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Genotypes of Candida albicans isolated from healthy individuals and their distribution in patients with oral candidiasis
J Infect Chemother DOI 10.1007/s10156-013-0626-5
ORIGINAL ARTICLE
Genotypes of Candida albicans isolated from healthy individuals and their distribution in patients with oral candidiasis Yuki Takagi • Hideo Fukano • Kazuo Shimozato Reiko Tanaka • Toshinobu Horii • Fumihiko Kawamoto • Toshio Kanbe
•
Received: 16 January 2013 / Accepted: 23 May 2013 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract For the study of Candida albicans genotypes involved in development of candidiasis, Candida albicans isolates were collected from healthy volunteers and patients with oral candidiasis and genotyped on the basis of 25S rDNA and microsatellite polymorphisms. In the microsatellite analysis using two microsatellite markers (CDC3 and CAI), 63 healthy volunteer isolates were classified into 35 genotypes (allelic relations to CDC3 alleles 1:2/CAI alleles 1:2), among which genotypes II (115:119/23:23), III (115:123/18:27), and V (123:127/ 32:41) were found at frequencies of 12.7 %, 7.9 %, and 7.9 %, respectively. In 68 oral candidiasis isolates
classified into 39 genotypes, genotypes II and III were identified in 4.4 % and 20.6 % of the isolates, respectively. The frequency of genotype III was higher in the candidiasis isolates than in the healthy isolates (p \ 0.05). These results suggest that genotype III C. albicans assigned by CDC3/CAI is related to the development of oral candidiasis. Keywords Candida albicans Genotype Oral candidiasis Healthy individuals Microsatellite 25S rDNA
Introduction This paper was recommended for submission to this journal by the chairperson at the conference of the 86th Annual Conference of The Japanese Association for Infectious Diseases. Y. Takagi H. Fukano K. Shimozato Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya, Japan R. Tanaka Medical Mycology Research Center, Chiba University, Chiba, Japan T. Horii Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Japan F. Kawamoto Department of Social and Environmental Medicine, Institute of Scientific Research, Oita University, Yufu, Japan T. Kanbe (&) Division of Molecular Mycology and Medicine, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan e-mail: [email protected]
Candidiasis occurs in both nonimmunocompromised and immunocompromised individuals. Oral candidiasis is an opportunistic fungal infection that occurs in immunocompromised hosts and is caused by several Candida species which inhabit the oral cavity as a commensal population. Candida albicans is the most important etiological species of candidiasis because this fungus was the species most frequently isolated from candidiasis patients [1]. Several molecular techniques have been used to study the genotypes of C. albicans, some of which have shown a relationship between genotypes and the development of candidiasis [2–8]. The genotypes of C. albicans derived from patients with vulvovaginal candidiasis reportedly differed from those of commensal C. albicans [9]. Further, C. albicans genotypes were related to the severity of vulvovaginal candidiasis [10]. Recently, we studied the major genotype distribution of C. albicans isolated from patients with and without candidiasis and reported that microsatellite-based genotyping of commensal C. albicans from the oral cavity of patients
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without candidiasis using CDC3 and CAI assigned five major genotypes (genotypes I, II, III, IV, and V). Of these, genotypes II and III C. albicans were commonly isolated from both commensal and infective sites of patients with skin cutaneous candidiasis, vulvovaginal candidiasis, and candidemia. In contrast, genotype V was identified only in the commensal isolates of patients without candidiasis [11]. Unfortunately, C. albicans isolates from patients with oral candidiasis and from a commensal site of healthy individuals have not been subjected to genotype analysis. Most cases of candidiasis are endogenously caused by commensal C. albicans in the oral cavity [12, 13]. Therefore, C. albicans isolates from patients with oral candidiasis are useful for understanding the relationship between genotypes II, III, and V and candidiasis. Additionally, C. albicans isolates from healthy individuals are necessary to understand the genotype distribution of commensal C. albicans. If genotype V C. albicans defined by CDC3/CAI is widely distributed in the oral cavity but is less likely to cause either superficial or systemic candidiasis, it is expected that genotype V is not identified in isolates form the infection site of patients with oral candidiasis. In this study, for the relationship between genotypes and candidiasis, genotypes of commensal C. albicans from healthy individuals were analyzed and their distributions were compared with those of isolates from patients with oral candidiasis.
