The landscape of the bacteriome and mycobiome at different stages of root caries and the cross-kingdom interactions of the core species

doi:10.21203/rs.3.rs-4853822/v1

Background

The unbalanced oral microbiome is considered the key pathogenic agent for root caries, the most common tooth disease in elderly individuals; however, the bacteriome and mycobiome at different stages of root caries, especially from the same individual, are not clear.

Results

In this study, superficial and deep root caries plaques from thirty patients with different levels of root caries simultaneously in the oral cavity were collected, whereas sound root surface plaques from their healthy teeth served as caries-free controls. The full-length sequencing results of 16S and 18S rDNA analysis via the self-controlled method on the basis of the same patients indicated that the different stages of root caries represented different microbiota, including Streptococcus mutans and Actinomyces sp. HMT448 in superficial root caries and Prevotella sp. in deep root caries. Candida albicans was the most abundant fungal species from all the plaques, while it showed strong interspecies interactions with S. mutans and Actinomyces sp. Their interactions were closely associated with the different stages of root caries. An in vitro model further confirmed that C. albicans was able to increase the growth, biofilm formation and cariogenicity of S. mutans and A. viscosus through the activation of the arginine biosynthesis pathway, indicating its key roles in the development of root caries.

Conclusion

Our study revealed the first landscape of the microbiome from different stages of root caries and indicated that targeting the interactions of core species may be a practical way to prevent and treat clinical root caries.

Streptococcus mutans

Actinomyces viscosus

Candida albicans

Root caries

Cariogenic virulence

High-throughput DNA sequencing

Oral diseases affect approximately 3.5 billion people worldwide, of which 2.3 billion people suffer from permanent tooth caries, the most common oral problem¹. Root caries refers to caries that occur at the cervical dentin of the tooth and are frequently observed in elderly individuals. With the global increase in the elderly population, root caries has become a frequently occurring ailment that poses a significant threat to the oral health and overall quality of life of the elderly². A meta-analysis revealed an annual root caries incidence of 18.25% (CI = 13.22%-23.28%) and an increase of 0.45% (CI = 0.37–0.53) in decayed/filled surfaces per year³. Root caries occur in a concealed location that is difficult to clean and progresses rapidly. Owing to the proximity of the caries lesion to the gingival margin, achieving adhesive bonding during restorative treatment is challenging; thus, it is more prone to trigger secondary complications such as pulpitis, apical periodontitis, and even tooth fracture and loss among the elderly population⁴.

Dental caries initiation is closely linked to dental plaque, and a comprehensive understanding of the oral microbiota holds pivotal importance in caries treatment⁵. Early investigations employed culture-based techniques to isolate and identify microorganisms present in root caries plaques, identifying predominant strains such as Streptococcus, Lactobacillus, and Actinomyces⁶. In recent years, the use of advanced molecular techniques that do not rely on culture has revealed the complex community of microorganisms linked to root caries⁷. By combining traditional culture methods with modern pyrosequencing, researchers have identified potential contributors to the development of root caries, including Propionibacterium acidifaciens, Streptococcus mutans, Prevotella multisaccharivorax, Lactobacillus crispatus, Lactobacillus fermentum, and Candida albicans⁸.

However, in individuals with root caries, the varying degrees of caries at different sites within the same oral cavity have often been overlooked in existing research. Previous studies have indicated that the microbial composition of cariogenic biofilms undergoes continuous alterations concurrently with the occurrence and progression of dental caries, which might be associated with variations in environmental pH at different stages of caries, alterations in nutritional and metabolic conditions, and fluctuations in oxygen availability⁹. However, few studies have focused on changes in microbial composition in different stages of coronal or root caries¹⁰. To investigate the bacteriome and mycobiome at different stages of root caries, a systematic clinical study is needed to reveal the mechanisms underlying root caries development.

In addition, much of the current research has focused primarily on comparing bacterial detection rates and relative abundances between intact root surfaces from healthy individuals and root carious lesions from patients; however, the variations in microbial composition between individuals hinder the observation of microbial community changes^8,11,12. To observe the interplay between key microorganisms across the different stages of root caries development, such as the transition from superficial to deep caries, thus enabling a systematic analysis of microbial composition and its contributory role in root caries development, plaque samples from sound root surfaces and superficial and deep root caries lesions located at different teeth within individuals manifesting varying degrees of root caries severity were collected in this study. Comparative analysis is conducted via high-throughput sequencing and paired comparisons to avoid the impact of individual differences. In vitro experiments and RNA sequencing were subsequently applied to investigate the interactions between the main microorganisms. This comprehensive exploration of potential cross-kingdom interactions provides novel insights for the prevention and targeted treatment of root caries, thereby providing significant clinical value and application prospects.

1. The demographic and clinical characteristics of the root caries volunteers

To compare the differences in microorganisms across the different stages of root caries development, we recruited 30 patients who were diagnosed with root caries and had both untreated deep and superficial root caries lesions on different teeth. For each patient, samples of carious dental plaque from deep (Group D) and superficial (Group S) root caries, along with dental plaque from the intact root surface of the same dentition (Group F), were collected. The patients were aged between 53 and 88 years, with an average age of 69.9 years. The gender proportion (male) was 50% (15/30). The demographic and clinical characteristics of the study subjects are listed in Table 1. The sample collection and testing steps are illustrated in Fig. 1a.

