Abstract
Background
Dental caries, a bacterial infectious disease related to biofilm, occurs due to an increase in the proportion of cariogenic bacteria over that of commensal bacteria. This study aimed to create an in vitro biofilm model consisting of S. sanguinis, S. gordonii, and S. mutans to study the effects of Berberine chloride hydrate (BH) on the structure and cariogenic features of multispecies biofilms.
Materials and methods
The biofilm formation and cariogenic characteristics of multispecies streptococci biofilms were observed. The microbial composition of multispecies biofilms was quantified using fluorescent in situ hybridization (FISH). Besides, transcriptome analysis was performed to determine the differential expression levels of genes in each strain in multispecies biofilms with or without BH. The demineralization activity of biofilms treated with BH was analyzed using micro-CT.
Results
BH had the highest efficacy against S. mutans, exhibiting lower minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) values relative to S. sanguinis and S. gordonii. BH inhibited the biofilm formation as well as EPS synthesis in multispecies biofilms, resulting in decreased demineralization on bovine enamel surfaces. In addition, after BH treatment, the abundance of S. sanguinis and S. mutans was reduced, whereas the abundance of S. gordonii increased.
Conclusion
BH revealed great potential for the management of cariogenic biofilm, indicating its possible application in dental caries therapy.
Keywords: Streptococcus gordonii, Streptococcus mutans, Streptococcus sanguinis, biofilms, Berberine chloride hydrate
Key messages
BH had the highest efficacy against S. mutans, exhibiting lower MIC and MBIC values relative to S. gordonii and S. sanguinis.
BH inhibited the biofilm formation as well as EPS synthesis in multispecies biofilms. The demineralizing activity at the biofilm/enamel interface was reduced in BH-treated multispecies biofilms.
BH changed the composition of multispecies biofilms, with S. gordonii outcompeted S. sanguinis and S. mutans.
Introduction
Dental caries, a bacterial infectious disease related to biofilm, arises from biofilm dysbiosis and causes economic and psychological burdens on society [1]. The Global Burden of Disease Study 2017 reported that there were 3.5 billion instances of oral diseases globally, with 2.3 billion of these cases being untreated caries in permanent teeth [2]. Dental plaque is a biofilm that includes diverse bacteria and an extracellular matrix [3]. In general, plaque biofilms contain a mix of pathogenic and non-pathogenic bacteria, with intricate interactions and competition among the microbial communities [4]. Biofilms typically maintain an ecological balance among their different species under healthy conditions [5], which can be disrupted by environmental influences and interactions between species, leading to a shift in the proportion of acidogenic and aciduric bacteria, such as Streptococcus mutans (S. mutans) [6]. A higher concentration of cariogenic bacteria in plaque biofilm enhances the synthesis of the extracellular matrix and acidifies the microenvironment, which results in enamel demineralization and caries formation [7,8]. Inhibiting the colonization and virulence of cariogenic bacteria is essential for the prevention of dental caries [9].
Dental plaque formation begins with oral streptococci, characterized by a specific timing and spatial arrangement crucial for dental biofilm development [10]. As is known, S. mutans is identified as a primary cariogenic bacterium causing dental caries, attributed to its acidogenic and aciduric properties, in addition to its capacity to create exopolysaccharides (EPS) that promote microbial attachment to teeth [6]. Once S. mutans initially attaches to the tooth surface, it develops microcolony structures surrounded by EPS [11]. The development of caries is driven by these microcolonies, which concentrate organic acids near the tooth surface, initiating demineralization [12,13]. Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis) are commonly found on caries-free tooth surfaces [14]. S. sanguinis and S. gordonii, prevalent inhabitants of the human oral cavity, are correlated with a decreased risk of dental caries and reduce caries risk by using an active arginine deiminase system to metabolize arginine [15]. Similarly, all three of these streptococcal strains are involved in the formation of dental plaque biofilm [16,17]. The biofilm model consisting of three species of streptococci is extensively utilized in caries studies [18,19]. S. sanguinis, S. mutans and S. gordonii have a relationship characterized by competition and antagonism, where an increase in the abundance of one species correlates with a decrease in the levels of the others [20]. During biofilm formation, S. sanguinis and S. gordonii compete with S. mutans for adherence to locations and nutrients, thus hindering S. mutans growth and preserving microbial equilibrium [21]. Thus, the intricate microecology and environment of plaque biofilm highlight an urgent need to find effective antibacterial agents. To manage dental caries ecologically, agents should selectively target cariogenic bacteria such as S. mutans, leaving the rest of the oral microbiome unaffected [22].