Materials and methods Subjects Commensal Candida species were isolated from 88 healthy volunteers (37 males and 51 females; mean age, 37.8 years) from the Aichi-Gakuin University School of Dentistry (HV group). Infective Candida species were isolated from 50 patients with oral candidiasis (7 males and 43 females; mean age, 70.5 years) (OC group). After the patients were diagnosed as oral candidiasis, 48 patients received treatment with amphotericin B syrup and the remaining patients were treated with fluconazole in the University Hospital. In all cases, the Candida species is isolated before treatment with antifungal agents. Isolation and identification of Candida species All volunteers from the HV and OC groups gargled 20 ml sterile distilled water, and the samples were centrifuged at 3,000 rpm for 10 min. The pellets were suspended in 400 ll sterile distilled water, and 30 ll of the suspension was spread onto a chromogenic agar (CHROMagar Candida; BD Co., Tokyo, Japan) plate. After stationary culture
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at 30 °C for 3–4 days, each colony with a different color (green, pink, blue, and white) was separately transferred onto another chromogenic agar plate and cultured for 2–3 days at 30 °C to isolate single colonies. To study the genotype homogeneity of C. albicans, 16 green colonies from each group were transferred onto new plates. Additionally, non-green colonies were also transferred to identify the Candida species. A total of 2,755 isolates (1,117 isolates from the HV group and 1,638 isolates from the OC group) were obtained. Species identification of the isolates was performed by polymerase chain reaction (PCR) targeting the DNA topoisomerase II gene (TOP2) [14, 15]. To identify Candida dubliniensis, primer sets specific to C. dubliniensis TOP2 and 25S rDNA (see following) were used [14]. Yeasts that were not identified by these PCRs are referred to as ‘‘other yeast species’’ in this article. Using multi-colony isolation, 472 strains of C. albicans were obtained from the HV group and 676 strains of C. albicans were obtained from the OC group. All the isolates were genotyped at the 25S rDNA and microsatellite levels (described below). The isolation, identification, and genotyping of Candida yeasts (described below) were carried out with informed consent from all the participants. Purification of genomic DNA Genomic DNA was purified from the isolates as previously described [11, 16]. Briefly, all isolates were cultured on agar plates containing 2 % glucose, 0.3 % yeast extract, and 1 % peptone for 3–4 days at 30 °C. The yeast cells were suspended in extraction buffer and the genomic DNA was extracted by vortexing with glass beads. After centrifugation, the genomic DNA was purified using a DNA purification kit (FastDNA Kit; Qbiogene, Carlsbad, CA, USA) as described previously [14]. 25S rDNA-based genotyping C. albicans genomic DNAs were amplified with the 25SIN primer set, and the nucleotide sequences of forward and reverse primers and PCR parameters were previously published [17]. From this PCR, C. albicans was divided into four groups by the size of the amplified DNA (450 bp for group A, 840 bp for group B, 450 and 840 bp for group C, and 1,080 bp for group E), and C. dubliniensis was assigned as group D with a 1,040-bp DNA band [17, 18]. Microsatellite-based genotyping Fragment analysis of the microsatellites of the C. albicans isolates was carried out using four microsatellite marker
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sets: CDC3, HIS3, CAI, and CAIII. The fluorescent dyes and nucleotide sequences of these markers and PCR parameters have been described previously [17]. The PCR products generated by each of the microsatellite marker sets were diluted 1:10–1:20 with distilled water, and 1 ll of the mixture and 0.2 ll of size standard (GeneScan-500 LIZ Size Standard; Applied Biosystems, Warrington, UK) were added to 20 ll hi-Di Formamide (Applied Biosystems). All samples were applied to an ABI Prism 310 Genetic Analyzer (Applied Biosystems) with a short capillary. The fragment sizes of the PCR products were identified using the GeneMapper ver. 4.0 software (Applied Biosystems). All isolates were genotyped on the basis of allelic relation (alleles 1:2). Genotypes of CDC3 and HIS3 use the size of DNA fragments. In contrast, genotypes of CAI and CAIII are presented as the number of repeated units in the DNA fragment; the DNA fragments of 252 and 113 bp correspond to 41 (for CAI) and 11 (for CAIII) repeated units, respectively [19, 20]. Genetic distances with multilocus genotypes and the phylogenetic relationships of the C. albicans isolates were analyzed using Populations (ver. 1.2.32) [21].
Table 1 Candida species isolated from healthy volunteers (HV) and patients with oral candidiasis (OC) Candida species
Numbers of HV and OC HV (n = 42)
OC (n = 50)
C. albicans
24
23
C. glabrata
2
1
C. parapsilosis II
0
1
C. dubliniensis
1
0
Other yeasts
4
4 6
Single species
Double species C. albicans ? C. glabrata
4
C. albicans ? C. parapsilosis II
0
1
C. albicans ? C, dubliniensis C. albicans ? C. tropicalis
1 0
0 1
C. albicans ? Other yeasts
5
6
C. glabrata ? C. parapsilosis II
1
0
C. parapsilosis I ? other yeasts
0
1
0
1
Triple species C. albicans ? C. glabrata ? C. dubliniensis
Results
C. albicans ? C. glabrata ? other yeast
0
2
C. albicans ? C. parapsilosis II ? other yeasts
0
2
Isolation of Candida species In this study, yeasts were isolated from 41 of 88 HVs (46.6 %), and 34 of these (81 %) had C. albicans. In contrast, C. albicans was found in 42 of 50 patients from the OC group (84 %) who presented yeasts on the chromogenic cultures (Table 1). In both groups, C. albicans constituted the majority of the isolates. Additionally, Candida glabrata was found in 7 HVs (16.7 %) and 11 OC patients (22 %) (Table 1). Candida parapsilosis I and II, C. dubliniensis, and Candida tropicalis II were isolated from the oral cavity of HV and/or OC participants, but the frequencies were remarkably lower than those of C. albicans and C. glabrata (Table 1). C. albicans was isolated as the sole species from 25 HVs (60.9 %) and from 23 OCs (46 %). C. glabrata and C. dubliniensis were also isolated as the sole species from 1 member each of the HV and OC groups (Table 1). In 10 HVs (23.8 %) and 19 OCs (38 %), C. albicans was isolated with other Candida species or other yeasts; for example, C. albicans was isolated with C. glabrata in 4 HVs (9.5 %) and 9 OCs (18 %) (Table 1). Genotype homogeneity of C. albicans in the oral cavity In this study, 472 and 676 C. albicans isolates were collected from 34 HVs and 42 OCs, respectively, by multi-
Table 2 25S rDNA-based groups of Candida albicans isolates
25S rDNA-based group
