MAJOR ARTICLE
Candida albicans Is Not Always the Preferential
Yeast Colonizing Humans: A Study in Wayampi
Amerindians
Cécile Angebault,1,2,3,6,7 Félix Djossou,9 Sophie Abélanet,3,6 Emmanuelle Permal,3,6 Mouna Ben Soltana,2
Laure Diancourt,5 Christiane Bouchier,4 Paul-Louis Woerther,1 François Catzeflis,8 Antoine Andremont,1
Christophe d’Enfert,3,6 and Marie-Elisabeth Bougnoux2,3,6,7
EA3964 Université Paris-Diderot “Résistance bactérienne dans les flores commensales,” Hôpital Bichat-Claude Bernard and 2Unité de ParasitologieMycologie, Service de Microbiologie, Hôpital Necker-Enfants-Malades, Assistance Publique - Hôpitaux de Paris, 3Unité Biologie et Pathogénicité
Fongiques and 4PF1 Génomique, Département Génomes et Génétique, and 5Pasteur Genopole, PF8 Génotypage des Pathogènes et Santé Publique,
Département Infection et Epidémiologie, Institut Pasteur, 6USC 2019, F-75015, INRA, 7Université Paris Descartes, Sorbonne Paris-Cité Paris, 8Institut des
Sciences de l’Evolution, UMR-5554 Centre National de Recherche Scientifique, Université Montpellier 2, Montpellier, France; and 9Centre Hospitalier de
Cayenne Andrée Rosemon, Cayenne, French Guiana
1
In industrialized countries Candida albicans is considered the predominant commensal yeast of the human intestine, with approximately 40% prevalence in healthy adults. We discovered a highly original colonization
pattern that challenges this current perception by studying in a 4- year interval a cohort of 151 Amerindians
living in a remote community (French Guiana), and animals from their environment. The prevalence of C. albicans was persistently low (3% and 7% of yeast carriers). By contrast, Candida krusei and Saccharomyces cerevisiae were detected in over 30% of carriers. We showed that C. krusei and S. cerevisiae carriage was of food or
environmental origin, whereas C. albicans carriage was associated with specific risk factors (being female and
living in a crowded household). We also showed using whole-genome sequence comparison that C. albicans
strains can persist in the intestinal tract of a healthy individual over a 4-year period.
Keywords.
intestinal colonization; yeasts; candida albicans; amerindians; whole-genome sequencing; MLST.
Candida albicans is by far the most abundant and significant species that colonize humans. Previous studies
showed that >80% of individuals colonized with yeast
in the oral, digestive, or vaginal mucosa are C. albicans
carriers [1–3]. Eventually, these commensal C. albicans
strains may cause life-threatening infections, especially
in immunocompromised or critically ill patients [4].
Colonization may happen at or around birth [5, 6] and
Received 24 March 2013; accepted 5 June 2013; electronically published 31 July
2013.
Presented in part: The 51st Interscience Conference on Antimicrobial Agents and
Chemotherapy, Chicago, Illinois, September 17–20, 2011; 11th American Society of
Microbiology Conference on Candida and Candidiasis, San Francisco, California,
March 29-April 2, 2012.
Correspondence: Marie-Elisabeth Bougnoux, MD, PhD, Unité Biologie et Pathologie Fongiques, Institut Pasteur, 25-28 rue du Dr Roux, 75015, France (bougnoux@
pasteur.fr).
The Journal of Infectious Diseases 2013;208:1705–16
© The Author 2013. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected].
DOI: 10.1093/infdis/jit389
transmission can occur within families or through
close physical contact [1, 7]. A limited number of
related strains of C. albicans can colonize one or several
mucosal membranes of a given individual [1, 2, 8]. The
long-term dynamics of C. albicans carriage in healthy
persons has not been studied yet, with only 1 study reporting short-term oropharyngeal colonization with
identical strains based on polymerase chain reaction
fingerprinting [2].
C. albicans is also a possible commensal microorganism of domestic and wild warm-blooded animals (eg,
birds), but studies on its prevalence in animal populations remain scarce [9–13]. There is no apparent environmental reservoir for C. albicans, and no terrestrial
life cycle has been described thus far [14, 15]. C. albicans can survive in water microcosms for a long period,
but its presence seems most often secondary to fecal
pollution [16, 17]. Therefore, it is widely believed that
human beings are the main reservoir of C. albicans and
that this species has coevolved with its host [18, 19].
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This hypothesis is supported by the distribution of the human
strain types and clades that point to a degree of localized evolution of C. albicans strains in different geographic niches [18,
20]. However, these assumptions can be biased because studies
on human yeast colonization have mostly been performed on
Westerners or populations living in industrialized areas. To
overcome this concern, we studied the biodiversity and longterm intestinal yeast colonization in a cohort of healthy Amerindians from a remote and traditional community that has unhindered access to health care and antimicrobial treatments
along with intestinal yeast carriage of wild and domestic
animals from their environment.
and answered a standard questionnaire at each sampling time.
The characteristics of the 151 volunteers included in the cohort
were not significantly different from those of the other adults
(Table 1). Both campaigns were approved by an Ethics Committee (Comité de Protection des Personnes Sud-Ouest et
Outre Mer III, 2006/0498-DGS, 2010-A00682-37).
In addition, rectal swab samples were obtained from 183
wild animals trapped along a 3000-m transect heading through
the nonanthropized primary forest [25] and 30 domestic
animals caught in the village in 2006. Samples from 15 local
fruits and 9 handmade manioc-fermented beverages (cachiri)
were collected in 2006 and 2010.
