Microbial Characteristics of the Extrinsic Black Stain in Primary Dentition

Abstract

Introduction and aims

Extrinsic black stain (EBS), a pigmented dental deposit prevalent in primary dentition, is challenging to remove, tends to recur rapidly, and poses aesthetic and psychological concerns. While the microbiota plays a key role in EBS formation, the specific microbial features remain poorly understood. The aim of the study was to explore the microbial characteristics of children aged 3-5 years with the EBS.

Methods

EBS (30) and healthy (30) supragingiva plaque samples were collected for comparative analysis. The microbial features of the 2 groups were investigated using Illumina MiSeq sequencing technology and verified by quantitative polymerase chain reaction (qPCR). Potential EBS-dominant genera were isolated and identified, followed by whole-genome sequencing and bio-informatic analysis.

Results

The qPCR results verified that Abiotrophia, Lautropia and Arachnia were significantly more enriched in the EBS group than that in the healthy group (P < .05). Four species of the above genera were isolated and identified, including E11004 (Abiotrophia defectiva), E3101(Lautropia mirabilis), E1715 (Arachnia propionica) and E10012 (Arachnia rubra). E3101 possessed the highest number of iron metabolism and biofilm-related genes among the isolates. Two genes, including 4-hydroxyphenylacetate hydroxylase (K23470) and 4-hydroxyphenylpyruvate dioxygenase (K05606), encoding key enzymes in the melanin synthesis pathway, were found in E3101, E1715 and E10012.

Conclusions

This study identifies E11004, E3101, E1715 and E10012 as species-level contributors to EBS, with genomic evidence implicating their potential roles in iron metabolism, biofilm formation and melanin production. These findings highlight the need for functional studies to elucidate their mechanistic roles in EBS pathogenesis.

Clinical significance

Identifying E11004, E3101, E1715 and E10012 as key contributors to EBS offers potential targets for novel preventive or therapeutic strategies, such as antimicrobial agents or biofilm inhibitors, to reduce recurrence and alleviate the aesthetic burden in affected children.

Introduction

Extrinsic black stain (EBS) is a common condition in primary dentition, with reported prevalence rates ranging from 2.4% to 26%.¹ Clinically, EBS typically presents as firmly adherent black or brown dots or lines along the tooth cervical margin. In severe cases, the deposits may extend into pits and fissures or even cover the entire crown surface. Unlike regular dental plaque, EBS cannot be removed through routine oral hygiene practices (e.g. brushing with toothpaste) and requires professional dental scaling for complete elimination.² Furthermore, EBS demonstrates a high recurrence rate,³ causing not only aesthetic concerns but also significant psychological distress for affected patients. Despite its clinical importance, the aetiology of EBS remains unclear. Elucidating the underlying causes and formation mechanisms of EBS is therefore crucial for developing effective preventive strategies and clinical management approaches.

The oral cavity constitutes a complex micro-ecosystem harbouring diverse microbial communities. Early studies by Bibby et al.4, 5, 6, 7 identified the primary microbial constituents of EBS were Gram-positive facultative anaerobic and anaerobic bacilli as well as Gram-positive facultative anaerobic cocci. Under the microscope, there was a cell-free and unstructured basement membrane similar to the acquired membrane, on which the rod-shaped bacteria were vertically aggregated and arranged, closely adhering to each other and lacking an intercellular matrix, forming a dense "network" structure. In the areas with fewer rod-shaped bacteria attached, amorphous calcareous deposits were present and covered by a large number of cocci. Therefore, the EBS is considered to be a special type of dental plaque.

To investigate the pathogenic microorganisms of EBS, before the 20th century, electron microscopy and in vitro culture technology were the main research methods. Researchers believed that filamentous bacteria or Actinomyces were the main pathogenic bacteria.^5,8, 9, 10, 11 Since 2015, researchers have adopted next-generation sequencing (NGS) technology, mainly targeting the V3-V4 hypervariable gene regions of the 16S rRNA gene to study the diversity, structure and correlations of microbial communities in EBS. Compared with the traditional isolation and culture techniques, the NGS technology is not dependent on microbial cultivability, enabling the detection of both non-viable and unculturable microorganisms while significantly enhancing sequencing speed and throughput. As a result, NGS has become the dominant sequencing technology in this area.¹²

A recent literature review revealed that certain bacterial genera exhibited higher abundance in EBS samples compared with healthy controls when analysed using NGS. These genera include Actinomyces, Cardiobacterium, Haemophilus, Corynebacterium, Fusestania, Treponema, Leptotrichia, Fusobacterium, Streptococcus, Neisseria, Arachnia, Rothia, Pseudomonas fluorescens, Aggregatibacter and Lautropia.13, 14, 15, 16, 17, 18, 19 However, no single oral microorganism was consistently identified across all these studies. Additionally, these investigations were limited to genus-level analysis, leaving the exact species composition and the mechanistic roles of potential EBS biomarkers unverified.

