Journal of Medical Microbiology Papers in Press. Published January 16, 2015 as doi:10.1099/jmm.0.000015
Journal of Medical Microbiology Multilocus sequence typing of Candida albicans isolates from Burn Intensive Care Unit (BICU) in Iran --Manuscript Draft-Manuscript Number:
JMM-D-14-00282R1
Full Title:
Multilocus sequence typing of Candida albicans isolates from Burn Intensive Care Unit (BICU) in Iran
Short Title:
MLST of Candida albicans from BICU in Iran
Article Type:
Standard
Section/Category:
Epidemiology
Corresponding Author:
Tahereh Shokohi, Ph.D Mazandaran University of Medical Sciences IRAN (ISLAMIC REPUBLIC OF)
First Author:
Mohammad H Afsarian
Order of Authors:
Mohammad H Afsarian Hamid Badali Teun Boekhout Tahereh Shokohi, Ph.D Farzad Katiraee
Abstract:
Burn intensive care unit patients are specifically exposed to deep-seated nosocomial infections caused by Candida albicans. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. Multilocus sequence typing is a powerful genotyping method for pathogenic microorganisms, including Candida albicans. Thirty clinical isolates of C. albicans obtained from 22 patients that admitted to the Burn Intensive Care Unit (BICU) from a burn hospital at Sari, Mazandaran state, Iran were epidemiologically studied by multilocus sequence typing (MLST). Seventy five variable nucleotide sites were found. Sixty two alleles were identified among the seven loci of the C. albicans isolates and one new allele was obtained. Eighteen Diploid Sequence Types (DSTs) were identified and among those 10 were new. These isolates belonged to nine clonal clusters (CCs) while two isolates occurred as singletons. Eleven (36.7%) isolates belonged to CC 124 after eBURST analysis and 13 isolates (43.3%) were assigned to clade 4. Approximately 17% of the 30 isolates belonged to clade 1 (CC 69 and CC766). Isolates from several patients with burns were found to be genetically related. Some patients yielded multiple isolates with identical DSTs suggest colonization or infection caused by cross contamination between patients. Therefore, isolates that show identical or similar allelic profiles are presumed to be identical or closely related and may use to evaluate the genetic relationships between isolates from a specific environment such as BICU.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): JMM_4rd Revision By Shokohi.doc
1
JMM 2014
2 3
Multilocus sequence typing of Candida albicans isolates from Burn
4
Intensive Care Unit (BICU) in Iran
5 6
Mohammad H. Afsarian1, 2, Hamid Badali1, Teun Boekhout3, Tahereh Shokohi1*, Farzad
7
Katiraee4
8
1
9
School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; 2Department of
Department of Medical Mycology and Parasitology/Invasive Fungi Research Center (IFRC), 3
10
Microbiology, Fasa University of Medical Sciences, Fasa, Iran;
11
Centre, Utrecht, The Netherlands, 4Department of Pathobiology, Division of Mycology,
12
School of Veterinary Medicine, University of Tabriz, Tabriz, Iran
13
*Corresponding Authors: Tahereh Shokohi; PhD
14
Address: 18Km of Khazarabad Road, School of Medicine, Mazandaran University of Medical
15
Sciences, Sari, Iran.
16
PO Box: 48175-1665, Sari, Iran
17
Email: [email protected]
18
Tel: +98 11 33543081-3 ext.2403; Fax: +98 11 33543248 (office)
19
Running title: MLST of Candida albicans from BICU in Iran
20 21 22 23 24 1
CBS, Fungal Biodiversity
25
Abstract:
26
Burn intensive care unit patients are specifically exposed to deep-seated nosocomial
27
infections caused by Candida albicans. Superficial carriage of C. albicans is a potential
28
source of infection and dissemination, and typing methods could be useful to trace the
29
different isolates. Multilocus sequence typing is a powerful genotyping method for pathogenic
30
microorganisms, including Candida albicans. Thirty clinical isolates of C. albicans obtained
31
from 22 patients that admitted to the Burn Intensive Care Unit (BICU) from a burn hospital at
32
Sari, Mazandaran state, Iran were epidemiologically studied by multilocus sequence typing
33
(MLST). Seventy five variable nucleotide sites were found. Sixty two alleles were identified
34
among the seven loci of the C. albicans isolates and one new allele was obtained. Eighteen
35
Diploid Sequence Types (DSTs) were identified and among those 10 were new. These isolates
36
belonged to nine clonal clusters (CCs) while two isolates occurred as singletons. Eleven
37
(36.7%) isolates belonged to CC 124 after eBURST analysis and 13 isolates (43.3%) were
38
assigned to clade 4. Approximately 17% of the 30 isolates belonged to clade 1 (CC 69 and
39
CC766). Isolates from several patients with burns were found to be genetically related. Some
40
patients yielded multiple isolates with identical DSTs suggest colonization or infection caused
41
by cross contamination between patients. Therefore, isolates that show identical or similar
42
allelic profiles are presumed to be identical or closely related and may use to evaluate the
43
genetic relationships between isolates from a specific environment such as BICU.
44 45
Keywords: MLST, Candida albicans, BICU, Candidemia
46 47 48 49 50 51 2
52
Introduction:
53
Fungal infections due to Candida species have increased during the last decades as these
54
yeasts emerged as important opportunistic fungal pathogen. Candida species can cause
55
various clinical manifestations ranging from superficial to systemic candidiasis either in
56
immunocompetent and immunocompromised hosts (Garcia-Hermoso et al. 2007; Odds et al.
57
2007; Pfaller & Diekema 2007; Fesharaki et al. 2013). Infections due to C. albicans are an
58
important cause of morbidity and mortality among hospitalized patients who have been
59
admitted to Intensive Care Units (ICUs) and in oncology wards worldwide (Bougnoux et al.
60
2006; Bougnoux et al. 2008; Horn et al. 2009; Tortorano et al. 2012; Sardi et al. 2013). Burn
61
patients are especially susceptible to Candida infections because they have most of the risk
62
factors for hospital-acquired fungal infections, i.e., cutaneous and vascular portals of entry
63
(inhabiting catheters, parenteral nutrition) and broad-spectrum antimicrobial therapy. In most
64
cases, colonization of C. albicans of the skin or mucosa occurs before invasion and systemic
65
infection and colonization may be a main reservoir for cross contamination. Therefore,
66
identification of the source of colonization and the transmission route of infection will
67
contribute to the prevention of nosocomial infections due to C. albicans among burn patients
68
(Bougnoux et al. 2004; Lee et al. 2013). Nosocomial candidiasis caused by C. albicans
69
requires a precise understanding of the epidemiology in different geographic regions using a
70
reliable typing system. Recently, several molecular typing techniques have been used to study
71
epidemiological relationships of C. albicans isolates such as randomly amplified polymorphic
72
DNA (Robert et al. 1995), restriction fragment length polymorphism (Vazquez et al. 1993),
73
Southern blot hybridization with discriminating probes (Myoung et al. 2011), amplified
74
fragment length polymorphism analysis (Borst et al. 2003) and microsatellites length
75
polymorphism (Garcia-Hermoso et al. 2007). The multilocus sequence typing (MLST)
76
technique has been extensively used because it yields high levels of resolution allowing it to
77
characterize a large number of isolates rapidly, and it does not require any subjective
78
interpretation of banding patterns. Moreover, MLST sequences can be stored and
79
characterized in multiple electronic formats, thus offering an unprecedented degree of
80
portability and accessibility to other users (website http://calbicans.mlst.net) (Bougnoux et al.
