Introduction

Removable orthodontic appliances play a crucial role in the correction of dental malocclusions and the restoration of masticatory function. However, their intricate structural design, particularly those fabricated from acrylic resins, poses significant challenges to optimal oral hygiene. The presence of retentive areas within these appliances facilitates microbial colonization and subsequent biofilm formation, which may contribute to oral infections and systemic complications1,2. Additionally, the inherent porosity of acrylic resins provides a favorable substrate for bacterial adhesion and proliferation, increasing the risk of conditions such as denture stomatitis and, in susceptible individuals, pulmonary aspiration pneumonia3,4.

Dental plaque, a complex and highly organized cariogenic biofilm, is initiated by primary colonizers, notably Streptococcus mutans. This bacterium rapidly adheres to the dental enamel surface and initiates biofilm formation through the synthesis of extracellular polysaccharides (EPS), primarily glucans, from dietary sucrose. Subsequent co-aggregation with other streptococci and related genera, including Lactobacillus and Actinomyces, facilitates biofilm maturation and augments cariogenicity. The acidic byproducts of bacterial fermentation, such as lactic acid, induce a localized decrease in pH at the tooth surface, driving the demineralization of dental enamel and culminating in dental caries5,6.

Conventional antimicrobial agents, such as chlorhexidine gluconate and fluoride, have been employed to mitigate oral health complications associated with intraoral appliances7. Chlorhexidine, a broad-spectrum cationic biocide, disrupts bacterial cell membrane integrity, while fluoride enhances enamel remineralization and inhibits bacterial metabolic activity. However, concerns regarding the prolonged use of chlorhexidine, including potential dental staining and gustatory alterations, persist. Furthermore, both agents exhibit limited efficacy against established biofilms, owing to the protective barrier conferred by the EPS matrix, which impedes penetration and reduces antimicrobial potency8,9. The EPS matrix, composed of polysaccharides, proteins, and extracellular DNA, significantly impedes drug diffusion. Furthermore, this matrix binds antimicrobial agents, consequently diminishing their bioavailability and therapeutic efficacy. Additionally, residual components resulting from EPS disruption may exhibit cytotoxic effects on oral tissues. These limitations necessitate the exploration of safer and more targeted anti-plaque agents.

Plant-derived products represent a promising avenue for the development of novel anti-plaque agents, offering a potentially safer and more biocompatible alternative to traditional antimicrobials. The red pitahaya fruit (Hylocereus polyrhizus L.), a source of betalain, a nitrogen-containing, water-soluble pigment, presents potential anti-biofilm, antioxidant, and other bioactive properties10. Betalain, specifically betacyanin, has demonstrated activity against Pseudomonas aeruginosa biofilms potentially through mechanisms involving membrane disruption and inhibition of respiratory enzymes11. Recent investigations have demonstrated the potential of betacyanin to inhibit biofilm formation, disrupt established biofilms, reduce bacterial adhesion, and decrease acid production by S. mutans, among other oral pathogens, without compromising bacterial viability12. While previous research11,12 has explored the anti-biofilm properties of Hylocereus polyrhizus and red spinach (Amaranthus dubius) betacyanins against various oral and non-oral pathogens, our study distinguishes itself by being the first to employ stringent transcriptomic analysis to elucidate the precise molecular mechanisms by which red pitahaya betacyanin fraction (BF) inhibits S. mutans biofilm formation. This unique combination of in-depth molecular insights with a robust in vitro denture plaque model allows for direct validation of BF’s anti-biofilm efficacy in a clinically relevant setup.

This study aims to elucidate the molecular mechanisms underlying the anti-biofilm activity of the red pitahaya fruit BF against S. mutans. Through the application of transcriptomic and network analysis, the study seeks to identify the specific genes and pathways targeted by BF, potentially revealing novel therapeutic strategies for the prevention of dental caries and the promotion of oral health. Furthermore, the efficacy of BF in inhibiting S. mutans dental plaque formation on acrylic denture materials was evaluated using a standardized in vitro model. To ensure standardized and reproducible conditions for the investigation of potential plaque inhibitors, this study employs a static monospecies biofilm model utilizing S. mutans. While this model simplifies the complex multispecies interactions observed in vivo, it allows for a focused investigation of the effects of treatment on a key early colonizer and provides a robust platform for mechanistic studies.

Materials and methods

Betacyanin preparation

Red pitahaya fruits (Hylocereus polyrhizus) with total soluble solids of 12–15 °Brix were purchased for betacyanin extraction12. Following pulp extraction, the solution was concentrated by rotary evaporation (40 °C) and freeze-dried for storage at − 80 °C. Semi-purification of the crude extract was performed using ion-exchange liquid chromatography12. This extract was termed betacyanin fraction. Individual betacyanin components were analysed using an Agilent 1260 Infinity HPLC system13, and their identities were further confirmed using an Agilent 1290 Infinity LC system coupled with an Agilent 6520 Accurate-Mass Q-TOF LC/MS12.