Although NGS can determine the relative abundance of microbial communities within samples, it cannot measure absolute abundance. Quantitative polymerase chain reaction (qPCR) remains the gold standard for quantifying the absolute microbial load, providing an accurate representation of the true number of microorganisms in a sample and enabling precise comparisons across different samples.²⁰ Therefore, integrating NGS with qPCR is essential to comprehensively characterise the microbial profile of EBS. Microbial genomics is used to obtain all the genome sequences of bacteria through high-throughput sequencing to study their whole structure and function and further identify their pathogenic genes and metabolic pathways.

This study had 2 primary objectives: (1) to explore the microbial characteristics of children with EBS by using a combined NGS and qPCR approach; and (2) to isolate and culture the species of the dominant genera and obtain their whole-genome sequencing features to further explore the potential coding genes associated with EBS formation.

Methods

Clinical sample collections

To examine the association between oral microbiome composition and structural characteristics in children with EBS, we recruited 30 children with extrinsic black tooth stain (EBS) as the experimental group (EG) and 30 healthy, EBS-free children as the control group (HG) from kindergartens in Kunming, China. Ethical approval was gained from the institutional review board of the Affiliated Stomatological Hospital of Kunming Medical University (KYKQ2020MEC006). Informed consent forms were obtained from all parents or guardians before enrolment. All participants met the following criteria: (1) age 3-5 years; (2) absence of oral diseases (including dental caries, gingivitis and mucosal diseases); (3) no dental restorations or orthodontic appliances; (4) no intake of iron supplements or professional fluoride treatment within the preceding 3 months; and (5) absence of systemic diseases. EBS and supragingival plaque samples were collected from the labial surfaces of the anterior tooth region for the EG and HG, respectively.

16S rRNA sequencing and analyses

Total bacterial DNA was extracted from the samples using the QIAamp DNA Mini Kit, following the manufacturer’s protocol. The DNA purity and concentration were verified using the NanoDrop 2000 spectrophotometer. The V3-V4 hypervariable regions of bacterial 16S rRNA genes were amplified using universal primers (338F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). The amplicons were sequenced on the Illumina MiSeq PE300 platform. QIIME (version 2.0) and The R Foundation for Statistical Computing (version 3.3.1) were used for sequence data analysis, including quality control and classification of the raw data.²¹ Annotation was performed according to the NCBI database. The following microbial community analyses were conducted: (1) the Chao 1, ACE, Shannon and Simpson indices were used to study the microbial alpha diversity; (2) the Principal Co-ordinates Analysis (PCoA) at the OTU level based on the Bray–Curtis distance was carried out to discriminate the microbial composition differences between the EG and HG; (3) the ANOSIM analysis was conducted for statistical validation of the differences; and (4) linear discriminant analysis (LDA) of effect size (LEfSe) was performed to define the microbial feature for each group, with the logarithmic LDA score threshold set at 3.0. Based on the results of the LEfse, Arachnia, Abiotrophia, Lautropia and F0332 were significantly enriched in the EG, and Gemella was significantly abundant in the HG. DNA counts of Gemella, Arachnia, Abiotrophia and Lautropia were quantified by qPCR using genus-specific primers (Table 1). F0332 was excluded because of the lack of reference sequences in public databases.

Table 1.

The Primer Sequences of the Target Genus

Genus	Forward primer	Reverse primer
Gemella	5′-GCTACTACTCCGTCTGCT-3′	5′- TTATTCGTATTGGTGCTG-3′
Arachnia	5′-AATACGTCGTCGCCCTCC-3′	5′- TTGTTCTGGTCCATGAACTGC-3′
Abiotrophia	5′- GTGCTAGAAGTGGCTAG-3′	5′-CGATCAAATTGCATACCTTC-3′
Lautropia	5′-GTCCTTTTCGTTCCCGCC-3′	5′-CAAGGCGACGATCTGTAGCTGG-3′