81
2003; Bougnoux et al. 2004). The purpose of this study is to investigate the MLST analysis of 3
82
clinical isolates of C. albicans recovered from patients with burns in a burn intensive care unit
83
(BICU) of a hospital at Sari city, Iran. It is also the first nationwide study into the genotypic
84
relationships of C. albicans isolates from Iran using the MLST method.
85 86
Materials and Methods
87
Thirty clinical isolates of C. albicans were collected from hospitalized patients at Sari city,
88
Iran. These strains were isolated from 22 burn patients with superficial colonization or
89
candidiasis, candiduria and candidemia that were admitted to the BICU of the burn hospital
90
from July 2011 to March 2012. The skin burns were sampled using sterile swabs (Becton,
91
Dickinson, USA) and samples cultured onto Sabouraud’s dextrose agar (SDA, Difco) medium
92
supplemented with chloramphenicol (SC). Blood samples were inoculated onto biphasic
93
fungal blood culture media containing brain heart infusion (BHI, Difco) broth and BHI Agar
94
and incubated for 10 days at 37 ºC. Blood cultures were sub-cultured on SC and CHROMagar
95
Candida (BioMérieux, Marcy l'Etoile, France). Five patients were sampled at different body
96
sites and different times: skin burns (hand), blood and urine of patient 2 (P2) were sampled
97
within a 7 day period; skin burns (hand & abdomen), blood and urine of P3 were sampled
98
within a 10 day period; skin burns (foot) and urine of P7 were sampled within a 7 day period;
99
skin burns of P13 including chest and scapula were sampled within a 5 day period; skin burns
100
(chest) and two blood samples of P17 were sampled within a 21 day period. MLST was used
101
to type the 30 clinical isolates as described previously (Bougnoux et al. 2003). Briefly,
102
genomic DNA was extracted using glass beads and the phenol/chloroform method (Yamada et
103
al. 2002) and stored at −20°C prior to use. MLST used seven housekeeping genes, AAT1a,
104
ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b (Bougnoux et al. 2003). PCR amplification
105
was performed for seven loci using a thermal cycler (Biorad-C1000) and described primers
106
(Bougnoux et al. 2003), followed by sequencing using an ABI 3730XL automatic sequencer
107
(Applied Biosystems, 3730/3730xl DNA Analyzers Sequencing, Bioneer, Korea). Sequence
108
data were manually aligned using MEGA 5.05 (http://www.megasoftware.net/) (Tamura et al.
109
2011) and Bioedit ver. 7.0.9 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Alignment,
110
BioEdit Sequence 2011) software packages. Heterozygosity was identified by the presence of
4
111
two peaks on both strands at the same loci and the consensus sequences for the seven loci of
112
all isolates were defined.
113
The number of alleles and diploid sequence types (DSTs) was determined by comparing the
114
sequences with those available in the C. albicans MLST database (http://calbicans.mlst.net).
115
Chromatograms for the isolates with new alleles and new DSTs were sent to the central
116
MLST database curator and new MLST numbers were assigned. Newly identified DSTs were
117
assigned
118
http://calbicans.mlst.net/sql/burstspadvanced.asp. Epidemiological relationships were assessed
119
by eBURST analysis (http://eburst.mlst.net/) (Feil et al. 2004) and DSTs of 30 isolates were
120
compared with those available (n = 2099) in the MLST database at June 2014. This algorithm
121
places all related isolates into groups called clonal clusters (CC: set of DSTs that are believed
122
to have descended from the same founding genotype). Clonal clusters were identified using a
123
reference DST for each clade according to previously described major C. albicans clades
124
(Odds et al. 2007; Odds 2010). Clonal clusters of isolates differ in sequence at only one of the
125
seven loci (Odds et al. 2007). The UPGMA dendrogram based on MLST sequence data was
126
drawn using CLC Sequence Viewer 6 software (http://www.clcbio.com/).
the
numbers
2240
to
2251
(except
2246
and
2248)
in
127 128
Results
129
Thirty epidemiologically related isolates of C. albicans were obtained from 22 burn patients
130
(age range: 2 – 60 years) admitted to the burn intensive care unit (BICU). C. albicans strains
131
were isolated from blood cultures (n=6), skin burns (n=19), urine (n=3), catheter (n=1) and
132
sputum (n=1). The MLST data of the 30 C. albicans isolates consisted of 2,883 nucleotides.
133
Seventy five (2.6%) nucleotide sites were found to be variable and heterozygous in at least
134
one isolate. Thus the seven sequenced genes yielded a total of 75 variable sites of which
135
VPS13 produced the highest number (n = 14) of polymorphic sites, while ACC1 produced the
136
lowest number (n = 6). Sixty two alleles were identified in the seven loci studied. The gene
137
VPS13 generated the highest number of alleles (n = 11), while ACC1 generated the lowest
138
number (n = 7). Among the alleles, one new allele was determined in the ADP1 locus (Allelic
139
number 122) and this was added to the MLST database (http://calbicans.mlst.net). Dated June
5
140
2014, from the 125 alleles present only allele number 122 of the ADP1 locus displayed a
141
polymorphism at nucleotide site 366 (Y instead of C).
142
Eighteen unique DSTs were obtained in the seven loci of the 30 Iranian C. albicans isolates
143
from
144
www.calbicans.mlst.net. Eight of these 18 DSTs had been previously identified and 10 novel
145
DSTs were added to the online database (http://calbicans.mlst.net). The eBURST program
146
was used for the analysis of the genotypic relationships among the MLST data of the 30
147
Iranian C. albicans strains using all available DSTs (n= 2099) in the MLST database. This
148
yielded 108 eBURST groups (CCs) and 707 singleton isolates. The 30 isolates in this study
149
were placed in 9 CCs while two isolates (DST 2095) were singletons. Eleven Iranian isolates,
150
i.e. 36.7%, recovered from eight patients belonged to CC 124, 4 isolates (13.3%) from four
151
patients belonged to CC 172, 3 isolates (10%) from two patients belonged to CC 69, 2 isolates
152
(6.7%) from two patients belonged to CC 918, 3 isolates (10%) from one patient belonged to
153
CC 601, 2 isolates (6.7%) from two patients belonged to CC 766, one isolate (3.3%) belonged
154
to CC 461 and one isolate (3.3%) belonged to CC 1865. However, according to the latest
155
classification by Odds et al. 2007 and Odds 2010, the taxonomical position for new DSTs
156
(2099 and 2102) were not clarified so far (Fig. 1).
157
Among 30 isolates of C. albicans collected from skin burns and blood cultures of 22 patients
158
in the same BICU, the MLST analysis showed that some isolates had the same DST (Fig. 1).
159
From 22 patients in this study, 19 isolates from skin burns or cutaneous candidiasis yielded 13
160
DSTs belonging to five clades (1, 4, 9, 12 and 15), of which seven DSTs belonged to clade 4
161
because some alleles were shared between seven DSTs. These results may suggest he
162
presence of microvariation due to microevolution of the genomes of epidemiologically related
163
strains of C. albicans in the BICU (Fig. 1). Three out of the seven strains isolated from
164
candidaemic patients with burns belonged to clade 4 (CC 124) and two of them originated
165
from multiple episodes of one patient (P17). Four other strains were assigned to four CCs (69,
166
918, 461 and 601) in four clades (1, 9, 11 and 12). Moreover, four isolates recovered from
167
candidaemic patients were identified as new DSTs (Fig. 1).
168
Some patients yielded multiple isolates with identical DSTs, four patients (P4, P9, P10 and
169
P12) yielded isolates identified as DST 172, two patients (P19 and P22) had DST 656, two
22
BICU
patients.