Table 1

Demographic and clinical characteristics of the included patients
Number	Gender	Age	NRT	RCT
1	M	74	32	7
2	M	68	20	6
3	F	58	28	5
4	F	81	22	9
5	F	67	21	7
6	M	88	15	9
7	F	59	25	3
8	M	69	20	4
9	F	75	20	8
10	M	68	24	5
11	F	80	22	11
12	F	61	26	7
13	F	74	24	10
14	M	65	23	8
15	F	65	30	12
16	M	61	16	6
17	F	64	28	4
18	F	62	15	6
19	M	62	17	8
20	M	73	25	5
21	M	66	21	9
22	M	70	13	3
23	M	76	14	6
24	F	78	14	9
25	F	75	12	6
26	M	80	22	11
27	F	75	27	9
28	M	53	19	3
29	M	86	24	8
30	F	65	24	8
Average(Mean ± SD)		69.93 ± 8.52	21.43 ± 5.23	7.07 ± 2.45

SD = standard deviation of the mean.

NRT = number of remaining teeth.

RCT = root caries teeth.

2. The different stages of root caries from the same patient presented different abundances of bacterial species

The plaques were then subjected to 16S and 18S full-length sequencing. In 16S rDNA high-throughput sequencing, a total of 570,651 sequences were obtained after processing, with an average sequence length of 1499.3 bp. A total of 263 operational taxonomic units (OTUs) were obtained. By BLAST against the Human Oral Microbiome Database (HOMD), 12 phyla, 26 classes, 38 orders, 60 families and 100 genera were detected in the samples. High-throughput 18S rDNA sequencing revealed 645,874 sequences after processing, with an average sequence length of 1855.8 bp, and 80 OTUs were obtained. By BLAST against the Silva 138 database, 25 phyla, 35 classes, 38 orders, 39 families and 42 genera were detected in the samples. The detailed sequence information is shown in Fig. S1 and Tables S1 and S2. Clustering was performed when the sequence similarity was > 97%. The Good’s coverage index is greater than 99%, indicating that the sequencing data are large and reliable enough to cover the information of most species in the sample.

The α-diversity and β-diversity analyses of the 16S rDNA revealed no significant differences in the species diversity and community structure of the bacteriome among the superficial caries (Group S), deep root caries (Group D) and caries-free teeth (Group F) (Fig. 1b and Table S3), whereas according to the α-diversity analysis of the 18S rDNA, the Shannon index and Simpson index of Group F were significantly lower than those of Groups S and D (P < 0.05) (Fig. 1c and Table S4), indicating a decreased eukaryotic diversity in caries plaques compared with healthy sound root surfaces. No significant differences in β diversity from 18S rDNA were observed among the three groups (Fig. 1c).

The distributions of the phyla in the three groups are shown in Fig. 1d, e. The results of Wilcoxon paired analysis at the genus level on the basis of the results of 16S rDNA sequencing are shown in Table S5. Compared to the caries-free samples, the abundances of Streptococcus, Actinomyces, Lactobacillus, and Campylobacter in the superficial caries samples were significantly greater, whereas the abundance of Leptotrichia in the superficial caries samples was significantly lower (P < 0.05) (Fig. 1h). Compared with superficial caries group, the abundances of Capnocytophaga and Leptotrichia in the deep caries group gradually decreased, whereas the relative abundances of Lactobacillus, Prevotella and Propionibacterium significantly increased (P < 0.05), indicating that Streptococcus, Actinomyces, Lactobacillus and Campylobacter are involved in the occurrence of root caries, whereas Prevotella, Propionibacterium and Lactobacillus are related to the progression of root caries (Fig. 1h).

The 16S rDNA sequencing results of Wilcoxon paired analysis at the species level (Fig. 1h, Table S6) revealed that the abundances of Campylobacter gracilis, Streptococcus mutans, Actinomyces sp. HMT448 and Prevotella deticola were significantly greater in superficial caries samples (Group S) (P < 0.05) than in caries-free samples (Group F), whereas compared with the superficial caries group (Group S), the abundance of Capnocytophaga ochracea was gradually lower in the deep root caries group (Group D) (P < 0.05). The abundances of Prevotella deticola, Propionibacterium acidifaciens and Lactobacillus gasseri also significantly increased in deep root caries (Group D) (P < 0.05).

Furthermore, linear discriminant analysis effect size (LEfSe) was carried out via Wilcoxon paired analysis. The differences in phyla, classes, orders, families, genera and species were analyzed (threshold value: LDA > 3.5) (Fig. 1g). Compared with those in Group F, the genera Streptococcus, Actinomyces, Lactobacillus, Bacillus, Propionibacterium, and Scardovia and the species Streptococcus mutans and Actinomyces sp. HMT448 were significantly enriched in Group S, whereas the species Prevotella sp. HMT300 was relatively more abundant in Group D than in Group S (LDA > 3.5, P < 0.05). Notably, the phylum Fusobacteriota was enriched in Group F, indicating that this phylum might be related to healthy root surface conditions.

According to the 18S rDNA analysis, Candida albicans was the most abundant fungal species from all the plaques, and the relative abundance in all three experimental groups was greater than 93% (Fig. 1f), indicating that C. albicans was the most important fungal species in the older oral cavity and that its interactions with bacterial species could be key for the development of root caries.

2. The correlations between C. albicans, S. mutans and Actinomyces sp. contributed to the occurrence and development of root caries.

To determine the cross-kingdom interactions in root caries lesions, 13 out of 30 cases were randomly selected to conduct Pearson correlation analysis between C. albicans and all the bacterial species. The 13 cases included 232 OTUs from 16S rDNA sequencing of 263 OTUs and 61 OTUs from 18S rDNA sequencing of 80 OTUs. The abundances of root caries-related species, including S. mutans, Actinomyces sp. HMT 448, P. acidifaciens, L. gasseri and Prevotella oris, were strongly positively correlated with the abundance of C. albicans (R > 0.3, P < 0.05) (Fig. 2), among which S. mutans and Actinomyces sp. HMT448 had relatively high average abundances, indicating that the interspecies interactions between C. albicans and these species were closely related to the occurrence and development of root caries.

