Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro

Abstract

Chinese

Preventing the development and recurrence of periodontal diseases often includes antimicrobial mouthrinses to control the growth of the periodontal pathogens. Most antimicrobials are nonselective, targeting the symbiotic oral species as well as the dysbiosis-inducing ones. This affects the overall microbial composition and metabolic activity and consequently the host–microbe interactions, which can be detrimental (associated with inflammation) or beneficial (health-associated). Consequently, guiding the antimicrobial effect for modulating the microbial composition to a health-associated one should be considered. For such an approach, this study investigated electrolyzed saline as a novel rinse. Electrolyzed saline was prepared from sterile saline using a portable electrolysis device. Multispecies oral homeostatic and dysbiotic biofilms were grown on hydroxyapatite discs and rinsed daily with electrolyzed saline (EOS). Corresponding positive (NaOCl) and negative (phosphate-buffered saline) controls were included. After 3 rinses, biofilms were analyzed with viability quantitative polymerase chain reaction and scanning electron microscopy. Supernatants of rinsed biofilms were used for metabolic activity analysis (high-performance liquid chromatography) through measuring organic acid content. In addition, human oral keratinocytes (HOKs) were exposed to EOS to test biocompatibility (cytotoxicity and inflammation induction) and also to rinsed biofilms to assess their immunogenicity after rinsing. Rinsing the dysbiotic biofilms with EOS could reduce the counts of the pathobionts (>3 log₁₀ Geq/mm² reduction) and avert biofilm dysbiosis (≤1% pathobiont abundance), leading to the dominance of commensal species (≥99%), which altered both biofilm metabolism and interleukin 8 (IL-8) induction in HOKs. EOS had no harmful effects on homeostatic biofilms. The scanning electron micrographs confirmed the same. In addition, tested concentrations of EOS did not have any cytotoxic effects and did not induce IL-8 production in HOKs. EOS showed promising results for diverting dysbiosis in in vitro rinsed biofilms and controlling key periopathogens, with no toxic effects on commensal species or human cells. This novel rinsing should be considered for clinical applications.

Keywords

periodontal disease(s)/periodontitis scanning electron microscopy (SEM)biocompatibility biofilm(s)microbial ecology microbiome

Introduction

The oral cavity is a complex resilient ecosystem inhabited by various microorganisms. Imbalances in the oral microbiome lead to a dysbiotic state characterized by altered composition and phenotype of oral biofilms. Biofilm accumulation on tooth surface exposes surrounding periodontal tissues to bacteria-secreted virulence factors, provoking tissue inflammation. Therefore, maintaining periodontal health requires biofilm control (Al-Radha et al. 2012). Mouthrinses can aid in preventing periodontal diseases when mechanical plaque control is challenging. In addition, using mouthrinses may enhance the effectiveness of periodontal therapy (Kim and Nam 2018).

Electrolyzed oxidizing solutions (EOSol) are generated through electrolyzing chloride-containing water, typically in the form of sodium chloride (NaCl), generating free chlorine in the form of hypochlorous acid (HOCl), hypochlorite ions (ClO–), and soluble chlorine gas (Cl₂), with broad-spectrum antibacterial activity (Huang et al. 2008; Dube and Jain 2018; Kim and Nam 2018). In addition, oxygen radicals and reactive hydrogen are also generated (Shimada et al. 2000; Ogunniyi et al. 2019; Cárdenas et al. 2022).

While the electrolysis parameters and the concentration of chloride ions in the original solution determine the concentration of the generated free chlorine in the EOSol, the pH after electrolysis affects the equilibrium between various free chlorine components (Takeda et al. 2020). At pH 7, about 75% of the free chlorine exists in form of HOCl, which is the most effective antibacterial form (Farah and Al-Haj Ali 2021). HOCl interacts with outer membrane lipids, producing chlorohydrin intermediates, increasing membrane permeability and destroying its function and structure. HOCl concentrations decrease when pH rises, with ClO^– dominating at ≈pH 9, as in NaOCl solutions, thereby increasing cellular toxicity and storage risks (Dube and Jain 2018).