Each
allelic
profile
6
query
has
been
submitted
to
170
patients (P5 and P13) DST 124 and one patient (P3) was infected with C. albicans isolates
171
belonging to two DST (i.e., 918 and 2093) (Table 1). Five patients (P2, P3, P7, P13 and P17)
172
were sampled at different body sites (Table 1), which four patients appeared to harbor just one
173
strain of C. albicans, whereas one patient (P3) yielded two different strains isolated from three
174
samples (blood, urine and skin burns) (Table 1).
175 176
Discussion
177
Multilocus sequence typing is a powerful and useful technique for understanding the
178
epidemiological and evolutionary relationships of pathogenic microorganisms (Taylor &
179
Fisher 2003; Urwin & Maiden 2003; Maiden 2006; Cliff et al. 2008; Odds & Jacobsen 2008;
180
Da Matta et al. 2010; Shin et al. 2011). Prevalence of C. albicans infections in the BICU of
181
the Zare hospital (Sari, Iran) led us to investigate the epidemiologically relationship of 30
182
isolates involved using the MLST method. Although the sample size of strains typed herein
183
was low, our study revealed 10 new DSTs, i.e. 33.3%, among 30 isolates. Three new DSTs
184
belonged to clade 4 (CC 124) as did a new singleton DSTs. The remainder of the isolates
185
belonged to various CCs. Twenty eight isolates were found to be located in 9 CCs by
186
eBURST analysis and two isolates were identified as singletons, thus revealing a high genetic
187
heterogeneity among the C. albicans population of these hospitalized patients.
188
Among a large assembly of strains collected worldwide, Odds and coworkers (Odds et al.
189
2007; Odds 2010) showed that C. albicans clade 1 (CC 69) was the most prevalent one
190
followed by clade 4 (CC 124), clade 3 (CC 344), clade 2 (CC 155) and clade 11 (CC 538),
191
respectively. In addition, they found that 34% of the Middle East isolates belonged to clade 1.
192
Ninety three percent of our isolates clustered within 6 of the 17 known clades and 63%
193
belonged to three of the 5 major clades (i.e. clades 1, 4 and 11) as defined previously (Odds
194
2010). In contrast, approximately 17% of our isolates belonged to clade 1 including CC 69
195
and CC 766, whereas 43% belonged to clade 4 including CC 124. Alastruey et al. (2013)
196
identified 37 DSTs of C. albicans including 17 new DSTs from Israel and they concluded that
197
most strains belonged to CC 124 in both candidemia and superficial candidiasis isolates
198
(Alastruey-Izquierdo et al. 2013). Notably none of our strains clustered in clade 2 (CC 155)
199
and clade 3 (CC344). In contrast, Gammelsrud et al. (Gammelsrud et al. 2012) analyzed 62 7
200
strains from Norway and found 32 DSTs from which 31% belonged to clade 2 (CC 155). Shin
201
et al. (Shin et al. 2011) found 58% new DSTs among 156 Korean isolates, and 15% of the
202
isolates clustered in clade 4 and 19% clustered in a new Asia-specific clade with P-distance ≤
203
0.035, while based on the P-distance ≤ 0.04 (Odds 2010), all isolates were assigned to clade 3.
204
In the present study, our results showed that none of the Iranian isolates belonged to this Asia-
205
specific clade.
206
Isolates obtained from different body sites of patients belonged to the same CC, for example
207
P2, P7, P13 and P17 (Fig. 1). This finding is in accordance with previous studies that reported
208
that C. albicans strains isolated from different body sites of a person are usually genetically
209
similar or identical (Bougnoux et al. 2006; Cliff et al. 2008), whereas two strains from P3
210
were assigned to two CC 69 and CC 918.
211
Some DSTs (656, 124 and 172) of C. albicans isolated from more than one patient in the same
212
BICU during one month; therefore, it seems that colonized patients are a main reservoir of C.
213
albicans in hospitals and the isolation of C. albicans with the same DST in more than one
214
patient may be provide evidence for cross contamination between patients which might be a
215
crucial factor for nosocomial infections (Bougnoux et al. 2003; Bougnoux et al. 2004;
216
Bougnoux et al. 2006).
217
Several closely related strains were isolated from patients. For example, the new DST 2093
218
differs from DST 69 only in locus ADP1 with one nucleotide different that may have arisen
219
from a single recombination event. Similarly DST 2101 differs from DST 766 only in the
220
locus ADP1 with one nucleotide different and four mentioned DSTs assigned to clade 1 (Fig.
221
1). Moreover three new DSTs, including 2092, 2096 and 2103, assigned to CC124 were
222
founded to be different in three loci with DSTs 124, 605 and 656 (Fig. 1). These findings may
223
suggest micro-evolutionary changes of a single strain occurring during adaptation to varying
224
environmental conditions (Bonfim-Mendonca et al. 2013; Paluchowska et al. 2014).
225
Cliff and coworkers showed evidence for the presence of an endemic strain in a ICU that was
226
isolated repeatedly from patients and staff, and suggested horizontal transmission of C.
227
albicans in the unit (Cliff et al. 2008). Bougnoux and colleagues using the MLST technique
228
showed frequent colonization of C. albicans in a person or in the whole family by genetically
229
closely related isolates that differed at one or several of the MLST loci (Bougnoux et al.
230
2006). 8
231
In conclusion, although a limited number of isolates was analyzed in the current study, a
232
significant genetic relationship was identified among epidemiologically related C. albicans
233
isolates from burn patients in Iran suggesting a likelihood of nosocomial spread. Isolates that
234
show identical or highly similar allelic profiles are presumed to be identical or closely related
235
and may use to evaluate the genetic relationships between isolates from a specific
236
environment such as BICU. To best of our knowledge, this study is the first MLST study
237
analyzing C. albicans clinical isolates from Iran, thus also improving the epidemiological
238
knowledge of C. albicans in this region.
239 240
Acknowledgments
241
This research was financially supported by a grant of the Mazandaran University of Medical
242
Science (No. 91-32), which we gratefully acknowledge. We would like to thank Nazanin Lotfi
243
and Seyede Zahra Nouranibaladezaei for technical assistance. We acknowledge the use of the
244
Candida albicans MLST database which is located at Imperial College London and is funded by
245
the Welcome Trust. We also thank the curator of MLST database, Marie-Elisabeth Bougnoux for
246
assigning our new alleles and DSTs to the C. albicans MLST database. T. B. was supported by
247
grant HPRP 5-298-3-086 from the Qatar National Research Fund, a member of Qatar foundation.
248
The statements herein are solely the responsibility of the authors.
249 250
Declaration of interest: All authors report no potential conflicts of interest. Only the
251
authors are responsible for the content and writing of the paper.