Culture, Isolation, and Identification of Yeasts
MATERIALS AND METHODS
Design, Study Site, and Population
We conducted 2 prevalence studies on yeast intestinal colonization in October 2006 and October 2010, using a cohort of 151
adult Wayampi Amerindians. The study was carried out in
Trois-Sauts, an isolated village in the Amazonian forest in
French Guiana. The population and lifestyle of the community
has been described elsewhere [21–23]. In brief, it is an ethnically homogeneous traditional community living on the Oyapock
River. The villagers share large huts and they use small areas
along the river for drinking and bathing. They eat local food
almost exclusively [24]. Resident paramedical officers provide
complete medical care to all villagers at the health post.
All adult villagers (N = 238) were asked to participate in the
2006 study, and 163 healthy volunteers (68.5%) who signed a
consent form were included (female-male ratio, 1.09; mean
age: 35.1 years); 151 of them agreed to participate again in
2010. Each volunteer provided a freshly passed fecal sample
Table 1. Comparison of the Adult Volunteers Included in the
Study With the Non included Adult Population
Characteristic
Sex, No. (%)
Female
Male
Volunteers
(n = 151)
.69
77 (51)
74 (49)
Age, mean (range), y
No. of life partners, No. (%)b
Non included
P
adults (n = 87) Valuea
34.4 (18–78)
47 (54)
40 (46)
33.3 (18–84)
0
20 (13)
18 (24)
1
2
123 (82)
7 (5)
55 (72)
3 (4)
.61
.14
No. of children, mean (range)
3.9 (0–13)
3.1 (0–9)
.06
Antibiotic courses per year,
mean (range)
0.9 (0–5)
0.8 (0–5)
.84
Analysis was performed using Fisher exact and Student t tests (2-sided;
significance level, .05).
a
b
Data missing in 12 subjects : 1 volunteer and 11 non included adults.
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A pea-sized amount of each human stool sample, rectal swab
samples collected from each animal, 2 pieces (1 × 1 cm) of each
fruit, and 1 mL of each cachiri were inoculated extemporaneously onto chloramphenicol-gentamicin Sabouraud agar slants
in screw-cap tubes (BioRad Laboratories) and kept at room
temperature (approximately 30°C) until they were sent to metropolitan France about 2 weeks later. The entire yeast growth
was then inoculated onto chromogenic yeast-specific plates
(BBL CHROMagar Candida; Becton Dickinson). Morphologically different colonies were further identified using MatrixAssisted Laser Desorption/Ionisation - Time of Flight mass
spectrometry (Andromas). When needed, molecular identification was performed by sequencing of the ITS1-ITS4 and NL1NL4 regions of ribosomal DNA [26].
Typing Methods
The C. albicans and C. krusei isolates were typed using the specific multilocus sequence typing (MLST) schemes [27, 28].
Allele and sequence type (ST) assignments were determined
using the C. albicans (http://calbicans.mlst.net/) and C. krusei
(http://pubmlst.org/ckrusei/) MLST databases. Phylogenetic
analysis was carried out using the unweighted pair group
method with arithmetic mean implemented in MEGA 5.0 software [29], as described elsewhere [20]. The C. albicans strains
were assigned to known C. albicans clades, as described elsewhere [30]. The genotypic diversity (D) in C. krusei, defined as
the probability that 2 strains taken at random had different STs,
was calculated using Multilocus software, version 1.3b (http
://www.agapow.net/software/multilocus-2/, P-M. Agapow and
A. Burt) [31]. The diversity index was calculated as 1 − D. Microsatellite polymorphisms were used for Saccharomyces cerevisiae strain typing [32]. Microsatellite sequences were analyzed
using GeneMapper software, version 4.0 (Applied Biosystems)
[32].
Whole-Genome Sequencing of C. albicans Isolates
Genomic DNA was extracted from C. albicans isolates, as described elsewhere [33]. Libraries were prepared using the
TruSeq DNA Sample Prep kit (Illumina), according to the
manufacturer’s recommendations. DNA was sheared by sonication to an average fragment length of 500 base pairs (bp). Illumina adapters were blunt-end ligated, and libraries were
amplified by polymerase chain reaction. Each sample was sequenced on an Illumina Genome Analyzer platform (Illumina;
HiSeq 2000). The 30–36-bp single-end reads obtained were
aligned using the Short Read Analysis Pipeline SHORE
(v0.5.0beta) [34] against the reference genome sequence of
strain SC5314 (assembly 21, CGD; http://www.candidagenome.
org). Consensus command was used to create consensus sequences from alignments and generate output files gathering
SNPs between the 2 isolates. From these data, we computed the
sequencing depth along the chromosomes, located SNPs, and
checked for loss of heterozygosity (LOH) events. The genome
sequence data are available on the National Center for Biotechnology Information BioSample database (accession numbers,
2 144 021 for H014_2006 strain [CEC2013] and 2 144 022 for
H014_2010 strain [CEC3943]).
Epidemiological Data
We used information recorded at the health post or obtained
from local informers. Demographics (age, sex, marital status,
and number of children), lifestyle and environmental data (size
of household, contacts with animals, daily activities, and babysitting of children under 5 years), and medical history (current
pregnancy, chronic disease, previous hospitalizations and operations, and systemic antimicrobial agent intake during the year
before sampling) were collected from volunteers in 2006 and
2010, using a standard data collection form.
Statistical Methods
We performed the epidemiological analyzes using R software
(version 2.13.0; http://www.cran.r-project.org). Univariate analysis of discrete variables was performed using 2-sided Pearson
χ2 and Fisher exact tests; Student’s t and Wilcoxon tests were
used to test continuous variables. All tests performed were 2
sided, and the significance level was set at 5%. All variables
with univariate P values <.20 were included in the multivariate
analysis, which was performed using descending stepwise logistic regression. The final logistic models were tested for statistical
stability using the RVAideMemoire package (http://cran.rproject.org/web/packages/RVAideMemoire/).