Due to safety and proven bactericidal effect, EOSol has been approved for sanitation and disinfection in food and agriculture industries in the United States, Japan, and Korea (Liao et al. 2020). Subsequently, it gained attention for medical applications, such as wound disinfection (Stroman et al. 2017; Mourad and Hobro 2020). Furthermore, EOSol has demonstrated oral antibiofilm activity comparable to that of NaOCl (ClO^– dominant), which is used clinically in periodontal treatments for irrigation despite its high pH (9-10) (Kamagate et al. 2005; Cheng et al. 2016; Yan et al. 2022). EOSol is a potential oral biofilm control rinse due to its biocompatibility and lack of toxicity to enamel or dentin at clinically relevant exposure times and no tissue-dissolving risks due to its near-neutral pH (Qing et al. 2006; Christensen et al. 2008; Morita et al. 2011). Therefore, this study evaluated the efficacy of EOSol generated from electrolyzed saline (EOS), in a preventive approach, to control key periodontal pathogens and divert dysbiosis in an in vitro periodontitis biofilm model. The biocompatibility of the EOS was also investigated.

Materials and Methods

Bacterial Strains, Media, and Culture Conditions

Fourteen key oral bacterial species were included in this study (Appendix Table 1) (Zayed et al. 2022, 2023). All the strains were maintained as described in the table.

Multispecies Communities and Biofilms

Multispecies bacterial communities were grown in a Biostat-B Twin bioreactor using Brain Hearth Infusion-2 (BHI-2) broth (Herrero et al. 2016). Biofilms were grown on standardized (12 mm diameter, 0.05 mm thickness) hydroxyapatite discs (HADs) (HiMed Inc.) microaerophilicaly for 48 h using bioreactor-derived communities. Two biofilm models were investigated: symbiotic and dysbiotic, simulating homeostatic healthy and periodontitis-associated biofilms, respectively (Zayed et al. 2023). Dysbiosis was induced using BHI-2 medium with higher concentrations of L-cysteine, which neutralize the H₂O₂ oxidative stress of the oral streptococci in the multispecies biofilms, leading to imbalances simulating neutralizing factors that initiate dysbiosis in the oral cavity. Four biological replicates were performed.

Preparation of EOS Rinse

A volume of 100 mL sterile saline was used to generate 1 batch of EOS using a portable EOS-generating machine. EOS was checked for its pH value and free chlorine concentrations using chlorine colorimetric test strips (Sigma-Aldrich). Free chlorine concentrations were adjusted to 200 ppm via dilution with Milli-Q water, and then pH was adjusted to 7.0 (corresponding to HOCl dominance) using HCl. Before each rinsing experiment, independent batches were prepared and maintained at room temperature for the experiment time. pH and free chlorine were monitored before each rinse. Phosphate-buffered saline (PBS) and sodium hypochlorite solution (0.25% NaOCl, pH 10.8) (Sigma-Aldrich) were used as negative and positive control rinses, respectively.

Biofilm Rinsing

Biofilms were rinsed using EOS, PBS, or NaOCl for 3 min while shaking at 230 rpm at room temperature (Zayed et al. 2022). Afterward, biofilms were washed with PBS and reincubated in fresh BHI-2 for symbiotic biofilms and BHI-2C for dysbiotic ones. Biofilms were rinsed daily and collected for analysis 24 h after each rinsing.

Biofilm Collection, DNA Extractions, and Viability Quantitative Polymerase Chain Reaction

Biofilms were collected from HAD surfaces using trypsin for 45 min at 37°C with shaking at 250 rpm. DNA extractions and viability quantitative polymerase chain reaction (v-qPCR) were performed using propidium monoazide-xx (PMAxx) treatment and a Qiagen DNA extraction kit according to the manufacturer’s instructions (Zayed et al. 2022). Strain-specific primers and probes were used for the quantitative polymerase chain reaction (qPCR) analysis, as indicated in Appendix Table 2.