252 253
References:
254 255 256 257 258 259 260 261 262 263
Alastruey-Izquierdo, A., Mandelblat, M., Ben Ami, R., Perlin, D. S., & Segal, E. (2013). Multilocus sequence typing of Candida albicans isolates from candidemia and superficial candidiasis in Israel. Med Mycol 51, 1-4. Alignment, BioEdit Sequence (2011). version 7.0. 9. Bonfim-Mendonca, P. d. S., Fiorini, A., Shinobu-Mesquita, C. S., Baeza, L. C., Fernandez, M. A., & Svidzinski, T. I. E. (2013). Molecular Typing of Candida albicans isolates from hospitalized patients. Rev Inst Med Trop São Paulo, 55, 385-391. 9
264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309
Borst, A., Theelen, B.,Reinders, E., Boekhout, T., Fluit A. C. and Savelkoul, P. H. M. (2003). Use of amplified fragment length polymorphism analysis to identify medically important Candida spp., including C. dubliniensis. J Clin Microbiol 41(4): 1357-1362. Bougnoux, M.-E., Aanensen, D. M., Morand, S., Théraud, M., Spratt, B. G., & d’Enfert, C. (2004). Multilocus sequence typing of Candida albicans: strategies, data exchange and applications. Infec Genet Evol 4, 243-252. Bougnoux, M.-E., Diogo, D., Francois, N., Sendid, B., Veirmeire, S., Colombel, J. F., Bouchier, C., Van Kruiningen, H., d’Enfert, C. & Poulain, D. (2006). Multilocus sequence typing reveals intrafamilial transmission and microevolutions of Candida albicans isolates from the human digestive tract. J Clin Microbiol 44, 1810-1820. Bougnoux, M.-E., Kac, G., Aegerter, P., d’Enfert, C., & Fagon, J.-Y. (2008). Candidemia and candiduria in critically ill patients admitted to intensive care units in France: incidence, molecular diversity, management and outcome. Intensive Care Med 34, 292-299. Bougnoux, M.-E., Tavanti, A., Bouchier, C., Gow, N., Magnier, A., Davidson, A., Maiden, M. C. J., d’Enfert, C. & Odds, F. (2003). Collaborative consensus for optimized multilocus sequence typing of Candida albicans. J Clin Microbiol 41, 5265-5266. Cliff, P., Sandoe, J., Heritage, J., & Barton, R. (2008). Use of multilocus sequence typing for the investigation of colonisation by Candida albicans in intensive care unit patients. J Hospital Infect 69, 24-32. Da Matta, D. A., Melo, A. S., Guimarães, T., Frade, J. P., Lott, T. J., & Colombo, A. L. (2010). Multilocus sequence typing of equential Candida albicans isolates from patients with persistent or recurrent fungemia. Med Mycol 48, 757-762. Feil, E. J., Li, B. C., Aanensen, D. M., Hanage, W. P., & Spratt, B. G. (2004). eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J bacteriol, 186, 1518-1530. Fesharaki, S. H., Haghani, I., Mousavi, B., Kargar, M.L., Boroumand, M., Anvari, M.S., Abbasi, K., Meis, J.F.,& Badali H. Endocarditis due to a co-infection of Candida albicans and Candida tropicalis in a drug abuser. J Med Microbiol 62, 1763-1767. Gammelsrud, K. W., Lindstad, B. L., Gaustad, P., Ingebretsen, A., Høiby, E. A., Brandtzaeg, P., & Sandven, P. (2012). Multilocus sequence typing of serial Candida albicans isolates from children with cancer, children with cystic fibrosis and healthy controls. Med Mycol 50, 619-626. Garcia-Hermoso, D., Cabaret, O., Lecellier, G., Desnos-Ollivier, M., Hoinard, D., Raoux, D., Costa, J. M., Dromer, F. & Bretagne, S. (2007). Comparison of microsatellite length polymorphism and multilocus sequence typing for DNA-based typing of Candida albicans. J Clin Microbiol 45, 3958-3963. 10
310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353
Horn, D. L., Neofytos, D., Anaissie, E. J., Fishman, J. A., Steinbach, W. J., Olyaei, A. J., Marr, K. A., Pfaller, M. A., Chang, C. H. & Webster, K. M. (2009). Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry. Clin Infect Dis 48, 1695-1703. Lee, H. G., Jang, J., Choi, J. E., Chung, D. C., Han, J. W., Woo, H., Jeon, W. & Chun, B. C. (2013). Blood stream infections in patients in the burn intensive care unit. Infect chemother, 45, 194-201. Maiden, M. C. (2006). Multilocus sequence typing of bacteria. Annu Rev Microbiol 60, 561588. Myoung, Y., Shin, J. H., Lee, J. S., Kim, S. H., Shin, M. G., Suh, S. P. and Ryang, D. W. (2011). Multilocus sequence typing for Candida albicans isolates from candidemic patients: comparison with Southern blot hybridization and pulsed-field gel electrophoresis analysis. Korean J Lab Med 31, 107-114. Odds, F. C. (2010). Molecular phylogenetics and epidemiology of Candida albicans. Future Microbiol 5, 67-79. Odds, F. C., Bougnoux, M.-E., Shaw, D. J., Bain, J. M., Davidson, A. D., Diogo, D., Jacobsen, M. D., Lecomte, M., Li, S. Y. & other authors (2007). Molecular phylogenetics of Candida albicans. Eukaryot Cell 6, 1041-1052. Odds, F. C., & Jacobsen, M. D. (2008). Multilocus sequence typing of pathogenic Candida species. Eukaryot Cell 7, 1075-1084. Paluchowska, P., Tokarczyk, M., Bogusz, B., Skiba, I., & Budak, A. (2014). Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland. Mem Inst Oswaldo Cruz, 109, 436-441. Pfaller, M., & Diekema, D. (2007). Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2, 133-163. Robert, F., Lebreton, F., Bougnoux, M., Paugam, A., Wassermann, D., Schlotterer, M., Tourte-schaefer, C. & Dupouy-Camet, J. (1995). Use of random amplified polymorphic DNA as a typing method for Candida albicans in epidemiological surveillance of a burn unit. J Clin Microbiol 33, 2366-2371. Sardi, J., Scorzoni, L., Bernardi, T., Fusco-Almeida, A., & Giannini, M. M. (2013). Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol, 62, 10-24. Shin, J. H., Bougnoux, M.-E., d'Enfert, C., Kim, S. H., Moon, C.-J., Joo, M. Y., Lee, K., Kim. M. N., Lee, H. S. & other authors (2011). Genetic diversity among Korean Candida 11
354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377
albicans bloodstream isolates: assessment by multilocus sequence typing and restriction endonuclease analysis of genomic DNA by use of BssHII. J Clin Microbiol 49, 2572-2577. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 28, 2731-2739. Taylor, J. W., & Fisher, M. C. (2003). Fungal multilocus sequence typing—it’s not just for bacteria. Curr Opin Microbiol 6, 351-356. Tortorano, A. M., Prigitano, A., Dho, G., Grancini, A., & Passera, M. (2012). Antifungal susceptibility profiles of Candida isolates from a prospective survey of invasive fungal infections in Italian intensive care units. J Med Microbiol, 61, 389-393. Urwin, R., & Maiden, M. C. (2003). Multi-locus sequence typing: a tool for global epidemiology. Trend Microbiol 11, 479-487. Vazquez, J. A., Sanchez, V., Dmuchowski, C., Dembry, L. M., Sobel J. D., & Zervos, M. J. (1993). Nosocomial acquisition of Candida albicans: an epidemiologic study. J Infec Dis 168, 195-201. Yamada, Y., Makimura, K., Merhendi, H., Ueda, K., Nishiyama, Y., Yamaguchi, H., & Osumi, M. (2002). Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts. Japan J Infect Dis 55, 122-125.