Recently, there has been growing interest in using natural compounds from plants to influence the initial phases of biofilm development, presenting a promising alternative approach [23]. Natural products, such as ursolic acid derived from berries, privet leaves, and paulownia leaves [24], curcumin derived from turmeric roots [25], and celastrol derived from the Tripterygium wilfordii plant [26], have significant anti-biofilm properties against multispecies biofilms. Berberine chloride hydrate (BH), obtained from the Chinese herb Coptis chinensis, has been utilized extensively within the traditional Chinese medical system for centuries [27]. Berberine has garnered significant global interest owing to its diverse molecular targets, safe clinical application, low toxicity at therapeutic doses, and its compatibility with numerous pharmaceutical drugs [28]. The chloride or sulfate salt of berberine is generally used for clinical purposes [29]. Berberine has been proven effective in both removing microbial biofilms and enhancing the effects of antibiotics [23]. In our earlier research [30], BH demonstrated effective anti-biofilm properties against S. mutans biofilm formation. However, to our knowledge, the antimicrobial and microecological effects of BH on multispecies biofilms of S. sanguinis, S. gordonii, and S. mutans have not been reported.
In this study, we created an in vitro model of a multispecies biofilm with S. sanguinis, S. mutans, and S. gordonii, examining the role of BH in altering the biofilm's composition and its cariogenic potential. Additionally, this study aimed to investigate the role of BH within an in vitro enamel demineralization model and to preliminarily elucidate the mechanisms underlying the modulation of multispecies biofilm formation mediated by BH.
Materials and methods
Bacterial strains, chemicals and compounds
Berberine chloride hydrate (BH) (99.82%) [31] was dissolved in distilled water to create a 1024 μg/mL solution and stored at −80 °C. BH was serially diluted two-fold to the concentrations of 512, 256, 128, 64 and 32 μg/mL. S. sanguinis (ATCC 10556), S. mutans UA159 (ATCC 700610), and S. gordonii (DL-1) were obtained from the lab. The bacteria were grown in brain heart infusion (BHI; Difco, Detroit, MI, USA) under anaerobic conditions at 37 °C.
Biofilm cultivation
S. sanguinis, S. gordonii, and S. mutans were grown anaerobically in BHI broth overnight and then diluted to 2 × 106 CFU/mL in 1% BHIS (BHI medium added with 1% sucrose) before the experiment.
For mono-species biofilm formation, bacteria were grown in 1% BHIS at a final concentration of 1 × 106 CFU/mL.
For multispecies biofilm formation, each species with 1:1:1 volume was mixed in 1%BHIS with or without BH to obtain a final concentration of 1 × 106 CFU/mL.
All the biofilm assays were performed under anaerobic conditions. Specifically, the culture plates were incubated inside an anaerobic workstation (Mart, MART AJ9023) continuously gassed with a mixture of 85% N₂, 10% CO₂ and 5% H₂. Bacteria cultured in broth without treatment served as the negative control. Chlorhexidine (CHX) served as the positive control for crystal violet staining.
Bacterial sensitivity analysis
Overnight bacterial cultures were standardized to 2 × 106 CFU/mL with BHI, then added with equal quantities of BH ranging from 512 μg/mL to 32 μg/mL, and incubated anaerobically at 37 °C for 24 h. The negative control consisted of their respective solvent with BHI broth. Subsequently, the OD 600 measurements were taken using a spectrophotometer (Tecan, Reading, Switzerland).
Biofilm susceptibility assays
The impact of BH on the development of single-species and multi-species biofilms was assessed using crystal violet staining. In short, biofilms were established as detailed above. After 24 h, the media was disposed out, and the dishes were rinsed with PBS. Then, methanol was added for 15 min, and the mixture was dried for 30 min. The biofilms underwent staining for 15 min with crystal violet (0.1%), followed by dissolving the dye in 95% ethanol. The absorbance was finally read at 595 nm using a spectrophotometer.