Bacteria associated with EBS

Isolation and culture

EBS samples were collected and preserved in a 30% glycerol buffer solution. Subsequently, a 1-mL aliquot of the EBS solution was immediately mixed with sodium pyrophosphate (1:1,000 dilution) to disperse the bacterial aggregates. The dilution was plated onto 3 selective media, including sheep blood agar, tryptic soy agar, and trypticase-yeast extract-glucose agar. These media were selected based on their demonstrated efficacy in cultivating the target strains in previous studies.22, 23, 24, 25 Plates were incubated under both aerobic and anaerobic conditions at 37 °C for 48-72 hours. Following incubation, distinct colonies were selected based on colony morphology including size, shape, colour, transparency, sheen, texture, and margin on solid media. Selected colonies were cultured onto fresh TSA plates to obtain pure isolates.

Molecular identification using the 16S rRNA gene sequencing

The genomic DNA was extracted using the Bacterial Genomic DNA Extraction Kit. PCR amplification and sequencing for the bacterial 16S rRNA gene were performed using universal primers 27F: 5’-AGAGAGTTTGATCCTGGCTCAG-3’ and 1492R: 5’-GGTTACCTTGTTACGACTT-3’. The Human Oral Microbiome Database (HOMD) was used for sequence comparison. Isolates showing ≥97% sequence similarity with reference strains in HOMD were considered to represent the same species. Selected isolates underwent comprehensive morphological observation using Gram staining, scanning electron microscopy and transmission electron microscopy.

Whole-genome sequencing and bioinformatics analyses

Genomic DNA of the obtained strains was extracted according to the Wizard Genomic DNA Purification Kit instructions, and the DNA purity was measured using NanoDrop2000. DNA concentration was determined using Quantus Fluorometer. The integrity of the extracted DNA was evaluated by 1% agarose gel electrophoresis. High-quality DNA was used for later sequencing library building. Whole-genome sequencing was performed using a dual-platform approach, the PacBio RS II for long-read sequencing and the Illumina platform for short-read sequencing. Raw data underwent quality clipping. Adapter sequences and non-AGCT bases at the 5′ ends were removed. Read ends with quality scores <Q20 were trimmed. Reads containing >10% N bases were discarded. Fragments <25 bp after trimming were excluded. The Unicycler software (version 0.5.0) was used to integrate Illumina and PacBio data for short-read-first hybrid assembly to obtain complete and accurate genome assembly results. Illumina reads were mapped to assembled genomes using Pilon software (version 1.23) for error correction.²⁶ In addition, the quality of the assembled genome sequences was evaluated using GC-depth distribution analysis and K-mer analysis. Furthermore, the gene assembly was evaluated using N50, which was required to be greater than 50% of the total genome length. Q30 was used to evaluate the base quality, requiring Q30 > 85%.

The 16S rRNA of the obtained strains was aligned against the NCBI nucleotide database. Phylogenetic analysis was performed using MEGA software (version 11.0). Kimura-2 model was built using neighbour-joining, maximum likelihood and maximum parsimony methods (1,000 bootstrap replicates).^{27, 28} Pan-Genomes Analysis Pipeline software (PGAP, version 6.0) was used to further validate the identity of the obtained strains. To gain a deeper understanding of genome functions, 6 databases, including the Non-Redundant Protein Database (NR), Swiss-Prot Protein Knowledgebase (Swiss-Prot database), Protein Families Database (Pfam), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG database), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes databases (KEGG), were employed to comprehensively explore genomic information.²⁹ In addition, the KEGG database was used to systematically analyse the biological function and metabolic pathways of genes. The potential genes related to EBS biofilm formation were screened using the Virulence Factors Database (VFDB).

Results

A total of 2,652,044 high-quality sequences with 430 OTUs were obtained from all the clinical samples, which were assigned to 12 phyla, 22 classes, 53 orders, 89 families and 173 genera. There were 387 OTUs in the EBS group, belonging to 12 phyla and 150 genera. The healthy group contained 376 OTUs belonging to 10 phyla and 138 genera. We observed no significant difference in the microbial richness and diversity between the HG and EG (P > .05, Table 2). The results of the PCoA (Figure 1A) showed that the 2 groups partially overlapped (R² = 0.0395, P = .013). This difference in community structure was further confirmed by the ANOSIM analysis (R² = 0.0427, P = .028).

Table 2.

The Alpha Diversity of the Two Groups

Group	Shannon index	Simpson index	Chao 1 index	ACE index
EG	3.73 ± 0.40	0.06 ± 0.03	278.35 ± 56.30	275.82 ± 56.68
HG	3.72 ± 0.26	0.05 ± 0.02	283.72 ± 38.92	278.85 ± 34.76
P	.94	.76	.67	.80

Each value was represented by mean ± SD. The independent samples t-test was used for the statistical analysis of alpha diversity indices differences between the EG and HG.