378 379 380 381 382 383 384 385 386 387 388 389 390 391 12
392
Table 1. Summarized the epidemiologically related isolates are recovered from the same patients at
393
different times in BICU 2011
2012
DST July
August
September
January
February
March
P3(2)
2093
(Blood and Abdomen burns) P3(1)
918
(Urine) P2(3)
601
(Hand burns, Blood and Urine) P7(2)
2095
(Foot burns and Urine) P5(1) (Chest burns)
124
P13(2) (Chest and Scapula burns) P17(3) (Chest burns)
605
and 2 Blood samples) P4(1) (Foot burns)
172
P9(1) (Hand burns)
P12(1) (Chest burns)
P10(1) (Hand burns)
656
394 395 396 397
P19(1)
P22(1)
(Hand
(Abdomen
burns)
burns)
Numbers in parentheses symbolize the number of times the DST was isolated from that patient in that month; P: patient
13
398 399 400 401
Figure. 1. Summarizes all information of the 30 Iranian C. albicans comprising the allelic number for each locus, DSTs, CCs, clades and it also illustrates the UPGMA dendrogram based on MLST data. The scale indicates p-distances (P
Journal of Medical Microbiology Multilocus sequence typing of Candida albicans isolates from Burn Intensive Care Unit (BICU) in Iran --Manuscript Draft-Manuscript Number:
JMM-D-14-00282R1
Full Title:
Multilocus sequence typing of Candida albicans isolates from Burn Intensive Care Unit (BICU) in Iran
Short Title:
MLST of Candida albicans from BICU in Iran
Article Type:
Standard
Section/Category:
Epidemiology
Corresponding Author:
Tahereh Shokohi, Ph.D Mazandaran University of Medical Sciences IRAN (ISLAMIC REPUBLIC OF)
First Author:
Mohammad H Afsarian
Order of Authors:
Mohammad H Afsarian Hamid Badali Teun Boekhout Tahereh Shokohi, Ph.D Farzad Katiraee
Abstract:
Burn intensive care unit patients are specifically exposed to deep-seated nosocomial infections caused by Candida albicans. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. Multilocus sequence typing is a powerful genotyping method for pathogenic microorganisms, including Candida albicans. Thirty clinical isolates of C. albicans obtained from 22 patients that admitted to the Burn Intensive Care Unit (BICU) from a burn hospital at Sari, Mazandaran state, Iran were epidemiologically studied by multilocus sequence typing (MLST). Seventy five variable nucleotide sites were found. Sixty two alleles were identified among the seven loci of the C. albicans isolates and one new allele was obtained. Eighteen Diploid Sequence Types (DSTs) were identified and among those 10 were new. These isolates belonged to nine clonal clusters (CCs) while two isolates occurred as singletons. Eleven (36.7%) isolates belonged to CC 124 after eBURST analysis and 13 isolates (43.3%) were assigned to clade 4. Approximately 17% of the 30 isolates belonged to clade 1 (CC 69 and CC766). Isolates from several patients with burns were found to be genetically related. Some patients yielded multiple isolates with identical DSTs suggest colonization or infection caused by cross contamination between patients. Therefore, isolates that show identical or similar allelic profiles are presumed to be identical or closely related and may use to evaluate the genetic relationships between isolates from a specific environment such as BICU.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): JMM_4rd Revision By Shokohi.doc
1
JMM 2014
2 3
Multilocus sequence typing of Candida albicans isolates from Burn
4
Intensive Care Unit (BICU) in Iran
5 6
Mohammad H. Afsarian1, 2, Hamid Badali1, Teun Boekhout3, Tahereh Shokohi1*, Farzad
7
Katiraee4
8
1
9
School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; 2Department of
Department of Medical Mycology and Parasitology/Invasive Fungi Research Center (IFRC), 3
10
Microbiology, Fasa University of Medical Sciences, Fasa, Iran;
11
Centre, Utrecht, The Netherlands, 4Department of Pathobiology, Division of Mycology,
12
School of Veterinary Medicine, University of Tabriz, Tabriz, Iran
13
*Corresponding Authors: Tahereh Shokohi; PhD
14
Address: 18Km of Khazarabad Road, School of Medicine, Mazandaran University of Medical
15
Sciences, Sari, Iran.
16
PO Box: 48175-1665, Sari, Iran
17
Email: [email protected]
18
Tel: +98 11 33543081-3 ext.2403; Fax: +98 11 33543248 (office)
19
Running title: MLST of Candida albicans from BICU in Iran
20 21 22 23 24 1
CBS, Fungal Biodiversity
25
Abstract:
26
Burn intensive care unit patients are specifically exposed to deep-seated nosocomial
27
infections caused by Candida albicans. Superficial carriage of C. albicans is a potential
28
source of infection and dissemination, and typing methods could be useful to trace the
29
different isolates. Multilocus sequence typing is a powerful genotyping method for pathogenic
30
microorganisms, including Candida albicans. Thirty clinical isolates of C. albicans obtained
31
from 22 patients that admitted to the Burn Intensive Care Unit (BICU) from a burn hospital at
32
Sari, Mazandaran state, Iran were epidemiologically studied by multilocus sequence typing
33
(MLST). Seventy five variable nucleotide sites were found. Sixty two alleles were identified
34
among the seven loci of the C. albicans isolates and one new allele was obtained. Eighteen
35
Diploid Sequence Types (DSTs) were identified and among those 10 were new. These isolates
36
belonged to nine clonal clusters (CCs) while two isolates occurred as singletons. Eleven
37
(36.7%) isolates belonged to CC 124 after eBURST analysis and 13 isolates (43.3%) were
38
assigned to clade 4. Approximately 17% of the 30 isolates belonged to clade 1 (CC 69 and
39
CC766). Isolates from several patients with burns were found to be genetically related. Some
40
patients yielded multiple isolates with identical DSTs suggest colonization or infection caused
41
by cross contamination between patients. Therefore, isolates that show identical or similar
42
allelic profiles are presumed to be identical or closely related and may use to evaluate the
43
genetic relationships between isolates from a specific environment such as BICU.
44 45
Keywords: MLST, Candida albicans, BICU, Candidemia
46 47 48 49 50 51 2
52
Introduction:
53
Fungal infections due to Candida species have increased during the last decades as these
54
yeasts emerged as important opportunistic fungal pathogen. Candida species can cause
55
various clinical manifestations ranging from superficial to systemic candidiasis either in
56
immunocompetent and immunocompromised hosts (Garcia-Hermoso et al. 2007; Odds et al.
57
2007; Pfaller & Diekema 2007; Fesharaki et al. 2013). Infections due to C. albicans are an
58
important cause of morbidity and mortality among hospitalized patients who have been
59
admitted to Intensive Care Units (ICUs) and in oncology wards worldwide (Bougnoux et al.
60
2006; Bougnoux et al. 2008; Horn et al. 2009; Tortorano et al. 2012; Sardi et al. 2013). Burn
61
patients are especially susceptible to Candida infections because they have most of the risk
62
factors for hospital-acquired fungal infections, i.e., cutaneous and vascular portals of entry
63
(inhabiting catheters, parenteral nutrition) and broad-spectrum antimicrobial therapy. In most
64
cases, colonization of C. albicans of the skin or mucosa occurs before invasion and systemic
65
infection and colonization may be a main reservoir for cross contamination. Therefore,
66
identification of the source of colonization and the transmission route of infection will
67
contribute to the prevention of nosocomial infections due to C. albicans among burn patients
68
(Bougnoux et al. 2004; Lee et al. 2013). Nosocomial candidiasis caused by C. albicans
69
requires a precise understanding of the epidemiology in different geographic regions using a
70
reliable typing system. Recently, several molecular typing techniques have been used to study
71
epidemiological relationships of C. albicans isolates such as randomly amplified polymorphic
72
DNA (Robert et al. 1995), restriction fragment length polymorphism (Vazquez et al. 1993),
73
Southern blot hybridization with discriminating probes (Myoung et al. 2011), amplified
74
fragment length polymorphism analysis (Borst et al. 2003) and microsatellites length
75
polymorphism (Garcia-Hermoso et al. 2007). The multilocus sequence typing (MLST)
76
technique has been extensively used because it yields high levels of resolution allowing it to
77
characterize a large number of isolates rapidly, and it does not require any subjective
78
interpretation of banding patterns. Moreover, MLST sequences can be stored and
79
characterized in multiple electronic formats, thus offering an unprecedented degree of
80
portability and accessibility to other users (website http://calbicans.mlst.net) (Bougnoux et al.
81
2003; Bougnoux et al. 2004). The purpose of this study is to investigate the MLST analysis of 3
82
clinical isolates of C. albicans recovered from patients with burns in a burn intensive care unit
83
(BICU) of a hospital at Sari city, Iran. It is also the first nationwide study into the genotypic
84
relationships of C. albicans isolates from Iran using the MLST method.
