Microbiome variability and role of Candida albicans in site-specific dental plaques in orthodontic adolescent patients with white spot lesions

ABSTRACT

White spot lesions (WSLs) are a common complication of orthodontic treatment. However, the cariogenic discrepancy in the supragingival microbiome between demineralized and non-demineralized surfaces and the influence of Candida albicans associated with WSLs remain unexplored. This study investigated the changes in supragingival microbiome of orthodontic adolescents with WSLs, encompassing both demineralized and non-demineralized sites, and explored C. albicans colonization in these patients. Supragingival plaques were collected from 29 orthodontic adolescents with WSLs (categorized into demineralized and non-demineralized groups based on the presence/absence of demineralization at sampling sites) and 23 healthy orthodontic adolescents. Supragingival microbiome composition was evaluated using 16S rRNA sequencing, and C. albicans colonization was identified using fungal culture methods. The supragingival microbiome on non-demineralized surfaces showed intermediate cariogenic potential between demineralized and healthy states, but closer to the demineralized state. C. albicans exhibited a propensity for colonization in WSLs patients without site-specificity. C. albicans influenced bacterial composition, with Streptococcus mutans significantly enriched on the demineralized surfaces of C. albicans-positive patients. In orthodontic adolescents with WSLs, non-demineralized surfaces showed microbiome shifts, necessitating interventions to promote a healthy microbiome. C. albicans can impact microbiome composition and potentially contribute to WSLs pathogenesis.

KEY MESSAGES

• In adolescent orthodontic patients with WSLs, the supragingival microbiome on non-demineralized surfaces shows cariogenic potential intermediate between demineralized and healthy states, but closer to the demineralized state.

• These patients are also more susceptible to C. albicans colonization, which influences the bacterial community composition and is not site-specific.

• Enrichment of S. mutans can be detected on the demineralized surfaces of C. albicans-positive patients, suggesting a potential interkingdom interaction between these two species at demineralized sites.

Introduction

Malocclusion, recognized by the World Health Organization as one of the three major oral diseases, affects up to 45% of the population [1,2]. However, the use of orthodontic appliances during fixed orthodontic treatment increases the difficulty of maintaining oral hygiene and is prone to complications such as white spot lesions (WSLs). The incidence of WSLs in fixed orthodontic patients can reach up to 50–70% [3], with adolescents, who represent the primary population in need of orthodontic interventions, being particularly susceptible [4,5]. WSLs have long-term implications for the dental health and aesthetics of orthodontic patients [3,6]. Although natural remineralization through saliva and localized fluoride application can facilitate the remineralization of demineralized enamel surfaces, the extent of enamel recovery is limited and depends on the lesion severity [7,8]. Thus, early assessments of WSLs risk are crucial for the control and prevention of WSLs progression [9–11].

Identifying significant predictive biomarkers is key to WSL prevention [8,12]. For example, low arginine metabolism capacity [13] and low plaque fluoride concentration can indicate the risk of dental caries [14]. Additionally, clinical characteristics such as thick and heavy dental plaques on occlusal surfaces also suggest caries susceptibility [15,16]. The onset of WSLs is associated with microecological dysbiosis of dental plaque on enamel surfaces, prompting increased attention to changes in pathogenic bacteria [17,18]. Recent high-throughput sequencing technologies have enabled the comprehensive analysis of oral microbiome changes during fixed orthodontic treatment [19,20]. Previous microbial analyses of plaque or saliva samples have demonstrated an increased abundance of caries-associated bacteria, such as Streptococcus species, and a decreased abundance of bacteria typically detected on healthy enamel surfaces, such as the Rothia genus, as treatment progresses [21]. Variations in caries-associated bacteria within the supragingival microbiome are considered potential predictors of dental caries and WSLs [22,23].

To ensure an adequate sample size when collecting plaque from patients with WSLs, most studies opt to pool dental plaque samples together from all tooth surfaces, including both demineralized and non-demineralized surfaces [24–27], or alternatively select saliva as the research sample [28–32]. Few studies have focused on specific demineralization sites for sample analysis without clarifying whether the entire dentition, including both demineralized and non-demineralized surfaces, is colonized by highly cariogenic plaques in patients with WSLs. The composition of the supragingival microbiome on non-demineralized enamel surfaces in these patients remains uncertain, particularly regarding whether it resembles a demineralized or relatively healthy state. When WSLs occur, the risk of caries on non-demineralized tooth surfaces remains uncertain. If the supragingival microbiome on non-demineralized surfaces resembles a demineralized state, patients may be more susceptible to demineralization at healthy tooth sites, necessitating interventions to promote a healthy microbiome. Conversely, if the supragingival plaque microbiome at these sites is closer to a healthy state, WSLs occurrence may exhibit site-specificity. Therefore, in addition to focusing on bacterial differences between orthodontic patients with typical WSLs and healthy individuals, investigating plaque conditions on both demineralized and non-demineralized tooth surfaces in orthodontic patients with WSLs is crucial for developing effective prevention and risk assessment strategies.

