Efficacy of hyaluronic acid on the inhibition of periodontal bridging organisms Fusobacterium nucleatum and Prevotella intermedia: An in vitro study

Abstract

Objective:

This in vitro study aimed to evaluate the antimicrobial effects of high-molecular-weight hyaluronic acid (HA) against two key periodontal bacteria, Prevotella intermedia and Fusobacterium nucleatum. These organisms play an important role in biofilm formation and disease progression in periodontitis.

Materials and Methods:

Standard laboratory strains of P. intermedia (ATCC 25611) and F. nucleatum (ATCC 25568) were cultured in anaerobic conditions. HA with different molecular weights (141 kDa, 757 kDa, and 1,300 kDa) was tested at concentrations from 0.5 to 2.0 mg/ml. The minimum inhibitory concentration was determined through serial dilution in thioglycollate broth. Each test was performed in triplicate. Turbidity after 48–72 h of incubation served as an indicator of bacterial growth. Statistical significance was analyzed using G*Power with α = 0.05 and 80% power.

Results:

HA showed concentration-dependent bacteriostatic effects. P. intermedia showed growth inhibition at 1 mg/ml, whereas F. nucleatum required 2 mg/ml for similar inhibition. Concentrations below these amounts did not inhibit growth. These findings suggest that HA may disrupt adhesion and nutrient uptake needed for bacterial growth and biofilm formation.

Conclusion:

High-molecular-weight HA shows potential as a nonantibiotic support agent in periodontal therapy by selectively inhibiting key anaerobic bacteria involved in biofilm development. Its two actions, antimicrobial and immunomodulatory, highlight its importance by breaking down pathogenic cooperation and supporting periodontal healing. Further research with multispecies biofilms, clinical isolates, and host interaction models is necessary for clinical application.

INTRODUCTION

Periodontitis is a chronic inflammatory disease that destroys the gingiva, periodontal ligament, and alveolar bone, driven primarily by a dysbiosis within the subgingival microbial community.[1] This dysbiosis promotes the formation and maturation of pathogenic biofilms that play a central role in the progression of periodontal tissue destruction.[2] The structured development of multispecies biofilms depends on key microbial interactions, including cell-cell adhesion, nutrient sharing, and spatial organization.[3] Hyaluronic acid (HA), a naturally occurring glycosaminoglycan found throughout connective tissues, is known for its physiological roles in hydration, matrix stability, and tissue turnover.[4] Its biological relevance has expanded into periodontal therapy, where HA demonstrates antimicrobial, anti-inflammatory, and wound-healing properties.[5]

Among the many microorganisms involved in periodontal infections, Prevotella intermedia and Fusobacterium nucleatum are of high significance because they serve as bridging organisms that facilitate coaggregation between early and late colonizers in dental plaque.[6] These organisms help microbial clusters attach and mature, enhancing biofilm stability and enabling colonization by more pathogenic anaerobes.[7] This bridging function accelerates periodontal destruction by promoting the survival and pathogenicity of late colonizers.[8]

Traditional periodontal therapies, including scaling, root planning, and adjunctive antimicrobials, often fail to fully eliminate subgingival biofilms, especially in deep pockets or anatomically complex areas.[9] Targeting bridging organisms such as P. intermedia and F. nucleatum, therefore, provides a strategic opportunity to disrupt biofilm integrity, hinder microbial succession, and potentially slow disease progression.[10]

However, despite the documented antibacterial and immunomodulatory potential of HA, along with its regenerative capacity, little is known about the effect of high-molecular-weight pure HA on the growth and biofilm activity of these bridging pathogens.[11] Studies that have investigated the potential effects of HA on P. intermedia and F. nucleatum in vitro could provide new insights into nonantibiotic approaches to managing periodontal infections and improving regenerative results. Most prior research on HA has targeted early colonizers such as Streptococcus mutans and Streptococcus sanguinis;[12] our study targets the bridging organisms that are essential to biofilm maturation and thus have not been previously studied in relation to HA.

MATERIALS AND METHODS

ATCC strains of P. (ATCC 25611) and F. nucleatum (ATCC 25568) were obtained from HiMedia Mumbai, India. Recombinant HA of low (141 kD), medium (757 kD), and high (1300 kD) molecular weight was suspended and dissolved in sterile distilled water at the concentrations of 0.5, 1.0, and 2.0 mg/mL. Three trials were conducted for each bacterial strain. Each trial consisted of 10 determinations: one for each concentration of each molecular weight HA preparation and one control without any HA.

Statistical analysis

A power analysis was established by G*power, version 3.0.1 (Franz Fauluniversität, Kiel, Germany). Three replicates of each sample would yield 80% power to detect significant differences, assuming an effect size of 0.30.

Sample size estimation

A power analysis was established by G*power, version 3.0.1 (Franz Fauluniversität, Kiel, Germany). A sample size of six samples (three samples per group) would yield 80% power to detect significant differences, assuming an effect size of 0.30 and a significance level of 0.05.