85 86
Materials and Methods
87
Thirty clinical isolates of C. albicans were collected from hospitalized patients at Sari city,
88
Iran. These strains were isolated from 22 burn patients with superficial colonization or
89
candidiasis, candiduria and candidemia that were admitted to the BICU of the burn hospital
90
from July 2011 to March 2012. The skin burns were sampled using sterile swabs (Becton,
91
Dickinson, USA) and samples cultured onto Sabouraud’s dextrose agar (SDA, Difco) medium
92
supplemented with chloramphenicol (SC). Blood samples were inoculated onto biphasic
93
fungal blood culture media containing brain heart infusion (BHI, Difco) broth and BHI Agar
94
and incubated for 10 days at 37 ºC. Blood cultures were sub-cultured on SC and CHROMagar
95
Candida (BioMérieux, Marcy l'Etoile, France). Five patients were sampled at different body
96
sites and different times: skin burns (hand), blood and urine of patient 2 (P2) were sampled
97
within a 7 day period; skin burns (hand & abdomen), blood and urine of P3 were sampled
98
within a 10 day period; skin burns (foot) and urine of P7 were sampled within a 7 day period;
99
skin burns of P13 including chest and scapula were sampled within a 5 day period; skin burns
100
(chest) and two blood samples of P17 were sampled within a 21 day period. MLST was used
101
to type the 30 clinical isolates as described previously (Bougnoux et al. 2003). Briefly,
102
genomic DNA was extracted using glass beads and the phenol/chloroform method (Yamada et
103
al. 2002) and stored at −20°C prior to use. MLST used seven housekeeping genes, AAT1a,
104
ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b (Bougnoux et al. 2003). PCR amplification
105
was performed for seven loci using a thermal cycler (Biorad-C1000) and described primers
106
(Bougnoux et al. 2003), followed by sequencing using an ABI 3730XL automatic sequencer
107
(Applied Biosystems, 3730/3730xl DNA Analyzers Sequencing, Bioneer, Korea). Sequence
108
data were manually aligned using MEGA 5.05 (http://www.megasoftware.net/) (Tamura et al.
109
2011) and Bioedit ver. 7.0.9 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Alignment,
110
BioEdit Sequence 2011) software packages. Heterozygosity was identified by the presence of
4
111
two peaks on both strands at the same loci and the consensus sequences for the seven loci of
112
all isolates were defined.
113
The number of alleles and diploid sequence types (DSTs) was determined by comparing the
114
sequences with those available in the C. albicans MLST database (http://calbicans.mlst.net).
115
Chromatograms for the isolates with new alleles and new DSTs were sent to the central
116
MLST database curator and new MLST numbers were assigned. Newly identified DSTs were
117
assigned
118
http://calbicans.mlst.net/sql/burstspadvanced.asp. Epidemiological relationships were assessed
119
by eBURST analysis (http://eburst.mlst.net/) (Feil et al. 2004) and DSTs of 30 isolates were
120
compared with those available (n = 2099) in the MLST database at June 2014. This algorithm
121
places all related isolates into groups called clonal clusters (CC: set of DSTs that are believed
122
to have descended from the same founding genotype). Clonal clusters were identified using a
123
reference DST for each clade according to previously described major C. albicans clades
124
(Odds et al. 2007; Odds 2010). Clonal clusters of isolates differ in sequence at only one of the
125
seven loci (Odds et al. 2007). The UPGMA dendrogram based on MLST sequence data was
126
drawn using CLC Sequence Viewer 6 software (http://www.clcbio.com/).
the
numbers
2240
to
2251
(except
2246
and
2248)
in
127 128
Results
129
Thirty epidemiologically related isolates of C. albicans were obtained from 22 burn patients
130
(age range: 2 – 60 years) admitted to the burn intensive care unit (BICU). C. albicans strains
131
were isolated from blood cultures (n=6), skin burns (n=19), urine (n=3), catheter (n=1) and
132
sputum (n=1). The MLST data of the 30 C. albicans isolates consisted of 2,883 nucleotides.
133
Seventy five (2.6%) nucleotide sites were found to be variable and heterozygous in at least
134
one isolate. Thus the seven sequenced genes yielded a total of 75 variable sites of which
135
VPS13 produced the highest number (n = 14) of polymorphic sites, while ACC1 produced the
136
lowest number (n = 6). Sixty two alleles were identified in the seven loci studied. The gene
137
VPS13 generated the highest number of alleles (n = 11), while ACC1 generated the lowest
138
number (n = 7). Among the alleles, one new allele was determined in the ADP1 locus (Allelic
139
number 122) and this was added to the MLST database (http://calbicans.mlst.net). Dated June
5
140
2014, from the 125 alleles present only allele number 122 of the ADP1 locus displayed a
141
polymorphism at nucleotide site 366 (Y instead of C).
142
Eighteen unique DSTs were obtained in the seven loci of the 30 Iranian C. albicans isolates
143
from
144
www.calbicans.mlst.net. Eight of these 18 DSTs had been previously identified and 10 novel
145
DSTs were added to the online database (http://calbicans.mlst.net). The eBURST program
146
was used for the analysis of the genotypic relationships among the MLST data of the 30
147
Iranian C. albicans strains using all available DSTs (n= 2099) in the MLST database. This
148
yielded 108 eBURST groups (CCs) and 707 singleton isolates. The 30 isolates in this study
149
were placed in 9 CCs while two isolates (DST 2095) were singletons. Eleven Iranian isolates,
150
i.e. 36.7%, recovered from eight patients belonged to CC 124, 4 isolates (13.3%) from four
151
patients belonged to CC 172, 3 isolates (10%) from two patients belonged to CC 69, 2 isolates
152
(6.7%) from two patients belonged to CC 918, 3 isolates (10%) from one patient belonged to
153
CC 601, 2 isolates (6.7%) from two patients belonged to CC 766, one isolate (3.3%) belonged
154
to CC 461 and one isolate (3.3%) belonged to CC 1865. However, according to the latest
155
classification by Odds et al. 2007 and Odds 2010, the taxonomical position for new DSTs
156
(2099 and 2102) were not clarified so far (Fig. 1).
157
Among 30 isolates of C. albicans collected from skin burns and blood cultures of 22 patients
158
in the same BICU, the MLST analysis showed that some isolates had the same DST (Fig. 1).
159
From 22 patients in this study, 19 isolates from skin burns or cutaneous candidiasis yielded 13
160
DSTs belonging to five clades (1, 4, 9, 12 and 15), of which seven DSTs belonged to clade 4
161
because some alleles were shared between seven DSTs. These results may suggest he
162
presence of microvariation due to microevolution of the genomes of epidemiologically related
163
strains of C. albicans in the BICU (Fig. 1). Three out of the seven strains isolated from
164
candidaemic patients with burns belonged to clade 4 (CC 124) and two of them originated
165
from multiple episodes of one patient (P17). Four other strains were assigned to four CCs (69,
166
918, 461 and 601) in four clades (1, 9, 11 and 12). Moreover, four isolates recovered from
167
candidaemic patients were identified as new DSTs (Fig. 1).
168
Some patients yielded multiple isolates with identical DSTs, four patients (P4, P9, P10 and
169
P12) yielded isolates identified as DST 172, two patients (P19 and P22) had DST 656, two
22
BICU
patients.
Each
allelic
profile
6
query
has
been
submitted
to
170
patients (P5 and P13) DST 124 and one patient (P3) was infected with C. albicans isolates
171
belonging to two DST (i.e., 918 and 2093) (Table 1). Five patients (P2, P3, P7, P13 and P17)
172
were sampled at different body sites (Table 1), which four patients appeared to harbor just one
173
strain of C. albicans, whereas one patient (P3) yielded two different strains isolated from three
174
samples (blood, urine and skin burns) (Table 1).