Scanning Electron Microscope Visualization

Scanning electron micrographs of the rinsed biofilms (hydrated) were taken using a scanning electron microscope (FEI XL30-FEG; FEI) at high vacuum with a 10-keV acceleration voltage. Biofilms were fixed for visualization as described earlier (Zayed et al. 2023). Afterward, biofilms were dried at room temperature and then coated with Pt/Pd 80/20 using a sputtering device (Quorum Q150T S; Quorum Technologies).

Metabolic Activity of Rinsed Biofilms

Twenty-four hours after dysbiotic biofilms were rinsed, supernatants were collected and filter-sterilized. Metabolic acids in the supernatants (butyric, propionic, acetic, formic, lactic, and succinic acids) were measured using an Agilent HPLC 1200 series equipped with a Bio-Rad Aminex HPX-87H Column and Bio-Rad Cation H Micro-Guard, with both columns heated to 40°C. Mobile phase consisted of 5 mM H₂SO₄ at a flowrate of 0.6 mL/min. Detection of compounds was established with a diode array detector at 210 nm and/or a refractive index detector at 40°C.

Human Oral Keratinocytes Immunogenic Response to Rinsed Biofilms

The human oral keratinocyte (HOK) cell line (HOK-18A) was grown as described earlier (Sliepen et al. 2009), using keratinocyte serum-free medium (KSFM), supplemented with bovine pituitary extract (25 µg/mL) and recombinant epidermal growth factor (5 ng/mL). HOKs were seeded in 24-well plates after silicon rings were placed at the well bottoms. After confluence, HADs were placed on the silicon ring, as a spacer between the HOKs and the biofilms, with the biofilms facing the cells. Two hours later, HADs, silicon rings, and media were removed. RNA from HOKs was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was then converted to complementary DNA (cDNA) using the PrimeScript first-strand cDNA Synthesis Kit (Takara). Expression of inflammatory genes, interleukin 8 (IL-8), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), was analyzed using qPCR with respect to housekeeping genes.

Biocompatibility of EOS

Serial dilutions (1:2), in supplement-free KSFM, of EOS and NaOCl were tested for biocompatibility with HOKs (Aherne et al. 2022). PBS was diluted similarly as a negative control.

For cytotoxicity testing, confluent cells were exposed to the test solutions for 9 min, corresponding to the total rinsing time of the 3-day experiment. Afterward, test solutions were removed, and an XTT-assay (Sigma Chemical Co.) was performed according to the manufacturer’s instructions.

For assessing the inflammatory response of HOK monolayers, cells were exposed to the testing solutions as abovementioned, and then fresh KSFM was added to the cells after removing solutions. Cells were incubated for 24 h, and then cell-free supernatants were collected to detect IL-8 production using the Human-IL-8 ELISA Kit (ThermoFisher Scientific) according to the manufacturer’s instructions.

Statistics

The normality of residuals was assessed using a normal quantile plot, and the equality of variability for different groups was assessed using a residual dot plot. Biofilm data were analyzed using a linear mixed model, with biological replicates as a random factor and rinsing agent, time point, and biofilm model as fixed factors. Contrasts were calculated using the fixed-factor estimates of the linear effects model and their variance-covariance matrix. Cytotoxicity, inflammation, and metabolic activity data were analyzed using analysis of variance (ANOVA) followed by Tukey’s HSD (honestly significant difference) test for multiple comparisons analysis, with a confidence level of 95% for each.

Results

Effect on Biofilm Bacterial Numbers and Composition (v-qPCR Results)