Fig. 1.

Comparative microbial profiles of the EBS and HG. A, Bacterial communities in different samples at the OTU level using the PCoA. Each sample was represented by a dot. The closer the distance between samples, the more similar the microbial community structure between them. The bacterial structures of the two groups partially overlapped (R² = 0.0395, P = .013). B, Relative abundance of the top 20 predominant bacteria at the genus level. C, Histogram of the LDA scores for abundant features between the EBS and healthy group. The length of the bar represents the impact. The threshold on the logarithmic LDA score for discriminate features was set to 3.0. qPCR quantification of the DNA counts of Abiotrophia (D-a), Lautropia (D-b), Arachnia (D-c) and Gemella (D-d) in the EG and HG. Independent samples t-test was employed to compare the differences in the DNA counts for the two groups comparison.

The 20 most abundant genera in both groups (Figure 1B) were Corynebacterium, Leptotrichia, Actinomyces, Capnocytophaga, Streptococcus, Neisseria, Saccharimonadaceae_ge, Arachnia, Selenomonas_3, Fusobacterium, F0332, Aggregatibacter, Lautropia, Veillonella, Porphyromonas, Prevotella_2, Cardiobacterium, Campylobacter, Comamonas and Tannerella. The results of the LEfSe analysis highlighted the potential biomarkers in different groups. At the genus level, Abiotrophia, Lautropia, F0332 and Arachnia were significantly abundant in EG while Gemella was significantly enriched in HG (P < .05) (Figure 1C). The qPCR results further confirm that Abiotrophia (EG: 4.355±0.6269 copies/ul(log) versus HG: 3.822 ± 0.7566 copies/ul (log), R² = 0.1720, P = .0043), Lautropia (EG: 5.802 ± 0.6235 copies/ul (log) versus HG:5.359 ± 0.5232 copies/ul(log), R² = 0.1331, P = .0042) and Arachnia (EG: 5.177 ± 0.4506 copies/ul(log) versus HG: 4.767 ± 0.4131 copies/ul(log), R² = 0.1885, P = .0005) were significantly enriched in the EG compared with the same in the HG, and the Gemella (EG: 4.443±0.7895 copies/ul (log) versus HG: 5.088±0.6411 copies/ul (log), R² = 0.1720, P = 0.0010) was significantly enriched in the HG compared with the same in the EG (Figure 1D).

Four isolated strains were successfully cultured and identified through 16S rRNA sequencing. The E11004 strain was related to Abiotrophia defectiva (A. defectiva), E3101 to Lautropia mirabilis (L. mirabilis), E1715 to Arachnia propionica (A. propionica) and E10012 to Arachnia rubra (A. rubra). Their similarities were 99.32%, 100%, 99.10% and 99.93%, respectively. As presented in the phylogenetic tree (Figure 2A), E11004 and A. defectiva ATCC 49176 are clustered in the branch of Abiotrophia. E3101 and L. mirabilis ATCC 51599 clustered in the branch of Lautropia. E1715 was clustered with A. propionica NBRC 14587. E10012 was clustered with the A. rubra SK-1. E1715 and E10012 both belonged to the same branch of Arachnia. The general genomic characteristics of E11004, E3101, E1715 and E10012 are shown in Table 3. The results of the pan-genomics (Figure 2B) indicated that E11004 and D14035481 shared 88.76% (1,603) of their genes. E3101 and NCTC 12852 had 99.68% (2,477) of their genes shared. The E1715 had 78.23% (2,267) homology compared with NCTC12967. E10012 and DSMZ 100122 exhibited a homology of 96% (2711). Phylogenetic and pan-gene analysis revealed that E11004, E3101, E1715 and E10012 belonged to A. defectiva, L. mirabilis, A. propionica and A. rubra, respectively. E1104 was grown under anaerobic conditions, whereas the other strains were aerobic. The Gram staining results showed that E11004, E1715 and E10012 were Gram positive and E3101 was Gram negative (Figure 2C). Their morphological characteristics after 2-3 days on TSA at 37 ℃ are shown in Fig. 2C-d, h, I, P.

Fig. 2.