175 176
Discussion
177
Multilocus sequence typing is a powerful and useful technique for understanding the
178
epidemiological and evolutionary relationships of pathogenic microorganisms (Taylor &
179
Fisher 2003; Urwin & Maiden 2003; Maiden 2006; Cliff et al. 2008; Odds & Jacobsen 2008;
180
Da Matta et al. 2010; Shin et al. 2011). Prevalence of C. albicans infections in the BICU of
181
the Zare hospital (Sari, Iran) led us to investigate the epidemiologically relationship of 30
182
isolates involved using the MLST method. Although the sample size of strains typed herein
183
was low, our study revealed 10 new DSTs, i.e. 33.3%, among 30 isolates. Three new DSTs
184
belonged to clade 4 (CC 124) as did a new singleton DSTs. The remainder of the isolates
185
belonged to various CCs. Twenty eight isolates were found to be located in 9 CCs by
186
eBURST analysis and two isolates were identified as singletons, thus revealing a high genetic
187
heterogeneity among the C. albicans population of these hospitalized patients.
188
Among a large assembly of strains collected worldwide, Odds and coworkers (Odds et al.
189
2007; Odds 2010) showed that C. albicans clade 1 (CC 69) was the most prevalent one
190
followed by clade 4 (CC 124), clade 3 (CC 344), clade 2 (CC 155) and clade 11 (CC 538),
191
respectively. In addition, they found that 34% of the Middle East isolates belonged to clade 1.
192
Ninety three percent of our isolates clustered within 6 of the 17 known clades and 63%
193
belonged to three of the 5 major clades (i.e. clades 1, 4 and 11) as defined previously (Odds
194
2010). In contrast, approximately 17% of our isolates belonged to clade 1 including CC 69
195
and CC 766, whereas 43% belonged to clade 4 including CC 124. Alastruey et al. (2013)
196
identified 37 DSTs of C. albicans including 17 new DSTs from Israel and they concluded that
197
most strains belonged to CC 124 in both candidemia and superficial candidiasis isolates
198
(Alastruey-Izquierdo et al. 2013). Notably none of our strains clustered in clade 2 (CC 155)
199
and clade 3 (CC344). In contrast, Gammelsrud et al. (Gammelsrud et al. 2012) analyzed 62 7
200
strains from Norway and found 32 DSTs from which 31% belonged to clade 2 (CC 155). Shin
201
et al. (Shin et al. 2011) found 58% new DSTs among 156 Korean isolates, and 15% of the
202
isolates clustered in clade 4 and 19% clustered in a new Asia-specific clade with P-distance ≤
203
0.035, while based on the P-distance ≤ 0.04 (Odds 2010), all isolates were assigned to clade 3.
204
In the present study, our results showed that none of the Iranian isolates belonged to this Asia-
205
specific clade.
206
Isolates obtained from different body sites of patients belonged to the same CC, for example
207
P2, P7, P13 and P17 (Fig. 1). This finding is in accordance with previous studies that reported
208
that C. albicans strains isolated from different body sites of a person are usually genetically
209
similar or identical (Bougnoux et al. 2006; Cliff et al. 2008), whereas two strains from P3
210
were assigned to two CC 69 and CC 918.
211
Some DSTs (656, 124 and 172) of C. albicans isolated from more than one patient in the same
212
BICU during one month; therefore, it seems that colonized patients are a main reservoir of C.
213
albicans in hospitals and the isolation of C. albicans with the same DST in more than one
214
patient may be provide evidence for cross contamination between patients which might be a
215
crucial factor for nosocomial infections (Bougnoux et al. 2003; Bougnoux et al. 2004;
216
Bougnoux et al. 2006).
217
Several closely related strains were isolated from patients. For example, the new DST 2093
218
differs from DST 69 only in locus ADP1 with one nucleotide different that may have arisen
219
from a single recombination event. Similarly DST 2101 differs from DST 766 only in the
220
locus ADP1 with one nucleotide different and four mentioned DSTs assigned to clade 1 (Fig.
221
1). Moreover three new DSTs, including 2092, 2096 and 2103, assigned to CC124 were
222
founded to be different in three loci with DSTs 124, 605 and 656 (Fig. 1). These findings may
223
suggest micro-evolutionary changes of a single strain occurring during adaptation to varying
224
environmental conditions (Bonfim-Mendonca et al. 2013; Paluchowska et al. 2014).
225
Cliff and coworkers showed evidence for the presence of an endemic strain in a ICU that was
226
isolated repeatedly from patients and staff, and suggested horizontal transmission of C.
227
albicans in the unit (Cliff et al. 2008). Bougnoux and colleagues using the MLST technique
228
showed frequent colonization of C. albicans in a person or in the whole family by genetically
229
closely related isolates that differed at one or several of the MLST loci (Bougnoux et al.
230
2006). 8
231
In conclusion, although a limited number of isolates was analyzed in the current study, a
232
significant genetic relationship was identified among epidemiologically related C. albicans
233
isolates from burn patients in Iran suggesting a likelihood of nosocomial spread. Isolates that
234
show identical or highly similar allelic profiles are presumed to be identical or closely related
235
and may use to evaluate the genetic relationships between isolates from a specific
236
environment such as BICU. To best of our knowledge, this study is the first MLST study
237
analyzing C. albicans clinical isolates from Iran, thus also improving the epidemiological
238
knowledge of C. albicans in this region.
239 240
Acknowledgments
241
This research was financially supported by a grant of the Mazandaran University of Medical
242
Science (No. 91-32), which we gratefully acknowledge. We would like to thank Nazanin Lotfi
243
and Seyede Zahra Nouranibaladezaei for technical assistance. We acknowledge the use of the
244
Candida albicans MLST database which is located at Imperial College London and is funded by
245
the Welcome Trust. We also thank the curator of MLST database, Marie-Elisabeth Bougnoux for
246
assigning our new alleles and DSTs to the C. albicans MLST database. T. B. was supported by
247
grant HPRP 5-298-3-086 from the Qatar National Research Fund, a member of Qatar foundation.
248
The statements herein are solely the responsibility of the authors.
249 250
Declaration of interest: All authors report no potential conflicts of interest. Only the
251
authors are responsible for the content and writing of the paper.