Moreover, fungi also represent a significant component of the oral microbiome, coexisting with viruses, protozoa, archaea, and bacteria [33]. Notably, Candida albicans is the most prevalent fungal species identified in children predisposed to dental caries [34]. The co-aggregation of C. albicans and Streptococcus mutans can lead to the formation of a robust and highly cariogenic biofilm on the enamel surface [34]. In vitro studies have suggested that clinical isolates of C. albicans can exacerbate WSLs severity, thereby reducing enamel hardness [35]. Therefore, in addition to investigating the changes in cariogenic bacteria within the supragingival microbiome, it is also crucial to consider the impact of C. albicans in patients with WSLs. As C. albicans primarily exists in saliva rather than dental plaque [36], the relative frequency of C. albicans colonization in saliva and dental plaque in patients with WSLs remains unclear. Further, in vivo research is needed to determine whether C. albicans preferentially colonizes dental plaque on demineralized surfaces or healthy tooth sites in adolescent orthodontic patients with WSLs and to elucidate its effects on supragingival microbiome composition.

Therefore, in this study, we used 16S rRNA gene sequencing technology to analyze differences in the supragingival microbiome between non-demineralized and demineralized tooth sites in adolescent orthodontic patients with -WSLs and compared these findings with those of healthy orthodontic patients. Furthermore, we investigated the detection rates of C. albicans in saliva and supragingival plaque at different sites as well as its impact on oral bacterial microbiome composition. We propose three null hypotheses: 1) in orthodontic patients with WSLs, the supragingival microbiome composition on non-demineralized surfaces does not differ significantly from that in healthy individuals; 2) C. albicans is not enriched in the saliva or dental plaque (including both demineralized and non-demineralized sites) of orthodontic patients with WSLs; and 3) the presence of C. albicans does not influence the composition of bacteria within the supragingival microbiome.

Patients/materials and methods

Patient recruitment

This study was approved by the Ethics Committee of Beijing Stomatological Hospital, Capital Medical University (CMUSH-IRB-KJ-YJ-2022–15). We recruited patients aged 11–18 years undergoing orthodontic treatment at the Department of Orthodontics, Beijing Stomatological Hospital. The WSLs experimental group comprised 29 patients, further categorized into WSLs-WSLs and WSLs-Health groups based on the presence or absence of demineralization at the sampling sites. The Control group comprised 23 patients. The inclusion criteria for the WSLs experimental group were as follows: (1) adolescents with complete natural dentition who underwent fixed orthodontic treatment for 6–12 months; (2) presence of WSLs in the anterior and premolar regions, with an average Enamel Demineralization Index (EDI) > 0.12 [37]. The Control group exhibited no signs of WSLs in the anterior and premolar regions. Both groups had healthy periodontal tissues characterized by a probing depth (PD) ≤ 3 mm, no periodontal attachment loss, and a Gingival Index (GI) ≤ 1. The exclusion criteria for both groups were: (1) the presence of active untreated caries beyond specified regions; the number of ‘decayed, missing due to caries, and filled teeth’ (DMFT) > 2; and (2) developmental dental disorders such as dental fluorosis, systemic diseases, infectious diseases, long-term medication use, or antibiotic treatment within the past 3 months.

Sample collection

Participants were instructed to abstain from oral hygiene practices for 12 h and to avoid eating or drinking for 2 h before their appointments. The non-stimulated saliva samples were collected for the experiment. During the collection, patients were positioned in a natural rest state with their mouths closed, heads lowered, while avoiding swallowing to allow saliva to accumulate in the mouth. Subsequently, saliva (0.5 mL) was collected in a 2 mL sterile centrifuge tube. In the WSLs experimental group, supragingival plaque samples were collected from the labial surfaces of 12 anterior teeth and eight premolars at sites exhibiting observable WSLs under saliva-moistened conditions. The collected plaque was then aggregated and designated as the WSLs-WSLs group (abbreviated as WSLs-W). Additionally, for these patients, dental plaque samples were collected from the labial surfaces of 12 anterior teeth and eight premolars at sites without WSLs, and designated as the WSLs-Health group (abbreviated as WSLs-H). In detail, for the WSLs-H group, we selected dental plaque from sites without demineralization, specifically targeting areas that were distant from sites existing WSLs. After the plaque was collected, the corresponding tooth surfaces were inspected in an initially wet state and again after 5 s of drying with a gentle air stream to ensure the sampling sites without WSLs. In the Control group, plaque was collected from healthy labial surfaces of 12 anterior teeth and eight premolars using a sterile periodontal scaler. After oral hygiene maintenance, clinical evaluations of WSLs and periodontal health were performed. The procedure was carried out by two well training orthodontists (H.Y. and HY.G.), who were accountable for the oral examination. Both had undergone strict training in sample collection protocols and EDI scoring criteria and methodologies.

Following collection, plaque samples were suspended in 1,000 µL of sterile 1× Tris-EDTA buffer and stored on ice. Care was taken to ensure that the periodontal scaler did not re-enter the oral cavity between the collections. The samples were promptly transported to the laboratory. Saliva samples were stored directly at − 80°C. Plaque samples were vortexed and divided into two 500 µL aliquots. One aliquot was centrifuged and resuspended in 1 mL 1× Tris-EDTA buffer (containing 20% glycerol) for Candida culture and identification, and the other aliquot was used for DNA extraction. All the samples were preserved at − 80°C.

DNA extraction, 16S rRNA sequencing, and processing

The DNA from supragingival plaque samples was extracted using the FastDNA® Spin Kit for Soil (MP Biomedicals, USA) according to the manufacturer’s protocol. DNA quality was assessed using 1% agarose gel electrophoresis, and DNA concentration and purity were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Extracted DNA served as a template for PCR amplification of the V1-V3 variable regions of the 16S rRNA gene using the primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 533 R (5’-TTACCGCGGCTGCTGGCAC-3’). Sequencing was performed on the Illumina MiSeq PE 300 platform (Shanghai Majorbio Biomedical Technology Co. Ltd.). Raw sequencing reads were submitted to the NCBI Sequence Read Archive database (accession number PRJNA1221216). Raw sequencing data were subjected to quality control using fastp (v0.19.6), and quality-filtered sequences were merged using FLASH software (v1.2.7) [38,39]. The DADA2 [40] plugin within the Qiime2 pipeline was used to denoise the optimized sequences [41]. Taxonomic classification of amplicon sequence variants was conducted using the Naïve Bayes classifier from Qiime2, with reference to the Human Oral Microbiome Database (HOMD) (v15.2).