Analysis

A priori: compute required sample size

Input: Number of groups. =2

Number of measurements = 3

Output: Total sample size = 6

As the sample size estimation is referenced from the master article (Bacteriostatic Effects of HA), it is therefore justified.

RESULTS

The assessment of the viability of the bacterial strains was done using the minimum inhibitory concentration test. Various concentrations of HA, ranging from 0.08% to 4 mg/ml, were tested on both organisms, P. intermedia and F. nucleatum. The two organisms showed sensitivity at different concentrations. F. nucleatum was sensitive at a concentration of 2 mg/ml, but when the concentration was lowered to between 1 mg/ml and 0.0075 mg/ml, it exhibited resistance, as shown in Table 1, Graph 1, and Figure 1. When P. intermedia) was exposed to HA at a concentration of 1 mg/ml, it was sensitive. However, when we used concentrations between 0.5 mg/ml and 0.0075 mg/ml, P. intermedia showed resistance, as shown in Table 1, Graph 2, and Figure 2. Thus, growth inhibition caused by HA on the two organisms revealed its bacteriostatic effect.

Table 1.

Antibacterial activity of hyaluronic acid test group against standard strains of Prevotella intermedia and Fusobacterium nucleatum

Pi – Prevotella intermedia; Fn – Fusobacterium nucleatum; mg/mL – milligram per milliliter; B – Broth; C – Control; O – Negative

Graph 1.

Fusobacterium nucleatum showing sensitivity at a concentration of 2 mg/ml. Fn – Fusobacterium nucleatum; mg/ml – Milligram per milliliter; B – Broth; C – Control; O – negative

Figure 1.

Minimum inhibitory concentration for Fusobacterium nucleatum at a concentration of 2 mg/mL. MIC – Minimum inhibitory concentration; Fn – Fusobacterium nucleatum; mg/ml – Milligram per milliliter

Graph 2.

Prevotella intermedia showing sensitivity at a concentration of 1 mg/ml. Pi – Prevotella intermedia; mg/ml – Milligram per milliliter; B – Broth; C – Control; O – negative

Figure 2.

Minimum inhibitory concentration for Prevotella intermedia at a concentration of 1mg/ml. MIC – Minimum inhibitory concentration; Pi – Prevotella intermedia; mg/ml – Milligram per milliliter

DISCUSSION

HA is a nonsulphated, high-molecular-weight glycosaminoglycan that is gaining attention for its potential to fight infections in periodontal therapy. This interest stems from its biocompatibility, anti-inflammatory effects, and ability to inhibit bacterial growth.[8] The current study showed that pure, high-molecular-weight HA inhibits two important periodontal pathogens, P. intermedia and F. nucleatum, in a concentration-dependent manner. The growth of P. intermedia was inhibited at 1 mg/mL, while F. nucleatum showed similar inhibition at 2 mg/mL. These results align with earlier research indicating that HA does not kill bacteria but can slow their growth by interfering with vital cellular processes.

Regarding Aggregatibacter actinomycetemcomitans, other studies have reported HA’s ability to inhibit this pathogen, especially at medium molecular weights and higher concentrations. This suggests that HA could reduce colonization by this pathogen, which is known to cause aggressive periodontitis. Similarly, Tannerella forsythia, a late-colonizing organism with strong protease and hemagglutinin activity, may be vulnerable to HA’s ability to prevent adhesion, although specific research on this is limited. Drawing from Pavesio et al. (1998), who found that HA coatings lower the adhesion of Staphylococcus epidermidis, we can reasonably suggest that similar steric and electrostatic effects reduce the adherence of Tannerella forsythia and Aggregatibacter actinomycetemcomitans to host and material surfaces.

Research by Pirnazar et al.[1] supports the idea that HA’s ability to fight bacterial changes depending on its molecular weight and concentration. While their study did not focus explicitly on P. intermedia or F. nucleatum, it provided a broader understanding of HA’s selective inhibition of Gram-negative anaerobes, which this study builds upon.[1]

In addition, Alharbi et al. and Binshabaib et al. investigated Porphyromonas gingivalis, an organism similar to P. intermedia, and supported the observed inhibition. Their study showed that HA, although less effective than azithromycin, performed better than chlorhexidine in limiting both planktonic and biofilm forms of Porphyromonas gingivalis. This indicates that HA might also affect related species like P. intermedia, which share similar harmful traits such as proteolytic activity and evasion of the immune system.[2,5]

The higher concentration of HA needed to inhibit F. nucleatum may be due to its key role in biofilm structure. F. nucleatum helps in coaggregation between early and late colonizers, making it more resistant to antibiotics. This resilience matches findings from Eick et al., who showed that while HA can reduce the survival of biofilm-embedded pathogens, it typically requires higher concentrations and longer exposure. These differences in susceptibility highlight the importance of understanding specific microbial processes and biofilm dynamics when evaluating treatment methods.[3]