3. C. albicans enhanced the cariogenic virulence of S. mutans, Actinomyces viscosus and their dual-species biofilms.

To investigate the contribution of cross-kingdom interactions to the progression of root caries, a single, dual and three-species biofilm model was built in vitro on the basis of the close relationships among S. mutans, Actinomyces sp. HMT448 and C. albicans. Our previous study confirmed the positive correlation between C. albicans and Actinomyces viscosus in clinical root caries samples and in mice ¹³; thus, A. viscosus was employed to represent the species Actinomyces sp. HMT448 in this study. The results of crystal violet staining (Fig. 3a) and cell counting (Fig. 3b) revealed that the biofilm formation ability of the three species was significantly greater than that of the single-species biofilms of S. mutans, A. viscosus and C. albicans (P < 0.05). The EPS production capacity of the three-species biofilm was also significantly greater than that of the two-species biofilm of two bacteria and the single-species biofilm of each of the three strains (P < 0.05) (Fig. 3c). To observe the biofilm structure, scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) were performed. SEM revealed that the three-species biofilms were larger and denser than the single- and dual-species biofilms were (Fig. 3d). Compared with the single- and dual-species biofilms, the quantitative fluorescence calculations revealed increased biomass of the three species in their mixed biofilms (Fig. 3e), indicating that the cross-kingdom interactions with C. albicans promoted the cariogenic virulence of these two bacterial species and their dual-species biofilms.

4. C. albicans activated arginine biosynthesis to promote the formation of S. mutans and A. viscosus dual-species biofilms.

To further identify the key pathway by which C. albicans synergistically interacts with S. mutans and A. viscosus, the transcriptomes of C. albicans from the three-species biofilms compared with those from the C. albicans single-species biofilm were analyzed. Five hundred eighty-four genes were differentially expressed in the three C. albicans species biofilms, of which 508 were upregulated and 76 were downregulated (Fig. S2). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis revealed that 69 genes were differentially expressed between the two groups, whereas C. albicans from the three-species biofilms were significantly enriched in the steroid biosynthesis pathway, nitrogen metabolism pathway, galactose metabolic pathway, phenylalanine metabolism pathway and arginine biosynthesis pathway (Fig. 4a). Since our previous research revealed that the arginine synthesis pathway of C. albicans is upregulated in root caries biofilm¹³, the effects of arginine on the growth of S. mutans and A. viscosus were evaluated. The addition of arginine significantly promoted the formation of dual-species biofilms of S. mutans and A. viscosus in a dose-dependent manner (Fig. 4b) and was similar to the effects of coculture with C. albicans (Fig. 4c).

Owing to the relatively poor oral hygiene behaviors observed in the elderly population, issues such as salivary gland impairment, gingival recession, and root surface exposure—each recognized as a risk factor for root caries—are widely prevalent. Dental plaque serves as the initial factor in the development of caries. Our analysis of α and β diversity revealed a significant overlap in microbiota species across the three groups, suggesting a similarity in the composition and structure of the microorganisms. Nonetheless, alterations in the relative abundance of a few species were observed, which is consistent with previous research^8,11. This observation aligns with the "ecological plaque hypothesis"¹⁴, which posits that caries is a result of shifts in the population balance of resident microbes rather than simply the presence of caries-associated species. Moreover, our investigation highlights the prevalence of C. albicans in root plaques and its potential interactions with cariogenic bacteria, which is consistent with emerging research on cross-kingdom interactions in caries pathogenesis^15–18. Furthermore, our in vitro experiments revealed the impact of C. albicans on biofilm formation and virulence factor expression in S. mutans. These findings underscore the complex nature of microbial interactions in root caries development and highlight potential therapeutic targets for disease intervention.

Variations in the oral environment, such as temperature, saliva flow rate, and pH, among other factors, have led to challenges in comparing the microbiome composition between caries patients and healthy individuals due to individual differences^8,11. Additionally, as root caries advance, changes in the carious environment, encompassing the root dentin structure, periodontal tissues, and anaerobic conditions, are likely to lead to alterations in the microbial population within carious lesions. In a study by Lima et al.^10, which involved sampling from different layers of an occlusal deep caries site, reverse-capture checkerboard analysis revealed no substantial differences in the microbiota. In contrast, our present study adopted a unique approach by recruiting patients with both superficial and deep root caries from various dentition sites, collecting dental plaque samples from each patient, and applying Wilcoxon paired comparisons to mitigate individual difference bias. This approach allowed us to discern the microbial transformation in different stages of root caries. This approach may explain why some prior studies reported slightly reduced community diversity in carious plaques compared with healthy caries-free plaques^19,20, whereas our study revealed no significant difference in diversity among the three groups.

Previous studies^8,11 have associated specific bacterial species, such as P. acidifaciens, S. mutans, Olsenella profusa, Lactobacillus crispatus, and L. gasseri, with root caries. Similarly, our study revealed changes in the abundance of these species during the progression of root caries. In our investigation, Actinomyces sp. HMT448 and S. mutans presented significant differences in abundance between caries-free and superficial caries samples, suggesting their potential role in root caries initiation. Moreover, the abundances of L. gasseri and Prevotella sp. substantially differed between superficial and deep caries samples, potentially indicating their involvement in the exacerbation of root caries. The prevalence of C. albicans within root plaques was prominently greater than that of other fungi according to our 18S rDNA high-throughput sequencing results. This finding aligns with earlier studies focused on early childhood caries (ECC)²¹ and middle-aged and elderly individuals with root caries²², further supported by recent research^23–25 highlighting the prevalence of C. albicans in the dental plaque biofilms of caries patients.