Fungal culture and identification of Candida species

Saliva (0.5 mL) and dental plaque samples were inoculated onto CHROMagar Candida-selective medium (CHROMagar, FRA). The samples were then incubated at 37°C for 72 h [42]. After 72 h, the medium was examined for fungal growth, and C. albicans colonies were identified based on their characteristic green colonies [43]. Preliminary identification of Candida species was based on colony morphology, size, color, and texture.

Following fungal culture, positive colonies were harvested using a sterile inoculation loop and transferred to 2 mL sterile centrifuge tubes for DNA extraction and rDNA internal transcribed spacer (ITS) region analysis [43]. ITS amplification was performed using the following primers: ITS4, 5’-TCCTCCGCTTATTGATATGC-3’ and ITS5, 5’-GGAAGTAAAAGTCGTAACAAGG-3’. PCR amplification was performed using an ABI GeneAmp 9700 thermocycler (Eppendorf, DE) according to the following program: 5 min pre-denaturation at 95°C, followed by 35 cycles (30 s at 95°C for denaturation, 30 s at 55°C for annealing, and 1 min at 72°C for extension), with a final extension at 72°C for 10 min [44]. PCR products were sequenced at the Beijing Genomics Institute. The resulting sequences were compared with homologous sequences in the GenBank database using BLAST to identify closely related fungal species.

In subsequent experiments, we further divided the samples into four subgroups based on the detection of C. albicans in the WSLs-W and WSLs-H groups. WSLs-W group samples were further divided into two subgroups: a Candida-positive WSLs subgroup (WSLs-W-CaP) and a Candida-negative WSLs subgroup (WSLs-W-CaN). Similarly, the WSLs-H group was subdivided into two subgroups: a Candida-positive non-demineralization subgroup (WSLs-H-CaP), and a Candida-negative non-demineralization subgroup (WSLs-H-CaN), allowing for further analysis of the effect of C. albicans on bacterial community composition in the context of WSLs.

Statistical analysis

Statistical analyses were performed using SPSS v24.0 software. The HOMD database (v15.2) and the Naïve Bayes classifier within Qiime2 were used for taxonomic analysis of amplicon sequence variants. Alpha diversity was assessed using Chao1, ACE, Simpson, and Shannon indices. Beta diversity was compared using principal coordinate analysis (PCoA), and intergroup differences were assessed using the ADONIS test. Differences in species among multiple groups were analyzed using the Kruskal – Wallis rank – sum test, followed by Dunn’s multiple-comparison test for post-hoc analysis. Statistical significance was set at p < 0.05.

Results

The non-demineralized dental surfaces in orthodontic adolescents with WSLs exhibit microbiome shifts

A total of 29 adolescent orthodontic patients with WSLs were included in this study (each patient was further categorized into WSLs-W and WSLs-H groups based on the presence or absence of demineralization at the sampling sites), along with 23 healthy adolescent orthodontic patients without demineralization (Control group). A total of 81 supragingival plaque samples were sequenced. The essential characteristics of the study participants are listed in Table 1. Clinical indicators revealed a significantly higher EDI for the WSLs-W and WSLs-H groups than for the Control group, confirming sample representativeness. Other variables, including patient age, orthodontic treatment duration, and gingival index, showed no statistically significant differences among the groups. Following sequencing, 5,634,459 raw reads were obtained from the 81 samples. After quality control filtering, 5,305,781 optimized sequences were retained. The DADA2 plugin of the Qiime2 pipeline was used to denoise the quality-controlled assembled sequences, resulting in 1,477,492 final sequences. High-throughput sequencing revealed 12 phyla, 27 classes, 46 orders, 78 families, 148 genera, and 500 species across 81 samples.

Table 1.

Mean (standard deviation) of basic information and clinical indicators.

Group	Age	Course of treatment month	EDI	GI
WSLs-W (n = 29)	14.17 (1.79)	8.93 (1.96)	0.40 (0.18)	0.29 (0.13)
WSLs-H (n = 29)	14.17 (1.79)	8.93 (1.96)	0.40 (0.18)	0.29 (0.13)
Control (n = 23)	13.91 (1.78)	8.87 (1.84)	0.00 (0.00)	0.25 (0.11)

To investigate differences in the species richness and diversity of the supragingival plaque microbiome among the WSLs-W, WSLs-H, and Control groups, we analyzed alpha diversity (Figure 1a). The ACE, Chao1, Simpson, and Shannon indices showed no statistically significant differences among the three groups, suggesting similar community richness and diversity (p > 0.05). A comparison of beta diversity using PCoA analysis is depicted in Figure 1b, where each dot represents a supragingival plaque sample. No significant differences in beta diversity were observed between WSLs-W and WSLs-H groups (p > 0.05), suggesting that within the same patient with WSLs, the supragingival plaque microbiome composition of demineralized and non-demineralized surfaces did not differ significantly. However, significant differences in beta diversity were observed between the WSLs-W and Control groups (p = 0.001), as well as between the WSLs-H and Control groups (p = 0.009), indicating that both demineralized and non-demineralized surfaces in patients with WSLs exhibited significantly different microbiome compositions compared with healthy individuals. Further analyses of community composition and microbial differences were conducted to explore changes in bacterial abundance in the supragingival plaque microbiome for the WSLs-W and WSLs-H groups compared to healthy controls.

