Selective Antimicrobial Effects of an Herbal Compound Rinse Against Multi-species Oral Biofilms

doi:10.21203/rs.3.rs-3775080/v1

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Selective Antimicrobial Effects of an Herbal Compound Rinse Against Multi-species Oral Biofilms

https://doi.org/10.21203/rs.3.rs-3775080/v1

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Background:

The growing utilization of oral rinses in dental and periodontal care has prompted the need for innovative products with selective antibacterial properties. This study evaluates the efficacy of an herbal compound rinse (StellaLife VEGA Oral Rinse, StellaLife Inc, IL) containing plant attenuations and propolis, in comparison to conventional antimicrobial oral rinses.

Materials and Methods:

Streptoccoccus oralis, Streptococcus gordonii DL1, Veillonella parvula, Fusobacterium nucleatum, and Porphyromonas gingivalis were cultured and treated with StellaLife VEGA Oral Rinse (SL), 0.12% chlorhexidine (CHX), and Listerine Cool Mint mouthwash. Planktonic bacterial growth was assessed through optical density measurements (OD600), and colony forming units (CFU) counts. A clinical ex vivo multi-species biofilm was used to evaluate antibiofilm effects through fluorescence biofilm tracking.

Results:

SL significantly inhibited the growth of F. nucleatum 12 hours after treatment and P. gingivalis 96 hours after treatment, while exhibiting lower toxicity toward commensal bacteria such as S. oralis, S. gordonii, and V. parvula compared to LIS or CHX. In the clinical ex vivo biofilm, StellaLife, CHX, and Listerine all showed significant antibiofilm effects, disrupting biofilm structure and reducing bacteria viability.

Conclusions:

SL’s selective action on oral bacteria, targeting pathogens while preserving commensal microbes, holds promise for selective preservation of eubiotic biofilms. This study demonstrates the potential of this herbal compound rinse as an effective aid to selectively combat particularly against periodontal pathogens. Additional clinical studies are warranted to validate these findings and explore their broader applications in oral health.

oral rinses

chlorhexidine

antimicrobial(s)

oral biofilms

periodontitis

gingivitis

Oral rinse (also called mouth rinses/mouth rinses) uses have significantly increased in the last couple of decades. The indications range from pre- and post-dental procedures specifically targeting oral bacterial control and healing, to adjunctive treatment of periodontal diseases and long-term maintenance as part of oral hygiene. Mouthwashes have has also gained increased attention considering their negative effect on commensal microbes, i.e., ‘good bacteria’ that are important for tissue-healthy homeostasis.¹ The concerns related to the mouthwash-related depletion of oral commensal bacteria extend beyond the scope of periodontal tissue healthy homeostasis to adverse systemic health effects. Specifically, the San Juan Overweight Adults Longitudinal Study revealed independent significant associations between use of mouthwash two or more times daily and hypertension as well as prediabetes/diabetes.² These associations were linked to the mouthwash-related depletion of commensal microorganisms, which act as modulators of the conversion of nitrate to nitric oxide thus reducing systemic inflammatory burden. ^{3, 4} This underlines the need for novel targeted mouthwash formulations that exhibit selective cytotoxicity rather than a ‘gunshot’ approach to “balance microbial communities” rather than eradicating them.¹

Various plant-based attenuations and dilutions have been shown to exhibit antimicrobial efficacy against periodontal pathogens. Mistry et al. assessed Azadirachta indica (Neem), Ocimum sanctum (Tulsi), Mimusops elelngi (Bakul), Tinospora cardifolia (Giloy) and chlorhexidine gluconate (CHX) on common endodontic pathogens like Streptococcus mutans, Enterococcus faecalis and Staphylococcus aureus.^{5, 6} The study concluded that methanolic extract of A. indica, O. sanctum, M. elengi, and T. cardifolia had considerable antimicrobial activity against S. mutans, E. faecalis and S. aureus similar to that of chlorhexidine gluconate. ⁶ A number of independent investigations have shown significant antimicrobial and antiviral effects of neem and Calendula officinalis as a possible antigingivitis agent.^5–15 In addition, Eichenesia was also shown to have antifungal properties and to exert positive effects on the immune system.^16–19 Lastly, propolis is a resin-like material made by bees from the buds of poplar and cone-bearing trees. Propolis has been shown to have a potential to prevent periodontal disease and was found to be non-toxic to gingival fibroblasts.^20–23