Mounting evidence^17,26,27 is strengthening our understanding of the intricate interplay between oral Candida and cariogenic bacteria, including Streptococcus, Actinomyces, Prevotella, and Lactobacillus^28–33. Our investigation aligns with these trends, revealing a positive correlation between the abundance of Actinomyces sp. HMT448, S. mutans, P. acidifaciens, L. gasseri, and P. oris and that of C. albicans. Earlier research¹¹ underscores the resemblance in core microorganisms of root caries, suggesting a potential role of these bacteria in triggering and advancing root caries.

Growing evidence suggests that bacterial‒fungal cross-kingdom interactions synergistically increase the cariogenic potential of multispecies biofilms^34–39. A recent RNA sequencing study⁴⁰ revealed significant upregulation of genes associated with metabolism, sugar transport, stress tolerance, invasion, and virulence in C. albicans isolated from root carious plaques compared with those in C. albicans from healthy root surfaces. A. viscosus emerges as a key player in the development of root surface caries, particularly during the initial stages of progression^41,42. Our earlier work highlighted mutual enhancement between A. viscosus and C. albicans through the arginine metabolism pathway¹³. In the present study, multiple carbohydrate and amino acid metabolic pathways, including the arginine biosynthesis pathway, were upregulated in C. albicans within the three-species biofilm. Previous research⁴³ also demonstrated that, by upregulating genes related to arginine biosynthesis, C. albicans promotes hyphal formation and immune system evasion, increasing virulence. While arginine concentrations above 1.5% can increase oral biofilm pH through the arginine deiminase system, Vaziriamjad et al. ⁴⁴ reported that lower arginine concentrations (below 0.17%) stimulate the growth of planktonic and biofilm forms of S. mutans. Our study adds to this understanding by showing that the addition of 0.2% arginine to the medium promotes the formation of dual-species biofilms of A. viscosus and S. mutans, comparable to the presence of C. albicans, which is consistent with the findings of Vaziriamjad et al.

In addition to physical interactions, a phenomenon of crossfeeding metabolism emerges between S. mutans and C. albicans. S. mutans relies on sucrose as a nutrient source, converting sucrose into fructose and glucose—nutrient substrates for C. albicans⁴⁵. Additionally, the presence of C. albicans significantly amplifies biochemical activities related to sugar transport systems, glycolysis, pyruvate degradation, and the tricarboxylic acid cycle in S. mutans ³³. This study revealed changes not only in the arginine biosynthesis pathway but also in multiple carbohydrate and amino acid metabolic pathways, including the galactose metabolism and nitrogen metabolism pathways. These pathways require further research and validation.

The composition and structure of the oral microecology of the same individual are stable, and the imbalance of species abundance in the microecology is the main factor for the occurrence of root caries. The cross-kingdom interaction between S. mutans, A. viscosus and C. albicans plays an important role in the occurrence of root caries. Furthermore, the cariogenic virulence of the multispecies biofilm was improved through the arginine biosynthesis pathway. The results of this study provide new ideas for understanding the cross-kingdom interaction between fungi and bacteria in root caries and for the prevention and treatment of clinical root caries.

1 Clinical sample analysis

1.1 Study population

This study was approved by the ethical board of the West China Hospital of Stomatology, Sichuan University, under protocol WCHSIRB-D-2020-072.

1.2 Inclusion Criteria and Exclusion Criteria

Inclusion criteria:

1. Aged between 50 and 90 years.

2. All patients were diagnosed with root caries and had both untreated deep and superficial root caries lesions on different teeth. Periapical radiographs revealed that the root apices of all teeth had completed development and that the depth of deep caries of the anterior or posterior teeth exceeded two-thirds of the depth of dentin.3. All patients were able to understand and were willing and able to read and sign the informed consent form.

Exclusion criteria:

1. Less than 8 teeth left

2. Gingival swelling and congestion 3. Periodontal abscess, > 4 mm periodontal pocket
4. Xerostomia
5. Poor oral hygiene or bad eating habits
6. Pulp or periapical diseases
7. Oral mucosal diseases
8. Oral and maxillofacial tumors and history of radiotherapy or chemotherapy
9. Systemic diseases (including hepatitis, tuberculosis, and diabetes)
10. Antibiotics were used within the past two months.

In total, 30 patients with root caries were enrolled at West China Hospital of Stomatology between July 2020 and March 2022, and informed consent was obtained from all patients.

1.3 Sample collection

The sample collection flow chart is shown in Fig. 1a. One clinician examined the cases under aseptic conditions. Plaque samples from the caries-free positions were obtained by scraping the cervical part of a healthy tooth with a sterile metal spoon excavator (Group F). For superficial (Group S) and deep root caries (Group D) samples, the selective removal of carious tissue was performed to collect samples from the deepest layer of caries⁴⁶. After local anesthesia (if necessary), superficial carious tissue was removed via a sterile spoon excavator, and samples from the deepest layer of the root caries were transferred to a sterile Eppendorf tube (EP tube) containing 1 mL Tris-EDTA buffer solution. The EP tubes with samples were numbered according to group and registration order and stored immediately at -80°C before high-throughput rDNA sequencing.

1.4 DNA extraction and amplification

16S and 18S rRNA gene amplicon sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). DNA was isolated from the contents via a MagPure Soil DNA LQ Kit (Magen, Guangdong, China) following the manufacturer’s instructions. DNA concentration and integrity were measured via a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the full-length hypervariable regions of the 16S and 18S rRNA genes was carried out in a 25 µl reaction mixture via universal primer pairs. The reverse primer contained a sample barcode, and both primers were connected with an Illumina sequencing adapter.