RESULTS
Yeast Carriage
The prevalence of intestinal yeast carriage in humans was 66%
(100 of 151 volunteers) in 2006 and 100% in 2010, with 13 and
17 different species identified, respectively (Figure 1 and Supplementary Table 1). Overall, 22 species were identified, but
only 8, including C. albicans, C. krusei, Candida tropicalis,
Candida guilliermondii, Candida orthopsilosis, S. cerevisiae,
Geotrichum candidum, and Rhodotorula sp, were found both in
2006 and 2010. The most predominant species were C. krusei
and S. cerevisiae, with similar high prevalence rates among the
carriers (30% in 2006 and >70% in 2010) (Figure 1). In contrast, C. albicans was rare. Its prevalence among the carriers
was 10 times lower than those observed for C. krusei and S. cerevisiae at each campaign (3% in 2006 and 7% in 2010).
The prevalence of intestinal yeast carriage in animals was
42% (90 of 213) in 2006, with 25 species identified (Supplementary Table 1). The 3 main species were C. tropicalis (36% of
carriers), Issatchenkia occidentalis (20%), and C. krusei (16%).
The prevalence of C. albicans was low (7%). Ten species were
shared by humans and animals (Figure 1).
Twelve fruit samples (80%) and all cachiri tested positive for
yeasts (Supplementary Table 1); S. cerevisiae was predominant
in fruits and cachiris, and C. krusei was isolated in cachiri
but no fruit. Neither fruits nor cachiri tested positive for
C. albicans.
Dynamics of C. krusei or S. cerevisiae Carriage in Humans
To investigate the unusual high prevalence of C. krusei and S.
cerevisiae in this population, we performed an epidemiological
risk factors analysis as well as a molecular comparison of isolates collected from humans, animals and alimentary products.
The epidemiological analysis revealed no significant differences
between C. krusei or S. cerevisiae carriers and noncarriers in
either 2006 or 2010 in terms of lifestyle environment, demographic data, or medical history (Table 2).
Thirty-six human, animal, and cachiri C. krusei strains isolated in 2006 were available for MLST analysis. Nineteen distinct STs were identified, of which 16 (84%) were singletons
and 3 were shared by several isolates (Figure 2). STGU150 was
shared by isolates from 6 humans, 5 wild animals (3 rodents
and 2 marsupials), and 1 cachiri sample; STGU152 was shared
by isolates from 5 humans and 1 domestic dog; and STGU156
was shared by 2 human isolates (Figure 2).
Interestingly, the predominant STGU150 was included in a
cluster with the closely related STGU151 and ST89. STGU150
differed from STGU151 and ST89 at 1 of 6 loci (TRP1 and
HIS3, respectively; Supplementary Table 2). Sequence analysis
of these loci showed that variations between the alleles resulted
from LOH events (Supplementary Table 2). The 2 other STs
shared by multiple isolates were also part of clusters with
closely related STs, differing only by LOH events (Supplementary Table 2). Microevolution of C. krusei isolates by LOH
event has never been described thus far. No epidemiological
link (family ties or household location) was found between the
carriers of identical or closely related strains, except for a
married couple (carriers H043 and H044) who shared
STGU152 strains (Figure 2A).
Intestinal Yeast Carriage in Amerindians
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Figure 1.
Distribution of yeast species isolated from human (2006 and 2010) and animal (2006) intestinal carriers.
Among all isolates collected in 2010, we typed the 21 isolates
from carriers colonized both in 2006 and in 2010 (double carriers). Only 1 double carrier (H034) was colonized twice with
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Angebault et al
strains from the same ST (STGU150), and 1 (H005) with
closely related strains (STGU150 and STGU151; Figure 2A and
2B and Supplementary Table 2). The remaining 19 double
Table 2.
Epidemiological Analysis of Risk Factors Associated With Candida krusei, Saccharomyces cerevisiae, and Candida albicans Carriage (vs. Non carriage) in 2006 and 2010a
C. krusei
Risk Factor
Volunteers
2006 (n = 151)
S. cerevisiae
C. krusei
S. cerevisiae
C. albicans b
Carriers
(n = 30)
Puc
Carriers
(n = 30)
Puc
Volunteers 2010
(n = 151)
Carriers
(n = 108)
Puc
Carriers
(n = 121)
Puc
Carriers
(n = 11)
Puc
34.0 (20–51)
.81
34.13 (18–62)
.90
39.1 (22–83)
39.0 (22–67)
1.0
38.8 (22–83)
.6
36.6 (28–53)
.34
.42
12
.22
74
50
61
.54
77
58
Multivariate Odds
Ratioc
Demographics
Age, mean (range), y
34.4 (18–79)
Sex
Male
74
17
Female
77
13
18
.37
60
1
.01
10
1.0
12 (2.1–226.0)
Marital status
Single
20
2
131
28
≤2
58d
9
3–5
53d
6
11
6–15
38d
14
4
Coupled
.37
6
.23
24
13
6
138
102
.05
10
.72
39
26
66
48
53
2
46
34
38
6
111
1
1.00
10
No. of children
.01
15
.19
.76
30
.81
3
.12
Lifestyle
Intestinal Yeast Carriage in Amerindians
No. of household
inhabitants
2–6
75
14
7–12
76
16
.84
17
.42
13
79
55
72
53
.72
63
1.00
58
1
<.01
10
Animals in household
Presence
123
21
.11
25
1.00
115
85
.29
94
.47
8
Dogs
88
14
.21
21
.40
55
38
.71
46
.53
3
.72
Chickens
93
14
.09
19
.68
106
7
1.00
8
1.00
8
1.00
.72
River
78e
13
.52
20
.10
59f
41
.71
47
1.00
2
.20
Cove
67
e
17
.09
12
.54
55f
40
1.00
45
.67
2
.21
Tap water
27e
6
.59
6
.79
93f
69
.45
74
1.00
9
.33
Drinking water
Duty
•
Hunting
NA
72
47
.15
59
.68
1
.01
JID 2013:208 (15 November)
Fishing
NA
125
87
.34
104
.06
11
.21
Manioc culture
NA
137
102
.02
107
.31
10
1.00
Cooking
NA
99
73
.45
78
.67
10
.10
Cachiri preparing
NA
80
59
.59
63
.69
11
<.01g
Pirogue driver
NA
87
60
.47
74
.10
5
.53
Community worker
9h
2
1.00
2
1.00
8
7
.44
6
.66
0
1.00
Babysitting children
≤5 y old
112i
24
.47
24
.81
107
74
.43
87
.65
8
1.00
130
92
.80
104
1.00
8
.18
•
Travel outside TroisSauts during past
yearj
NA
1.0
13.8 (2.5–258.7)
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Table 2 continued.