v-qPCR results of symbiotic (homeostatic) biofilms

After 3 rinses, PBS-rinsed biofilms contained ≈7.03 log₁₀ Geq/mm², while EOS- and NaOCl-rinsed ones contained ≈6.26 and 4.15 log₁₀ Geq/mm², respectively (Fig. 1A). The 2 subcommunities, pathobionts and commensals, were reduced relative to PBS control by ≈4.77 and ≈2.8 log₁₀ Geq/mm² respectively, after 3 NaOCl rinses (Fig. 1B, C). However, after EOS rinsing, commensals were much less reduced (≈0.67 log₁₀ Geq/mm² decrease), while pathobionts were similarly reduced as in the NaOCl-rinsed biofilms (≈4 log₁₀ Geq/mm² decrease). Although the ecology of NaOCl-rinsed biofilms was not significantly changed due to its equal effect on both pathobionts and commensal subcommunities, an increase in the abundance of the cariogenic Streptococcus sobrinus was observed in the pathobionts subcommunity, which was not observed for the EOS-rinsed biofilms. Affecting periodontal pathogens more than other bacteria, EOS rinsing significantly shifted biofilm ecology to more homeostasis (Fig. 1D), with ≈99% commensal abundance starting after 1 rinse. However, the streptococci commensals significantly dominated both EOS- and NaOCl-rinsed biofilms, while PBS-rinsed biofilms were dominated by Veillonella parvula (Fig. 1E).

v-qPCR results of dysbiotic (periodontitis-associated) biofilms

EOS-rinsed biofilms had a ≈1.39 log₁₀ Geq/mm² decrease in the biofilm microbial load relative to control biofilms, while NaOCl rinsing caused a 2.4 times greater decrease in the microbial load (a decrease of ≈3.28 log₁₀ Geq/mm²) (Fig. 2A). More specifically, daily EOS rinsing decreased the total pathobionts by ≈2.17 log₁₀ Geq/mm² compared to PBS rinsing. However, commensals were not significantly affected (≈6.12 vs. ≈6.95 log₁₀ Geq/mm² for EOS and PBS, respectively). In contrast, NaOCl rinsing had a more pronounced effect (P < 0.05) on the commensals (≈4.18 log₁₀ Geq/mm²) despite having comparable total pathobiont counts (≈2.45 log₁₀ Geq/mm²).

The ecology of the rinsed biofilms differed between those rinsed with PBS, NaOCl, and EOS. Pathobionts (mainly periopathogens) constituted ≈70% of the PBS-rinsed biofilms, whereas NaOCl-rinsed ones contained only ≈9% pathobionts (mainly periopathogens), and EOS-rinsed biofilms had a significant shift in the biofilm species composition, with no detected pathobionts (cariogenic or periopathogens), with 100% dominance of the streptococci commensals.

Scanning Electron Microscopy Visualization of Recovered Biofilms

Scanning electron microscopy (SEM) at 20,000× was used to compare biofilms after 3 rinses. The SEM micrographs confirmed the v-qPCR data. PBS-treated biofilms were dominated by either cocci-shaped cells (symbiotic homeostatic biofilm model) or rod-shaped cells and filaments of long rods (dysbiotic periodontitis biofilm model) (Fig. 3). NaOCl- and EOS-rinsed biofilms had less microbial load and rod-shaped cells, which was more obvious in the dysbiotic periodontitis biofilm model. At higher magnification (40,000×), the effect of the rinsing on the bacterial membranes was more apparent in NaOCl-rinsed biofilms.

Metabolic Activity of Rinsed Biofilms

Twenty-four hours after rinsing the dysbiotic biofilms, metabolic acids were measured in the cell-free supernatants (Fig. 4A). EOS- and NaOCl-rinsed biofilms exhibited lower metabolic activities than PBS-rinsed ones, which was associated with decreased production of butyric, propionic, formic, and acetic acids and increased production of succinic and lactic acids (P < 0.05). The metabolic activity per bacterial cell was 10 and 320 times higher in EOS- and NaOCl-rinsed biofilms than in PBS-rinsed ones, respectively.

Immunogenicity of Rinsed Biofilms

The HOK monolayers were exposed to symbiotic homeostatic biofilms rinsed with PBS, EOS, or NaOCl, followed by cellular RNA extraction and reverse transcription qPCR (RT-qPCR). The relative fold change of IL-8, IL-6, and TNF-α gene expression was calculated (Fig. 4B). No changes in IL-6 and TNF-α production were observed in HOKs exposed to EOS- or NaOCl-rinsed biofilms. In contrast, IL-8 production increased significantly when the HOKs were exposed to the NaOCl-rinsed biofilms, despite the lower microbial load and pathobiont abundance compared to PBS-rinsed biofilms.