We further modified the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay [31] to assess the mature biofilm metabolic activity. Biofilms were formed by anaerobically incubating bacterial cultures with 1% BHIS at 37 °C for 24 h. Following the removal of the media, BH was applied to the plates. Twenty-four hours later, the supernatant of the plate was eliminated, and MTT solution (0.5 mg/mL) was added and incubated for 1 h at 37 °C under dark. After removing the supernatants, followed by the addition of 100% DMSO, the samples were then incubated under dark for 20 min. Finally, a measurement of the absorbance at 540 nm was taken.
Biofilm imaging
Multispecies biofilms were formed as described above, and their respective solvents with broth were used as the negative control.
For scanning electron microscope (SEM) analysis, the biofilm was cultivated for 24 h at 37 °C, then washed twice with PBS after removing the supernatant, followed by the fixation of the biofilm with 2.5% glutaraldehyde for 12 h. After that, a graded ethanol series was used to dry the biofilms. After applying gold sputtering, the coverslips were analyzed with an SEM (ZEISS, Oberkochen, Germany).
EPS staining involved the addition of 2.5 μM Alexa Fluor 647-labelled dextran when the biofilm was initiated. Following a 24-h period, the supernatant was extracted, and distilled water was used to clean the plates. Afterwards, the sample was stained with 2.5 μM SYTO9 green dye for 15 min in the dark.
For dead/live staining, following the formation of a 24 h-old biofilm, the medium was removed, and the wells were washed using distilled water. Subsequently, the dishes were then filled with an equal volume mixture of SYTO9 and propidium iodide (Invitrogen, USA) and left in darkness for 15 min.
For fluorescent in situ hybridization (FISH) staining [32], biofilms were initially exposed to 4% paraformaldehyde for 12 h. Subsequently, after drying at 46 °C, the biofilms were added with a lysozyme solution (30mg/mL) for 20 min at 37 °C. Next, biofilms underwent a stepwise dehydration process with 50%, 80% and 100% ethanol, followed by drying for 10 min at 46 °C. The biofilms were subsequently incubated in the dark for 90 min at 46 °C using a hybridization buffer with 2 nM of each designated probe (Table S1). Then, the warmed wash solution was used to wash the biofilm at 48 °C for 15 min.
An LSM 980 (Zeiss, Oberkochen, Germany) was employed to observe all the biofilm images listed above with a 20 × oil immersion lens. And the 3D structures of biofilms were quantitatively analyzed utilizing Image J COMSTAT software.
Quantitative evaluation of water-insoluble EPS
As described earlier, multispecies biofilms were established. After rinsing the pellet twice with PBS, it was centrifuged for 10 min at 4,000 × g, then dissolved in 0.1 M NaOH and kept at 37 °C for 2 h. The EPS precipitation was obtained by passing the supernatant through a 0.22 μm nitrocellulose membrane filter after centrifugation for 10 min. After incubating the mixture overnight with three volumes of pre-chilled 95% ethanol, the supernatant was subsequently combined with 5% phenol and concentrated sulfuric acid (H2SO4) at a volumetric ratio of 1:1:5. Next, the microplate reader was employed to determine the absorbance at 492 nm.
Determining lactic acid and pH values
Following 24 h of biofilm establishment, the biofilms were washed using PBS. Then, each well received 1.5 ml of buffered peptone water (BPW) with 0.2% sucrose, and the plate was subsequently incubated at 37 °C for 3 h with 5% CO2 to produce acid. A lactate assay kit (Jiancheng, Nanjing, China) was used to examine the supernatants, and the rate of absorption was measured at 530 nm.
A pH electrode (Mettler Toledo, Zurich, Switzerland) was used to measure the pH of the supernatant from multispecies biofilms at 3, 6, 12 and 24 h.