Culturation and genomics of microbial biomarkers of EBS in primary dentition. Phylogenetic trees of the E11004 (A-a), E3101 (A-b), E1715 (A-c) and E10012 (A-d). (The phylogenetic tree displayed the genetic relationship between strains E11004, E3101, E1715 and E10012 and other closely related species. Comparing the 16S rRNA region sequences of different species which were retrieved from the NCBI database. Accession numbers of genome were indicated in parentheses. Sequences were aligned by scraper using default parameters. Phylogenetic inferences were obtained using the neighbour-joining method and the MEGA version 11.0 software. Bootstrap values were calculated from 1,000 replicates. T symbol indicated “Type Strain” that was used as a reference. The homologous genes’ Venn diagram of the Abiotrophia defectiva (B-a), Lautropia mirabills (B-b), Arachnia propionica B-c) and Arachnia rubra (B-d) using the pan-genomics analysis. (Different-coloured circles represented different strains. The numbers in the cross sections between circles indicated the number of homologous genes between strains. The numbers in the circular uncrossed sections indicated the number of genes unique to the strain.) The morphological characteristics of E11004, E3101, E1715 and E10012 strains under SEM, TEM and direct visual observation when grown on TSA solid media for 2-3 days (C-a: E11004, smooth surface, with no convexity, irregular edges, opaque opalescent colony; C-b, c: E11004, obtuse round ends and approximately spherical shape, 0.5-1 μm in diameter with complete cell wall, no flagella; C-d: E11004, Gram-positive; C-e: E3101, surfaces with folds, protrusions, irregular edges, opaque yellowish colonies; C-f, g: E3101, near-spherical cocci, gram-negative cell wall morphology, small spheres (polysaccharides) and a bundle of flagella attached on the surface, small septa divided the cells, electron-dense materials within the internal cell region, thick cementing layer between cells, the individual cell size around 1 μm in diameter; C-h: E3103, Gram-positive; C-i: E1715, Smooth surface, tapered convex, regular edges, opaque round light brown or white colonies; C-j, k: E1715, Rod-shaped bacilli with enlarged ends, around 0.5 μm in diameter and 2-20 μm in length, mediators because of the cell membranes invagination, without capsules, flagella or fimbriae; C-l: E1715, Gram-negative; C-m: E10012, Round pink or white colonies, with a smooth surface, tapered convex and regular edges; C-n, o: E10012, Coccobacillus with hollow terminal, approximately spherical, sporulation free, polycrystalline, around 0.4-0.8 μm in diameter and 0.8-1.0 μm in length, no capsule, flagella or fimbria; C-p: E10012, Gram-positive).

Table 3.

Assembly Statistics, KO ID, Iron and Biologically Related Genes of the Microbial Species Associated With EBS

Genomic features	E11004	E3101	E1715	E10012
Genome length (bp)	2045142	3167435	3307556	3329618
N50 (bp)	8483	9387	9750	8966
GC content (%)	46.81	65.54	63.47	64.21
Accession No.	PRJNA1080312	PRJNA1080314	PRJNA1080315	PRJNA1080316
Iron Transport Systems (NR, Swiss-Prot, Pfam, EggNOG, GO, KEGG)	Efe system:efeU, efeB, efeO	Efe system:efeU, efeB, efeO(2) Feo system:feoB, feoA Siderophore-dependent iron ABC transport system :afuA, fhuE Siderophore-independent iron ABC transport system :fecA, fecB(2), fecC, fecD, fecR, fecE	Feo system:feoB (2), feoA Siderophore-independent iron ABC transport system:yclO, yclN, fagD, btuC, fepG(3), fepD(2),	Feo system:feoB, feoA Siderophore-independent iron ABC transport system:yclO, yclN, fagD(2), btuC, fepG(3), fepD(4), fepC(4), torC,
Melanin-related genes KO ID (KEGG)	N/A	K23470	K05606	K05606
Biofilm-associated genes in VFDB	Motility:flgJa Adherence:chpAb, rpoNd Biofilm:algZc, algIc, mucDc, mucPc, vpsCc, bopDe (2)	Motility:motYa, motBa, flgLa, flgHa, flgGa, flgDa, flhCa, flhDa, fliDa, fliOa, fliQa, fliRa, fleNa,d, fleSa,d Adherence:crcc, rpoSd, rpoNd, pilGb,d, pilHb,d, pilJb,d, pilZb,d, pilY1b,d, pilRb,d, pilSb,d, pilQb,d, pilMb,d, fapDg, chpAb, fimVa,b Biofilm:vpsIc, adeGd(2), adeHd, mucDc, mucPc, algWc, algCc, algZc, algRc (2), algDc Regulation:cdpAc,d(7) Effector delivery system:VC0395_RS15460	Motility: PA1459 d, fliIa Adherence:flpFb (2), crcd, pilHb,d Biofilm:vpsGc, bopDe, algRc, algWc, algDc, vpsIc, algUc Regulation:bvgAd (2)	Motility:cheBf Adherence:flpFb (2), crcd Biofilm:vpsIc (2), vpsGc, bopDe (2), algDc, algIc, algWc Regulation:bvgAd

^aEncoding genes related to flagella.