Figure 1.

Comparison of alpha and beta diversities of microbial communities among the WSLs-W, WSLs-H and Control groups. (a) Four indices, ACE, Chao1, Simpson, and Shannon, were selected to compare the alpha diversity. (b) Principal coordinate analysis (PCoA) was used to analyze beta diversity, and the ADONIS test was applied to evaluate statistical distinctions (*p < 0.05, **p < 0.01, ***p < 0.001).

To further elucidate the differences in microbial composition among the WSLs-W, WSLs-H, and Control groups, core genera (relative abundance > 1.0%) were analyzed. Figure 2a illustrates that the core genus composition remained consistent across the three groups. The ten most dominant genera were Leptotrichia, Saccharibacteria_TM7_G-1, Actinomyces, Streptococcus, Corynebacterium, Capnocytophaga, Prevotella, Selenomonas, Veillonella, and Fusobacterium. Despite the overall similarity in core genus composition, the WSLs-W group exhibited elevated relative abundance of Actinomyces, Prevotella, Selenomonas, and Veillonella. In contrast, the Control group exhibited a higher relative abundance of Corynebacterium, Saccharibacteria_TM7_G-1, and Neisseria. The relative abundances of the genera in the WSLs-H group were generally situated between those of the WSLs-W and Control groups.

Figure 2.

Microbial composition analysis among the WSLs-W, WSLs-H and Control groups at the genus level. (a) Microbial composition at the genus level. (b) The Kruskal–Wallis rank–sum test demonstrated significant differences in the genera among the WSLs-W, WSLs-H, and Control groups (taking the top 1–8 genera ranged by relative abundance). (c) After Kruskal – Wallis rank – sum test, post – hoc Dunn’s test was performed to conduct pairwise comparisons among the multiple groups. The genera were selected from the top 1–8 genera with significant differences, specifically those associated with caries or commonly detected on healthy tooth surfaces (*p < 0.05, **p < 0.01, ***p < 0.001).

Based on the obtained community abundance data, the significance of the differences in relative abundance across groups was evaluated at the genus and species levels. The top eight genera and 32 species with the highest relative abundances were analyzed (Figures 2b,c and 3). Remarkably, at the genus level, Prevotella, previously associated with caries development, exhibited significant differences in abundance between the WSLs-W and Control groups (p < 0.001), and between the WSLs-H and Control groups (p < 0.05), but not between the WSLs-W and WSLs-H groups (p > 0.05). This suggested Prevotella enrichment in both WSLs-W and WSLs-H groups, with a relatively higher abundance in the WSLs-W group. Selenomonas, also linked to caries development, showed an increased relative abundance in the WSLs-W group compared to the Control group (p < 0.05), whereas the WSLs-H group showed no significant differences from either of the other two groups (p > 0.05). This suggested Selenomonas enrichment in the WSLs-W group, with their abundances in the WSLs-H group intermediate between the other two groups. Correspondingly, certain species displayed differences in abundance at the species level (Figure 3). Prevotella denticola and Prevotella pallens showed significantly higher relative abundances in the WSLs-W group than in the Control group (p < 0.05), but not between the WSLs-H group and the other two groups (p > 0.05). This indicates that P. denticola and P. pallens were enriched in the WSLs-W group, with their relative abundances in the WSLs-H group intermediate between the other two groups. Furthermore, the abundance of Prevotella melaninogenica, Prevotella salivae, and Selenomonas sputigena also exhibited significant differences between the WSLs-W and Control groups (p < 0.001), as well as between the WSLs-H and Control groups (p < 0.05), but not between the WSLs-W and WSLs-H groups (p > 0.05). This indicated greater enrichment of these bacteria in the WSLs-W and WSLs-H groups, with relatively higher abundance in the WSLs-W group.

Figure 3.

Microbial composition analysis among the WSLs-W, WSLs-H and Control groups at the species level. (a) The Kruskal – Wallis rank – sum test demonstrated significant differences in the species among the WSLs-W, WSLs-H, and Control groups (taking the top 1–32 species ranged by relative abundance). (b) post – hoc Dunn’s test was performed to conduct pairwise comparisons among the multiple groups. The species were selected from the top 1–32 species with significant differences, specifically those associated with caries or commonly detected on healthy tooth surfaces (*p < 0.05, **p < 0.01, ***p < 0.001).

In addition, certain bacteria typically enriched on healthy tooth surfaces, such as Corynebacterium exhibited significant differences in relative abundance between the WSLs-W and Control groups, as well as between the WSLs-W and WSLs-H groups (p < 0.05), but not between the Control and WSLs-H groups (p > 0.05) (Figure 2b,c). At the species level, Corynebacterium durum even showed a higher abundance within the WSLs-H group (p < 0.05) (Figure 3), indicating significant enrichment of the genus Corynebacterium and the species C. durum in the Control and WSLs-H groups. Other healthy tooth surface-associated bacteria, such as Rothia and Lautropia and its species Lautropia mirabilis exhibited significant differences in relative abundance between the WSLs-W and Control groups, with higher abundance in the Control group (p < 0.01). However, no significant differences were observed between the WSLs-H group and the other two groups (p > 0.05), suggesting an intermediate relative abundance in the WSLs-H group. At the species level, Rothia aeria exhibited significant differences in abundance between the WSLs-W and Control groups (p < 0.001), as well as between the WSLs-H and Control groups (p < 0.05). No significant differences were observed between the WSLs-W and WSLs-H groups (p > 0.05). This suggests that R. aeria was more likely to be enriched in the Control group, but showed similar relative abundances in the WSLs-W and WSLs-H groups, although that in the WSLs-H group was higher.