Single group A
Numbers of HV and OC HV
OC
18
21
B
7
8
C
0
8
A?B
1
1
A?C
3
2
B?C
5
2
Double group
HV healthy volunteers, OC oral candidiasis
colony isolation (10–16 colonies/individual). All the 1,148 C. albicans isolates were subjected to genotype homogeneity studies by 25S rDNA- and microsatellite-based genotyping. 25S rDNA-based genotyping divided C. albicans isolates into three groups, among which group A C. albicans constituted the majority in both the HV and OC groups, followed by group B and C C. albicans (Table 2). Group A C. albicans was found in 22 HVs (64.7 %) and 24 patients (57.1 %). The multi-colony analysis of C. albicans from individual HV and OC members showed that a single
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genotype of C. albicans was found in 25 HVs (73.5 %) and 37 patients (88.1 %), and the remaining participants presented 2 genotypes of C. albicans (groups A ? B, A ? C, or B ? C) at the 25S rDNA level (Table 2). No group E C. albicans was found in this study. C. albicans isolates obtained from HVs and OCs that presented 10–16 colonies were used to study genotype homogeneity in the oral cavity of HV and OC individuals. All the isolates were divided into three groups (groups A, B, and C) on the basis of 25S rDNA, and each group was then classified on the basis of the combination of allelic relationships to four microsatellite markers (CDC3/CAI/ HIS3/CAIII) (Fig. 1). In the HV group, 16 individuals (47.1 %) showed two genotypes of C. albicans, and 13 individuals (38.2 %) had a single genotype of C. albicans. The number of C. albicans genotypes in the OC individuals was the same as that from the HV group. In the OC group, a single genotype of C. albicans was found in 26 (61.9 %) patients; this frequency was higher than that of the HV group (p \ 0.05). On the other hand, two genotypes of C. albicans were found in 12 (28.6 %) patients; this frequency was lower than that of the HV group (p \ 0.05). C. albicans genotype distribution of isolates from the HV and OC groups To compare C. albicans genotype distribution between the HV and OC groups, C. albicans isolates from 4 HVs and 5 OCs who presented 1 or 2 colonies were also used for this study, in addition to the isolates from HVs and OCs who presented multiple colonies (10–16 colonies). Sixty-three isolates from 38 HVs and 68 isolates from 47 OCs were subjected to this study, grouped by allelic relationships of CDC3 and CAI. The isolates of the HVs were classified into 35 allelic relationships. Of these, three allelic relationships, 115:119/23:23, 115:123/18:27, and 123:127/ 70
Frequency (%)
60 50
32:41 (CDC3 alleles 1:2/CAI alleles 1:2), accounted for 12.7 % (8 isolates), 7.9 % (5 isolates), and 7.9 % (5 isolates) of 63 isolates from the HV group, respectively (Table 3). These allelic relationships corresponded to genotypes II, III, and V in a previous report [11]. Therefore, we referred to the 115:119/23:23, 115:123/18:27, and 123:127/32:41 allelic relations as genotype II, III, and V, respectively. In contrast, the isolates from the patients were classified into 39 allelic relationships, and genotypes II and III were assigned to 3 (4.4 %) and 14 (20.6 %) of 68 isolates from the OC group; however, genotype V C. albicans was not found (Table 3). The frequency of genotype II was slightly higher in the HV isolates than in the OC isolates (p [ 0.05). In contrast, the frequency of genotype III C. albicans in the OC group was higher than that in the HV group (p \ 0.05). The allelic relationships corresponding to genotypes I and IV were not found in any isolates from this study. When CDC3/CAI-based genotypes were combined with 25S rDNA groups, genotypes II, III, and V C. albicans of the HV isolates (18 isolates) and genotypes II and III C. albicans of the OC isolates (17 isolates) were classified into six and four types, respectively (Fig. 2). Genotypes (CDC3/CAI-25S rDNA) II-A and III-A were found equally in the HV and OC group. The genotype III-C was clearly more frequent in the OC group than the HV group. Additionally, all the genotype V–C C. albicans were found only in the HV group. Genotype II, III, and V C. albicans isolates (total, 35 isolates) were further classified by allelic relationships to additional microsatellite markers, HIS3 and CAIII. These isolates were classified into 21 types: genotypes II, III, and V showed independent clusters and were divided into 9 (types 1–9, genotype II), 9 (types 10–18, genotype III), and 3 (types 19–21, genotype V) types, respectively (Fig. 2). Types 4 and 12 were common to both HV and OC groups. Types 14, 16, 17, and 18, which were group C, were found only in the OC group. In contrast, types 19, 20, and 21, which were also group C, were found only in the HV group.
40
Discussion
30 20 10 0
1
2
3
4
No. of genotypes
Fig. 1 Comparison of heterogeneity of Candida albicans genotype between commensal and infective sites. Horizontal axis indicates different genotype numbers of C. albicans in healthy volunteers (HV) (open columns) and patients with oral candidiasis (OC) (solid columns). Vertical axis indicates frequencies of each genotype
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Candida albicans constitutes the majority of the isolates from each group, and it was co-isolated with other Candida or yeast species from 19 patients of the OC group (38 %), a slightly higher frequency than that from the HV group (23.8 %). This result suggests that Candida species other than C. albicans act as endogenous pathogens and are involved in the development of oral candidiasis. In this study, C. glabrata was frequently co-isolated with C. albicans. Oral candidiasis caused by C. albicans was
J Infect Chemother Table 3 Genotypes of Candida albicans and their distributions in HV and OC groups Genotype and number of isolates
Genotype and number of isolates
Allelic relationship (alleles 1:2)
Group
Allelic relationship (alleles 1:2)
Group
CDC3
CAI
HV
OC
CDC3
CAI
HV
95:111 99:115
31:31
1
115:123
28:29
1
27:35
2
115:123
29:29
3
99:115 103:115
51:51 18:27
1
115:123 115:123
29:31 33:33
1 2
103:119
23:23
107:123
18:27
1
1
115:123
33:39
2
115:123
34:34
111:115
21:28
1
3
115:123
34:50
1
111:115
28:28
2
111:115
29:33
1
115:123
34:52
1
115:123
36:36
111:115
33:42
1
115:123
36:55
111:115
33:43
115:115
18:27
1
2
115:123
52:52
1
1
115:127
33:33
115:115
23:23
2
119:119
23:23
2
115:115
23:30
115:115
23:32
1
1
119:119
28:41
3
119:119
30:32
115:115
25:25
1
119:123
28:33
115:115
25:30
2
115:115 115:115
25:41 27:27
1
115:115
28:37
115:119
15:23
115:119
23:23
115:119
23:33
115:119 115:119 115:119
23:45
115:119
1
1
OC
2
2 2 1 1
2 1
119:123
28:34
1 1
119:123 119:127
41:41 39:40
1
1
123:123
32:33
1
2
1
123:123
34:34
8 (12.7)
3 (4.4)
123:123
34:50
1
1
1
123:123
37:39
2
23:37
1