Biofilm preparation and RNA extraction

Streptococcus mutans (ATCC 25175) was cultured anaerobically in Brain Heart Infusion (BHI) broth at 37 °C under 5% CO2. These conditions mimic the oxygen-limited environment of the oral cavity, promoting optimal growth of this oral pathogen. Biofilms were prepared and BF treatment treated according to an established methodology, as previously described12. Streptococcus mutans suspensions, adjusted to 0.5 McFarland standard (OD600 of 0.1) with 1% glucose, were added to 96-well plates and treated with equal volumes of BF (final concentrations of 15 mg/mL) for 24 h at 37 °C to facilitate biofilm formation. After incubation, the wells were aspirated and delicately washed with distilled water to remove loosely adherent cells. The collected biofilms then proceeded to the rigorous RNA extraction process for transcriptomic analysis. The total RNA was extracted from detached biofilms using Direct-zol RNA Purification Kits (Zymo Research) followed by a rigorous quality control process. RNA concentration (Qubit Fluorometer 4.0), purity (Implen NanoQuant Spectrophotometer), and integrity (LabChip GX Touch™ Nucleic Acid Analyzer) were assessed. Library preparation involved Illumina Stranded Total RNA Prep with Ribo-Zero Plus, which efficiently depletes ribosomal RNA (rRNA) to enhance mRNA recovery. High-quality sequencing was performed on the Illumina NovaSeq 6000, with raw data processing, alignment, and quantification via Illumina DRAGEN. For the analysis, the reference genome Streptococcus mutans UA159 (serotype c) was used. Biological duplicates were used for both the control and BF-treated groups to ensure statistical robustness. Alignment of the quality-controlled reads to the reference genome was also performed using Illumina DRAGEN, which is optimized for efficiency and accuracy. Post-alignment, gene quantification was conducted by counting reads mapped to each gene using DRAGEN’s built-in quantification module. The average alignment rate across all samples was consistently high, exceeding 90%, with robust mapping percentages indicating excellent coverage of the S. mutans transcriptome.

RNA sequence analysis

Differential expression (DE) analysis was conducted with DESeq2, employing a negative binomial model and Benjamini–Hochberg FDR correction (initial analysis p < 0.05, stringent analysis, p value < 0.01). Gene Ontology (GO) enrichment analysis was conducted using the ShinyGO tool14 to identify enriched biological processes and molecular functions in differentially expressed genes (DEGs) following AF or BF treatment. Initial analysis utilized a p value threshold of < 0.05, while a more stringent false discovery rate (FDR) cutoff < 0.01 was applied in the revised analysis using ShinyGO to enhance the reliability of results. KEGG pathway analysis, performed using the clusterProfiler15 R package, was employed to determine the statistical enrichment of DEGs within the KEGG pathway database16,17,18 and elucidate potentially impacted metabolic pathways. To further investigate the relationships among DEGs, protein–protein interaction (PPI) network analysis was conducted using the STRING database, allowing for the visualization of key protein clusters and interaction patterns associated with AF or BF treatment.

In vitro dental plaque model and assessment

An established in vitro dental plaque model19 was employed to assess the inhibitory effects of BF on plaque formation. Artificial dentures were disinfected with 50 g/L sodium hypochlorite for 24 h, then tyndallized by heating to 80 °C for 10 min, followed by a further wash in sterile distilled water to establish a sterile baseline20. To simulate the acquisition of the acquired pellicle, a crucial protein layer that precedes biofilm development, dentures were preconditioned in artificial saliva (prepared according to McKnight-Hanes and Whitford protocols)21 at 37 °C for 4 h.

Monospecies cultures of S. mutans were prepared by inoculating single colonies from BHI agar into sucrose-enriched BHI broth and incubating overnight (37 °C, shaking at 150 strokes/min). The bacterial suspensions were adjusted to a 0.5 McFarland standard for standardization. Following artificial saliva conditioning, dentures were inoculated with bacterial culture anaerobically [4–6 h (initial attachment phase), 37 °C] and rinsed with sterile distilled water to remove loosely-adherent cells. The inoculated dentures were subsequently incubated in artificial saliva supplemented with 5% sucrose and BF (3.75 mg/mL) or distilled water (negative control) for 48 h to facilitate plaque development.

Plaque coverage evaluation

To assess plaque coverage, dentures were rinsed with sterile water to remove unbound bacteria and stained with 0.25% methylene blue disclosing solution (Wako Pure Chemical Industries Ltd) for 1 min. The excess stain was removed by rinsing again with sterile water. Standardized images of the dentures (mucosal, right, and left polished surfaces) were captured using a white-light system (Lightbox S; Suntech Co, Ltd) and a digital camera (70D; Canon).

ImageJ software (National Institutes of Health) was used for image analysis to quantify plaque coverage. Denture images were calibrated using a reference ruler and the “Set Scale” function. The denture surface area was outlined and measured in square centimeters, followed by the selection and measurement of the plaque area after applying thresholding if necessary. To calculate percentages, denture and plaque areas were converted to pixels using the pixel resolution obtained during calibration. The percentage plaque was calculated as the plaque pixels area to denture pixel area, providing a quantitative measure of plaque coverage on each denture surface.