Transcriptome analysis
Biofilms were grown with or without BH, as previously described. The total RNA was extracted as previously described [30]. Enriched mRNA samples were used to produce cDNA libraries via the TruSeqTM RNA sample preparation kit (Illumina, San Diego, CA). A second-generation high-throughput sequencing platform was used to pool and sequence different libraries. For further analysis of gene expression patterns, genes with a fold change exceeding 2.0 and a P-value under 0.05 were selected.
Micro-CT scanning and analysis
Bovine enamel specimens were prepared as described above [31]. The incisor teeth from bovines were sectioned into 5 mm × 5 mm pieces with a 3 mm thickness using a hard tissue microtome (EXAKT 300CP, Germany). The silicon carbide sandpaper with grits of 600, 800, 1500 and 2000 was used to polish the enamel blocks.
The blocks were treated with sterile artificial saliva (Leagene, Beijing, China) for 2 h. The blocks were processed with 0.9% sterile saline before being transferred to fresh plates containing BH and BHI for a 24-h incubation period. This process was repeated daily for 7 days. After the biofilms were eliminated by sonication, the blocks were washed with PBS. The micro-CT (µCT 50, Scanco Medical AG, Switzerland) was employed to measure the mineral density and demineralization depth in five randomly selected 50 µm × 50 µm areas, using Mimics Research 20.0.
Statistical analysis
Every experiment was carried out separately three times. GraphPad Prism version 9.0 was used for data analysis. All the data were first assessed for normality using the Shapiro–Wilk test (P > 0.05 was considered to indicate a normal distribution). To assess significant differences across various samples, a one-way ANOVA and Tukey's post hoc test were applied, considering a P-value below 0.05 as statistically significant.
Results
Berberine chloride hydrate (BH) showed different antimicrobial effects against three streptococcal species
BH exhibited different bactericidal activity against S. sanguinis, S. mutans, and S. gordonii planktonic cells (P < 0.01, Fig S1). For S. mutans, the MIC of BH was 64 μg/mL. For S. sanguinis and S. gordonii, the minimum inhibitory concentration (MIC) of BH was 256 μg/mL and 128 μg/mL, respectively. Among the diverse strains, BH demonstrated lower MIC values and demonstrated greater efficacy against S. mutans.
BH showed good antimicrobial activity on multispecies streptococci biofilms
To visualize the influence of BH on multispecies streptococcal biofilm, crystal violet staining was conducted. As shown in Figure 1A (P < 0.05), BH exhibited different bactericidal activity against S. sanguinis, S. mutans, and S. gordonii biofilm. In the presence of 128 μg/mL, 256 μg/mL, 64 μg/mL, and 128 μg/mL BH, respectively, the biomass of S. sanguinis, S. gordonii, S. mutans, and their multi-species biofilms were significantly reduced. The effects of CHX on the multispecies streptococci biofilms are shown in Figure S2. Similarly, BH demonstrated greater efficacy in preventing S. mutans biofilm formation, with lower minimum biofilm inhibitory concentration (MBIC) values compared to the other two bacteria. Besides, we used the MTT assay to explore the impact of BH on established biofilms. As shown in Figure 1B (P < 0.05), the treatment with BH (256 μg/mL) began to decrease the metabolic activities markedly in pre-established biofilm among these three streptococcal species and multispecies streptococcal biofilms.
Figure 1.
BH suppresses multispecies streptococcal biofilms. (A) Effect of BH on S. mutans, S. sanguinis, S. gordonii biofilms, along with their cariogenic multispecies biofilms. (B) Effect of BH on S. mutans, S. sanguinis, S. gordonii mature biofilms, along with their mature multispecies biofilms. *P < 0.05, **P < 0.01, ***P < 0.001.
The SEM analysis consistently demonstrated that the treatment with BH (128 μg/mL) significantly decreased the total biomass of the multispecies biofilm (Figure 2A). The dead/live bacteria ratio (P < 0.05, Figure 2B and C) was elevated, and the thickness of the biofilm (P > 0.05, Figure 2B and D) was decreased after 128 μg/mL of BH was applied, as indicated by fluorescent staining. These findings were consistent with the biofilm development assay results.
Figure 2.