1.5 Library construction and sequencing

The amplicon quality was visualized via gel electrophoresis. The PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter Co., USA) and quantified via a Qubit dsDNA assay kit.

The concentrations were then adjusted for sequencing. Sequencing was performed on an Illumina NovaSeq 6000 with two paired-end read cycles of 250 bases each. (Illumina Inc., San Diego, CA; OE Biotech Company; Shanghai, China).

1.6 Bioinformatic analysis

Paired-end reads were preprocessed via Trimmomatic software⁴⁷ to detect and cut off ambiguous bases (N). Low-quality sequences with average quality scores less than 20 were also removed via the sliding window trimming approach. After trimming, paired-end reads were assembled via FLASH software⁴⁸. The parameters of the assembly were as follows: 10 bp of minimal overlap, 200 bp of maximum overlap and a 20% maximum mismatch rate. The sequences were further denoised as follows: reads with ambiguous, homologous sequences or below 200 bp were discarded. Reads with 75% of bases above Q20 were retained via QIIME software (version 1.8.0) ⁴⁹. Then, reads with chimeras were detected and removed via VSEARCH⁵⁰. Clean reads were subjected to primer sequence removal and clustering to generate operational taxonomic units (OTUs) via VSEARCH software with a 97% similarity cutoff of ⁵⁰. The representative read of each OTU was selected via the QIIME package. All representative reads were annotated and blasted against the Human Oral Microbe Database (HOMD) and Silva database Version 138 via the RDP classifier (confidence threshold was 70%) ⁵¹.

Alpha diversity was defined as the mean species diversity in sites (within-community) or habitats at a more local scale. Alpha diversity is the number of bacterial taxa and the proportional representation of each taxon per sample. The ACE, Chao, Shannon, and Simpson indices of alpha diversity were tested via the Wilcoxon rank-sum test. To examine the differences in alpha diversity, the data were analyzed via Bayesian methods via the limma package in R software (v2.2.0). Beta diversity was determined via principal component analysis (PCoA) via a weighted UniFrac matrix via R software (v2.2.0). To avoid the influence of individual differences on the results, the relative abundance of each taxon was compared between groups via the Wilcoxon paired test at the genus and species levels, and the significance tests described above used an alpha value of 0.05. On the basis of the results of the Wilcoxon paired test, the linear discriminant analysis effect size (LEfSe) method was used to compare the taxonomy abundance spectrum, and an LDA score greater than 3.5 was considered significantly different.

The sample data of 13 patients were randomly selected, and Pearson correlation analysis between C. albicans and microbial abundance in the 16S sequencing results was performed at the species level. The significance level was α = 0.05, and the absolute value of the correlation coefficient R greater than 0.3 was considered correlated.

2 In vitro three-species biofilm model

2.1 Microbial strains and growth conditions

S. mutans (UA159), A. viscosus (ATCC 19246) and C. albicans (SC 5314, ATCC MYA2876) were obtained from the State Key Laboratory of Oral Diseases, Sichuan University. S. mutans strains were subsequently grown in brain heart infusion medium (BHI) broth (Difco, Sparks, MD, USA). A. viscosus was grown on BHY medium (brain heart infusion medium containing 5 g/L yeast extract) anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37°C, and C. albicans was grown on yeast extract peptone dextrose (YPD) medium aerobically at 37°C. The coculture mixture (S. mutans and A. viscosus 10⁶ CFU/mL, C. albicans 10⁴ CFU/mL)⁵² was grown on yeast nitrogen base (YNB) supplemented with Na₂HPO₄-NaH₂PO₄, N-acetylglucosamine, casamino acids, and sucrose media anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37°C⁵³.

2.2 Crystal violet assay and CFU counts

In 6-well plates, 24-h biofilms were produced (single-species groups: 4 mL of C. albicans, S. mutans, and A. viscosus for each well; dual-species groups: 2 mL of C. albicans + 2 mL of A. viscosus, 2 mL of C. albicans + 2 mL of S. mutans, and 2 mL of S. mutans + 2 mL of A. viscosus for each well; three-species groups: 1.3 mL of C. albicans, S. mutans and A. viscosus for each well) under stationary conditions in YNBB medium anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37°C. The total biomass of each biofilm was quantified via a crystal violet assay as previously described¹³. The biofilms were sequentially fixed with methanol and stained with 0.1% (wt/vol) crystal violet for 15 to 30 min. The suspension was removed, and the cells were resolubilized with 33% (vol/vol) glacial acetic acid. The total biomass was determined from the OD600 nm of the suspension. To quantify the viable cells in biofilms, CFU counts were performed as described previously¹³. Briefly, the biofilms were scraped off and resuspended in an equal volume of phosphate buffer solution (PBS). The suspensions were then serially diluted 1:10, and viable biofilm cells were quantified via CFU counts after the proper dilutions were plated on BHI agar and incubated for 24 h.

2.3 Scanning electron microscopy observation and fluorescence in situ hybridization observation

Twenty-four-hour biofilm samples were produced on sterile glass slides at the bottom of each well of 24-well plates (single species groups: 2 mL of C. albicans, S. mutans, and A. viscosus for each well; dual species groups: 1 mL of C. albicans + 1 mL of A. viscosus, 1 mL of C. albicans + 1 mL of S. mutans, and 1 mL of S. mutans + 1 mL of A. viscosus for each well; three-species groups: 0.67 mL of C. albicans, S. mutans and A. viscosus for each well) under stationary conditions in YNBB medium anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37°C. SEM analysis was carried out as previously described¹³. Each sample was observed via SEM imaging (FEI, Hillsboro, USA) at 1,000× magnification. The FISH procedure was performed as previously described ^13, and the samples were observed with an Eclipse FV1000 inverted confocal laser scanning microscope (Olympus Corporation, Japan). The sequences of oligonucleotide probes ^13,54 are listed in Table S6. The probes were synthesized by Sangon Biotech (Shanghai, China).