252 253
References:
254 255 256 257 258 259 260 261 262 263
Alastruey-Izquierdo, A., Mandelblat, M., Ben Ami, R., Perlin, D. S., & Segal, E. (2013). Multilocus sequence typing of Candida albicans isolates from candidemia and superficial candidiasis in Israel. Med Mycol 51, 1-4. Alignment, BioEdit Sequence (2011). version 7.0. 9. Bonfim-Mendonca, P. d. S., Fiorini, A., Shinobu-Mesquita, C. S., Baeza, L. C., Fernandez, M. A., & Svidzinski, T. I. E. (2013). Molecular Typing of Candida albicans isolates from hospitalized patients. Rev Inst Med Trop São Paulo, 55, 385-391. 9
264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309
Borst, A., Theelen, B.,Reinders, E., Boekhout, T., Fluit A. C. and Savelkoul, P. H. M. (2003). Use of amplified fragment length polymorphism analysis to identify medically important Candida spp., including C. dubliniensis. J Clin Microbiol 41(4): 1357-1362. Bougnoux, M.-E., Aanensen, D. M., Morand, S., Théraud, M., Spratt, B. G., & d’Enfert, C. (2004). Multilocus sequence typing of Candida albicans: strategies, data exchange and applications. Infec Genet Evol 4, 243-252. Bougnoux, M.-E., Diogo, D., Francois, N., Sendid, B., Veirmeire, S., Colombel, J. F., Bouchier, C., Van Kruiningen, H., d’Enfert, C. & Poulain, D. (2006). Multilocus sequence typing reveals intrafamilial transmission and microevolutions of Candida albicans isolates from the human digestive tract. J Clin Microbiol 44, 1810-1820. Bougnoux, M.-E., Kac, G., Aegerter, P., d’Enfert, C., & Fagon, J.-Y. (2008). Candidemia and candiduria in critically ill patients admitted to intensive care units in France: incidence, molecular diversity, management and outcome. Intensive Care Med 34, 292-299. Bougnoux, M.-E., Tavanti, A., Bouchier, C., Gow, N., Magnier, A., Davidson, A., Maiden, M. C. J., d’Enfert, C. & Odds, F. (2003). Collaborative consensus for optimized multilocus sequence typing of Candida albicans. J Clin Microbiol 41, 5265-5266. Cliff, P., Sandoe, J., Heritage, J., & Barton, R. (2008). Use of multilocus sequence typing for the investigation of colonisation by Candida albicans in intensive care unit patients. J Hospital Infect 69, 24-32. Da Matta, D. A., Melo, A. S., Guimarães, T., Frade, J. P., Lott, T. J., & Colombo, A. L. (2010). Multilocus sequence typing of equential Candida albicans isolates from patients with persistent or recurrent fungemia. Med Mycol 48, 757-762. Feil, E. J., Li, B. C., Aanensen, D. M., Hanage, W. P., & Spratt, B. G. (2004). eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J bacteriol, 186, 1518-1530. Fesharaki, S. H., Haghani, I., Mousavi, B., Kargar, M.L., Boroumand, M., Anvari, M.S., Abbasi, K., Meis, J.F.,& Badali H. Endocarditis due to a co-infection of Candida albicans and Candida tropicalis in a drug abuser. J Med Microbiol 62, 1763-1767. Gammelsrud, K. W., Lindstad, B. L., Gaustad, P., Ingebretsen, A., Høiby, E. A., Brandtzaeg, P., & Sandven, P. (2012). Multilocus sequence typing of serial Candida albicans isolates from children with cancer, children with cystic fibrosis and healthy controls. Med Mycol 50, 619-626. Garcia-Hermoso, D., Cabaret, O., Lecellier, G., Desnos-Ollivier, M., Hoinard, D., Raoux, D., Costa, J. M., Dromer, F. & Bretagne, S. (2007). Comparison of microsatellite length polymorphism and multilocus sequence typing for DNA-based typing of Candida albicans. J Clin Microbiol 45, 3958-3963. 10
310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353
Horn, D. L., Neofytos, D., Anaissie, E. J., Fishman, J. A., Steinbach, W. J., Olyaei, A. J., Marr, K. A., Pfaller, M. A., Chang, C. H. & Webster, K. M. (2009). Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry. Clin Infect Dis 48, 1695-1703. Lee, H. G., Jang, J., Choi, J. E., Chung, D. C., Han, J. W., Woo, H., Jeon, W. & Chun, B. C. (2013). Blood stream infections in patients in the burn intensive care unit. Infect chemother, 45, 194-201. Maiden, M. C. (2006). Multilocus sequence typing of bacteria. Annu Rev Microbiol 60, 561588. Myoung, Y., Shin, J. H., Lee, J. S., Kim, S. H., Shin, M. G., Suh, S. P. and Ryang, D. W. (2011). Multilocus sequence typing for Candida albicans isolates from candidemic patients: comparison with Southern blot hybridization and pulsed-field gel electrophoresis analysis. Korean J Lab Med 31, 107-114. Odds, F. C. (2010). Molecular phylogenetics and epidemiology of Candida albicans. Future Microbiol 5, 67-79. Odds, F. C., Bougnoux, M.-E., Shaw, D. J., Bain, J. M., Davidson, A. D., Diogo, D., Jacobsen, M. D., Lecomte, M., Li, S. Y. & other authors (2007). Molecular phylogenetics of Candida albicans. Eukaryot Cell 6, 1041-1052. Odds, F. C., & Jacobsen, M. D. (2008). Multilocus sequence typing of pathogenic Candida species. Eukaryot Cell 7, 1075-1084. Paluchowska, P., Tokarczyk, M., Bogusz, B., Skiba, I., & Budak, A. (2014). Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland. Mem Inst Oswaldo Cruz, 109, 436-441. Pfaller, M., & Diekema, D. (2007). Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2, 133-163. Robert, F., Lebreton, F., Bougnoux, M., Paugam, A., Wassermann, D., Schlotterer, M., Tourte-schaefer, C. & Dupouy-Camet, J. (1995). Use of random amplified polymorphic DNA as a typing method for Candida albicans in epidemiological surveillance of a burn unit. J Clin Microbiol 33, 2366-2371. Sardi, J., Scorzoni, L., Bernardi, T., Fusco-Almeida, A., & Giannini, M. M. (2013). Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol, 62, 10-24. Shin, J. H., Bougnoux, M.-E., d'Enfert, C., Kim, S. H., Moon, C.-J., Joo, M. Y., Lee, K., Kim. M. N., Lee, H. S. & other authors (2011). Genetic diversity among Korean Candida 11
354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377
albicans bloodstream isolates: assessment by multilocus sequence typing and restriction endonuclease analysis of genomic DNA by use of BssHII. J Clin Microbiol 49, 2572-2577. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 28, 2731-2739. Taylor, J. W., & Fisher, M. C. (2003). Fungal multilocus sequence typing—it’s not just for bacteria. Curr Opin Microbiol 6, 351-356. Tortorano, A. M., Prigitano, A., Dho, G., Grancini, A., & Passera, M. (2012). Antifungal susceptibility profiles of Candida isolates from a prospective survey of invasive fungal infections in Italian intensive care units. J Med Microbiol, 61, 389-393. Urwin, R., & Maiden, M. C. (2003). Multi-locus sequence typing: a tool for global epidemiology. Trend Microbiol 11, 479-487. Vazquez, J. A., Sanchez, V., Dmuchowski, C., Dembry, L. M., Sobel J. D., & Zervos, M. J. (1993). Nosocomial acquisition of Candida albicans: an epidemiologic study. J Infec Dis 168, 195-201. Yamada, Y., Makimura, K., Merhendi, H., Ueda, K., Nishiyama, Y., Yamaguchi, H., & Osumi, M. (2002). Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts. Japan J Infect Dis 55, 122-125.
378 379 380 381 382 383 384 385 386 387 388 389 390 391 12
392
Table 1. Summarized the epidemiologically related isolates are recovered from the same patients at
393
different times in BICU 2011
2012
DST July
August
September
January
February
March
P3(2)
2093
(Blood and Abdomen burns) P3(1)
918
(Urine) P2(3)
601
(Hand burns, Blood and Urine) P7(2)
2095
(Foot burns and Urine) P5(1) (Chest burns)
124
P13(2) (Chest and Scapula burns) P17(3) (Chest burns)
605
and 2 Blood samples) P4(1) (Foot burns)
172
P9(1) (Hand burns)
P12(1) (Chest burns)
P10(1) (Hand burns)
656
394 395 396 397
P19(1)
P22(1)
(Hand
(Abdomen
burns)
burns)
Numbers in parentheses symbolize the number of times the DST was isolated from that patient in that month; P: patient
13
398 399 400 401
Figure. 1. Summarizes all information of the 30 Iranian C. albicans comprising the allelic number for each locus, DSTs, CCs, clades and it also illustrates the UPGMA dendrogram based on MLST data. The scale indicates p-distances (P