In addition to the aforementioned genera, certain species closely associated with caries development exhibited statistically significant differences in their relative abundance (Figure 3). For instance, Streptococcus anginosus was significantly more abundant in the WSLs-W group than in the Control group (p < 0.01), but not between the WSLs-H group and the other two groups (p > 0.05). This indicated a clear enrichment of S. anginosus in the WSLs-W group, with its abundance in the WSLs-H group intermediate between the other two groups. The abundance of Leptotrichia sp._HMT_498, Actinomyces sp._HMT_448, and Streptococcus mutans were significantly different between the WSLs-W and Control groups, as well as between the WSLs-H and Control groups (p < 0.05), but not between the WSLs-W and WSLs-H groups (p > 0.05). This suggested that these species were more abundant in the WSLs-W and WSLs-H groups, with a relatively higher abundance in the WSLs-W group. In contrast, bacteria associated with healthy conditions showed significant differences. Streptococcus sanguinis and Streptococcus cristatus; clade_578 exhibited significantly higher abundance in the Control group than in the WSLs-W group (p < 0.05), whereas the WSLs-H group showed no significant differences from the other two groups (p > 0.05). Leptotrichia sp._HMT_215 and Leptotrichia sp._HMT_225 demonstrated significantly different abundances between the WSLs-W and Control groups, as well as between the WSLs-H and Control groups (p < 0.05), whereas no significant differences were observed between the WSLs-W and WSLs-H groups (p > 0.05). This suggested that Leptotrichia sp._HMT_215 and Leptotrichia sp._HMT_225 were more likely to be enriched in the Control group, but showed similar relative abundances in the WSLs-W and WSLs-H groups.

C. albicans is more likely to colonize the oral cavities of orthodontic patients with WSLs, without site specificity

Among the 29 adolescents with WSLs undergoing fixed orthodontic treatment, nine patients tested positive for Candida in the culture. These positive samples displayed green colonies on CHROMagar Candida-selective medium. Furthermore, the detection sites for Candida in these patients with WSLs did not exhibit site specificity (Table 2). Candida was detected in saliva samples and in supragingival plaque collected from both demineralized and non-demineralized tooth surfaces in all nine patients, yielding a total of 27 Candida-positive samples. In contrast, the remaining patients with WSLs and all patients in the Control group showed negative Candida culture results for both supragingival plaques and saliva (Table 2). Subsequent rDNA ITS analysis identified all 27 positive samples from demineralized surfaces, non-demineralized surfaces, and saliva samples as C. albicans. This finding suggests that C. albicans colonization is more prevalent in patients with WSLs than in healthy individuals, with a colonization rate of up to 31%. Additionally, in the oral cavities of patients testing positive for C. albicans, colonization was observed on both demineralized and non-demineralized surfaces as well as in saliva, indicating no site specificity. The samples were categorized into the WSLs-W-CaP (n = 9), WSLs-W-CaN (n = 20), WSLs-H-CaP (n = 9), and WSLs-H-CaN (n = 20) subgroups for further analysis to explore the impact of C. albicans on microbial communities in patients with WSLs.

Table 2.

Comparison of C. albicans detection rates among different groups n (%).

Group	C. albicans detection rate in plaque	C. albicans detection rate in saliva
WSLs-W (n = 29)	9 (31)	9 (31)
WSLs-H (n = 29)	9 (31)	9 (31)
Control (n = 23)	0 (0)	0 (0)

Colonization of C. albicans influences the supragingival plaque microbiome composition in orthodontic patients with WSLs

Alpha diversity analysis of the supragingival plaque microbiome revealed (Figure 4a) no statistically significant differences in the ACE, Chao1, Simpson, or Shannon indices among the four groups (p > 0.05), suggesting that C. albicans detection did not affect microbiome richness and diversity. PCoA analysis of the overall composition of bacterial communities indicated significant differences in beta diversity between the WSLs-W-CaP and WSLs-W-CaN groups (p = 0.003), as well as between the WSLs-W-CaP and WSLs-H-CaN groups (p = 0.006) (Figure 4b). These findings indicate that the supragingival plaque microbiome composition on demineralized surfaces in Candida-positive patients with WSLs differed significantly from that of Candida-negative patients with WSLs, including both demineralized and non-demineralized surfaces.

Figure 4.

Comparison of alpha and beta diversities of microbial communities among the WSLs-W-CaP, WSLs-W-CaN, WSLs-H-CaP, and WSLs-H-CaN subgroups. (a) Four indices, ACE, Chao1, Simpson, and Shannon, were selected to compare the alpha diversity. (b) Principal coordinate analysis (PCoA) was used to analyze beta diversity, and the ADONIS test was applied to evaluate statistical distinctions (*p < 0.05, **p < 0.01, ***p < 0.001).