Results of the stained plaque coverage image assessment were expressed as mean ± standard deviation. To assess the variations between the test groups, a one-way analysis of variance (ANOVA) test was employed. Additionally, Tukey’s multiple comparison post-test was utilized to compare the means. A p value less than 0.01 indicated a statistically significant difference.

Ethics statement

This study was conducted in vitro using bacterial cultures; therefore, no human or animal subjects were involved, and ethical approval was not required.

Results and discussion

Transcriptomic analysis of differentially expressed genes

Transcriptomic analysis demonstrates a profound and reproducible impact of BF on S. mutans gene expression, supported by an average alignment rate consistently exceeding 90% and robust mapping percentages, indicating excellent and reliable coverage of the S. mutans transcriptome. This is evident in the clear separation of treated and control groups in the principal component analysis (PCA) plot (Fig. 1A). The first principal component (PC1), accounting for 99.13% of the total variance, identified BF treatment as the primary determinant of differential gene expression. Volcano plot analysis (Fig. 1B) further illustrated the magnitude of BF-induced transcriptomic changes, revealing a significant number of DEGs. Specifically, 170 genes were upregulated and 240 genes were downregulated in response to BF. Employing a stringent statistical threshold (p < 0.01), 57 genes exhibited significant upregulation, while 77 genes displayed significant downregulation. This observation suggests that BF elicits a robust and comprehensive modulation of S. mutans gene expression, impacting a substantial proportion of the transcriptome.

Fig. 1
figure 1

Transcriptomic profiling of S. mutans in response to BF. (A) Principal component analysis (PCA) differentiating gene expression profiles between BF and control samples (B) Volcano map of all significant DEGs, screened based on p value < 0.01. Red plots represent up-regulated genes. Blue plots represent down-regulated genes. (C) Heatmap visualizing the expression patterns of significant DEGs (p value < 0.01, − 2 < Log2FC > 2); the red colour indicates increased gene expression, and the green indicates decreased gene expression.

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Hierarchical clustering and heatmap visualization (Fig. 1C) demonstrated a high degree of concordance in gene expression patterns across biological replicates, both under less stringent (p < 0.05, |log2 fold change|> 2) and more stringent (p < 0.01, |log2 fold change|> 2) statistical criteria. This reproducibility underscores the reliability of the observed BF-mediated gene expression changes. Furthermore, a consistent core set of DEGs was identified across both analyses, with 56 upregulated and 77 downregulated genes at p < 0.01, and 57 upregulated and 77 downregulated genes at p < 0.05. The convergence of these results, irrespective of the statistical threshold, reinforces the biological relevance of these DEGs and their potential as critical targets for elucidating the molecular mechanisms of BF’s anti-biofilm activity. Notably, the consistent downregulation of a specific subset of genes, regardless of the statistical rigor applied, strongly implicates these genes as pivotal mediators of S. mutans physiology and potential targets for BF-mediated biofilm inhibition. Further functional characterization of these core DEGs will be critical to fully understand the molecular basis of BF’s efficacy.

Downregulated genes

A stringent transcriptomic analysis (p value < 0.01, log2 fold change < − 2) of BF-treated S. mutans biofilms, corroborating less stringent analysis (p value < 0.05, log2 fold change < − 2) (Supplementary Table S1), revealed key insights into the mechanisms underlying BF’s anti-biofilm activity.

The consistent downregulation of genes involved in sucrose-dependent pathways was observed across both analyses. This includes significant suppression of pfkB (phosphofructokinase) (log2FC = − 2.67) and fructose-specific PTS transporter subunits (EIIC, log2FC = − 3.13; IIA, log2FC = − 2.61), suggesting that BF robustly interferes with sugar processing and energy production. This strongly suggests that BF’s anti-biofilm action stems from disrupting sucrose metabolism, a critical pathway for S. mutans adherence. Specifically, the downregulation of these genes likely reduces the bacterium’s ability to transport and metabolize sucrose, limiting the production of extracellular polysaccharides (EPS) essential for biofilm formation.

Additionally, consistent downregulation of LysM peptidoglycan-binding domain-containing protein (log2FC = − 2.63), involved in cell wall adhesion, and the two-component system ciaR/ciaH (log2FC = − 4.03/− 4.02), which regulates surface adhesin expression, reinforces BF’s ability to inhibit both sucrose-dependent and -independent adherence mechanisms.