Clinical studies by Eick et al.[3] have also shown HA’s added value in periodontal therapy. In split-mouth trials after scaling and root planning, HA treatment led to faster decreases in sulcus bleeding and fluid flow and analyses of periodontal microbiota showed lower prevalence of Porphyromonas gingivalis. Reviews have indicated significant microbiological improvements with HA used alongside traditional therapies.[3]

The mechanisms underlying HA’s bacteriostatic effects are complex. The negative charge of HA might disrupt the outer membrane of Gram-negative bacteria, affecting nutrient transport and metabolic functions. HA can also physically block bacterial adhesion and biofilm formation. Its ability to bind to host cell receptors such as CD44 adds an immunomodulatory layer by boosting leukocyte recruitment, cytokine release, and extracellular matrix remodeling. These immune responses may indirectly impact bacterial survival, enhancing HA’s potential for treatment in inflamed periodontal conditions.[4]

High-molecular-weight HA (1.5 MDa) significantly suppressed Porphyromonas gingivalis virulence-associated genes (fimA, mfa1, hagA, rgpA, and kgp), demonstrating concentration-dependent inhibition. Compared to controls, HA exhibited superior gene downregulation relative to chlorhexidine but lower than azithromycin, indicating its potential antimicrobial and anti-virulence activity.[6]

HA’s known anti-inflammatory and tissue-repairing properties support its antimicrobial effects. It eliminates reactive oxygen species, controls matrix metalloproteinase activity, and neutralizes inflammatory signals. Cross-linked high-molecular-weight HA structures have been used effectively in guided tissue regeneration and bone grafting, underscoring its ability to promote healing.[9]

Despite these encouraging results, there are notable limitations. Most studies focus on mono-species and planktonic models, which do not capture the spatial complexities and interactions found in real periodontal biofilms. Pathogens such as Porphyromonas gingivalis, P. intermedia, F. nucleatum, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans often live in structured, mixed communities.[10] Recent ecological models stress the need for biofilm experiments that consider quorum sensing, spatial variations, and metabolic interactions. Furthermore, the impact of HA on complex host-microbe systems is not well understood. Combining different host cell types, such as epithelial cells, fibroblasts, and immune cells, in model systems could provide insights into HA’s role in healing, immune response, and microbial control. In addition, many in vitro studies rely on standard laboratory strains; clinical isolates, with their varied characteristics and antibiotic resistance, might behave differently.

From a formulation perspective, it is necessary to compare linear high-molecular-weight HA, cross-linked HA, and combinations with antimicrobials or scaffolds to determine the best delivery methods. Evaluating dosage, duration of action, and breakdown in situ will help translate these findings into clinical practice.

Finally, controlled clinical trials evaluating relevant clinical factors, such as probing pocket depth, attachment gain, bleeding, and microbial profiles, are crucial. Important questions remain: What concentrations and molecular weights of HA provide the best antimicrobial and healing effects? Can HA decrease the need for antibiotics in periodontal treatment? Longitudinal randomized controlled trials should explore these questions.

In summary, high-molecular-weight HA shows consistent effects against various periodontal pathogens, such as Porphyromonas gingivalis, P. intermedia, F. nucleatum, and potentially Tannerella forsythia and Aggregatibacter actinomycetemcomitans. Its combination of direct microbial inhibition, prevention of adhesion, suppression of harmful traits, immune system modulation, and tissue regeneration supports its potential as an addition to treatment. However, thorough research involving mixed biofilms, clinical isolates, and host–pathogen interactions is essential for effective integration into periodontal care.

CONCLUSION

The treatment of periodontitis is challenging because complex subgingival biofilms persist. Bridging organisms such as P. intermedia and F. nucleatum are critical for microbial aggregation and biofilm maturation. These organisms increase the stability and harmful potential of polymicrobial communities, making them hard to treat with standard therapies. Therefore, it is important to target these species to disrupt biofilm structure and stop disease progression.

HA is a biocompatible, high-molecular-weight glycosaminoglycan that offers a new two-in-one approach by providing both antimicrobial and anti-inflammatory effects. This study showed that HA inhibits P. intermedia and F. nucleatum in a concentration-dependent manner. It likely does this by interfering with adhesion, nutrient uptake, and biofilm development. In addition, HA promotes tissue regeneration by modulating inflammatory responses and improving extracellular matrix repair.

Although HA shows promise in periodontal therapy, further research and clinical studies are needed to assess its effectiveness in multispecies biofilm models and with clinical bacterial isolates. Understanding how it interacts with complex host-microbe relationships and optimizing delivery systems will be crucial. Overall, HA may be a valuable additional agent that improves treatment outcomes and reduces the need for traditional antimicrobials in periodontics.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.