C. krusei
•
Risk Factor
Angebault et al
Having a child at school
outside Trois-Sautsj
Volunteers
2006 (n = 151)
Carriers
(n = 30)
S. cerevisiae
Puc
Carriers
(n = 30)
Puc
NA
C. krusei
Volunteers 2010
(n = 151)
39
S. cerevisiae
C. albicans b
Carriers
(n = 108)
Puc
Carriers
(n = 121)
Puc
Carriers
(n = 11)
Puc
31
.22
29
.35
5
.15
Multivariate Odds
Ratioc
Medical history during
past year
Pregnancyk
Hospitalization
Surgery
Serious medical
event
Antibiotic use
9
3
.17
1
1.00
5
4
1.00
3
.32
1
.52
24
4
.79
7
.26
17h
13
.78
11
.11
1
1.00
12
2
1.00
3
.71
5h
3
.62
4
1.00
0
1.00
9
2
1.00
2
1.00
15h
9
.37
11
.50
1
1.00
67
13
1.00
18
.07
55h
45
.04
43
.68
3
.75
3
0
1.00
0
1.00
11
8
1.00
7
.23
3
.20
108
82
.04
8
1.00
7
.23
Intestinal cocolonization
C. albicans
C. krusei
30
S. cerevisiae
30
3
.20
121
82
.04
Abbreviation: NA, Not available.
a
Data represent numbers of subjects, unless otherwise indicated.
b
Due to the restricted number of subjects carrying C. albicans in 2006, the epidemiological analysis for risk factors was performed only in 2010.
c
Pu : Univariate P value. Univariate analyses were performed using Pearson χ2, Fisher exact, Wilcoxon, or Welch tests (α = .05). All variables with Pu <.20 were included for multivariate descending stepwise logistic
regressions. No variable was significantly associated with C. krusei or S. cerevisiae carriage in multivariate analyses, neither in 2006, nor in 2010 (data not shown). For C. albicans, only Odds Ratio of the significant variables
participating in the final multivariate model are given with their 95% confidence interval.
d
Data missing in 2 subjects.
e
Data missing in 8 subjects.
f
Data missing in 5 subjects.
g
Cachiri preparing was not included in the logistic regression, because this variable was strongly correlated with sex, which was already taken into consideration in the model.
h
Data missing in 1 subject.
i
Data missing in 5 subjects.
j
Most frequent locations for travel and schools outside of Trois-Sauts: Camopi, Saint-Georges, and Cayenne.
k
Among 77 women.
Figure 2. Dendrogram (A) of the phylogenetic relationships between 36 strains of Candida krusei isolated from humans, animals and cachiri in 2006 on
the basis of their sequence types (ST), associated with s second dendrogram (B) presenting the phylogenetic relationships between 21 strains isolated in
2010 from humans who were already carriers in 2006 (double carriers). The topology trees are unrooted UPGMA trees constructed with arithmetic averages
and the matrix of distances in Mega 5.0 [29]. Numbers at nodal points indicate bootstrap values ( percentages) for 1000 replications. The scale indicates p
distance. Human strains are identified by the prefix H, domestic animal strains by DA, wild animal strains by WA, and cachiri strains by Cach. The main
tree (left) presents 27 human C. krusei strains, 8 animal strains (DA05, a dog from the village; WA22, WA52, and WA59, 3 rodents trapped in the village;
WA45, WA29, WA49, and WA114, 1 rodent and 3 marsupials, respectively, captured in the forest), and 1 cachiri strain. The complementary tree (right) presents the 21 human C. krusei strains in 2010 originating from double carriers. The STs corresponding to each branch are indicated below or next to the
branch. Double carrier strains (2006 and 2010) are joined by gray dotted arrows when they are from completely different STs, by gray full arrows when
they are from presumably related STs (1 loss of heterozygosity event), and by black full arrows when they are from identical STs.
carriers (90%) had isolates with distinct STs (Figure 2A and
2B). Notably, the diversity of the 21 isolates from 2010 was
lower than that observed in 2006 (diversity index, 0.62 and
0.95, respectively), with >50% of the 2010 isolates sharing the
predominant STGU150 (Figure 2B). Finally, STGU150 was
also found in 4 of 5 isolates of C. krusei collected from cachiri
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in 2010. The last cachiri isolate belonged to the closely related
STGU151. Altogether, these results suggested a common environmental source for the C. krusei carriage.
The diversity of the S. cerevisiae strains was assessed by
microsatellite typing [32] of 15 strains isolated in 2006 from
8 humans, 4 cachiris, 2 animals, and 1 fruit and showed
12 types, with 1 cachiri and 3 human strains sharing a common
pattern (data not shown). The identity of S. cerevisiae microsatellite patterns between human and cachiri strains suggested a
possible foodborne origin.