123:123
39:39
2
23:42
1
123:127
28:41
1
2
123:127
32:32
1
23:46
2
123:127
32:41
5 (7.9)
115:119
23:50
1
123:127
36:39
1
115:119
23:51
1
123:127
39:40
2
115:123
18:27
5 (7.9)
123:127
40:40
115:123
19:27
1
123:127
41:41
115:123
21:23
123:135
23:42
14 (20.6) 3
1 1 1
1 1 1
HV healthy volunteers, OC oral candidiasis
accompanied by C. glabrata in several cases. It is important to learn the involvement of C. glabrata in oral candidiasis for accurate therapy because C. glabrata generally has low susceptibility to the azole class of antifungal therapies [1]. Recently, several studies have reported a relationship between C. glabrata pathogenicity and genotype [22, 23]. The C. albicans isolates from HVs showed three major allelic relationships to CDC3/CAI, which corresponded to genotypes II, III, and V reported by Takagi et al. [11]. Of these three major genotypes, genotypes II and III were the major genotypes found in the OC group. The remaining
genotype, genotype V, was not observed for any isolate from this group. These results coincided with these in the previous study that the major genotype distributions of C. albicans isolated from patients with and without candidiasis were compared. Genotypes II and III C. albicans were commonly isolated from patients with skin candidiasis, vulvovaginal candidiasis, and candidemia. In contrast, genotype V C. albicans was not found [11]. These results suggested that the genotype V C. albicans defined by CDC3/CAI was distributed widely in the oral cavity but was less likely to cause either superficial candidiasis or systemic candidiasis. This study strongly supports the idea
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J Infect Chemother Fig. 2 Dendrogram of genotypes II, III, and V of C. albicans based on four microsatellite markers
that the genotype V C. albicans is not involved in the development of candidiasis because genotype V C. albicans was not isolated from the oral cavity of patients with oral candidiasis although this genotype is the major genotype in the oral cavity of healthy individuals. Shimizu et al. [13] used the microsatellite markers HIS3 and CAI to genotype C. albicans and reported that the genotype distribution of C. albicans was not identical in commensal and infective lesions. Additionally, in the previous study, genotype I (111:115/33:41) and IV (115:123/33:40) C. albicans were included in the isolates from patients with and without candidiasis; however, they were not found in either the HV or OC group in this study. Genotypes I and IV C. albicans may not be involved in the development of oral candidiasis. Previously, the microsatellite markers CDC3 and CAI were used to genotype C. albicans from the bloodstream, and genotypes II and III C. albicans were shown to have the ability to cause candidemia [11]. In contrast, Dalle et al. [24] used the microsatellite marker CEF3 to genotype C. albicans and concluded that C. albicans from the bloodstream and non-bloodstream samples were genetically heterogeneous. When microsatellite analysis was combined with 25S rDNA-based genotyping, genotype II C. albicans was classified into groups A and B, and group B was found only in the HV group. The genotype III C. albicans was classified into groups A and C, and group C was only identified in the OC group. All genotype V isolates were classified into group C. The group A C. albicans was found equally in both the HV and OC groups. These results suggest that the A group isolates were widely distributed in both HV
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and OC individuals, and these isolates have the ability to cause candidiasis. The genotype II C. albicans isolates grouped into ‘‘B’’ and genotype V isolates grouped into ‘‘C’’ were less able to cause candidiasis. Additionally, the microsatellite analysis using four markers combined with 25S rDNA-based typing divided the three major genotypes into 21 types, of which type 14 (genotype III/group C) was found only in the OC group, and types 20 and 21 (genotype V/group C) were found only in the HV group. This result suggests that a comparison of biological properties of these types is useful to understand the development of candidiasis and the virulence factors of this fungus. She et al. [9] reported that 25S rDNA and alanine aminotransferase (ALT)-based genotypes of C. albicans were genetically different in cutaneous and vaginal candidiasis. Fan et al. [10] used a microsatellite method to genotype C. albicans from patients with vaginal candidiasis and reported that the genotype correlated with the severity of candidiasis. In a previous study, the frequency of genotype III C. albicans from patients with vaginal candidiasis was lower than that from other groups [11]. Hattori et al. [12] reported that genotypes of C. albicans from the oral cavity were similar to those from infective sites of an individual; however, they did not use commensal C. albicans from the vagina. C. albicans can be isolated from the vagina of healthy females [1]. Thus, it is important to analyze genotypes of C. albicans isolated from the vagina. Studying the genotype homogeneity of C. albicans isolates from an individual showed that heterogeneous genotypes (from one to four genotypes) were found in both groups. The frequency of a single genotype of C. albicans
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was clearly higher in the OC group than in the HV group (p \ 0.05). In the HV group, two genotypes of C. albicans were isolated more frequently than a single genotype. Shimizu et al. [13] reported that the genotypes of multiple C. albicans isolates from infection sites of an individual patient with superficial candidiasis were generally homogeneous compared with that of oral isolates. Dalle et al. [25] analyzed genotypes of C. albicans collected serially from patients with acute leukemia and observed that C. albicans isolates had identical genotype profiles in all body locations during the isolation period. They concluded that C. albicans genotypes were rarely altered by antifungal agent pressure. It has been reported that several genotypes of C. albicans were isolated from different locations of the vagina of one female [26]. This finding suggests the involvement of a selection force for the development of superficial candidiasis. However, the selection force is unknown. Unfortunately, we did not analyze genotypes of C. albicans isolated serially from patients and healthy individuals in this study. For this, a longitudinal study of C. albicans multi-colony genotyping in individuals is necessary. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Educations, Culture, Sports, Science and Technology of Japan (#21405002 to R.T. and #22406011 to F.K). Conflict of interest of interest.