2.4 Anthrone-sulfuric acid assay.

The water-insoluble EPS production ability of the 24-h biofilms was analyzed via an anthrone-sulfuric acid assay. In 24-well plates, 24-h biofilms were produced (single-species groups: 2 mL of C. albicans, S. mutans, and A. viscosus for each well; dual-species groups: 1 mL of C. albicans + 1 mL of A. viscosus, 1 mL of C. albicans + 1 mL of S. mutans, and 1 mL of S. mutans + 1 mL of A. viscosus for each well; three-species groups: 0.67 mL of C. albicans, S. mutans and A. viscosus for each well) under stationary conditions in YNBB medium anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37°C. The biofilms were resuspended, and the precipitates were obtained and washed with sterile water to remove the water-soluble EPS. Then, each water-insoluble EPS sample was extracted with 0.4 M NaOH under agitation for 2 h. Three milliliters of 0.2% anthrone-sulfuric acid reagent were mixed into each supernatant sample and then incubated in a water bath at 95°C for 6 min. The water-soluble EPS production ability was determined by OD625 nm.

2.5 RNA sequencing and data analysis

Total RNA from three-species biofilms and single-species biofilms of C. albicans was extracted with TRIzol reagent (Invitrogen, Carlsbad, USA). RNA purity and quantification were evaluated via a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed via an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The libraries were subsequently constructed via the VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The libraries were sequenced on an Illumina NovaSeq 6000 platform, and 150 bp paired-end reads were generated. Approximately 50 raw reads for each sample were generated. Raw reads in fastq format were first processed via fastp ^55, and the low-quality reads were removed to obtain the clean reads. Then, approximately 46 clean reads for each sample were retained for subsequent analyses. The clean reads were mapped to the reference genome via HISAT2⁵⁶. Differential gene expression analysis was performed via fragments per kilobase per million (FPKM) values. A Q value < 0.05 and a fold change > 2 or a fold change < 0.5 were set as the thresholds for significantly differentially expressed genes (DEGs). DEGs were regarded as upregulated if their expression levels in three-species biofilm samples were greater than those in the C. albicans single-species biofilm samples, and vice versa. DEGs were subjected to functional enrichment analyses via Gene Orthology (GO) and KEGG annotations.

3 Statistical analysis

For the detection of clinical samples, differences between the relative abundance of each two groups were compared via paired Wilcoxon analysis. For the in vitro experiments, differences among multiple groups were compared via one-way ANOVA and post hoc Tukey’s multiple comparisons after a homogeneity test of variance with Levene’s test. Statistical analysis was performed via SPSS software (version 20.0; IBM Corp., Armonk, USA) with a significance level of 0.05, and then all figures were generated via GraphPad Prism7 software (version 7.00 for Windows; GraphPad Prism, Inc., La Jolla, USA).

Ethics approval and consent to participate

This study was approved by the ethical board of the West China Hospital of Stomatology, Sichuan University, under protocol WCHSIRB-CT-2020-072. The approval document and consents are uploaded in related file.

Consent for publication

not applicable

Funding

This work was supported by the National Natural Science Foundation of China (82071111).

Availability of data and materials

The original data can be obtained by requesting it from the corresponding author.

Competing interests

The authors declare that they have no competing interests

Authors' contributions

MJ, KX, DF, YC, YW, XY, HZ, YY, LY and YL performed the clinical diagnosis and collected the clinical characteristics and samples from the participants. MJ, KX, DF and YC contributed to data visualization. MJ, KX, YC and DF conducted the in vitro experiments and the statistical analysis. MJ, BR and LZ contributed to the concept and interpretation of the data. MJ wrote the original draft. All the authors made substantial contributions to the revision of the manuscript and approved the final manuscript.

Acknowledgements

We thank OE Biotech Co., Ltd (Shanghai, China) for providing 16S and 18S full-length rDNA sequencing and bioinformatics analysis. We appreciate eceshi (www.eceshi.com) for the FISH analysis.