Recently, an herbal compound rinse based on these homeopathic attenuations and propolis has been introduced and found to promote gingival wound healing on the transcriptional and translational levels.^{24, 25} Further, it was found to have virtually no cytotoxicity to host oral cells as compared to existing antimicrobial rinses, which are generally cytotoxic and may delay wound healing as in the case of chlorhexidine. However, there is no available information on the antimicrobial effects of this mouthwash. Therefore, in this study we compared its antibacterial and antibiofilm effects against commonly used rinses, such as 0.12% chlorhexidine and Listerine.

Bacterial Strains and Culture Conditions

The following oral bacterial species were evaluated: Streptoccoccus oralis (ATCC 35037), Streptococcus gordonii DL1, Veillonella parvula PK1910, Fusobacterium nucleatum (ATCC 25586), and Porphyromonas gingivalis (ATCC 33277). All bacteria were cultivated anaerobically (85% N₂, 10% CO₂ and 5% H₂) at 37°C. S. oralis and S. gordonii were struck from -80°C stocks onto Brain Heart Infusion (BHI) agar (BD Bacto) plates while V. parvula was struck onto BHI agar plates supplemented with 0.6% (v/v) sodium DL-lactate (BHIL). F. nucleatum was struck onto premade Brucella agar base (HiMedia Laboratories) containing 10 mg/L hemin and 10 mg/L vitamin K1 and supplemented with 5% defibrinated sheep blood (Hardy Diagnostics). Agar plates were incubated for 48-72 hrs prior to inoculating single colonies of S. oralis, S. gordonii, and F. nucleatum in 5 mL of BHI broth supplemented with 0.1 µg/mL vitamin K1 and 5 µg/mL hemin. V. parvula was inoculated in BHIL broth. P. gingivalis was directly inoculated (50 µL) from frozen stocks into 5 mL of supplemented BHI broth. All bacteria were grown until late-log or early stationary phase was reached based on optical density measurements at 600 nm wavelength (OD600) before treatment. Culture of all strains was performed under BSL2 conditions and was approved by the institutional biosafety committee. Additionally, this study incorporated a clinically isolated multi-species sample of periodontitis, comprising a diverse range of over thirty bacterial taxa to enhance clinical relevance and explore the extent of antibiofilm effects.²⁶ Detailed isolation and culture methods for the ex vivo ecological biofilm have been previously described.^{26, 27}

Assessment of Oral Rinsing Treatment on Planktonic Bacterial Growth

The following oral rinses were studied to evaluate their efficacy against neutralizing planktonic oral bacterial growth: 0.12% chlorhexidine gluconate, Listerine Cool Mint mouthwash, and StellaLife Oral Care Peppermint Rinse. 1x phosphate buffered saline (PBS) served as a negative control for oral rinsing treatment. Based on OD600 readings, 5 x 10⁸ CFU/mL of all monocultures and multispecies bacteria were harvested and pelleted by centrifugation at 13,000 x g for 4 min. The supernatant was discarded, and bacterial pellets were resuspended for in 100 µL of oral rinse for 1 min by vigorous pipetting. Afterward, bacterial resuspension in oral rinsing treatment was diluted 10-fold in 1x PBS (900 µL) twice immediately, and 30 µL of 100-fold diluted bacterial resuspension was inoculated in 3 mL of respective growth medium. Bacterial growth post rinsing treatment was monitored periodically via OD600 measurements and by plating 10-fold serial dilutions of bacterial medium. Based on each bacterial species growth times, S. oralis and S. gordonii growth were evaluated after 12, 24, and 36 h while V. parvula and F. nucleatum were evaluated after 12, 24, 36, and 48 h. P. gingivalis was evaluated after 48, 96, 128, 168, and 216 h while multispecies bacteria were evaluated after 12, 24, 48 and 96 h.