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11.
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The authors declare that they have no conflict
16.
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ORIGINAL ARTICLE
Genotypes of Candida albicans isolated from healthy individuals and their distribution in patients with oral candidiasis Yuki Takagi • Hideo Fukano • Kazuo Shimozato Reiko Tanaka • Toshinobu Horii • Fumihiko Kawamoto • Toshio Kanbe
•
Received: 16 January 2013 / Accepted: 23 May 2013 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract For the study of Candida albicans genotypes involved in development of candidiasis, Candida albicans isolates were collected from healthy volunteers and patients with oral candidiasis and genotyped on the basis of 25S rDNA and microsatellite polymorphisms. In the microsatellite analysis using two microsatellite markers (CDC3 and CAI), 63 healthy volunteer isolates were classified into 35 genotypes (allelic relations to CDC3 alleles 1:2/CAI alleles 1:2), among which genotypes II (115:119/23:23), III (115:123/18:27), and V (123:127/ 32:41) were found at frequencies of 12.7 %, 7.9 %, and 7.9 %, respectively. In 68 oral candidiasis isolates
classified into 39 genotypes, genotypes II and III were identified in 4.4 % and 20.6 % of the isolates, respectively. The frequency of genotype III was higher in the candidiasis isolates than in the healthy isolates (p \ 0.05). These results suggest that genotype III C. albicans assigned by CDC3/CAI is related to the development of oral candidiasis. Keywords Candida albicans Genotype Oral candidiasis Healthy individuals Microsatellite 25S rDNA
Introduction This paper was recommended for submission to this journal by the chairperson at the conference of the 86th Annual Conference of The Japanese Association for Infectious Diseases. Y. Takagi H. Fukano K. Shimozato Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya, Japan R. Tanaka Medical Mycology Research Center, Chiba University, Chiba, Japan T. Horii Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Japan F. Kawamoto Department of Social and Environmental Medicine, Institute of Scientific Research, Oita University, Yufu, Japan T. Kanbe (&) Division of Molecular Mycology and Medicine, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan e-mail: [email protected]
Candidiasis occurs in both nonimmunocompromised and immunocompromised individuals. Oral candidiasis is an opportunistic fungal infection that occurs in immunocompromised hosts and is caused by several Candida species which inhabit the oral cavity as a commensal population. Candida albicans is the most important etiological species of candidiasis because this fungus was the species most frequently isolated from candidiasis patients [1]. Several molecular techniques have been used to study the genotypes of C. albicans, some of which have shown a relationship between genotypes and the development of candidiasis [2–8]. The genotypes of C. albicans derived from patients with vulvovaginal candidiasis reportedly differed from those of commensal C. albicans [9]. Further, C. albicans genotypes were related to the severity of vulvovaginal candidiasis [10]. Recently, we studied the major genotype distribution of C. albicans isolated from patients with and without candidiasis and reported that microsatellite-based genotyping of commensal C. albicans from the oral cavity of patients
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without candidiasis using CDC3 and CAI assigned five major genotypes (genotypes I, II, III, IV, and V). Of these, genotypes II and III C. albicans were commonly isolated from both commensal and infective sites of patients with skin cutaneous candidiasis, vulvovaginal candidiasis, and candidemia. In contrast, genotype V was identified only in the commensal isolates of patients without candidiasis [11]. Unfortunately, C. albicans isolates from patients with oral candidiasis and from a commensal site of healthy individuals have not been subjected to genotype analysis. Most cases of candidiasis are endogenously caused by commensal C. albicans in the oral cavity [12, 13]. Therefore, C. albicans isolates from patients with oral candidiasis are useful for understanding the relationship between genotypes II, III, and V and candidiasis. Additionally, C. albicans isolates from healthy individuals are necessary to understand the genotype distribution of commensal C. albicans. If genotype V C. albicans defined by CDC3/CAI is widely distributed in the oral cavity but is less likely to cause either superficial or systemic candidiasis, it is expected that genotype V is not identified in isolates form the infection site of patients with oral candidiasis. In this study, for the relationship between genotypes and candidiasis, genotypes of commensal C. albicans from healthy individuals were analyzed and their distributions were compared with those of isolates from patients with oral candidiasis.