Zhou, M. et al. Mortality, morbidity, and risk factors in China and its provinces, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet (London, England) 394, 1145-1158, doi:10.1016/s0140-6736(19)30427-1 (2019).
Cai, J., Palamara, J., Manton, D. J. & Burrow, M. F. Status and progress of treatment methods for root caries in the last decade: a literature review. Aust Dent J 63, 34-54, doi:10.1111/adj.12550 (2018).
Hariyani, N., Setyowati, D., Spencer, A. J., Luzzi, L. & Do, L. G. Root caries incidence and increment in the population - A systematic review, meta-analysis and meta-regression of longitudinal studies. J Dent 77, 1-7, doi:10.1016/j.jdent.2018.06.013 (2018).
Hayes, M., Brady, P., Burke, F. M. & Allen, P. F. Vol. 33 299-307 (2016).
Xing, H., Liu, H. & Pan, J. High-Throughput Sequencing of Oral Microbiota in Candida Carriage Sjogren's Syndrome Patients: A Pilot Cross-Sectional Study. J Clin Med 12, doi:10.3390/jcm12041559 (2023).
Ellen, R. P., Banting, D. W. & Fillery, E. D. Longitudinal microbiological investigation of a hospitalized population of older adults with a high root surface caries risk. J Dent Res 64, 1377-1381, doi:10.1177/00220345850640121001 (1985).
Cleaver, L. & Garnett, J. A. How to study biofilms: technological advancements in clinical biofilm research. Front Cell Infect Microbiol 13, 1335389, doi:10.3389/fcimb.2023.1335389 (2023).
Chen, L. et al. Extensive Description and Comparison of Human Supra-Gingival Microbiome in Root Caries and Health.(Research Article). PLoS ONE 10, e0117064, doi:10.1371/journal.pone.0117064 (2015).
Takahashi, N. & Nyvad, B. The role of bacteria in the caries process: ecological perspectives. J Dent Res 90, 294-303, doi:10.1177/0022034510379602 (2011).
Lima, K. C., Coelho, L. T., Pinheiro, I. V., Rôças, I. N. & Siqueira, J. F., Jr. Microbiota of dentinal caries as assessed by reverse-capture checkerboard analysis. Caries Res 45, 21-30, doi:10.1159/000322299 (2011).
Jiang, Q., Liu, J., Chen, L., Gan, N. & Yang, D. The Oral Microbiome in the Elderly With Dental Caries and Health. Front Cell Infect Microbiol 8, 442, doi:10.3389/fcimb.2018.00442 (2018).
Du, Q. et al. Candida albicans promotes tooth decay by inducing oral microbial dysbiosis. The ISME journal 15, 894-908, doi:10.1038/s41396-020-00823-8 (2021).
Xiong, K. et al. The Arginine Biosynthesis Pathway of Candida albicans Regulates Its Cross-Kingdom Interaction with Actinomyces viscosus to Promote Root Caries. Microbiol Spectr 10, e0078222, doi:10.1128/spectrum.00782-22 (2022).
Marsh, P. D. Dental diseases--are these examples of ecological catastrophes? Int J Dent Hyg 4 Suppl 1, 3-10; discussion 50-12, doi:10.1111/j.1601-5037.2006.00195.x (2006).
Liu, Y. et al. Candida albicans CHK1 gene regulates its cross-kingdom interactions with Streptococcus mutans to promote caries. Appl Microbiol Biotechnol 106, 7251-7263, doi:10.1007/s00253-022-12211-7 (2022).
Deng, L. et al. Cross-kingdom interaction of Candida albicans and Actinomyces viscosus elevated cariogenic virulence. Arch Oral Biol 100, 106-112, doi:10.1016/j.archoralbio.2019.02.008 (2019).
Ji, M., Fu, D., Liao, G. & Zou, L. A Bibliometric Analysis of Studies on Root Caries. Caries Res 57, 32-42, doi:10.1159/000529050 (2023).
Montelongo-Jauregui, D. & Lopez-Ribot, J. L. Candida Interactions with the Oral Bacterial Microbiota. J Fungi (Basel) 4, doi:10.3390/jof4040122 (2018).
Preza, D. et al. Bacterial profiles of root caries in elderly patients. J Clin Microbiol 46, 2015-2021, doi:10.1128/jcm.02411-07 (2008).
Xiao, C., Ran, S., Huang, Z. & Liang, J. Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing. Front Microbiol 7, 1145, doi:10.3389/fmicb.2016.01145 (2016).
de Carvalho, F. G., Silva, D. S., Hebling, J., Spolidorio, L. C. & Spolidorio, D. M. P. Presence of mutans streptococci and Candida spp. in dental plaque/dentine of carious teeth and early childhood caries. Arch Oral Biol 51, 1024-1028, doi:10.1016/j.archoralbio.2006.06.001 (2006).
Shen, S., Samaranayake, L., Yip, H. & Dyson, J. Bacterial and yeast flora of root surface caries in elderly, ethnic Chinese. Oral Dis 8, 207-217, doi:10.1034/j.1601-0825.2002.01796.x (2002).
Nobile, C. J. & Johnson, A. D. Candida albicans Biofilms and Human Disease. Annu Rev Microbiol 69, 71-92, doi:10.1146/annurev-micro091014-104330 (2015).
Li, J. et al. Actinomyces and Alimentary Tract Diseases: A Review of Its Biological Functions and Pathology. Biomed Res Int 2018, 3820215, doi:10.1155/2018/3820215 (2018).
Dige, I. & Nyvad, B. Candida species in intact in vivo biofilm from carious lesions. Arch Oral Biol 101, 142-146, doi:10.1016/j.archoralbio.2019.03.017 (2019).
Klinke, T. et al. Acid production by oral strains of Candida albicans and lactobacilli. Caries Res 43, 83-91, doi:10.1159/000204911 (2009).
Diaz, P. I. et al. Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model. Infect Immun 80, 620-632, doi:10.1128/IAI.05896-11 (2012).
Janus, M. M. et al. Candida albicans alters the bacterial microbiome of early in vitro oral biofilms. J Oral Microbiol 9, 1270613, doi:10.1080/20002297.2016.1270613 (2017).
Xiao, J. et al. Association between Oral Candida and Bacteriome in Children with Severe ECC. J Dent Res 97, 1468-1476, doi:10.1177/0022034518790941 (2018).
Wu, T. et al. Cellular Components Mediating Coadherence of Candida albicans and Fusobacterium nucleatum. J Dent Res 94, 1432-1438, doi:10.1177/0022034515593706 (2015).