^bEncoding genes related to fimbriae.

^cEncoding genes related to regulating secretion and production of EPS.

^dEncoding genes related to regulating the physiological processes, such as biofilm formation, motility and virulence.

^eEncoding genes related to the group behavior of bacteria, host–cell interactions and environmental adaptability.

^fEncoding genes regulated the methylation status of chemotactic receptors, thereby influencing the chemotactic behaviour and motility of bacteria.

^gEncoding genes responsible for the adhesion between bacteria and the stability of the biofilm structure.

The numbers in parentheses represent the number of encoding genes.

For iron transport–related genes (Table 3), E11004 had only 3 Efe ferrous transport systems. No ferric transport system–encoding genes were found in E11004. However, the rest of the strains had a Feo ferrous transport system. E1715 and E10012 had only a siderophore-independent ABC transport system strains. E3101 had both siderophore-dependent and siderophore-independent ABC transport systems.

KEGG annotation results are shown in Table 3. We found 2 genes, 4-hydroxyphenylacetate hydroxylase (K23470) and 4-hydroxyphenylpyruvate dioxygenase (K05606), encoding key enzymes in the melanin synthesis pathway in E3101, E1715 and E10012.

We screened the genes related to biofilm formation in the VFDB, which were scattered across 4 categories including mobility, adherence, biofilm, regulation and effector delivery system. E3101 was predicted to have the largest number of genes associated with biofilms among the 4 isolated strains.

Discussion

The present study advances our understanding of the microbial ecology of EBS by identifying E11004, E3101, E1715 and E10012 as species-level contributors, with genomic evidence linking them to iron metabolism, biofilm formation and melanin synthesis. While these findings provide novel insights, several limitations warrant careful consideration and contextualisation within the evolving landscape of EBS research.

The modest sample size and cross-sectional design limited the generalisability of our findings and preclude causal inferences. Even though statistical significance was achieved for key genera (e.g. Abiotrophia and Lautropia), larger longitudinal cohorts are needed to confirm their stability as EBS-associated biomarkers and to track microbial dynamics during stain recurrence.

Notably, Actinomyces belongs to Actinobacteria has been implicated as EBS contributors in prior work, particularly species such as Actinomyces naeslundii.^15,16 However, this genus was not detected in our study. These discrepancies may arise from differences in stain severity, age groups, geographic factors or competitive microbial interactions with the core EBS microbiota. Interestingly, our study did identify Arachnia, another member of Actinobacteria, as being associated with EBS. In addition, Streptococcus is a well-recognised cariogenic pathogen, and some evidence suggests EBS may inhibit dental caries development.^16,18,30 However, the association between Streptococcus and EBS has not been confirmed.¹

Analyses of the Chao 1, ACE, Shannon and Simpson indices revealed that microbial richness and evenness were similar between the EG and HG. These results are aligned with prior studies by Li et al.¹³ and Chen et al.¹⁵ Moreover, 14 out of the top 20 predominant genera in this research (Corynebacterium, Leptotrichia, Actinomyces, Capnocytophaga, Neisseria, Arachnia, Selenomonas, Fusobacterium, Aggregatibacter, Lautropia, Veilonella, Prevotella, Cardiobacterium, Tannerella) were in agreement with previous studies, indicating a relatively steady microbial community in the EBS group.¹

For the β diversity, PCoA analysis revealed that the bacterial communities of the 2 groups were significantly different. The ANOSIM analyses further verified the microbiota composition differences between groups. The distinct composition of the EBS patients indicated that they could have specific bacterial community structures. However, the partial overlap in PCoA and modest ANOSIM R² suggested that microbial composition alone could not fully explain EBS aetiology. Host factors (e.g. salivary iron levels, immune response) and environmental variables (e.g. diet, oral hygiene practices) likely interact with the microbiome to influence pigment deposition. Future work should integrate metabolomic or host transcriptomic data to unravel these multifactorial interactions.

