bold and DSTs shared between
two isolates obtained from same or different patients from Kuwait are underlined.
Multiple isolates from only two patients (patient no. 3 and 38) showed little or no variations
The genetic association between the DSTs of C. tropicalis isolates from Kuwait was
determined by construction of an unrooted phylogenetic tree based on MLST data. The
dendrogram (Fig 1) showed that most (55 of 63, 87%) of the isolates were dispersed as unrelated
singletons belonging to a single unique DST while only four DSTs were shared, each among
two isolates. Majority of the isolates differed at two or more loci. No obvious relationship was
found between DSTs and specimen types or patient's nationality. Among the four cluster
DSTs (DST238, DST678, DST681 and DST688), DST688 actually involved two isolates from
related clinical specimens from the same patient. The remaining three clusters included C.
tropicalis isolates obtained from similar clinical specimens from two unrelated patients in each
case. The clinical and epidemiological data from the patients yielding cluster isolates were
compared to ascertain if the cluster isolates represented cross-transmission of infection and
are presented in Table 3. The data showed that cluster isolates in each case were isolated at
different time points which varied from >3 months to >5 months. Furthermore, the isolates
were obtained from patients that were hospitalized in different wards in two of the three
The population structure of C. tropicalis isolates from Kuwait was further studied by
determining genetic relationship between the 63 isolates from Kuwait with 804 isolates (as of
January 2017) in the central data library of C. tropicalis MLST database by using the eBURST and
minimal spanning tree algorithm of the BioNumerics software. The minimal spanning tree is
depicted in Fig 2. Of the 59 DSTs detected among 63 isolates, 49 DSTs were grouped into
seven groups (groups 1, 9, 20, 28, 30, 34 and 40), and 10 DSTs were identified as singletons.
Interestingly, 43 of 49 (88%) clonal group isolates belonged to eBURST group 1 that also
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Fig 1. An UPGMA-derived dendrogram based on allelic profile of 6 gene fragments from 63 C. tropicalis isolate from
Kuwait. Similarity is presented in percentages using the scale bar in the upper left corner. The columns from left to right
include, isolate number, MLST-based diploid sequence type (DST), clinical specimen yielding the isolate, data of isolation,
hospital ward/unit where the patients were housed and eBURST group. Repeat isolates from the same patient are indicated by
alphabets within brackets and DSTs for cluster isolates are shown by an asterisk (*) before the DST.
8 / 16
included most of the isolates from other geographical locations in the data base. Furthermore,
most of the isolates from Kuwait clustered with isolates from several Asian (China, Taiwan
and South Korea) countries while few isolates also clustered with C. tropicalis isolates from the
United Kingdom and Brazil (Fig 2).
This study is the first MLST analysis to characterize clinical C. tropicalis isolates from a Middle
Eastern country and compared the patterns of variation with those in the MLST database
representing isolates from other geographic regions. A total of 63 isolates obtained from 54
patients were analyzed. Consistent with earlier reports, most candiduria patients had several
underlying conditions for Candida infections including age older than 65 years, urinary
drainage devices, urinary obstruction (e.g. prostate hyperplasia) and/or kidney disease, prior use of
broad-spectrum antibiotics and diabetes mellitus, however, unlike other studies, male patients
outnumbered females [14±18]. Furthermore, all candidemia cases were associated with
All 63 isolates were phenotypically identified by Vitek 2 yeast identification system and
genotypically by PCR amplification of rDNA by using novel C. tropicalis-specific (CTROPF
and CTROPR) primers as C. tropicalis. The ITS sequencing data from five selected isolates
identified three haplotypes showing genotypic heterogeneity among C. tropicalis isolates.
Three ITS haplotypes were also identified among 48 C. tropicalis isolates while four haplotypes
were identified for D1/D2 domains of rDNA in a recent study from Beijing, China [
ITS region sequences show greater variation than D1/D2 domain sequences within rDNA [
]. It is, therefore, probable that sequencing of a larger number of C. tropicalis isolates from
Kuwait and other geographical locations may identify additional ITS haplotypes as it was
recently observed for clinical C. dubliniensis isolates [39±41].
The AST data obtained by using the Vitek 2 AST system which is considered comparable to
the reference broth microdilution method [
] showed that all 63 C. tropicalis isolates were
uniformly susceptible to amphotericin B, fluconazole, voriconazole, caspofungin and
micafungin with MIC values showing little variation among the isolates. Thus, antifungal drug
resistance was not a serious concern among C. tropicalis isolates at Al-Amiri Hospital in Kuwait.