The suppression of F0F1 ATP synthase subunit C (log2FC = − 2.07), crucial for pH homeostasis, further supports the disruption of energy metabolism. Notably, the consistent downregulation of transcriptional regulators Rgg/GadR/MutR (log2FC = − 3.29), ciaR (log2FC = − 4.03), and ciaH (log2FC = − 4.02), all involved in acid response mechanisms, across both analyses, suggests potential targets for combination therapy. The concomitant suppression of stress-related proteins YggT (log2FC = − 2.11), YlbG (log2FC = − 2.04), and NAD(P)/FAD-dependent oxidoreductases (log2FC = − 2.37) further reinforces the idea that BF could synergistically enhance the efficacy of acid-focused anti-plaque strategies by inhibiting biofilm resilience. This observation aligns with earlier in vitro bioassay studies demonstrating dose-dependent acid inhibition by betacyanin.

Collectively, these findings strongly indicate that BF impedes initial bacterial attachment by disrupting cell wall components and compromises the structural integrity of S. mutans biofilms, making them more susceptible to external stressors. The potent anti-biofilm activity of betacyanin extends beyond its effects on cell wall synthesis. BF treatment also consistently downregulated key enzymes involved in peptidoglycan processing and cell division, including S24 family peptidase (log2FC = − 3.61), serine hydrolase (log2FC = − 3.20), and the septum formation initiator protein (log2FC = − 3.83). These enzymes play pivotal roles in cell wall synthesis and division, and their downregulation likely compromises cell wall integrity and hinders bacterial growth within the biofilm.

BF’s ability to downregulate a diverse range of transcriptional regulators in S. mutans underscores its potent anti-biofilm potential. The consistent downregulation of the ciaR/ciaH two-component system (log2FC = − 4.03/− 4.02) in both the less stringent (p < 0.05) and stringent (p < 0.01) analyses, which is crucial for S. mutans’ stress response, biofilm formation, and acid tolerance by regulating the expression of surface adhesins, highlights its pivotal role. Furthermore, the consistent downregulation of Rgg/GadR/MutR (log2FC = − 3.29) and MarR (log2FC = − 2.27), in both analyses, emphasizes their importance in S. mutans’ survival and virulence. These regulators are involved in acid tolerance, quorum sensing, and virulence factor production, respectively, and their suppression by BF likely impairs the bacterium’s communication, stress response, and pathogenic potential.

The impact of BF extends beyond core biofilm pathways, with the downregulation of regulators like sdpR (log2FC = − 2.22), comGA/GC/GF (log2FC = − 2.15/− 2.04/− 2.0), hdrR (log2FC = − 2.01), and lacR & lacD (log2FC = − 2.94 and − 2.62), all of which were significant in the less stringent analysis and remained consistent in the stringent analysis. This demonstrates the compound’s ability to disrupt a wide array of regulatory networks. These regulators are involved in diverse cellular processes, including biofilm maturation, competence, oxidative stress response, and sugar metabolism, suggesting that betacyanin exerts a comprehensive inhibitory effect on S. mutans physiology.

The transcriptomic data, analysed at both p < 0.05 and p < 0.01 significance levels, clearly demonstrates BF’s ability to downregulate multiple transcriptional regulators in S. mutans. This disruption of key regulatory networks, which control various aspects of bacterial physiology including stress response, communication, and virulence, coupled with its previously established effects on acid tolerance, cell wall integrity, and adherence, reinforces the potential of BF as a promising candidate for developing novel antibiofilm treatments against S. mutans.

Upregulated genes

While BF primarily induces a downregulation of genes, the upregulation of specific genes elucidates the S. mutans adaptive response (Supplementary Table S2). A consistent upregulation of genes involved in glycolysis, gluconeogenesis, and arginine metabolism across both the less stringent (p < 0.05) and stringent (p < 0.01) analyses indicates a pronounced metabolic adaptation to BF-induced stress, likely osmotic and oxidative stress. Key enzymes within these pathways, including class II fructose-bisphosphate aldolase (log2FC = 2.2), glucose-1-phosphate adenylyl transferase (log2FC = 2.55), LytS (log2FC = 2.2), glycogen synthesis enzymes GlgA (log2FC = 2.12), GlgD (log2FC = 2.42), and GlgB (log2FC = 2.6), acetylornithine transaminase (log2FC = 2.05), ArgB (log2FC = 2.26), and ArgH (log2FC = 2.52), demonstrate the importance of these metabolic shifts, likely aimed at maintaining energy production and cellular homeostasis, after BF exposure.

Similarly, the significant upregulation of multiple phosphotransferase system (PTS) transporters (sugar uptake systems), including glucitol/sorbitol (log2FC = 2.08) and beta-glucoside (log2FC = 2.18) transporters, persisted in the stringent analysis. Notably, the pronounced increase in cellobiose-specific PTS transporter subunits IIA and IIB (log2FC = 6.42 and 6.99, respectively) further underscores the bacterium’s potential reliance on cellobiose as an alternative sugar source under BF-induced stress.