Fluorescence microscopy analysis antibiofilm effects on clinical ex vivo multi-species biofilm

To study antibiofilm effects, ex vivo multi-species biofilms grown in modified SHI media were used in this study as previously described.²⁶ Briefly, for biofilm formation, frozen samples stocked in -80°C were thawed and directly inoculated in SHI broth. Then, 1 mL aliquots of 5 x 10⁷ CFU/ml were added into sterile 24-well plates and incubated anaerobically at 37 °C for 24 hours. Subsequently, to determine antibiofilm effects, multi-species biofilms were washed with sterile deionized water and then stimulated with 1 mL PBS, SL, CHX, LIS, and 70% isopropyl alcohol (IPA) for 1 min, the biofilm with PBS was used as control. IPA is a broad spectrum bactericidal, tuberculocidal, fungicidal, and virucidal agent when at a 60%-90% concentration.²⁸ After stimulation, the 24-well plate was washed with deionized water to remove residual solutions. The biofilms were stained with Syto9 and Propidium Iodide (Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit) according to manufacturer’s instruction. After staining, biofilms were gently washed once with deionized water to remove dyes. Fluorescent imaging was performed using 10× and 20× objective magnification on a KEYENCE BZ-X800 fluorescence microscope. 3D reconstruction was performed in ImageJ.

Statistical Analysis

A standard two-way ANOVA test was used for statistical analyses in Graph Pad Prism 8.4.1., and significance was determined at a P value of ≤ 0.05 followed by post-hoc testing. When multiple timepoints were assessed, repeated measures ANOVA models were implemented for each condition and timepoints followed by appropriate post hoc tests.

Antibacterial Effects

To assess the comparative antibacterial effects of the herbal StellaLife (SL) mouthwash, we treated planktonic bacteria with SL, 0.12% chlorhexidine (CHX), Listerine (LIS), or sterile phosphate-buffered saline (PBS). Figure 1A presents the growth curves of the tested bacteria, measured by optical density (OD) at a wavelength of 600nm at different time points, with comparisons made against the PBS-treated control group (Figure 1B). In the growth curves, blue highlights indicate early colonizing or commensal bacteria, while red highlights represent pathogenic bacteria and multispecies samples. It is evident that SL does not inhibit commensal bacterial growth as compared to PBS. In contrast, CHX impedes the growth of S. oralis and V. parvula up to 12 hours post-treatment, and stunts growth of S. gordonii, which does not recover even after 24 hours post-treatment. Similarly, LIS eliminates S. oralis below the limit of detection up to 24 hours after treatment, stunts the growth of S. gordonii 24 hours after treatment, and impedes V. parvula growth until recovery after 36 hours.

For the red-highlighted pathogenic bacteria, SL significantly suppresses growth up to 12 hours after treatment for F. nucleatum and 96 hours for P. gingivalis, similar to CHX and LIS as compared to PBS control. However, CHX maintains greater suppression of F. nucleatum up to 36 hours post-treatment, and both CHX and LIS eliminates P. gingivalis growth below the limit of detection up to 216 hours after treatment. When treating the more complex multispecies composition, SL has a total microbial growth profile similar to PBS control. CHX retards growth up to 96 hours after treatment, which occurs to a greater extent for LIS.

Figure 2A depicts viable bacterial cell counts using colony forming units (CFU) at different time points after treatment. These results align closely with the optical density data, demonstrating that SL generally exhibits lower toxicity toward commensal bacteria such as S. oralis, S. gordonii, and V. parvula compared to LIS or CHX. SL only significantly inhibits the growth of S. oralis 8 hours after treatment, with recovery observed at the 12-hour time point (Figure 2B). Otherwise, SL does not significantly affect the growth of commensal bacteria. LIS, on the other hand, completely prevents the growth of S. oralis and stunts the growth S. gordonii at all time points after treatment and delays V. parvula recovery until 24 hours after treatment. CHX significantly reduces the growth of S. oralis 8 hours after treatment, stunts S. gordonii recovery at all time points, and has no effect on V. parvula.