Materials and methods Subjects Commensal Candida species were isolated from 88 healthy volunteers (37 males and 51 females; mean age, 37.8 years) from the Aichi-Gakuin University School of Dentistry (HV group). Infective Candida species were isolated from 50 patients with oral candidiasis (7 males and 43 females; mean age, 70.5 years) (OC group). After the patients were diagnosed as oral candidiasis, 48 patients received treatment with amphotericin B syrup and the remaining patients were treated with fluconazole in the University Hospital. In all cases, the Candida species is isolated before treatment with antifungal agents. Isolation and identification of Candida species All volunteers from the HV and OC groups gargled 20 ml sterile distilled water, and the samples were centrifuged at 3,000 rpm for 10 min. The pellets were suspended in 400 ll sterile distilled water, and 30 ll of the suspension was spread onto a chromogenic agar (CHROMagar Candida; BD Co., Tokyo, Japan) plate. After stationary culture
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at 30 °C for 3–4 days, each colony with a different color (green, pink, blue, and white) was separately transferred onto another chromogenic agar plate and cultured for 2–3 days at 30 °C to isolate single colonies. To study the genotype homogeneity of C. albicans, 16 green colonies from each group were transferred onto new plates. Additionally, non-green colonies were also transferred to identify the Candida species. A total of 2,755 isolates (1,117 isolates from the HV group and 1,638 isolates from the OC group) were obtained. Species identification of the isolates was performed by polymerase chain reaction (PCR) targeting the DNA topoisomerase II gene (TOP2) [14, 15]. To identify Candida dubliniensis, primer sets specific to C. dubliniensis TOP2 and 25S rDNA (see following) were used [14]. Yeasts that were not identified by these PCRs are referred to as ‘‘other yeast species’’ in this article. Using multi-colony isolation, 472 strains of C. albicans were obtained from the HV group and 676 strains of C. albicans were obtained from the OC group. All the isolates were genotyped at the 25S rDNA and microsatellite levels (described below). The isolation, identification, and genotyping of Candida yeasts (described below) were carried out with informed consent from all the participants. Purification of genomic DNA Genomic DNA was purified from the isolates as previously described [11, 16]. Briefly, all isolates were cultured on agar plates containing 2 % glucose, 0.3 % yeast extract, and 1 % peptone for 3–4 days at 30 °C. The yeast cells were suspended in extraction buffer and the genomic DNA was extracted by vortexing with glass beads. After centrifugation, the genomic DNA was purified using a DNA purification kit (FastDNA Kit; Qbiogene, Carlsbad, CA, USA) as described previously [14]. 25S rDNA-based genotyping C. albicans genomic DNAs were amplified with the 25SIN primer set, and the nucleotide sequences of forward and reverse primers and PCR parameters were previously published [17]. From this PCR, C. albicans was divided into four groups by the size of the amplified DNA (450 bp for group A, 840 bp for group B, 450 and 840 bp for group C, and 1,080 bp for group E), and C. dubliniensis was assigned as group D with a 1,040-bp DNA band [17, 18]. Microsatellite-based genotyping Fragment analysis of the microsatellites of the C. albicans isolates was carried out using four microsatellite marker
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sets: CDC3, HIS3, CAI, and CAIII. The fluorescent dyes and nucleotide sequences of these markers and PCR parameters have been described previously [17]. The PCR products generated by each of the microsatellite marker sets were diluted 1:10–1:20 with distilled water, and 1 ll of the mixture and 0.2 ll of size standard (GeneScan-500 LIZ Size Standard; Applied Biosystems, Warrington, UK) were added to 20 ll hi-Di Formamide (Applied Biosystems). All samples were applied to an ABI Prism 310 Genetic Analyzer (Applied Biosystems) with a short capillary. The fragment sizes of the PCR products were identified using the GeneMapper ver. 4.0 software (Applied Biosystems). All isolates were genotyped on the basis of allelic relation (alleles 1:2). Genotypes of CDC3 and HIS3 use the size of DNA fragments. In contrast, genotypes of CAI and CAIII are presented as the number of repeated units in the DNA fragment; the DNA fragments of 252 and 113 bp correspond to 41 (for CAI) and 11 (for CAIII) repeated units, respectively [19, 20]. Genetic distances with multilocus genotypes and the phylogenetic relationships of the C. albicans isolates were analyzed using Populations (ver. 1.2.32) [21].
Table 1 Candida species isolated from healthy volunteers (HV) and patients with oral candidiasis (OC) Candida species
Numbers of HV and OC HV (n = 42)
OC (n = 50)
C. albicans
24
23
C. glabrata
2
1
C. parapsilosis II
0
1
C. dubliniensis
1
0
Other yeasts
4
4 6
Single species
Double species C. albicans ? C. glabrata
4
C. albicans ? C. parapsilosis II
0
1
C. albicans ? C, dubliniensis C. albicans ? C. tropicalis
1 0
0 1
C. albicans ? Other yeasts
5
6
C. glabrata ? C. parapsilosis II
1
0
C. parapsilosis I ? other yeasts
0
1
0
1
Triple species C. albicans ? C. glabrata ? C. dubliniensis
Results
C. albicans ? C. glabrata ? other yeast
0
2
C. albicans ? C. parapsilosis II ? other yeasts
0
2
Isolation of Candida species In this study, yeasts were isolated from 41 of 88 HVs (46.6 %), and 34 of these (81 %) had C. albicans. In contrast, C. albicans was found in 42 of 50 patients from the OC group (84 %) who presented yeasts on the chromogenic cultures (Table 1). In both groups, C. albicans constituted the majority of the isolates. Additionally, Candida glabrata was found in 7 HVs (16.7 %) and 11 OC patients (22 %) (Table 1). Candida parapsilosis I and II, C. dubliniensis, and Candida tropicalis II were isolated from the oral cavity of HV and/or OC participants, but the frequencies were remarkably lower than those of C. albicans and C. glabrata (Table 1). C. albicans was isolated as the sole species from 25 HVs (60.9 %) and from 23 OCs (46 %). C. glabrata and C. dubliniensis were also isolated as the sole species from 1 member each of the HV and OC groups (Table 1). In 10 HVs (23.8 %) and 19 OCs (38 %), C. albicans was isolated with other Candida species or other yeasts; for example, C. albicans was isolated with C. glabrata in 4 HVs (9.5 %) and 9 OCs (18 %) (Table 1). Genotype homogeneity of C. albicans in the oral cavity In this study, 472 and 676 C. albicans isolates were collected from 34 HVs and 42 OCs, respectively, by multi-
Table 2 25S rDNA-based groups of Candida albicans isolates
25S rDNA-based group
Single group A
Numbers of HV and OC HV
OC
18
21
B