BF treatment elicited a pronounced stress response in S. mutans, as evidenced by the consistent upregulation of genes involved in oxidative stress defence, detoxification, and potential damage repair. Specifically, the upregulation of a glutathione-binding-like protein (log2FC = 2.29), ABC transporter components (permease subunit and substrate-binding protein, log2FC = 2.47 and 2.59, respectively), HAD family phosphatase (log2FC = 2.07), and thioesterase domain-containing protein (log2FC = 2.61) was observed across both the less stringent (p < 0.05) and stringent (p < 0.01) analyses, indicative of the robustness of this stress response. However, the upregulation of an alpha/beta fold hydrolase was observed only in the less stringent analysis (p < 0.05), suggesting that its induction may be contingent upon a less severe stress level.

The increased expression of transcriptional regulators, such as MerR (log2FC = 2.25) and sigma-70 family RNA polymerase sigma factor (log2FC = 2.24), suggests the activation of broader stress response networks, consistent with the less stringent analysis. Similarly, the presence of upregulated transposases (IS30, log2FC = 2.19; IS982, log2FC = 2.42; ISAs1, log2FC = 3.54) and a restriction-modification enzyme remained significant, regardless of the significance threshold, indicating potential BF-induced genomic instability, potentially leading to increased mutation rates or altered gene expression patterns.

Furthermore, the upregulation of genes involved in diverse metabolic pathways, including acyltransferase (log2FC = 2.08), methyltransferase (log2FC = 2.15), glycyl-radical enzyme activating protein (log2FC = 2.27), and AAA family ATPase (log2FC = 2.33), aligns with previously observed bacterial responses to various environmental stressors. This metabolic response, also observed in the less stringent analysis, indicates that BF exposure induces a comprehensive stress response in S. mutans. This prompts the activation of adaptive mechanisms to maintain metabolic function and ensure survival, but these adaptations, while promoting short-term survival, may ultimately hinder long-term biofilm formation by diverting resources away from biofilm matrix production and maintenance.

Gene ontology enrichment analysis

GO enrichment analysis of DEGs between BF-treated and control S. mutans biofilms revealed a significant enrichment of biological processes related to carbohydrate and energy metabolism (Fig. 2A). Notably, the processes of glycogen metabolism and energy reserve exhibited the highest degree of enrichment, with a fold enrichment of 52.1, indicating that BF disrupts bacterial energy storage and utilization. Furthermore, significant enrichment was observed in pathways related to cell wall biosynthesis and integrity, specifically those involving lipoteichoic acid (fold enrichment = 37.9) and teichoic acid (fold enrichment = 32.1). These findings suggest that BF likely compromises cell wall integrity by interfering with the synthesis of these essential cell wall constituents, which are critical for maintaining bacterial structural integrity and mediating interactions with the surrounding environment, thereby impacting biofilm formation and maintenance.

Fig. 2
figure 2

GO enrichment analysis of DEGs between BF and control: (A) biological process GO terms, (B) relationships between enriched biological processes. The pathways (nodes) are connected if sharing 20% (default) or more genes. Darker nodes show more significantly enriched gene sets. Bigger nodes represent larger gene sets. Thicker edges represent more overlapped genes.

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A significant influence on glucan and polysaccharide metabolism and biosynthesis, both essential for biofilm matrix formation, was also evident. The enrichment of broader carbohydrate metabolism-related processes further underscores the extensive influence of BF on sugar metabolism within S. mutans. The enrichment of higher-level GO terms, such as cellular macromolecule metabolic process, suggests that BF’s effects extend beyond carbohydrate metabolism, affecting diverse metabolic pathways critical for bacterial growth and survival. The interconnectedness of these enriched biological processes is visually depicted in Fig. 2B, robustly illuminated the dominant cluster centered on carbohydrate metabolism, encompassing processes involved in energy production, storage, utilization, cell wall biosynthesis, and polysaccharide metabolism.

GO enrichment analysis of cellular components (Fig. 3A) revealed that the cytosol was the most significantly enriched location, with a fold enrichment of 5.4, reflecting its central role in key metabolic processes. This observation suggests that BF may exert its effects by disrupting critical cytosolic processes, including protein synthesis, metabolism, and signalling. The significant overlap in genes associated with the terms “cellular anatomical entity”, “cytoplasm”, and “intracellular” (Fig. 3B) indicates that BF’s effects on cellular structure and function are not isolated but rather represent a broader cellular response. The higher enrichment of the cytosol relative to these broader terms suggests a pronounced impact on cytosolic processes.

Fig. 3
figure 3

GO enrichment analysis of DEGs between BF and control: (A) cellular components GO terms, (B) relationships between enriched cellular components. The pathways (nodes) are connected if sharing 20% (default) or more genes. Darker nodes show more significantly enriched gene sets. Bigger nodes represent larger gene sets. Thicker edges represent more overlapped genes.