Regarding pathogenic bacteria, SL significantly inhibits the growth of F. nucleatum 12 hours after treatment and P. gingivalis 216 hours after treatment. LIS eliminates F. nucleatum and P. gingivalis at all time points after treatment. CHX effectively suppresses growth for F. nucleatum up to 24 hours after treatment and P. gingivalis 216 hours after treatment.

Spot plating was performed on multispecies bacteria, as shown in Figure 2C. Interestingly, different phenotypes of colony-forming units (CFUs) were observed, including black-pigmented CFUs, in the control group treated with PPBS alongside non-black CFUs (Fig. 2C-iii). The total number of CFUs and the number of black-pigmented CFUs were recorded for further analysis, as the black-pigmented colonies typically represent more periodontopathic species in the oral cavity (Fig. 2C-i). Figure 2C-ii depicts the CFU count (diluted by 1000-fold) of black-pigmented CFUs 24 hours and 48 hours after treatment, revealing that at 48 hours, substantial growth of black pigmented CFU was noted in the control group (PBS), which was significantly reduced by the use of LIS, CHX and SL (all post-hoc p-val<0.05).

Antibiofilm effects on clinical ex vivo multi species biofilm

In this study, clinical ex vivo samples were utilized to create a complex multi-species biofilm, aiming to evaluate the antibiofilm effects of SL. The established biofilm from the clinical samples was subjected to treatment with different agents, including PBS, 0.12% CHX, LIS, SL, and isopropyl alcohol (IPA). To assess the surface coverage and viability of the stained multi-species biofilms, fluorescence microscopy was employed. Figure 3A displays the results of live/dead staining conducted 24 hours after treatment, where live bacteria were stained with SYTO® 9 green fluorescence and dead bacteria were stained with propidium iodide red fluorescence.

As can be seen in the images in Figure 3A, it is evident that the group treated with PBS exhibits a higher ratio of live bacteria, while the group treated with IPA shows a higher ratio of dead bacteria. In comparison, the groups treated with CHX, LIS, and SL display a relatively balanced ratio of live and dead bacteria. Notably, the LIS-treated group seems to have a higher ratio of dead bacteria.

Additionally, Figure 3B provides 3D reconstructions generated from the corresponding merged fluorescent channels. These reconstructions show the disruption of biofilm spatial structure and the reduction of residual bacteria following treatment. The groups with a higher presence of green fluorescence indicate that the biofilm remains intact, whereas the groups treated with IPA, LIS, and SL exhibit disrupted or flatter patterns consistent with biofilm reduction.

The presented research study offers a comprehensive evaluation of the effectiveness of SL, a novel homeopathic herbal compound mouthwash, against a spectrum of oral bacteria encompassing both periodontal pathogens and commensal organisms. This discussion will delve into several critical aspects, including the selective antibacterial properties of SL, its influence on periodontal pathogens, and the potential for future therapeutic approaches involving probiotics. The investigation revealed the remarkable selective targeting of pathogenic oral bacteria by SL. The growth curves and colony-forming unit (CFU) counts, as presented in Figs. 1A and 2A, depict the mouthwash’s ability to inhibit the proliferation of pathogenic bacteria while minimizing its impact on commensal microbes. This result is similar to a recent study, which evaluated four antibacterial agents and their effects on human gingival fibroblasts; CHX inhibited growth of all bacterial species including S. mutans, S. sanguis, F. nucleatum, and P. gingivalis at different dilutions while SL inhibited bacterial growth at 1/4 dilutions.²⁹ This selective action promising as it contributes to the selective retention of health-related commensal bacteria.^{30, 31}