This could be due to relatively low antifungal consumption for prophylactic or empiric use as
there are no hematology or oncology wards in our hospital. The findings are similar to two
other studies from China and Taiwan which showed that resistance to fluconazole and other
antifungal agents was rare in C. tropicalis isolates [
]. However, they are contrary to
several other reports documenting the presence of resistance to azoles and/or other antifungal
agents among >20% of clinical C. tropicalis isolates from some geographical locations
9 / 16
Fig 2. Minimum spanning tree showing relationship of 63 C. tropicalis isolates from Kuwait with 804 isolates from
other countries available from the MLST website as of January 2017. Each circle corresponds to a unique genotype,
and lines between circles represent relative distance between isolates. The sizes of the circles correspond to the number of
isolates of the same genotype (DST). Connecting lines correspond to the number of allele differences between genotypes,
with a solid thick line connecting genotypes that differ in one locus, a solid thin line connecting genotypes that differ in
twothree loci, a dashed line connecting genotypes that differ in four loci, and a dotted line connecting genotypes that differ in
more than four loci.
including some locations in China [44±50]. Resistance to antifungal agents in Candida spp.
develops during therapy as a result of selective drug pressure [
5, 6, 12, 42, 45, 48
one recent report has suggested that applications of triazole fungicides in indoor/outdoor
environment may have selected resistant strains of C. tropicalis which were passed onto human
hosts in Hainan, China, a scenario similar to the opportunistic human fungal pathogen
Aspergillus fumigatus . Our data, however, do not support this scenario as triazole-resistant A.
fumigatus have previously been isolated readily from both, environmental and clinical
specimens [51±53] but azole resistance in Candida spp. has rarely been detected in Kuwait [30, 41,
54±56]. The data further showed that only one isolate (Kw45-15) was resistant to 5-flucytosine
even though this drug is rarely used in Kuwait. Similar to our results, all C. tropicalis isolates
from candidemia patients in the Spanish study were also susceptible to fluconazole and other
antifungals while one isolate was resistant to 5-flucytosine . On the contrary, in a survey of
fungemia cases due to C. tropicalis conducted in Paris, France, and among serial C. tropicalis
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isolates from ICU patients in Taipei, Taiwan, ~35% of the isolates were resistant to
The MLST identified 144 polymorphic sites with maximum number (n = 40) of sites
obtained for SAPT2 among of 63 C. tropicalis isolates from Kuwait. Furthermore, the
maximum typing efficiency (number of genotypes/polymorphic site) was obtained with XYR1
while the lowest typing efficiency was obtained with SAPT2. Previous studies from other
geographical locations have reported variations in the number of polymorphic sites with different
loci exhibiting maximum number of polymorphic sites as well as variations in typing
efficiencies [23, 26, 45±47, 49]. These findings suggest genetic variations in C. tropicalis strains at
different geographical locations. The MLST data further showed that C. tropicalis isolates from
Kuwait contained abundant worldwide alleles for most loci but also exhibited novel genetic
variations as 22 new alleles were also identified [http://pubmlst.org/ctropicalis/].
C. tropicalis isolates in Kuwait exhibited high genetic diversity as MLST identified 59 DSTs
among 63 isolates including 54 new DSTs. All five (DST94, DST99, DST114, DST168 and
DST238) previously described DSTs are rare and include only 2±5 isolates from China,
Taiwan, UK, Colombia and Brazil. Although DST168, previously reported only from Taiwan,
was found in Kuwait, DST164 which was also detected in Taiwan and was previously shown to
be associated with 5-flucytosine resistance [
] was not detected in our study and the single
5-flucytosine-resistant isolate (Kw45-15) from Kuwait belonged to a new DST (DST719).
Furthermore, DST238, which was shared between two isolates originating from vaginal and
percutaneous endoscopic gastrostomy specimens in Kuwait, has previously been described for
two bloodstream isolates from Brazil [
]. Our data reinforce previous observations suggesting
lack of association between different DSTs with antifungal resistance, specimen type or
geographical location and support both shared and unique features among geographic
populations of C. tropicalis [
23, 26, 46, 49
Among the seven patients yielding multiple isolates from different anatomic sites at the same
or nearly same time, isolates from only two patients (patient no. 3 and 38) belonged to identical
or highly related (with five or all 6 identical loci) DSTs while multiple isolates from the
remaining five patients belonged to unrelated (with 3 or 4 different loci) or highly unrelated (with 5 or
all 6 different loci) DSTs indicating microvariation as well as macrovariation in the nucleotide
polymorphisms of the six loci. Other MLST studies involving multiple isolates from individual
patients have also reported that some patients maintain the same C. tropicalis clone while
isolates in other patients either undergo microvariation in one or two genes yielding related DSTs
or macrovariation in nearly all six genes yielding unrelated and different DSTs [
23, 43, 46, 47
The MLST data from Brazil also showed that C. tropicalis isolates from the catheter were
genetically different than the bloodstream isolate(s) in eight of nine patients indicating that different
clonal populations co-exist within the same host . Taken together, these findings suggest
that C. tropicalis isolates undergo genetic alterations in the six genes used for MLST under the
application of host and other (such as antifungal) stresses. This is in contrast to C. albicans
where sequential/multiple clinical isolates from various anatomical sites of the same patient
have been shown by MLST analyses to be genetically identical or highly similar [56, 58±62].