To further investigate the impact of C. albicans enrichment on supragingival plaque microbiome composition in the context of WSLs, we analyzed the differences in the relative abundances of core genera (relative abundance > 1.0%) among the WSLs-W-CaP, WSLs-W-CaN, WSLs-H-CaP, and WSLs-H-CaN subgroups (Figure 5a). At the genus level, the composition of core genera (relative abundance > 1.0%) was largely consistent across groups, with the ten most dominant genera being Leptotrichia, Actinomyces, Streptococcus, Saccharibacteria_TM7_G-1, Prevotella, Corynebacterium, Capnocytophaga, Selenomonas, Veillonella, and Fusobacterium. Although the core genus composition was similar, the relative abundances of Actinomyces, Prevotella, and Veillonella were higher in the WSLs-W-CaP subgroup. We further analyzed the differences in the supragingival plaque microbiome, focusing on the five genera and species with significant differences in abundance (Figure 5b–d). At the genus level, three genera exhibited statistically significant differences among the four groups (Figure 5b). Peptidiphaga and Propionibacterium were the most abundant in the WSLs-W-CaP subgroup, indicating their greater enrichment on demineralized surfaces of Candida-positive patients. Atopobium also showed higher enrichment on demineralized surfaces, but exhibited the lowest abundance in the WSLs-H-CaP subgroup (p < 0.05). At the species level, Actinomyces sp._HMT_448 was also most prominently enriched on the demineralized surfaces of Candida-positive samples (p < 0.05).

Figure 5.

Microbial composition analysis among the WSLs-W-CaP, WSLs-W-CaN, WSLs-H-CaP, and WSLs-H-CaN subgroups. (a) Microbial composition at the genus level. (b) The genera with significant differences among the WSLs-W-CaP, WSLs-W-CaN, WSLs-H-CaP, and WSLs-H-CaN subgroups (taking the top 1–5 genera ranged by relative abundance). (c) The species with significant differences among the WSLs-W-CaP, WSLs-W-CaN, WSLs-H-CaP, and WSLs-H-CaN subgroups (taking the top 1–5 species ranged by relative abundance). (d) Statistical comparison of Streptococcus mutans relative abundance among groups (*p < 0.05, **p < 0.01, ***p < 0.001).

Notably, our results confirmed that, when categorizing samples into three groups – WSLs-W, WSLs-H, and Control groups – the abundance of S. mutans was significantly higher on both demineralized and non-demineralized enamel surfaces in patients with WSLs (Figure 3b). However, when categorizing the samples into four subgroups based on C. albicans detection – WSLs-W-CaP, WSLs-W-CaN, WSLs-H-CaP, and WSLs-H-CaN subgroups – the abundance of S. mutans varied significantly (Figure 5d). S. mutans showed the highest abundance in the WSLs-W-CaP subgroup, with a significant difference compared to both the WSLs-W-CaN and WSLs-H-CaN subgroups (p < 0.05). Thus, in contrast to orthodontic patients with WSLs who tested negative for C. albicans (including both demineralized and non-demineralized surfaces), S. mutans was significantly more enriched on the demineralized surfaces of patients that tested positive for C. albicans. However, on non-demineralized enamel surfaces, C. albicans did not significantly affect S. mutans abundance, as no significant difference was observed between the non-demineralized enamel surfaces of C. albicans positive and negative WSLs patients (p > 0.05) (Figure 5d). This suggests that on the demineralized surfaces of patients who tested positive for C. albicans, an enrichment of S. mutans can be detected, indicating a potential interkingdom interaction between these two species at demineralized sites, which may exacerbate the severity of WSLs.

Discussion

WSLs have a high incidence rate in patients undergoing fixed orthodontic treatment [3]. In this study, we explored the differences in the supragingival microbiome between demineralized and non-demineralized surfaces in patients with WSLs, as well as healthy tooth surfaces in orthodontic patients without WSLs. Interestingly, our findings revealed that in adolescent orthodontic patients with WSLs, the supragingival microbiome on non-demineralized surfaces exhibits a cariogenic potential intermediate between demineralized and healthy states, but closer to the demineralized state. Furthermore, we examined site-specific colonization of C. albicans in the oral cavity of patients with orthodontic WSLs, an aspect that has received limited attention in previous studies. Our results confirmed that C. albicans colonization was more prevalent in patients with WSLs, but did not exhibit site specificity. Additionally, the presence of C. albicans influenced the composition of the supragingival plaque microbiome. Based on these findings, all the three null hypotheses were rejected.

Microbial diversity reflects the health of supragingival plaque communities. Beta diversity analysis revealed significant changes in the supragingival plaque microbiome on both demineralized and non-demineralized tooth surfaces in patients with WSLs compared with healthy patients. Analysis of the microbial community composition of supragingival plaques showed enrichment of acidogenic- or aciduric-dominated bacteria in the WSLs-W and WSLs-H groups. For example, the cariogenic potential of Prevotella has been increasingly substantiated [45], and the overexpression of collagenases for proteolytic metabolism in this species can aggravate caries [46]. Additionally, some Prevotella species produce acids, lowering the local pH of plaque biofilms and are often enriched in patients with high carbohydrate consumption [47]. For example, P. melaninogenica is more frequently observed in patients with high sugar intake [26] and is often enriched in supragingival plaques of patients with WSLs [25,31]. Moreover, certain Prevotella species, such as P. pallens, P. denticola, and P. salivae, enriched on demineralized tooth surfaces, have been used to design caries risk assessment models, exhibiting high predictive accuracy for conditions such as early childhood caries [22,23,46]. In addition to Prevotella, Selenomonas tends to aggregate on demineralized tooth surfaces [48]. At the species level, S. sputigena is strongly associated with the development of early childhood caries [49], suggesting that its increased abundance during orthodontic treatment warrants further investigation.