Based on the results of the LEfSe and qPCR, we concluded that Abiotrophia, Lautropia, and Arachnia are associated with the EBS. To investigate the functional roles of the target microbial genera further, we isolated and cultured the corresponding bacterial strains. There are 5 known species under these strains, namely A. defectiva, L. mirabilis, L. dentalis, A. propionicum and A. rubra. In our study, E11004, E3101, E1715 and E10012 were isolated from patients with EBS while we failed to successfully cultivate L. dentalis, resulting in the lack of its genomic information. Although we could analyse the reference genome of standard L. dentalis strains from gene banks, such data could not be directly correlated with EBS because bacterial genomes constantly evolve in response to environmental changes. Our study focused solely on the successfully obtained strains and could not preclude the possible role of L. dentalis in EBS formation mechanisms. We would conduct targeted isolation and mechanistic studies of L. dentalis in follow-up research. Furthermore, Zhang et al.¹⁸ reported through microbial diversity analysis that L. mirabilis, rather than L. dentalis, was the species associated with EBS. Therefore, the strains we obtained still maintain a certain representativeness.

According to the present understanding of the EBS physiochemical characteristics, we explored the pathogenic genes of the potential biomarkers from 3 perspectives: microbial iron uptake, melanin metabolism and biofilm formation. Many researchers believed that EBS might consist of insoluble iron compounds.^31,32 Iron is an essential nutrient for most bacteria. Iron deficiency might affect biofilm formation, bacterial metabolism and growth. However, microbiota cannot produce iron by themselves. They can only transport iron from the environment into cells for further biological use. Insoluble ferric (Fe^III) and soluble ferrous (Fe^II) were two forms of iron under study. In previous studies, the absorption peaks of iron, ferrous ions, iron sulphide and ferrous sulphide were detected in EBS samples.^4,6,31, 32, 33, 34 Furthermore, using inductively coupled plasma mass spectrometry, Zhang et al. found that the concentration of iron in the EBS was significantly higher than that in plaque.³² Additionally, some researchers found that during orthodontic treatment, sulphate-reducing bacteria in the patient's oral cavity could react with iron ions from metal brackets to produce black precipitates.³⁵ Therefore, the formation of EBS may be closely related to iron.

To uptake iron, bacteria have a series of iron transport systems for Fe^II and Fe^III. In low-oxygen environments, iron is found primarily in its reduced form, Fe^II. The Fe^II transport systems include Efe system, Feo system and FetMP system, among others.36, 37, 38 E11004 was anaerobic. Therefore, it was not surprising that E11004 had only the Fe^II transport system. The Efe Fe^II transport system probably was its main iron transportation system for survival and proliferation. The Feo system was detected in E3101, E1715 and E10012. This system has been shown to play a role in the colonisation or virulence of some pathogens. The Fe^III transport system is composed mainly of a siderophore-dependent ABC transport system and a siderophore-independent ABC transport system.³⁹ In this study, no Fe^III transport system was found in E11004. However, we have identified two distinct siderophore-associated ABC transport systems in E3101. This system was the main Fe^III transport system for the majority of bacteria, which promoted efficient iron acquisition by secreting siderophores in iron-scarce environments.⁴⁰ In summary, E11004 probably could transport iron in the environment for its basic life activities. Furthermore, because iron is related to bacterial enzyme activity, energy metabolism, biofilm formation and other life processes, E3101 has the potential to transport ferric iron in iron-scarce environments and could transport ferrous iron in low-oxygen environments. Therefore, it was speculated that the E3101 bacteria had strong adaptability, high competitive ability and pathogenicity in EBS formation.

There are 3 main ways for microorganisms to synthesise melanin: tyrosinase, HGA (homogentisate acid) and DHN (dihydroxynaphthalene) pathways. Tyrosinase and HGA pathways require tyrosine as the precursor material to synthesise melanin, and the DHN pathway requires malonic acid monoacyl-coenzyme A as a precursor material to synthesise melanin.^41,42 No relevant genes encoding the aforementioned precursors were detected in any of the 4 strains. However, genes encoding for key enzymes in the homogentisic acid (HGA) pathway were identified in E3101, E1715 and E10012. These enzymes catalyse the hydroxylation of tyrosine and other aromatic compounds to form dibenzoquinones and hydroquinone derivatives, which subsequently undergo spontaneous polymerisation into melanin-like polymers. Given the absence of melanin precursor-related genes in E3101, E1715 and E10012, we hypothesised that these bacteria acquire precursors from the micro-environment. The target strain likely functions as an intermediate producer of the key enzymes in the HGA pathway, which catalyses the conversion of environmental precursors into black pigments.