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GO enrichment analysis of molecular functions (Fig. 4A) revealed significant enrichment of kinase activity (fold enrichment = 5.7) and transferase activity (fold enrichment = 4.4), suggesting a substantial impact of BF on protein phosphorylation, signal transduction, and the transfer of functional groups. Furthermore, significant enrichment of various binding-related molecular functions, including anion binding (e.g., binding of chloride or phosphate ions, influencing enzyme activity), small molecule binding (e.g., binding of metabolites or signalling molecules, affecting metabolic pathways or cellular communication), nucleotide binding (e.g., binding of AMP, GMP, CMP, TMP), and ATP binding (crucial for energy-dependent processes), suggests that BF interferes with essential cellular processes, such as enzyme activity, signal transduction, and DNA replication. The central “binding” node in the interactive plot (Fig. 4B) visually confirms this interconnectedness, suggesting that BF’s interaction with various molecules may modulate the activity of enzymes involved in diverse cellular processes, including phosphorylation and the transfer of functional groups.

Fig. 4
figure 4

GO enrichment analysis of DEGs between BF and Control: (A) Molecular function GO terms, (B) Relationships between enriched molecular function. The pathways (nodes) are connected if sharing 20% (default) or more genes. Darker nodes show more significantly enriched gene sets. Bigger nodes represent larger gene sets. Thicker edges represent more overlapped genes.

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Protein–protein interaction network analysis

K-means clustering analysis of the PPI network of DEGs in BF-treated biofilm revealed a complex network of interconnected proteins (Fig. 5). The largest cluster, consisting of 53 genes, was highly enriched for proteins involved in essential cellular functions. Within this cluster, a significant impact was observed on energy metabolism, as evidenced by the downregulation of genes such as atpH (encoding ATP synthase F1, H+ transporting, alpha subunit), pfkB (6-phosphofructokinase), and lacD (tagatose 1,6-bisphosphate aldolase). This disruption of key metabolic enzymes indicates a compromised ability of the bacteria to generate ATP via oxidative and substrate-level phosphorylation, thereby impacting bacterial growth and survival.

Fig. 5
figure 5

STRING-based analysis of protein–protein interaction (PPI) network analysis in BF-treated S. mutans.

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Furthermore, the predominant gene cluster exhibited marked enrichment for cell wall biosynthesis and maintenance components. Notably, this included the dlt operon (dltA, dltB, dltC, dltX), governing d-alanylation of teichoic acids, a modification vital for Gram-positive bacterial cell wall integrity and virulence. d-alanylation, by reducing cell wall negativity, confers resistance to cationic antimicrobial peptides and promotes biofilm formation22. Consequently, BF-mediated downregulation of these genes weakens the cell wall’s protective barrier, increasing bacterial vulnerability to environmental stressors. Furthermore, the cluster encompassed peptidoglycan metabolism genes, such as lytS (autolysin) and htrA (serine protease), which participate in cell wall turnover and protein quality control/stress response, respectively. Disruption of htrA alters surface expression of extracellular proteins, including glucan-binding protein B, glucosyltransferases, fructosyltransferase, and glycolytic enzymes, impacting biofilm architecture23.

The predominant gene cluster identified pertains to carbohydrate metabolism, encompassing fruC, fruD, glgA, glgB, glgC, and glgD, crucial for fructose transport and glycogen dynamics. BF-mediated downregulation of these genes diminishes bacterial capacity for fructose utilization and glycogen synthesis, thereby compromising energy generation and storage. Consequently, reduced growth and biofilm formation ensue. Notably, disruption of glycogen metabolism consistently precipitates increased biofilm formation across all mutant strains24. This cluster also encompasses genes related to virulence (ciaH, ciaR) and competence (com genes), indicating that BF not only perturbs fundamental cellular processes but also impedes bacterial pathogenicity and environmental adaptation. The interconnected nature of these proteins within the cluster suggests a concerted downregulation of these crucial functions by BF. Subsequent diminutive clusters, spanning two to nine genes, encompassed loci related to DNA repair and recombination, such as greA and recX, cell division and wall maintenance, including ftsZ and ftsA, and a diverse array of functions, notably amino acid biosynthesis, transport, and regulation.

Pathway enrichment analysis

KEGG pathway enrichment analysis of BF-treated S. mutans biofilms has revealed a significant perturbation of bacterial metabolism (Fig. 6). It is observed that the most significantly enriched pathway was glycolysis/gluconeogenesis (p = 1.17E−06), followed in significance by pyruvate metabolism and starch and sucrose metabolism. These pathways, which are central to energy production, carbohydrate metabolism, and exopolysaccharide (EPS) synthesis, indicate that BF exerts a disruptive influence on metabolic processes that are critical for biofilm formation and survival. The enrichment of “microbial metabolism in diverse environments”, fructose and mannose metabolism, and the phosphotransferase system (PTS) further underscores the extensive impact of BF on carbohydrate utilization.

Fig. 6
figure 6

KEGG pathway analysis in BF versus control, ranked by p value.

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Furthermore, significant enrichment was observed in amino acid biosynthesis pathways (arginine, valine, leucine, isoleucine), pantothenate and CoA biosynthesis, 2-oxocarboxylic acid metabolism, amino sugar and nucleotide sugar metabolism, and the biosynthesis of secondary metabolites. These findings indicate that BF exerts a disruptive influence on a wide range of metabolic pathways, including not only those implicated in energy production and carbohydrate metabolism, but also those involved in cell wall synthesis, and virulence factor production.