Dynamics of C. albicans Carriage
To investigate whether the rare carriers of C. albicans isolates
presented specific sociodemographic, lifestyle or medical characteristics, we performed a statistical analysis using only the
2010 data. Indeed, in 2006, the number of carriers (n = 3) did
not allow such analysis. Inhabitants of crowded households
and women were at significantly higher risk for intestinal carriage of C. albicans (odds ratio, 17.5 [95% confidence interval
(CI), 2.9–38.5] and 12.9 [95% CI, 2.2–248.7], respectively;
Table 2).
To describe the molecular and epidemiological links between
isolates, we typed all C. albicans isolates (14 human and 6
animal isolates) collected in 2006 and 2010. Twelve STs were
identified, including 9 singletons (75%) and 3 STs shared by
multiple isolates (ST602, ST90, and ST1133; Figure 3). These
12 STs were assigned to 5 of the 18 C. albicans clades, known to
structure the C. albicans population [20, 30] (Figure 3). Eleven
strains spread in 4 STs belonged to clade 8. Notably, 3 of these
STs were closely related: ST1029 and ST1951 differed from
ST602 by LOH events at 1 of the 7 MLST loci (AAT1a and
SYA1, respectively; Supplementary Table 3). In clade 3, similarly, 2 of 4 STs (ST1952 and ST344) differed from each other by
1 LOH event.
Interestingly, we noticed epidemiological links between the 7
ST602 isolates. Four originated from 3 women (of which 1,
H014, was colonized in 2006 and 2010) related through sisterand mother-in-law relationships and living in adjacent houses.
The 3 remaining isolates came from 2 chickens and 1 rodent
captured close to the house of the H014 carrier. Altogether,
these findings suggested interhuman as well as human-animal
cross-transmission. In contrast, no epidemiological link was
Figure 3. Phylogenetic relationships between 20 strains of Candida albicans isolated from humans and animals in 2006 and 2010. The topology tree is
an unrooted UPGMA tree constructed with arithmetic averages and the matrix of distances in Mega 5.0 [20, 29]. Numbers at nodal points indicate bootstrap values ( percentages) for 1000 replications. The scale indicates p distance. Strains collected in 2006 are indicated using with asterisks, and 2010
strains with circles. Human strains are identified by the prefix H, domestic animal ( poultry) strains by DA, and wild animal strains by WA (WA38, WA10,
WA114, and WA06, a rodent trapped in the village, a rodent and a marsupial both captured in the forest, and a bird trapped along the river, respectively).
The sequence type (ST) is indicated in front of each corresponding branch. Clades are presented graphically with brackets.
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Table 3. Characteristics and Comparison of Whole-Genome Sequencing From H014_2006 and H014_2010 Strains (ST602), Isolated 4
Years Apart From Digestive Tracts of the Same Individuals
Characteristic
Chr1
a
Chr2
Chr3
Chr4
Chr5
Chr6
Chr7
ChrR
Total
No. of bases in genome of SC5314 strain
3 188 548 2 232 035 1 799 406 1 603 443 1 190 928 1 033 530 949 616 2 286 389 14 283 895
No. of bases sequenced in strains
H014_2006
3 187 109 2 232 025 1 799 391 1 601 921 1 189 487 1 031 587 948 887 2 285 378 14 275 785
H014_2010
3 187 116 2 232 028 1 799 405 1 601 952 1 189 518 1 031 589 948 889 2 285 375 14 275 872
No. of bases compared between the 2 strains 3 187 013 2 231 660 1 799 377 1 601 170 1 188 787 1 031 553 948 213 2 285 103 14 272 876
No. of identical positions
3 186 999 2 231 655 1 799 369 1 601 166 1 188 780 1 031 548 948 207 2 285 094 14 272 818
No. of SNPs
14
5
8
4
7
5
6
9
58
Abbreviations: Chr, chromosome; SNPs: single-nucleotide polymorphisms.
a
Assembly 21 (CGD; http://www.candidagenome.org).
found between the 3 other pairs of identical or closely related
strains (Figure 3).
Evolution of C. albicans Genome During Persistent Carriage
Evolution of the C. albicans genome during persistent intestinal
carriage in humans has never been investigated. Here, we performed whole-genome sequencing of the 2 ST602 isolates
(H014_2006 and H014_2010) identified in the same individual
(H014) at a 4-year interval. For H014_2006 and H014_2010,
respectively, 77X and 100X coverage of the C. albicans SC5314
reference genome [35] was obtained. These high levels of coverage allowed for an accurate resolution of the diploid assembly
of the 2 whole genomes. Comparison of 14 272 876 bases
(99.92% of the reference genome) between the 2 isolates revealed 58 single-nucleotide polymorphisms (SNPs) (Table 3).
The local sequencing depth at each SNP ranged from 29X to
123X for H014_2006 and from 21X to 121X for H014_2010
(mean coverage difference from 14X) and allowed a high
degree of confidence. These SNPs were evenly distributed over
all chromosomes and also on each chromosome (Figure 4).
Twelve SNPs corresponded to a LOH in H014_2010 relative to
H014_2006, 35 were due to a LOH in H014_2006 relative to
H014_2010, and 11 were due to a change of the bases composing the heterozygous pair (Figure 4). All 58 SNPs were isolated
point mutations, and no long-range LOH events were detected.
DISCUSSION
In the Wayampi community of Trois Sauts, we found an unexpected colonization pattern that persisted over time and was
characterized by high rates of overall intestinal yeast carriage, a
high diversity among yeast species, but rare C. albicans carriage.
First, C. krusei and S. cerevisiae were the most frequent yeast
species recovered among yeast carriers both in 2006 and 2010.