The cariogenic characteristics of multispecies biofilms were inhibited by BH. (A) The representative SEM images of multispecies streptococcal biofilms. (B) The typical multispecies streptococcal biofilm CLSM pictures. (C) The proportion of dead to live bacteria in the biofilms. (D) The thickness of the biofilms. *P < 0.05, **P < 0.01, ***P < 0.001.
BH reduced the cariogenic characteristics of multispecies streptococcal biofilms
The synthesis of EPS and the production of lactic acid in multispecies biofilms were quantified to assess the impacts of BH on the cariogenic properties of these biofilms. To further explain the effects of BH on EPS production, the CLSM results revealed that, when provided with 64 μg/mL of BH and 128 μg/mL of BH, the EPS synthesis of the biofilm was suppressed compared to that of the control (P < 0.05, Figure 3A and B). Thus, the ratio between EPS and bacteria was reduced following treatment with BH at 64 μg/mL and 128 μg/mL (P < 0.001, Figure 3A and C). Consistently, the phenol/H2SO4 assay demonstrated that BH at a concentration of 128 μg/mL greatly decreased the water-insoluble EPS of multispecies biofilms (P < 0.001, Figure 3D). Additionally, at 128 μg/mL, BH also significantly lowered the production of lactic acid (P < 0.001, Figure 3E) and decreased the pH values (Figure 3F) relative to those of the control group.
Figure 3.
The cariogenic characteristics of multispecies streptococcal biofilms were inhibited by BH. (A) The representative CLSM images of multispecies streptococcal biofilms. (B) Quantitative measurement of bacteria and EPS present in the biofilms. (C) The percentage of EPS compared to bacteria in the biofilms. (D) Quantitative measurement of water-insoluble EPS in biofilms. (E) Lactic acid production of multispecies biofilm. (F) pH values of the culture mediums in multispecies biofilm. *P < 0.05, **P < 0.01, ***P < 0.001.
BH altered the microbiota composition of the multispecies streptococcal biofilm
As shown in Figure 4, BH treatment caused a decrease in the biomass of S. sanguinis and S. gordonii, and S. mutans within multispecies biofilm (P < 0.05, Figure 4A and B). Furthermore, when compared to the control group, BH treatment at 128 μg/mL significantly reduced the relative abundance of S. mutans and S. sanguinis (Figure 4C). However, an increase in the relative abundance of S. gordonii was observed following treatment with 128 μg/ml of BH (Figure 4A and C), suggesting that BH selectively targets S. mutans and S. sanguinis within multispecies streptococcal biofilms.
Figure 4.
BH alters the microbiota composition of multispecies streptococcal biofilms. (A) FISH images representing multispecies biofilms. (B) Measurement of the levels of S. gordonii, S. mutans, and S. sanguinis in multispecies biofilms. (C) Assessment of the proportions of S. gordonii, S. mutans, and S. sanguinis in multispecies biofilms. *P < 0.05, **P < 0.01, ***P < 0.001.
Transcriptome analysis of the BH-treated multispecies streptococcal biofilm
The BH-treated group and the control group were clearly differentiated, according to principal component analysis (PCA) (Figure S3). The differentially expressed genes (DEGs) of S. gordonii, S. sanguinis, and S. mutans were identified in both the BH-treated group and the control group. In the BH-treated S. mutans strain, 371 genes were significantly downregulated, and 271 genes were upregulated compared to the controls. For S. sanguinis, 134 genes exhibited upregulated expression, and 54 genes were downregulated. Additionally, 302 genes had downregulated expression and 497 genes had increased expression for S. gordonii (Figure 5A, Table S2). To learn more about the biological effects of BH on multispecies streptococcal biofilms, we performed GO annotation enrichment of DEGs. As shown in Figure S4, the differential genes of the three bacteria were more consistently distributed in the three categories of biological processes (BP), cellular components (CC) and molecular functions (MF). Among them, the significantly changed genes were mainly involved in BP categories, such as cellular process and metabolic process. In the CC category, the genes of the cell and cell parts were significantly changed. In the MF category, the significantly changed genes were involved in the catalytic and binding processes.
Figure 5.
Transcriptome analysis of the BH-treated multispecies streptococcal biofilms. (A) Volcano plot showing genes with differential expression. (B) KEGG enrichment analysis.