A comparative analysis of KEGG pathway enrichment results obtained using less stringent (p < 0.05) and stringent (p < 0.01) p value cutoffs has revealed that the stringent analysis delineated a more comprehensive impact of BF on S. mutans metabolism. While the less stringent analysis primarily highlighted pathways related to carbohydrate metabolism and transport, the stringent analysis also brought to light affected pathways including glycolysis/gluconeogenesis, pyruvate metabolism, and amino acid biosynthesis. This demonstrates that BF disrupts carbohydrate metabolism and impairs other core metabolic processes essential for bacterial growth and survival.

Specifically, starch and sucrose metabolism was significantly enriched (p value = 0.000131), affecting 82 genes. These carbohydrates serve as major energy sources for S. mutans and are also crucial precursors for the synthesis of EPS, which forms the structural backbone of the biofilm matrix. Consequently, it may be inferred that the disruption of carbohydrate metabolism by BF impairs biofilm formation and virulence19. The enrichment of “microbial metabolism in diverse environments” (p = 0.000168, 14 genes) suggests that BF affects various metabolic pathways essential for S. mutans’ adaptation and survival under diverse conditions, potentially impairing the bacteria’s ability to thrive in the oral cavity and contributing to its anti-biofilm effect. Moreover, BF broadly affected carbohydrate utilization in S. mutans, as evidenced by the enriched pathways of fructose and mannose metabolism and the PTS. These pathways are crucial for the uptake and phosphorylation of sugars, further highlighting BF’s interference with the bacteria’s ability to utilize carbohydrates effectively25.

The impact of BF extended to amino acid metabolism, with significant enrichment in arginine biosynthesis (p = 0.0001839, 11 genes), valine, leucine, and isoleucine biosynthesis (p = 0.0298, 8 genes), and biosynthesis of amino acids (p = 0.03258, 5 genes). Arginine is not only essential for bacterial growth but also for the synthesis of virulence factors26. Additionally, the perturbation of pantothenate and CoA biosynthesis (14 genes), a pathway vital for energy production and fatty acid metabolism, suggests a further disruption of energy metabolism and cellular homeostasis27.

The influence of BF further extended to broader metabolic processes, including 2-oxocarboxylic acid metabolism (p = 0.000491, 13 genes), a pathway involved in the tricarboxylic acid (TCA) cycle, and amino sugar and nucleotide sugar metabolism (p = 0.021452, 9 genes), which are crucial for cell wall synthesis and biofilm matrix formation. It may be inferred that BF may weaken the bacterial cell wall and further impair biofilm formation. Additionally, the downregulation of 6 genes in the biosynthesis of secondary metabolites could affect the production of various virulence factors and signalling molecules.

KEGG pathway enrichment analysis of BF on S. mutans biofilms (Table 1) revealed a profound and diverse impact on bacterial metabolism when analysed with a stringent p value cutoff (p < 0.01). This stringent analysis demonstrated significant enrichment of pathways primarily involved in carbohydrate metabolism and transport, including starch and sucrose metabolism, the phosphotransferase system (PTS), and fructose and mannose metabolism. Beyond these, the analysis further highlighted the disruption of other core metabolic processes essential for bacterial growth and survival, such as glycolysis/gluconeogenesis, pyruvate metabolism, and various amino acid biosynthesis pathways (arginine, valine, leucine, and isoleucine). Additionally, broader cellular functions like the two-component system and ABC transporters were also significantly affected, underscoring BF’s comprehensive inhibitory effects on crucial bacterial metabolic and signalling networks.

Table 1 List of KEGG pathway enriched in BF-treated S. mutans.
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Effects of BF on denture plaque formation in vitro

To rigorously assess the anti-biofilm potential of BF, as indicated by prior in vitro bioassays12 and transcriptomic analyses, a standardized in vitro model utilizing acrylic denture substrates was implemented to simulate the oral cavity environment. Acrylic dentures, selected for their uniform and reproducible surface characteristics, offered a controlled platform for quantitative biofilm assessment. Consistent with established methodologies, mono-species bacterial cultures alone were insufficient to elicit robust biofilm formation. However, preconditioning the denture surfaces with artificial saliva, thereby mimicking the acquired pellicle, followed by bacterial culture inoculation, resulted in substantial biofilm development, achieving > 80% surface coverage within 48–72 h. This observation underscores the critical role of salivary glycoproteins in facilitating bacterial adhesion, a phenomenon corroborated by studies utilizing saliva-conditioned bovine enamel28.