This finding was unexpected because S. cerevisiae is usually reported to be a rare colonizer in healthy humans [3, 36, 37], and
C. krusei is almost never found as a commensal microorganism,
except among patients receiving antifungal drugs [38]. In our
study, the high prevalence of C. krusei carriage was not associated with consumption of antifungal agents at either the individual or population level. Actually, our epidemiological and
molecular results suggested that the carriage of C. krusei and S.
cerevisiae might be of foodborne origin, associated with a
common environmental source, such as plants or water [39,
40]. These findings were consistent with those reported elsewhere by authors who described C. krusei and S. cerevisiae as
fermentative yeasts found in the environment [41, 42].
In addition, among the large diversity of yeasts isolated in
this community, we noticed the absence of Candida glabrata,
usually one of the most frequent colonizing yeast in humans
after C. albicans in Europe and North America [1, 3, 6], and the
quasi-absence of Candida parapsilosis, which was replaced by 2
other species from the parapsilosis complex (Lodderomyces
elongisporus and C. orthopsilosis) [43].
The most striking result of our study was the unexpectedly
low rate of C. albicans in this population. Indeed, <7% of yeast
carriers in Trois-Sauts were C. albicans carriers. This is highly
divergent with the results of most previous studies, in which C.
albicans carriers represented >80% of healthy yeast carriers [1–
3]. Although no data are available regarding the C. albicans carriage rate among healthy persons living in French Guiana, C.
albicans remains the most prevalent species isolated at birth in
hospitalized infants in Cayenne (C. Azenave, personal communication). However, similar unusual low C. albicans carriage
rates have already been reported in other remote populations
[37, 44]. In rural Chinese persons, oral C. albicans carriage
ranked only fourth in prevalence (9% of yeast carriers) [37],
whereas in women from northern Thailand overall yeast carriage rate was high (approximately 90%), but C. albicans carriage was low (20% of carriers) [44]. In our study, we found 2
specific risk factors associated with C. albicans carriage: being
female and living in a crowded household. This is consistent
with previous reports showing that female sex and intrafamilial
cross-transmission are associated with C. albicans carriage [1,
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1713
Figure 4. Comparison of whole-genome sequences of the 2 Candida albicans ST602 strains isolated 4 years apart from the only persistent carrier
(H014_2006 and H014_2010). Overall number of positions compared is indicated at the right of each chromosome (Chr) in italics. All point mutations
(n = 58) identified are mapped on the C. albicans chromosomes by a thin line, their position on each chromosome is indicated under the line, and bases involved in the mutation are indicated above the line. The 12 SNPs corresponding to a LOH in H014_2010 relative to H014_2006 are shown in green, the 35
SNPs due to a LOH in H014_2006 relative to H014_2010 in blue and the 11 SNPs due to a change in the bases composing the heterozygous pair a in red.
Centromeres are indicated by a thick black line.
2, 7, 36, 45]. Taken together, these results strongly suggest that
commensal carriage of C. albicans in humans depends on their
lifestyle or environmental constraints.
Because Amerindian volunteers were sampled twice at a 4year interval, we were able to evaluate the long-term dynamics
of C. albicans carriage. The single individual colonized twice
with C. albicans carried isolates from the same ST, with only
minute differences in their genome. Even though various conditions encountered within the host’s environment, such as
high temperature or oxidative stress, are known to favor genetic
changes in C. albicans [46], our finding showed an almost
perfect maintenance of heterozygosity and suggested a high
degree of genomic stability during intestinal commensalism.
This finding shows that persistent carriage may exist in
humans, a fact that had never been demonstrated before.
However, we cannot exclude the possibility that our observation reflects the sampling of only 1 isolate at each sampling
time and that the sampled individual harbored a mixed population of clonally related isolates with different extents of
genomic rearrangements.
1714
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Angebault et al
Finally, we presented the first description of the intestinal
yeast flora of humans and animals living in the same environment. First, we found similar patterns of yeast colonization in
animals and in humans, with rare C. albicans but frequent C.
krusei carriage, suggesting that animals, at least the species
sampled, are not a reservoir of C. albicans. Second, we noticed
that species diversity in animals was higher than in humans.
Indeed, the animals carried various environmental yeasts,
mainly fermentative species such as Issatchenkia occidentalis or
yeast species that are commensals of the insect gut [40, 47].
Then, focusing on C. albicans carriage among humans and
animals, we observed that the majority of C. albicans strains
were from clade 8, a clade known to be predominant in South
America and more frequently associated with C. albicans
strains carried by wild animals [13, 20]. We also noticed that 4
of 6 animals (wild and domestic) carried C. albicans isolates
from STs identical or closely related to those found in humans.
In a Amerindian community where animals and humans live
in very close proximity to each other, this finding suggests possible cross-transmission of C. albicans strains between humans
and animals. Although the number of C. albicans animal carriers is small, our results do not seem to confirm a trend toward
genetic separation between human and animal C. albicans
strains [10, 13, 48].
Taken together, in this population living isolated in a very
specific environment, the intestinal yeast colonization pattern
was not similar to that described in populations living in industrialized areas. This could be related to specific genetic traits
present is this highly homogeneous community [23]. Studies
on C. albicans carriage among families reported substantial
fluctuations in the rate of carriage.[1, 2]. A second hypothesis is
that the global microbiota (bacteria and yeasts) of Wayampi
Amerindians is different from that of other communities owing
to genetic traits, lifestyle, and diet or environmental constraints.
In a recent study, Yatsunenko et al [49] compared the bacterial
diversity of the intestinal microbiota in 3 markedly different
settings (Amerindians from Venezuela, Malawian rural volunteers, and urban US citizens) and observed great differences in
bacterial assemblages and even functional gene repertoires.
Finally, our findings, as well as those of Xu and Mitchell [37] or
Reichart et al [44], may reflect an ancestral human-yeast association. C. albicans might not be a “normal” commensal microorganism of the human flora that coevolved with humans
throughout the ages [15, 18, 19], but a more recently acquired
commensal microorganism selected because of its ability to
become a persistent colonizer in a westernized environment
[15, 50].