7
8
C
0
8
A?B
1
1
A?C
3
2
B?C
5
2
Double group
HV healthy volunteers, OC oral candidiasis
colony isolation (10–16 colonies/individual). All the 1,148 C. albicans isolates were subjected to genotype homogeneity studies by 25S rDNA- and microsatellite-based genotyping. 25S rDNA-based genotyping divided C. albicans isolates into three groups, among which group A C. albicans constituted the majority in both the HV and OC groups, followed by group B and C C. albicans (Table 2). Group A C. albicans was found in 22 HVs (64.7 %) and 24 patients (57.1 %). The multi-colony analysis of C. albicans from individual HV and OC members showed that a single
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genotype of C. albicans was found in 25 HVs (73.5 %) and 37 patients (88.1 %), and the remaining participants presented 2 genotypes of C. albicans (groups A ? B, A ? C, or B ? C) at the 25S rDNA level (Table 2). No group E C. albicans was found in this study. C. albicans isolates obtained from HVs and OCs that presented 10–16 colonies were used to study genotype homogeneity in the oral cavity of HV and OC individuals. All the isolates were divided into three groups (groups A, B, and C) on the basis of 25S rDNA, and each group was then classified on the basis of the combination of allelic relationships to four microsatellite markers (CDC3/CAI/ HIS3/CAIII) (Fig. 1). In the HV group, 16 individuals (47.1 %) showed two genotypes of C. albicans, and 13 individuals (38.2 %) had a single genotype of C. albicans. The number of C. albicans genotypes in the OC individuals was the same as that from the HV group. In the OC group, a single genotype of C. albicans was found in 26 (61.9 %) patients; this frequency was higher than that of the HV group (p \ 0.05). On the other hand, two genotypes of C. albicans were found in 12 (28.6 %) patients; this frequency was lower than that of the HV group (p \ 0.05). C. albicans genotype distribution of isolates from the HV and OC groups To compare C. albicans genotype distribution between the HV and OC groups, C. albicans isolates from 4 HVs and 5 OCs who presented 1 or 2 colonies were also used for this study, in addition to the isolates from HVs and OCs who presented multiple colonies (10–16 colonies). Sixty-three isolates from 38 HVs and 68 isolates from 47 OCs were subjected to this study, grouped by allelic relationships of CDC3 and CAI. The isolates of the HVs were classified into 35 allelic relationships. Of these, three allelic relationships, 115:119/23:23, 115:123/18:27, and 123:127/ 70
Frequency (%)
60 50
32:41 (CDC3 alleles 1:2/CAI alleles 1:2), accounted for 12.7 % (8 isolates), 7.9 % (5 isolates), and 7.9 % (5 isolates) of 63 isolates from the HV group, respectively (Table 3). These allelic relationships corresponded to genotypes II, III, and V in a previous report [11]. Therefore, we referred to the 115:119/23:23, 115:123/18:27, and 123:127/32:41 allelic relations as genotype II, III, and V, respectively. In contrast, the isolates from the patients were classified into 39 allelic relationships, and genotypes II and III were assigned to 3 (4.4 %) and 14 (20.6 %) of 68 isolates from the OC group; however, genotype V C. albicans was not found (Table 3). The frequency of genotype II was slightly higher in the HV isolates than in the OC isolates (p [ 0.05). In contrast, the frequency of genotype III C. albicans in the OC group was higher than that in the HV group (p \ 0.05). The allelic relationships corresponding to genotypes I and IV were not found in any isolates from this study. When CDC3/CAI-based genotypes were combined with 25S rDNA groups, genotypes II, III, and V C. albicans of the HV isolates (18 isolates) and genotypes II and III C. albicans of the OC isolates (17 isolates) were classified into six and four types, respectively (Fig. 2). Genotypes (CDC3/CAI-25S rDNA) II-A and III-A were found equally in the HV and OC group. The genotype III-C was clearly more frequent in the OC group than the HV group. Additionally, all the genotype V–C C. albicans were found only in the HV group. Genotype II, III, and V C. albicans isolates (total, 35 isolates) were further classified by allelic relationships to additional microsatellite markers, HIS3 and CAIII. These isolates were classified into 21 types: genotypes II, III, and V showed independent clusters and were divided into 9 (types 1–9, genotype II), 9 (types 10–18, genotype III), and 3 (types 19–21, genotype V) types, respectively (Fig. 2). Types 4 and 12 were common to both HV and OC groups. Types 14, 16, 17, and 18, which were group C, were found only in the OC group. In contrast, types 19, 20, and 21, which were also group C, were found only in the HV group.
40
Discussion
30 20 10 0
1
2
3
4
No. of genotypes
Fig. 1 Comparison of heterogeneity of Candida albicans genotype between commensal and infective sites. Horizontal axis indicates different genotype numbers of C. albicans in healthy volunteers (HV) (open columns) and patients with oral candidiasis (OC) (solid columns). Vertical axis indicates frequencies of each genotype
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Candida albicans constitutes the majority of the isolates from each group, and it was co-isolated with other Candida or yeast species from 19 patients of the OC group (38 %), a slightly higher frequency than that from the HV group (23.8 %). This result suggests that Candida species other than C. albicans act as endogenous pathogens and are involved in the development of oral candidiasis. In this study, C. glabrata was frequently co-isolated with C. albicans. Oral candidiasis caused by C. albicans was
J Infect Chemother Table 3 Genotypes of Candida albicans and their distributions in HV and OC groups Genotype and number of isolates
Genotype and number of isolates
Allelic relationship (alleles 1:2)
Group
Allelic relationship (alleles 1:2)
Group
CDC3
CAI
HV
OC
CDC3
CAI
HV
95:111 99:115
31:31
1
115:123
28:29
1
27:35
2
115:123
29:29
3
99:115 103:115
51:51 18:27
1
115:123 115:123
29:31 33:33
1 2
103:119
23:23
107:123
18:27
1
1
115:123
33:39
2
115:123
34:34
111:115
21:28
1
3
115:123
34:50
1
111:115
28:28
2
111:115
29:33
1
115:123
34:52
1
115:123
36:36
111:115
33:42
1
115:123
36:55
111:115
33:43
115:115
18:27
1
2
115:123
52:52
1
1
115:127
33:33
115:115
23:23
2
119:119
23:23
2
115:115
23:30
115:115
23:32
1
1
119:119
28:41
3
119:119
30:32
115:115
25:25
1
119:123
28:33
115:115
25:30
2
115:115 115:115
25:41 27:27
1
115:115
28:37
115:119
15:23
115:119
23:23
115:119
23:33
115:119 115:119 115:119
23:45
115:119
1
1
OC
2
2 2 1 1
2 1
119:123
28:34
1 1
119:123 119:127
41:41 39:40
1
1
123:123
32:33
1
2
1
123:123
34:34
8 (12.7)
3 (4.4)
123:123
34:50
1
1
1
123:123
37:39
2
23:37
1
123:123
39:39
2
23:42
1
123:127
28:41
1
2
123:127
32:32
1
23:46
2
123:127
32:41
5 (7.9)
115:119