In addition to Prevotella and Selenomonas, no statistically significant differences in Streptococcus abundance were detected among the three groups at the genus level. This result may be attributed to the varied roles of different Streptococcus species during the occurrence of WSLs [50], the correlation of Streptococcus with health and demineralization states differing by species. In the present study, S. mutans was relatively abundant in patients with WSLs. Insoluble glucans synthesized from sucrose by S. mutans are critical for plaque biofilm matrix formation, aiding bacterial colonization on tooth surfaces, and maintaining an acidic environment conducive to the growth of cariogenic bacteria [51]. Consequently, S. mutans is an important biomarker for predicting caries risk [52]. Moreover, S. anginosus was notably enriched in teeth with WSLs. In the presence of sucrose, S. anginosus synthesizes substantial amounts of extracellular polysaccharides that promote bacterial adhesion and biofilm formation. Additionally, S. anginosus exhibits acid tolerance comparable to that of S. mutans and possesses a high acid production ability [53], with its increased abundance serving as an indicator of active caries [54]. Conversely, in this study, S. sanguinis as well as S. cristatus;clade_578 exhibited higher abundance in the Control group, consistent with previous research findings [50,55]. Both S. cristatus and S. sanguinis belong to the Streptococcus mitis group [56], are typically isolated from healthy dental surfaces, and possesses antagonistic activity against caries pathogens [57]. Mitis groups such as S. sanguinis can release hydrogen peroxide, which can diffuse to adjacent bacteria within the supragingival microbiome, thereby inhibiting the growth of several pathogens, including the cariogenic S. mutans. Similar to Streptococcus, the relevance of Leptotrichia in healthy and WSLs states varies among species [58–60]. Certain Leptotrichia species exhibit greater membrane transport capabilities and actively metabolize carbohydrates [61,62]. Some Leptotrichia species can synthesize extracellular polysaccharides and various cariogenic organic acids, which are potentially related to the pathogenesis of WSLs [62]. For example, L. sp._HMT_498, enriched in caries-active children [25], was more prevalent in the WSLs ecological niche in this study. Conversely, L. sp._HMT_215 and L. sp._HMT_225 were more enriched on healthy tooth surfaces. Previous studies have shown that these species are more prevalent on tooth surfaces of healthy individuals, although their exact roles still remain unclear [24,63].

Additionally, Corynebacterium and C. durum were significantly enriched in the Control and WSLs-H groups. Other bacteria, such as Lautropia, L. mirabilis, Rothia, and R. aeria, were more abundant in the Control group. These findings align with previous studies, suggesting these bacteria may contribute to the stability of a healthy supragingival microbiome [61,64]. Previous studies have suggested that these bacteria are enriched on healthy tooth surfaces owing to their intrinsic metabolic characteristics. Corynebacterium plays a crucial role in supragingival plaque by providing a structural framework for bacterial aggregation and formation of a symbiotic biofilm community through complex interactions with health-associated bacteria [65,66]. Moreover, Corynebacterium can increase the pH of dental plaque by metabolizing the organic acids produced by other microbes, such as lactic and acetic acids [25]. Corynebacterium also dominates the plaques of caries-free children, with C. durum exhibiting higher relative abundance [25,55]. Lautropia, found in higher abundance in dental plaque of caries-free patients, exhibits high levels of signaling ability, exogenous biodegradation, and metabolic capacity, which are beneficial for dental health [61,67,68]. L. mirabilis was also relatively more abundant in caries-free adolescents [25,55]. Similarly, Rothia is associated with proper dental health [45], and its abundance in saliva negatively correlates with lactic acid production following sucrose rinsing [69]. Rothia species have been identified as part of the oral microbiome in healthy individuals [70]. R. aeria is frequently identified in individuals with low susceptibility to caries [55,70].

Exploring changes in the supragingival plaque microbiome is crucial for preventing orthodontic WSLs and developing risk-assessment strategies. Orthodontic appliances complicate the maintenance of oral hygiene, leading to increased plaque accumulation. Under conditions of a high-carbohydrate diet and poor oral hygiene, the metabolism of the supragingival plaque microbiome changes, further shifting the microbial ecology towards an imbalanced state [47]. Previous studies have suggested that microbial changes can be detected before the onset and exacerbation of certain oral diseases, indicating that alterations in the oral microbiome may precede clinical symptoms. The oral microbiome may be altered before visible damage occurs on the tooth surface [71,72]. Our study confirmed that the relative abundance of cariogenic bacteria within the supragingival plaque microbiome on non-demineralized surfaces was intermediate between that on demineralized and healthy surfaces but closer to that on demineralized surfaces. Conversely, bacteria associated with healthy states were more abundant in the Control group, with similar relative abundances in the WSLs-W and WSLs-H groups but were generally higher in the WSLs-H group. These findings suggest that for adolescents with WSLs undergoing orthodontic treatment, the supragingival plaque microbiome on non-demineralized surfaces exhibits a cariogenic potential between that of demineralized and healthy states, but closer to the demineralized state. Thus, we propose that the presence of WSLs may indicate that the microbiome on non-demineralized surfaces is closer to a demineralized state, necessitating interventions to shift the microbiome towards a healthy state.