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary data
are the sole responsibility of the authors. Questions or messages regarding
errors should be addressed to the author.
Notes
Acknowledgments. We thank the villagers for their help and their
warm welcome and Gilles Peroz for excellent technical assistance. We thank
Jean-Luc Beretti for his technical support on Matrix-Assisted Laser Desorption/Ionisation - Time of Flight mass spectrometry use.
Financial support. The ERAES project was supported in part by the
Agence Française de Sécurité Sanitaire de l’Environnement et du Travail
(contracts ES-05-01 and EST-09-21), the Agence Nationale pour la Recherche (contract 05-9-114), the Institut National de la Santé et de la Recherche
Médicale (INSERM; contracts C06-18 and C10-19), the Centre National de
Référence “Résistance bactérienne dans les flores commensales,” the Fondation pour la Recherche Médicale (grants FDM20090615761 and
FDM20100619023 to C. A.), and the French government’s Investissement
d’Avenir program, Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (grant ANR-10-LABX-62-IBEID).
Potential conflict of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
References
1. Bougnoux M-E, Diogo D, François N, et al. Multilocus sequence typing
reveals intrafamilial transmission and microevolutions of Candida albicans isolates from the human digestive tract. J Clin Microbiol 2006;
44:1810–20.
2. Kam AP, Xu J. Diversity of commensal yeasts within and among
healthy hosts. Diagn Microbiol Infect Dis 2002; 43:19–28.
3. Xu J, Boyd CM, Livingston E, Meyer W, Madden JF, Mitchell TG.
Species and genotypic diversities and similarities of pathogenic yeasts
colonizing women. J Clin Microbiol 1999; 37:3835–43.
4. Eggimann P, Garbino J, Pittet D. Epidemiology of Candida species infections in critically ill non-immunosuppressed patients. Lancet Infect
Dis 2003; 3:685–702.
5. Qi QG, Hu T, Zhou XD. Frequency, species and molecular characterization of oral Candida in hosts of different age in China. J Oral Pathol
Med 2005; 34:352–6.
6. Issa SY, Badran EF, Akl KF, Shehabi AA. Epidemiological characteristics of Candida species colonizing oral and rectal sites of Jordanian
infants. BMC Pediatr 2011; 11:79.
7. Pierson DL, Mehta SK, Magee BB, Mishra SK. Person-to-person transfer of Candida albicans in the spacecraft environment. J Med Vet
Mycol 1995; 33:145–50.
8. Lockhart SR, Fritch JJ, Meier AS, et al. Colonizing populations of
Candida albicans are clonal in origin but undergo microevolution
through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing. J Clin Microbiol 1995; 33:1501–9.
9. Mancianti F, Nardoni S, Ceccherelli R. Occurrence of yeasts in psittacines droppings from captive birds in Italy. Mycopathologia 2002;
153:121–4.
10. Jacobsen MD, Bougnoux M-E, d’ Enfert C, Odds FC. Multilocus sequence typing of Candida albicans isolates from animals. Res Microbiol
2008; 159:436–40.
11. Brito EHS, Fontenelle ROS, Brilhante RSN, et al. The anatomical distribution and antimicrobial susceptibility of yeast species isolated from
healthy dogs. Vet J 2009; 182:320–6.
12. Costa AKF, Sidrim JJC, Cordeiro RA, Brilhante RSN, Monteiro AJ,
Rocha MFG. Urban pigeons (Columba livia) as a potential source
of pathogenic yeasts: a focus on antifungal susceptibility of Cryptococcus strains in Northeast Brazil. Mycopathologia 2010; 169:
207–13.
13. Wrobel L, Whittington JK, Pujol C, et al. Molecular phylogenetic analysis of a geographically and temporally matched set of Candida albicans
isolates from humans and nonmigratory wildlife in central Illinois. Eukaryot Cell 2008; 7:1475–86.
14. Gow NAR, van de Veerdonk FL, Brown AJP, Netea MG. Candida albicans morphogenesis and host defence: discriminating invasion from
colonization. Nat Rev Microbiol 2012; 10:112–22.
15. Rosenbach A, Dignard D, Pierce JV, Whiteway M, Kumamoto CA. Adaptations of Candida albicans for growth in the mammalian intestinal
tract. Eukaryot Cell 2010; 9:1075–86.
16. Chaieb K, Kouidhi B, Zmantar T, Mahdouani K, Bakhrouf A. Starvation survival of Candida albicans in various water microcosms. J Basic
Microbiol 2011; 51:357–63.
17. Valdes-Collazo L, Schultz AJ, Hazen TC. Survival of Candida albicans
in tropical marine and fresh waters. Appl Environ Microbiol 1987;
53:1762–7.
18. Odds FC. Molecular phylogenetics and epidemiology of Candida albicans. Future Microbiol 2010; 5:67–79.
19. Lott TJ, Fundyga RE, Kuykendall RJ, Arnold J. The human commensal
yeast, Candida albicans, has an ancient origin. Fungal Genet Biol 2005;
42:444–51.
20. Odds FC, Bougnoux M-E, Shaw DJ, et al. Molecular phylogenetics of
Candida albicans. Eukaryot Cell 2007; 6:1041–52.
21. Grenet K, Guillemot D, Jarlier V, et al. Antibacterial resistance, Wayampis Amerindians, French Guyana. Emerging Infect Dis 2004;
10:1150–3.
Intestinal Yeast Carriage in Amerindians
•
JID 2013:208 (15 November)
•
1715
22. Woerther P-L, Angebault C, Lescat M, et al. Emergence and dissemination of extended-spectrum beta-lactamase-producing Escherichia coli
in the community: lessons from the study of a remote and controlled
population. J Infect Dis 2010; 202:515–23.