Furthermore, the EBS has been characterised as a distinct form of dental plaque.¹ Its formation is typically initiated by bacterial flagella–mediated motility toward tooth surfaces.⁴³ Among the studied strains, E3101 possessed the highest number of flagella assembly genes (flgL, flgH, flgG, flgD, motB, flhC, flhD, fliD, fliO, fliQ, fliR, fleN, fleS), with flagella structures confirmed by electron microscopy. This genomic and phenotypic consistency suggests functional flagellar systems that likely contribute to biofilm formation. In contrast, E11004 and E1715 each carried only one flagella gene (flgJ and fliI, respectively), and no flagella structures were observed microscopically, indicating these genes were either non-functional or insufficient for flagella assembly. E10012 completely lacked flagella-related genes, correlating with its non-flagellated phenotype. Beyond flagella motility, bacterial surface attachment was also mediated by fimbriae through twitching motility.⁴³ All 4 strains harboured fimbriae assembly genes (pilGHJZRSQM, pilY1, chpA, rpoSN). However, no fimbriae structures were detected by standard electron microscopy or transmission electron microscopy. This apparent discrepancy might reflect the ultrastructural challenges of visualising these thin (3-5 nm) appendages, requiring higher-resolution imaging techniques for definitive observation.

Following initial surface attachment, bacteria release extracellular polymeric substances (EPS) through signal-regulated systems,⁴⁴ enabling tight and irreversible adhesion to tooth surfaces and subsequent microcolony development. Multiple genes encoding EPS were found in E11004(algZI, vpsC, mucDP), E3101(algWCRZD, vpsI, mucDP), E1715 (algRWDU, vpsGI)and E10012(algWDI, vpsGI). These genetic profiles indicate robust biofilm-forming potential. Additionally, EPS regulatory genes were identified in E11004 (bopD), E3101 (adeGH, cdpA, crc), E1715 (bopD, PA1459, crc) and E10012 (bopD, crc). Notably, E1715 and E10012 possessed bvgA, encoding a key biofilm initiation regulator.⁴⁵ E3101 carried cdpA, which encoded a phosphodiesterase that modulates biofilm development through multiple pathways including self-aggregation, flagellar synthesis and motility.⁴⁶ Therefore, E1715 and E10012 likely initiated biofilm formation. Strain E3101 exhibited a particularly strong biofilm-forming capacity, potentially influencing multiple plaque developmental stages. However, the functional roles of the identified genes (e.g. those encoding melanin synthesis enzymes) remain hypothetical and require experimental validation. Future studies should focus on conducting in vitro biofilm assays or generating knockout mutants to confirm these putative functions.

This study provides a genomic foundation for EBS pathogenesis, highlighting iron acquisition, biofilm regulation and melanin synthesis as key pathways. Although limitations exist, these findings invite mechanistic and translational research to transform EBS management. By integrating multi-omics data and experimental validation, future work could unlock targeted therapies to mitigate the aesthetic and psychological burden of this enigmatic condition.

Conclusions

This study characterises the microbial aetiology of EBS in children, identifying Abiotrophia, Lautropia and Arachnia as genus-level biomarkers. Four species from these genera were isolated from EBS samples, including E11004, E3101, E1715 and E10012. These strains showed high similarity to A. defectiva ATCC 49176, L. mirabilis ATCC 51599, A. propionica NBRC 14587 and A. rubra SK-1, respectively, with genomic analyses revealing iron transport systems, biofilm-associated genes, and genes encoding key enzymes for melanin formation—all of which implicate their potential roles in EBS pathogenesis. Functional distinctions emerged: E3101 may regulate multiple stages of EBS development, whereas E1715 and E10012 likely contribute to initiation. Critically, E3101, E1715 and E10012 harboured genes encoding melanin synthesis enzymes. This work advances EBS microbiology by translating taxonomic profiling into actionable therapeutic hypotheses. However, mechanistic validation through in vitro biofilm assays, genetic pathways verification and animal models is essential to confirm these roles.

Author contributions

J.L., G.L.and S.Z.: conceptualization, methodology, interpretation of data and critically revised manuscript. X.B. and X.D.: data collection, data analysis, interpretation of data, writing an original draft of the manuscript. Q.W. and W.Z.: data analysis. All authors discussed, commented, and revised the manuscript.

Conflict of Interests

None declared.