23:50
1
123:127
36:39
1
115:119
23:51
1
123:127
39:40
2
115:123
18:27
5 (7.9)
123:127
40:40
115:123
19:27
1
123:127
41:41
115:123
21:23
123:135
23:42
14 (20.6) 3
1 1 1
1 1 1
HV healthy volunteers, OC oral candidiasis
accompanied by C. glabrata in several cases. It is important to learn the involvement of C. glabrata in oral candidiasis for accurate therapy because C. glabrata generally has low susceptibility to the azole class of antifungal therapies [1]. Recently, several studies have reported a relationship between C. glabrata pathogenicity and genotype [22, 23]. The C. albicans isolates from HVs showed three major allelic relationships to CDC3/CAI, which corresponded to genotypes II, III, and V reported by Takagi et al. [11]. Of these three major genotypes, genotypes II and III were the major genotypes found in the OC group. The remaining
genotype, genotype V, was not observed for any isolate from this group. These results coincided with these in the previous study that the major genotype distributions of C. albicans isolated from patients with and without candidiasis were compared. Genotypes II and III C. albicans were commonly isolated from patients with skin candidiasis, vulvovaginal candidiasis, and candidemia. In contrast, genotype V C. albicans was not found [11]. These results suggested that the genotype V C. albicans defined by CDC3/CAI was distributed widely in the oral cavity but was less likely to cause either superficial candidiasis or systemic candidiasis. This study strongly supports the idea
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J Infect Chemother Fig. 2 Dendrogram of genotypes II, III, and V of C. albicans based on four microsatellite markers
that the genotype V C. albicans is not involved in the development of candidiasis because genotype V C. albicans was not isolated from the oral cavity of patients with oral candidiasis although this genotype is the major genotype in the oral cavity of healthy individuals. Shimizu et al. [13] used the microsatellite markers HIS3 and CAI to genotype C. albicans and reported that the genotype distribution of C. albicans was not identical in commensal and infective lesions. Additionally, in the previous study, genotype I (111:115/33:41) and IV (115:123/33:40) C. albicans were included in the isolates from patients with and without candidiasis; however, they were not found in either the HV or OC group in this study. Genotypes I and IV C. albicans may not be involved in the development of oral candidiasis. Previously, the microsatellite markers CDC3 and CAI were used to genotype C. albicans from the bloodstream, and genotypes II and III C. albicans were shown to have the ability to cause candidemia [11]. In contrast, Dalle et al. [24] used the microsatellite marker CEF3 to genotype C. albicans and concluded that C. albicans from the bloodstream and non-bloodstream samples were genetically heterogeneous. When microsatellite analysis was combined with 25S rDNA-based genotyping, genotype II C. albicans was classified into groups A and B, and group B was found only in the HV group. The genotype III C. albicans was classified into groups A and C, and group C was only identified in the OC group. All genotype V isolates were classified into group C. The group A C. albicans was found equally in both the HV and OC groups. These results suggest that the A group isolates were widely distributed in both HV
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and OC individuals, and these isolates have the ability to cause candidiasis. The genotype II C. albicans isolates grouped into ‘‘B’’ and genotype V isolates grouped into ‘‘C’’ were less able to cause candidiasis. Additionally, the microsatellite analysis using four markers combined with 25S rDNA-based typing divided the three major genotypes into 21 types, of which type 14 (genotype III/group C) was found only in the OC group, and types 20 and 21 (genotype V/group C) were found only in the HV group. This result suggests that a comparison of biological properties of these types is useful to understand the development of candidiasis and the virulence factors of this fungus. She et al. [9] reported that 25S rDNA and alanine aminotransferase (ALT)-based genotypes of C. albicans were genetically different in cutaneous and vaginal candidiasis. Fan et al. [10] used a microsatellite method to genotype C. albicans from patients with vaginal candidiasis and reported that the genotype correlated with the severity of candidiasis. In a previous study, the frequency of genotype III C. albicans from patients with vaginal candidiasis was lower than that from other groups [11]. Hattori et al. [12] reported that genotypes of C. albicans from the oral cavity were similar to those from infective sites of an individual; however, they did not use commensal C. albicans from the vagina. C. albicans can be isolated from the vagina of healthy females [1]. Thus, it is important to analyze genotypes of C. albicans isolated from the vagina. Studying the genotype homogeneity of C. albicans isolates from an individual showed that heterogeneous genotypes (from one to four genotypes) were found in both groups. The frequency of a single genotype of C. albicans
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was clearly higher in the OC group than in the HV group (p \ 0.05). In the HV group, two genotypes of C. albicans were isolated more frequently than a single genotype. Shimizu et al. [13] reported that the genotypes of multiple C. albicans isolates from infection sites of an individual patient with superficial candidiasis were generally homogeneous compared with that of oral isolates. Dalle et al. [25] analyzed genotypes of C. albicans collected serially from patients with acute leukemia and observed that C. albicans isolates had identical genotype profiles in all body locations during the isolation period. They concluded that C. albicans genotypes were rarely altered by antifungal agent pressure. It has been reported that several genotypes of C. albicans were isolated from different locations of the vagina of one female [26]. This finding suggests the involvement of a selection force for the development of superficial candidiasis. However, the selection force is unknown. Unfortunately, we did not analyze genotypes of C. albicans isolated serially from patients and healthy individuals in this study. For this, a longitudinal study of C. albicans multi-colony genotyping in individuals is necessary. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Educations, Culture, Sports, Science and Technology of Japan (#21405002 to R.T. and #22406011 to F.K). Conflict of interest of interest.
8.
9.
10.
11.
12.
13.
14.
15.
The authors declare that they have no conflict
16.
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