Montelongo-Jauregui, D., Srinivasan, A., Ramasubramanian, A. K. & Lopez-Ribot, J. L. An In Vitro Model for Candida albicans(-)Streptococcus gordonii Biofilms on Titanium Surfaces. J Fungi (Basel) 4, doi:10.3390/jof4020066 (2018).
Xiao, J. et al. Candida albicans and Early Childhood Caries: A Systematic Review and Meta-Analysis. Caries Res 52, 102-112, doi:10.1159/000481833 (2018).
He, J. et al. RNA-Seq Reveals Enhanced Sugar Metabolism in Streptococcus mutans Cocultured with Candida albicans within Mixed-Species Biofilms. Front Microbiol 8, 1036, doi:10.3389/fmicb.2017.01036 (2017).
Xu, H. et al. Streptococcal coinfection augments Candida pathogenicity by amplifying the mucosal inflammatory response. Cell Microbiol 16, 214-231, doi:10.1111/cmi.12216 (2014).
Falsetta, M. L. et al. Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect Immun 82, 1968-1981, doi:10.1128/iai.00087-14 (2014).
Harriott, M. M. & Noverr, M. C. Candida albicans and Staphylococcus aureus form polymicrobial biofilms: effects on antimicrobial resistance. Antimicrob Agents Chemother 53, 3914-3922, doi:10.1128/aac.00657-09 (2009).
Lombardi, L. et al. Characterization of the Candida orthopsilosis agglutinin-like sequence (ALS) genes. PLoS One 14, e0215912, doi:10.1371/journal.pone.0215912 (2019).
Bowen, W. H., Burne, R. A., Wu, H. & Koo, H. Oral Biofilms: Pathogens, Matrix, and Polymicrobial Interactions in Microenvironments. Trends Microbiol 26, 229-242, doi:10.1016/j.tim.2017.09.008 (2018).
Zhang, Q. et al. Inhibitory Effect of Lactobacillus plantarum CCFM8724 toward Streptococcus mutans- and Candida albicans-Induced Caries in Rats. Oxid Med Cell Longev 2020, 4345804, doi:10.1155/2020/4345804 (2020).
Ev, L. D., Dame-Teixeira, N., Do, T., Maltz, M. & Parolo, C. C. F. The role of Candida albicans in root caries biofilms: an RNA-seq analysis. J Appl Oral Sci 28, e20190578, doi:10.1590/1678-7757-2019-0578 (2020).
Douglas, C. W. Bacterial-protein interactions in the oral cavity. Adv Dent Res 8, 254-262, doi:10.1177/08959374940080021901 (1994).
Liu, T., Gibbons, R. J., Hay, D. I. & Skobe, Z. Binding of Actinomyces viscosus to collagen: association with the type 1 fimbrial adhesin. Oral Microbiol Immunol 6, 1-5, doi:10.1111/j.1399-302X.1991.tb00443.x (1991).
Jiménez-López, C. et al. Candida albicans induces arginine biosynthetic genes in response to host-derived reactive oxygen species. Eukaryot Cell 12, 91-100, doi:10.1128/ec.00290-12 (2013).
Vaziriamjad, S., Solgi, M., Kamarehei, F., Nouri, F. & Taheri, M. Evaluation of L-arginine supplement on the growth rate, biofilm formation, and antibiotic susceptibility in Streptococcus mutans. Eur J Med Res 27, 108, doi:10.1186/s40001-022-00735-7 (2022).
Ellepola, K. et al. Multiomics Analyses Reveal Synergistic Carbohydrate Metabolism in Streptococcus mutans-Candida albicans Mixed-Species Biofilms. Infect Immun 87, doi:10.1128/iai.00339-19 (2019).
Zheng, J. et al. Microbiome of Deep Dentinal Caries from Reversible Pulpitis to Irreversible Pulpitis. Infect Immun 45, 302-309 e301, doi:10.1016/j.joen.2018.11.017 (2019).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120, doi:10.1093/bioinformatics/btu170 (2014).
Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30, 460-465, doi:10.1038/nbt.2170 (2012).
Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7, 335-336, doi:10.1038/nmeth.f.303 (2010).
Rognes, T., Flouri, T., Nichols, B., Quince, C. & Mahe, F. VSEARCH: a versatile open source tool for metagenomics. PeerJ 4, e2584, doi:10.7717/peerj.2584 (2016).
Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73, 5261-5267, doi:10.1128/AEM.00062-07 (2007).
Krzyściak, W. et al. Effect of a Lactobacillus Salivarius Probiotic on a Double-Species Streptococcus Mutans and Candida Albicans Caries Biofilm. Nutrients 9, doi:10.3390/nu9111242 (2017).
Deng, L. et al. Cross-kingdom interaction of Candida albicans and Actinomyces viscosus elevated cariogenic virulence. Arch Oral Biol 100, 106-112, doi:https://doi.org/10.1016/j.archoralbio.2019.02.008 (2019).
Lyu, X. et al. Ursolic acid inhibits multispecies biofilms developed by Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii. Arch Oral Biol 125, 105107, doi:10.1016/j.archoralbio.2021.105107 (2021).
Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultrafast all-in-one FASTQ preprocessor. Bioinformatics 34, i884-i890, doi:10.1093/bioinformatics/bty560 (2018).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12, 357-360, doi:10.1038/nmeth.3317 (2015).

No competing interests reported.

SupplementaryMaterial.docx

The landscape of the bacteriome and mycobiome at different stages of root caries and the cross-kingdom interactions of the core species

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Introduction

Results

1. The demographic and clinical characteristics of the root caries volunteers

Discussion

Conclusion

Materials and methods

1 Clinical sample analysis

1.1 Study population

1.2 Inclusion Criteria and Exclusion Criteria

1.3 Sample collection

1.4 DNA extraction and amplification

1.5 Library construction and sequencing

1.6 Bioinformatic analysis

2.1 Microbial strains and growth conditions

2.2 Crystal violet assay and CFU counts

2.5 RNA sequencing and data analysis

3 Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1