Nonetheless, the SL mouthwash was particularly effective against pathogenic bacteria, such as Fusobacterium nucleatum and Porphyromonas gingivalis, as evidenced in Fig. 1. These findings corroborate its potential to serve as a robust tool for managing and preventing the progression of periodontal diseases. Notably, the CFU counts in Fig. 2A illustrate that SL preserves the commensal bacteria, especially Streptococcus oralis, Streptococcus gordonii, and Veillonella parvula. In short, SL is equally effective as LIS and CHX in preventing the growth of P. gingivalis up to 216 hours after treatment, but F. nucleatum shows faster recovery compared to CHX or LIS at 18 hours after treatment. An independent study also provided similar results where SL was bacteriostatic up to the 1/4th dilution, while CHX remained bactericidal up to the 1/64 dilution.²⁹ More importantly, SL was not cytotoxic to human gingival fibroblasts (HGF) and did not affect HGF proliferation at any dilution when CHX was cytotoxic at all dilutions.²⁹ Similar results have been previously reported by Zhou et al. in which SL was cytocompatible to cells critical to oral wound healing and promoted fibroblast migration and differentiation when compared to CHX.²⁴ This is a significant advantage as commensal bacteria contribute to averting opportunistic infections and maintaining oral homeostasis.³²

Overall, this study highlights the importance of formulating oral health treatments that specifically target pathogenic bacteria while preserving commensal microflora. In this context, the utilization of probiotics in oral health may hold great potential.³³ Probiotics are living microorganisms that, when administered in adequate quantities, can confer a health benefit on the host.³⁴

Future therapeutic methods may encompass the incorporation of probiotics in combination with commensal-sparing mouthwashes to stimulate the growth and dominance of beneficial commensal bacteria within the oral microbiome.³⁵ This could be achieved by introducing specific strains of probiotics that have demonstrated a positive influence on oral health. Probiotics have the potential to maintain a balanced oral microbiome, dysbiosis reversal, and host modulation, contributing to the prevention of conditions such as periodontal disease and peri-implantitis.^{35, 36}

In conclusion, this article demonstrates the potential of SL as an efficacious and selective tool for managing oral bacterial growth and biofilm formation, particularly against periodontal pathogens. Furthermore, the exploration of future therapeutic methods involving probiotics should be pursued to enhance the balance and health of the oral microbiome, potentially preventing and treating oral diseases effectively. Additional research and clinical studies are warranted to validate these findings and explore the full scope of their applications in oral health.

Author contribution: GK designed the study, interpreted the data and drafted the manuscript. YWT and DS collected and analyzed the data and drafted the manuscript.

Conflict of interest and financial relationships: All authors report no conflicts of interest or financial relationships impacting this manuscript. This work was partially sponsored by a unrestricted research grant by StellaLife Inc.

Grant support: This work was partially sponsored by an unrestricted research grant by StellaLife Inc.

Key Findings: Findings suggest that the herbal compound rinse may aid in the clinical management of oral bacterial growth and biofilm formation in periodontitis. Importantly this homeopathic rinse demonstrated selective action on oral bacteria targeting pathogens while preserving the commensal microbiota to maintain a eubiotic oral microbiome.

Ethical Approval

not applicable

Funding

This work was partially sponsored by an unrestricted research grant by StellaLife Inc.

Availability of data and materials

Data supporting this study are included within the article.

Acknowledgements

none

Conflicts of Interest

none

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No competing interests reported.

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Version 1

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You are reading this latest preprint version

Selective Antimicrobial Effects of an Herbal Compound Rinse Against Multi-species Oral Biofilms

Status:

Version 1

Abstract

Background:

Materials and Methods:

Results:

Conclusions:

Figures

Introduction

Materials and Methods

Bacterial Strains and Culture Conditions

Assessment of Oral Rinsing Treatment on Planktonic Bacterial Growth

Fluorescence microscopy analysis antibiofilm effects on clinical ex vivo multi-species biofilm

Statistical Analysis

Results

Antibacterial Effects

Antibiofilm effects on clinical ex vivo multi species biofilm

Discussion

Declarations

References

Additional Declarations

Status:

Version 1