23. Ruimy R, Angebault C, Djossou F, et al. Are host genetics the predominant determinant of persistent nasal Staphylococcus aureus carriage in
humans? J Infect Dis 2010; 202:924–34.
24. Hurault JM, Grenand F, Grenand P. Indiens de Guyane: Wayana et
Wayampi de la forêt. Paris: Autrement Éditions, 1998.
25. Catzeflis F. A survey of small non-volant mammals inhabiting
Wayampi Amerindian houses in French Guiana. Mammalia 2012;
76:327–30.
26. Leaw SN, Chang HC, Sun HF, Barton R, Bouchara J-P, Chang TC.
Identification of medically important yeast species by sequence analysis
of the internal transcribed spacer regions. J Clin Microbiol 2006;
44:693–9.
27. Bougnoux M-E, Tavanti A, Bouchier C, et al. Collaborative consensus
for optimized multilocus sequence typing of Candida albicans. J Clin
Microbiol 2003; 41:5265–6.
28. Jacobsen MD, Gow NAR, Maiden MCJ, Shaw DJ, Odds FC. Strain
typing and determination of population structure of Candida krusei by
multilocus sequence typing. J Clin Microbiol 2007; 45:317–23.
29. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.
MEGA5: Molecular Evolutionary Genetics Analysis using maximum
likelihood, evolutionary distance, and maximum parsimony methods.
Mol Biol Evol 2011; 28:2731–9.
30. Shin JH, Bougnoux M-E, d’ Enfert C, et al. Genetic diversity among
Korean Candida albicans bloodstream isolates: assessment by multilocus sequence typing and restriction endonuclease analysis of genomic
DNA by use of BssHII. J Clin Microbiol 2011; 49:2572–7.
31. Agapow P, Burt A. Indices of multilocus linkage disequilibrium. Mol
Ecol Notes 2001; 1:101–2.
32. Legras J-L, Ruh O, Merdinoglu D, Karst F. Selection of hypervariable
microsatellite loci for the characterization of Saccharomyces cerevisiae
strains. Int J Food Microbiol 2005; 102:73–83.
33. Selmecki A, Bergmann S, Berman J. Comparative genome hybridization reveals widespread aneuploidy in Candida albicans laboratory
strains. Mol Microbiol 2005; 55:1553–65.
34. Ossowski S, Schneeberger K, Ott F, Hagmann J, Bender S, Scotland A.
SHORE manual. Version 0.5.0beta. http://www.animalgenome.org/
bioinfo/resources/manuals/SHORE-README.pdf. Accessed 28 August
2012.
35. Jones T, Federspiel NA, Chibana H, et al. The diploid genome sequence
of Candida albicans. Proc Natl Acad Sci U S A 2004; 101:7329–34.
1716
•
JID 2013:208 (15 November)
•
Angebault et al
36. Ghannoum MA, Jurevic RJ, Mukherjee PK, et al. Characterization of
the oral fungal microbiome (mycobiome) in healthy individuals. PLoS
Pathog 2010; 6:e1000713.
37. Xu J, Mitchell TG. Geographical differences in human oral yeast flora.
Clin Infect Dis 2003; 36:221–4.
38. Mann PA, McNicholas PM, Chau AS, et al. Impact of antifungal prophylaxis on colonization and azole susceptibility of Candida species.
Antimicrob Agents Chemother 2009; 53:5026–34.
39. Medeiros AO, Kohler LM, Hamdan JS, Missagia BS, Barbosa FAR,
Rosa CA. Diversity and antifungal susceptibility of yeasts from tropical
freshwater environments in Southeastern Brazil. Water Res 2008;
42:3921–9.
40. Trindade RC, Resende MA, Silva CM, Rosa CA. Yeasts associated with
fresh and frozen pulps of Brazilian tropical fruits. Syst Appl Microbiol
2002; 25:294–300.
41. Omemu AM, Oyewole OB, Bankole MO. Significance of yeasts in the
fermentation of maize for ogi production. Food Microbiol 2007;
24:571–6.
42. Nuobariene L, Hansen AS, Jespersen L, Arneborg N. Phytase-active
yeasts from grain-based food and beer. J Appl Microbiol 2011;
110:1370–80.
43. Tavanti A, Davidson AD, Gow NAR, Maiden MCJ, Odds FC.
Candida orthopsilosis and Candida metapsilosis spp. nov. to replace
Candida parapsilosis groups II and III. J Clin Microbiol 2005;
43:284–92.
44. Reichart PA, Khongkhunthian P, Samaranayake LP, Yau J, Patanaporn V,
Scheifele C. Oral Candida species and betel quid-associated oral lesions
in Padaung women of Northern Thailand. Mycoses 2005; 48:132–6.
45. Yang YL, Leaw SN, Wang AH, Chen HT, Cheng WT, Lo HJ. Characterization of yeasts colonizing in healthy individuals. Med Mycol 2011;
49:103–6.
46. Forche A, Abbey D, Pisithkul T, et al. Stress alters rates and types of
loss of heterozygosity in Candida albicans. mBio 2011; 2:e00129–11.
47. Rao R, Bhadra B, Shivaji S. Isolation and characterization of xylitolproducing yeasts from the gut of colleopteran insects. Curr Microbiol
2007; 55:441–6.
48. Edelmann A, Krüger M, Schmid J. Genetic relationship between
human and animal isolates of Candida albicans. J Clin Microbiol 2005;
43:6164–6.
49. Yatsunenko T, Rey FE, Manary MJ, et al. Human gut microbiome
viewed across age and geography. Nature 2012; 486:222–7.
50. White SJ, Rosenbach A, Lephart P, et al. Self-regulation of Candida
albicans population size during GI colonization. PLoS Pathog 2007; 3:
e184.