The efficacy of BF and chlorhexidine (0.2%), a validated positive control, in mitigating denture biofilm accumulation was quantitatively evaluated (Fig. 7A). Digital image analysis and subsequent quantification of biofilm coverage revealed significant biofilm formation in the untreated control group, whereas BF treatment resulted in a statistically significant reduction in biofilm accumulation. Specifically, BF treatment elicited a 79% reduction in stained biofilm area compared to the control (p < 0.05) (Fig. 7B). While chlorhexidine exhibited the most pronounced inhibitory effect, BF demonstrated substantial anti-biofilm activity, validating its potential as a natural therapeutic agent for oral biofilm management. These findings are consistent with the previously observed anti-biofilm properties of BF in in vitro assays12 and transcriptomic analyses, and extend their applicability to a clinically relevant, acquired pellicle-mediated biofilm model. The capacity of BF to impede biofilm accumulation on denture substrates in the presence of an acquired pellicle underscores its potential for the development of novel anti-biofilm formulations for oral hygiene applications, demonstrating efficacy under conditions that closely approximate the oral environment.

Fig. 7
figure 7

Assessment of plaque deposition on dentures. (A) Representative images of denture surfaces after staining and digitization: Untreated (Negative control); BF-treated (3.75 mg/mL); Treated with chlorhexidine (0.2%) (Positive control). (B) Mean percentage plaque coverage on denture for each treatment group.

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The observed anti-biofilm activity of BF can be attributed to its complex phytochemical composition. Phyllocactin, the predominant betacyanin constituent, possesses potent antioxidant properties29, potentially disrupting biofilm development by scavenging reactive oxygen species (ROS), which are critical for biofilm matrix maintenance and bacterial signalling30. Furthermore, the structural diversity of BF, encompassing betanin, hylocerenin, and isobetanin, suggests a synergistic mechanism of action, enabling interactions with multiple bacterial targets and a comprehensive disruption of biofilm architecture. This hypothesis is supported by findings demonstrating the enhanced efficacy of red spinach betacyanin, particularly amaranthine, against Staphylococcus aureus11, underscoring the structure-dependent specificity of betacyanins in targeting diverse bacterial species. Betacyanins known to disrupt cellular membrane integrity, leading to a loss of pH gradient, decreased ATP levels, and disruption of the proton motive force, ultimately inducing bacterial cell death within the biofilm, which aligns with the observed reduction in biofilm formation and bacterial adhesion in the presence of BF11. The betalamic acid core structure may also play a crucial role in the anti-biofilm activity of betacyanins, potentially interacting with specific bacterial proteins involved in adhesion and quorum sensing, thereby disrupting biofilm formation and maintenance processes12.

Transcriptomic analyses further elucidated the mechanistic basis of these effects. BF exerted a pleiotropic impact on S. mutans metabolism, disrupting energy production at multiple metabolic nodes, including glycolysis/gluconeogenesis, pyruvate metabolism, and pantothenate and CoA biosynthesis. Furthermore, BF downregulated genes involved in amino acid metabolism, cell wall biosynthesis, and secondary metabolite production, all of which are critical for virulence and biofilm formation. These observations are consistent with the established paradigm that disruption of bacterial metabolism and cell wall integrity are pivotal strategies for inhibiting biofilm formation31. These findings are congruent with previous research on natural products for oral care, such as pomegranate extract and green tea extract, which have demonstrated anti-biofilm effects16,32,33. Notably, BF significantly reduced biofilm accumulation without inducing tooth discoloration, a common adverse effect associated with certain plant-based extracts, such as green tea. This absence of discoloration represents a significant advantage for oral hygiene applications, enhancing patient compliance and acceptance.

Conclusions

The present study demonstrates BF as a significant anti-biofilm agent against S. mutans, a key etiological agent in dental plaque formation. Stringent transcriptomic analysis (p < 0.01) revealed that BF exerts a broad inhibitory effect by targeting essential bacterial processes, including energy metabolism, cell wall integrity, and multiple stress response pathways. Specifically, BF disrupts glycolysis and the tricarboxylic acid (TCA) cycle, both of which are crucial for ATP production and bacterial survival, ultimately leading to diminished bacterial growth and biofilm formation. Furthermore, BF weakens cell wall integrity by downregulating genes involved in teichoic acid and peptidoglycan biosynthesis, thereby compromising the structural integrity of the biofilm. The ability of S. mutans to withstand environmental challenges within the biofilm is also compromised by BF. These findings were corroborated by in vitro anti-plaque assays, which demonstrated a significant reduction in plaque accumulation on acrylic dentures following BF treatment.

The identification of specific genes and pathways affected by BF provides insights into its mechanisms of action, highlighting its potential as a clinically relevant and safe alternative to conventional anti-plaque agents. While further validation, such as qRT-PCR for key DEGs, would enhance these findings, the robust evidence from transcriptomic and in vitro analyses underscores the promise of betacyanin as a natural therapeutic agent to combat oral biofilms. Although this investigation utilized a highly valuable mono-species S. mutans biofilm model for focused mechanistic studies, it is acknowledged that this model does not fully replicate the complex, multispecies interactions, dynamic flow, and diverse nutritional environment characteristic of in vivo oral biofilms. This work offers a strong foundation for future research aimed at elucidating precise molecular interactions and optimizing BF’s use in promoting oral health.