The phylogenetic tree based on MLST data showed that most (55 of 63, 87%) of the isolates
were unique strains while only four DSTs were shared, each among two isolates. The clinical
and epidemiological data from the patients yielding cluster isolates showed that cluster isolates
in each case were isolated at different time points. Furthermore, the isolates were obtained
from patients hospitalized in different wards in two of the three clusters. These findings
suggest that intrahospital transmission of C. tropicalis does not occur or occurs rarely among
hospitalized patients in Kuwait. Intrahospital transmission of C. albicans was also not found in a
recent study involving candidemia patients in Kuwait [
11 / 16
The data presented in this study have shown that most C. tropicalis strains were isolated from
patients whose mean age was >65 years. Common underlying conditions detected among
patients yielding C. tropicalis isolates included urinary catheter, treatment with
broad-spectrum antibiotics, diabetes mellitus and RT/GIT abnormalities. Most candiduria cases had
urinary catheter, ureteric stent or suprapubic catheters. All isolates were susceptible to five of six
antifungal drugs and only one isolate was resistant to 5-flucytosine. MLST identified 59 DSTs
including 54 newly-identified DSTs. C. tropicalis isolates from multiple sites of same patient
usually belonged to different DSTs. Interestingly, 56 of 57 isolates from 48 patients belonged to
unique genotypes. Our data show diverse origins of C. tropicalis infections in Kuwait as most
isolates were unique strains, however, microvariation and macrovariation were also noted
among C. tropicalis isolates recovered from the same patients. Only six isolates from six
patients belonged to three DSTs (clusters), however, C. tropicalis strains in each cluster were
isolated >3 months apart. There was no obvious correlation between cluster isolates with
respect to the time of isolation and/or hospital ward of their origin. The study has also
provided information on MLST-based DSTs found among clinical C. tropicalis isolates for the
first time from a Middle Eastern country. New DSTs have been added to the growing list in
the MLST central database that will be useful for further studies on genetic relationship and
global molecular epidemiology of C. tropicalis strains.
S1 Table. Source of isolation, clinical characteristics and MLST data for 63 C. tropicalis
isolates obtained from 54 patients analyzed in this study.
S2 Table. Salient features of various oligonucleotide primers used during MLST of C.
tropicalis isolates in this study.
S3 Table. Antifungal drug susceptibility testing data and the minimum inhibitory
concentration (MIC) range, MIC50 and MIC90 values for 63 C. tropicalis isolates.
S1 Fig. Agarose gel of PCR products using C. tropicalis-specific (CTROPF and CTROPR)
primers and genomic DNA from reference strains of C. albicans (Lane CA), C. dubliniensis
(Lane CD), C. tropicalis (Lane CT), C. viswanathii (Lane CV), C. parapsilosis (Lane CP), C.
orthopsilosis (Lane CO), L. elongisporus (Lane LE), C. glabrata (Lane CG), C. nivariensis
(Lane CN) and C. bracarensis (Lane CB).
S2 Fig. An UPGMA-derived dendrogram with Tamura-Nei parameters based on ITS
regions of rDNA sequence data from five C. tropicalis isolates from Kuwait together with
reference C. tropicalis strain ATCC 750.
Conceptualization: Suhail Ahmad, Ziauddin Khan.
Data curation: Khaled Al-Obaid, Mohammad Asadzadeh, Ziauddin Khan.
12 / 16
Formal analysis: Khaled Al-Obaid, Mohammad Asadzadeh, Suhail Ahmad, Ziauddin Khan.
Funding acquisition: Suhail Ahmad, Ziauddin Khan.
Investigation: Khaled Al-Obaid, Mohammad Asadzadeh.
Methodology: Khaled Al-Obaid, Mohammad Asadzadeh.
Project administration: Suhail Ahmad, Ziauddin Khan.
Resources: Suhail Ahmad, Ziauddin Khan.
Software: Mohammad Asadzadeh.
Supervision: Ziauddin Khan.
Validation: Mohammad Asadzadeh.
Visualization: Mohammad Asadzadeh.
Writing ± original draft: Khaled Al-Obaid, Mohammad Asadzadeh, Suhail Ahmad, Ziauddin
Writing ± review & editing: Khaled Al-Obaid, Mohammad Asadzadeh, Suhail Ahmad,
13 / 16
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