Based on the KEGG annotation analysis (Figure S5), the significantly altered genes were primarily engaged in metabolism pathways, especially carbohydrate metabolism, among the three strains. Further KEGG enrichment analysis (Figure 5B) showed that the ribosome was enriched for significantly changed genes among all of the three strains. In addition, carbon metabolism in S. mutans and aminoacyl-rRNA biosynthesis in S. sanguinis were also enriched with significantly changed genes.
BH attenuated the biofilm-induced demineralization of tooth enamel
The ability of oral biofilm to demineralize is linked to its potential to cause caries. Therefore, we developed a biofilm from saliva on bovine tooth enamel and utilized micro-CT to examine how BH influences the demineralization process facilitated by the biofilm. Then, we created multispecies streptococcal biofilm on bovine tooth enamel. We found that the lesion depth (P < 0.05, Figure 6A and B) was lower and the mineral densities in demineralized areas (P > 0.05, Figure 6A and C) were higher after BH treatment compared to the control group.
Figure 6.
BH prevents the multispecies streptococcal biofilm-induced demineralization of bovine tooth enamel. (A) Micro-CT images of bovine tooth enamel. (B) The measured depths of demineralization (n = 9). (C) The measured mineral densities (n = 9). *P < 0.05.
Discussion
Dental plaque biofilm is crucial for the initiation of dental caries. It is now understood that caries result from an imbalance in microecology, characterized by the development of multispecies biofilms with a predominance of pathogenic bacteria [33]. Recently, the ecological plaque theory has stated that microbial disequilibrium is more closely linked to dental caries [1,9]. Oral biofilms containing well-balanced oral cariogenic streptococci (e.g. S. mutans) and antibacterial compounds-producing (e.g. S. gordonii) are not cariogenic [14,34]. In caries studies, the multispecies biofilm model, which consists of pathogenic bacteria such as S. mutans and commensal bacteria such as S. sanguinis and S. gordonii, has been widely applied [7,32,35]. Consequently, the present study focused on assessing the impact of BH on a multispecies biofilm containing S. sanguinis, S. mutans and S. gordonii.
The present study indicates that BH exerts a better inhibitory effect on the planktonic growth and biofilm formation of S. mutans compared to S. sanguinis and S. gordonii. The inhibitory trends of BH were consistent with those of CHX, suggesting that BH has a stronger inhibitory effect on oral cariogenic bacteria than on oral commensal bacteria. Besides, BH at a concentration of 128 μg/mL, which exceeds the level needed to inhibit S. mutans formation, can significantly inhibit multispecies biofilm formation. This demonstrates that multispecies biofilms may be more resistant to BH than single-species biofilms. Moreover, our findings showed that BH inhibited the planktonic growth of S. gordonii without impeding its biofilm formation. We therefore hypothesize that the inhibitory effect of BH on biofilm formation in various bacterial species may not be directly linked to bacterial proliferative activity. Besides, we found that BH can also inhibit the metabolic activity of mature multispecies biofilms, but the influence of BH on biofilm eradication is weaker than its effect on inhibiting biofilm formation.
Oral biofilm formation involves multiple steps, including bacterial adherence, EPS generation, and biomass accumulation [36]. EPS can contribute to dental caries via enhancing microbial adhesion, producing a 3D framework that limits diffusion, supplying fermentable sugars, and establishing acidic conditions [37,38]. In our study, both the EPS and the EPS-to-bacteria ratio were significantly reduced in this study when BH was added, along with a disruption of biofilm structure, when compared to the control group. These results indicate that the bacteria would decrease EPS synthesis to survive when they were under tremendous stress.