Previous studies have primarily focused on the impact of oral bacterial composition. However, emerging evidence suggests that interactions between bacteria and fungi play a crucial role in the transition from healthy to diseased states [48]. In this study, we assessed the colonization rate of C. albicans in supragingival plaque and found that it was enriched in the oral cavities of patients with WSLs, with a colonization rate of up to 31%. Moreover, in patients with WSLs, C. albicans was detected on both demineralized and non-demineralized tooth surfaces, as well as in the saliva. In clinical practice, saliva sample collection is generally faster and more convenient than plaque sampling. Therefore, detection of C. albicans in saliva can serve as a predictive factor for WSLs risk, indicating the need for increased attention in these patients. There are multiple potential sources of C. albicans infection in the oral cavity. For instance, studies have demonstrated that C. albicans transmission occurs mostly vertically at birth from mother to infant [73,74], and C. albicans can be detected in the oral cavity in early life [75]. In addition to vertical transmission, exogenous sources such as hospitals and neonatal units may also contribute to the infection of C. albicans [74,76]. Furthermore, C. albicans present in parents or relatives who have close contact with children can be transmitted to their oral cavity [77]. Additionally, C. albicans may also be sourced from food such as snacks and cheese [78,79]. Typically, the supragingival microbiome maintains dynamic equilibrium in the oral cavity. However, overgrowth of microbes such as C. albicans can disrupt this balance. Under certain conditions, C. albicans forms biofilms and synergistically interacts with other microbes, thereby promoting their pathogenicity [80]. The enrichment of C. albicans can affect the bacterial community composition of the supragingival plaque.

In this study, the demineralized surfaces of patients who tested positive for C. albicans showed enrichment of S. mutans, suggesting a potential interkingdom interaction between these two species at demineralized sites. Co-aggregation of C. albicans and S. mutans is critical for their symbiosis on the enamel surface and plays a pivotal role in biofilm formation [81,82]. Under co-culture conditions, C. albicans exhibits accelerated growth [83]. When S. mutans initially adheres to the enamel surface, glucosyltransferase type I activity increases, utilizing sucrose as a substrate to synthesize insoluble glucans and mediate sucrose-dependent adhesion between S. mutans and the enamel surface [84,85]. These adhesion sites then serve as binding sites for late-colonizing microbes such as C. albicans [85]. Mannan and β-glucan in C. albicans provide binding sites for glucosyltransferases, and the structure of extracellular polysaccharides secreted by S. mutans may be influenced by β-1,3-glucan from C. albicans [81,86]. The result of the fluorescence in situ hybridization experiment further confirms a significant biogeographical association between C. albicans and S. mutans. C. albicans exhibits a hyphal morphology, while S. mutans clusters in proximity to the hyphal structures of C. albicans, indicating their ability to form co-aggregates with specific structures within dental plaque [87]. In addition to promoting adhesion, C. albicans and S. mutans also exhibit metabolic and virulence interactions. These interactions are particularly significant for exacerbating WSLs’ progression and severity. In the high-sucrose diet model, rats co-infected with C. albicans and S. mutans demonstrate a more rapid progression of caries and more severe lesions compared to those infected solely with S. mutans [86]. Co-cultivation of the two species significantly increases the total bacterial load and upregulates the expression of several virulence-associated genes and proteins [88,89]. For instance, compared to biofilms formed by S. mutans alone, biofilms composed of both S. mutans and C. albicans exhibit significantly elevated expression of genes such as gtfB/C, which are related to the carbohydrate metabolism and transportation of S. mutans, thus enhancing sugar utilization. Additionally, C. albicans influences the expression of genes associated with the CiaRH or ComDE two-component systems in S. mutans, thereby affecting its acid tolerance, adhesion, and biofilm formation capabilities [88]. The findings of this study suggest that such interspecies interactions may occur on demineralized enamel surfaces in patients with WSLs, emphasizing the need for particular attention during fixed orthodontic treatment. While focusing on cariogenic bacteria, it is imperative to address the detection of C. albicans simultaneously.

The supragingival plaque microbiome exhibits a certain degree of variability among different patients. One limitation of this study was its cross-sectional design, which cannot ascertain the changes in non-demineralized tooth surface of the patient compared with that before treatment. Moreover, another limitation of this study is its relatively small sample size, which should be addressed in future studies. Although statistically significant trends were observed, the impact of C. albicans on demineralized tooth surfaces only represents a preliminary exploration. In addition, our results indicated that A. sp. _HMT_448 is likely to accumulate on the demineralized enamel surfaces of C. albicans-positive patients; however, research on this interaction is limited, further research is needed to elucidate the mechanisms underlying the interactions between C. albicans and bacteria and to develop targeted preventive strategies.

Conclusions

This study revealed that adolescent orthodontic patients with WSLs exhibit a microbiome on non-demineralized surfaces that more closely resembles a demineralized state, highlighting the need for interventions to promote a healthier microbiome. Moreover, C. albicans exhibited a higher colonization rate (approximately 31%) in patients with WSLs without site specificity. The colonization of C. albicans influenced the bacterial community composition in patients with WSLs, with C. albicans-positive individuals showing significant enrichment of S. mutans on demineralized surfaces compared with C. albicans-negative patients. These findings offer novel insights for developing disease prevention strategies and improving the risk assessment approaches for orthodontic WSLs.

Funding Statement

This work was supported by International Orthodontic Foundation Young Grant under Grant number [2023IOFY14]; Beijing Hospitals Authority Youth Programme under Grant number [QML20231504]; Beijing Hospitals Authority Clinical medicine Development of special funding support under Grant number [ZLRK202330]; Young Scientist Program of Beijing Stomatological Hospital, Capital Medical University under Grant number [YSP202312].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

This study was approved by the Ethics Committee of the Beijing Stomatological Hospital (CMUSH-IRB-KJ-YJ-2022–11).