Managing the balance among oral streptococci to promote beneficial bacteria while suppressing pathogenic bacteria could be an effective strategy for maintaining oral microecology and preventing cavities [26,39,40]. The shift in microflora composition will reflect their propensity to develop in relation to health conditions or caries risk [19,41]. The S. mutans that arises in multispecies biofilms usually indicates increased acidogenicity, which may lead to caries [42,43]. Dental health is typically connected with a higher abundance of commensal early colonizers, including S. sanguinis and S. gordonii [44,45]. In the current study, the composition of multispecies biofilms changed, with S. gordonii outcompeting S. sanguinis and S. mutans when BH (128 μg/mL) was provided. Lyu et al. [24] showed that ursolic acid, a natural compound found in various plants, decreased the proportion of S. mutans in mixed-species biofilms while significantly increasing the proportion of non-cariogenic bacteria, aligning with our findings. Nobbs et al. [34] revealed that S. gordonii exhibits a naturally higher competitiveness in interspecific antagonism concerning binding site occupation. Furthermore, S. gordonii exerts a weak inhibitory effect on the growth of S. sanguinis [46]. We hypothesize that the composition changes in multispecies biofilms might result from interactions between different species as well as the conditions imposed by antibacterial agents. When treated with BH, S. mutans was significantly inhibited, while S. gordonii acquired a relative competitive advantage over S. sanguinis and became dominant. Above all, we speculate that BH enhanced the peroxidase-producing capabilities of S. gordonii within multispecies biofilms, resulting in a microbial consortium with improved competitive advantages against the opportunistic proliferation of S. mutans, thereby contributing to caries management.
To further investigate the impact of BH on the gene expression levels of each strain in multispecies biofilms, the transcriptome analysis of a single strain within multispecies biofilms was performed, respectively, with or without BH treatment. We found that BH treatment led to an elevation in the downregulated genes of S. mutans and an increase in the upregulated genes of other commensal streptococci in mixed biofilms. These findings indicate that BH can significantly inhibit S. mutans gene expression in multispecies biofilms, further suggesting its good clinical application potential. Further analysis revealed that BH can interfere with carbohydrate metabolism and composition. Numerous oral bacteria exhibit saccharolytic properties and can efficiently absorb substantial quantities of glucose derived from the host's diet or other carbohydrates. Elevated glucose levels in the surrounding area alter the metabolic products of commensal species, leading to the production of more potent organic acids such as lactate [47]. Combined with our results, we found that the lactic acid production of mixed biofilms was decreased when treated with BH, which suggests that the decrease in lactic acid production within multispecies biofilms may be caused by affected carbohydrate metabolism via BH. The ribosome, a critical component of genetically programmed protein synthesis, serves as a key target for antibiotics. A diverse array of chemically distinct compounds inhibits bacterial proliferation by interfering with protein synthesis through targeting the ribosome [48]. Based on our results, BH may modulate ribosome function in multiple bacterial species within multispecies biofilms, highlighting the need for further studies to elucidate its effects on ribosome function and the underlying molecular mechanisms.
To assess the demineralizing activity of BH-treated biofilms, the mineral density in demineralized areas was measured. Micro-CT is widely recognized as the gold standard for in vitro caries validation because of its non-destructive, 3D imaging of lesion depth [49]. Following earlier research, a biofilm enamel model was utilized to examine how BH affects the cariogenic potential of biofilm [50]. Furthermore, because bovine teeth are readily available and have a similar structure to human teeth, enamel samples from cows were employed in this study [51]. Our results showed that the demineralizing activity at the biofilm/enamel interface was reduced in BH-treated multispecies biofilms.
Our study has certain limitations. Currently, the mechanisms by which BH inhibits multispecies biofilms have only been preliminarily investigated through transcriptomic sequencing. Further research is required to elucidate these mechanisms comprehensively. Furthermore, future in vivo experiments on BH could enhance its potential for clinical application.
Conclusions
In summary, this research revealed that BH prevented biofilm formation as well as EPS synthesis in multispecies biofilms composed of S. sanguinis, S. gordonii, and S. mutans, altered its microbial structure. The demineralizing activity at the biofilm/enamel interface was reduced in BH-treated multispecies biofilms. Above all, BH revealed great potential for the management of cariogenic biofilm, indicating its possible application in dental caries therapy.
Supplementary Material
Funding Statement
This study was supported by the National Natural Science Foundation of China (No. 81970928).
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
All data generated or analyzed during this study are included in this article.
Ethics approval
Not applicable.
Supplementary material
Supplemental data for this article can be accessed at https://doi.org/10.1080/20002297.2026.2732737.
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