Biocompatibility of EOS

On HOK monolayers, the cytotoxic effects of PBS, EOS, and NaOCl were examined along with their serial dilutions in KSFM (up to 1:16). Exposure to PBS or the rinsing concentration of EOS had no effect on the viability of the exposed cells in comparison to naive, unexposed cells, and the same was observed for PBS and EOS dilutions (Fig. 5A). However, exposure to NaOCl decreased cell viability to less than 10%. Except for the final dilution (1:16 dilution), increasing concentrations of NaOCl significantly decreased the HOK monolayers’ viability.

The loss of cell viability prevented the IL-8 detection. PBS and EOS did not affect IL-8 production in comparison to naive cells at any of the tested concentrations (Fig. 5B). At 1:4 NaOCl dilution (≈65% cell viability), IL-8 was detected at significantly higher concentrations than the naive cells in the surviving HOKs. With increasing dilutions, the detected IL-8 decreased but was still greater than with EOS at the same dilutions (until 1:16).

Discussion

Despite their effectiveness in reducing dental plaque, currently used mouthrinses, including chlorhexidine-based ones, have been found to disrupt microbial communities, leading to microbial resistance (Brookes et al. 2020; Zayed et al. 2022). This in vitro study tested EOS for potential use as a mouthrinse for both healthy homeostatic (symbiotic) and periodontitis (dysbiotic) oral microbial conditions in comparison to negative control (PBS) and positive control (0.25% NaOCl) rinses. The primary antimicrobial component produced by electrolyzing saline is HOCl, which is also produced by the immune system to combat pathogens (Mourad and Hobro 2020; Farah and Al-Haj Ali 2021). HOCl damages bacterial cell walls and membranes, increasing membrane permeability, causing intracellular material (protein and DNA) leakage (Chen and Wang 2022).

Both EOS (200 ppm) and 0.25% NaOCl (2,500 ppm) decreased viable microbial loads in both symbiotic and dysbiotic oral biofilms. It has been reported that acidic/neutral electrolyzed water (HOCl-based antimicrobial) is more effective in preventing bacterial aggregation (antibiofilm properties) at lower concentrations than NaOCl (OCl^–-based antimicrobial) (Sauer et al. 2009). Low concentrations of stabilized HOCl reduced viability in various in vitro oral biofilm models (Aherne et al. 2022). Clinically, NaOCl products decrease supragingival biofilms, gingival inflammation, and bleeding upon probing. Patients combining rinsing with nonsurgical periodontal treatment encountered a decrease in the plaque index (Galván et al. 2014).

At clinically relevant exposure times, the biofilm species composition shifted to beneficial bacteria after EOS rinse. Biofilm SEM micrographs supported these findings. Similarly, microscopically, HOCl decreased periodontitis- and halitosis-related spirochetes and filamentous bacteria in supra- and subgingival plaque (Kim and Nam 2018).

The overgrowth of the periopathogens Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia was effectively controlled in the EOS-rinsed dysbiotic biofilms, compared to both PBS- and NaOCl-rinsed ones. EOS inhibited these 4 periopathogens in vitro and significantly reduced salivary bacteria when used as an oral rinse (Lee and Choi 2006). More important, the periopathogens decrease was accompanied by dominance of the commensal streptococci in the EOS-rinsed biofilms. Effectiveness of HOCl solutions against periopathogens was previously reported (Lee and Choi 2006). However, the observed selectivity of EOS for periopathogens over commensals in oral biofilm communities, as shown in this study, has not yet been reported. This can be attributed to the fact that most of the periopathogens are Gram-negative bacteria, whereas the streptococci commensals are Gram-positive. In contrast to chlorhexidine, Gram-negative bacteria are more susceptible to irreversible HOCl damage than Gram-positive bacteria due to the sulfur and heme content of their membranes (Castillo et al. 2015; Aherne et al. 2022). Damaging bacterial outer membranes, which are necessary for cell aggregation and adhesion, reduce the colonization potential of Gram-negative bacteria (Qi et al. 2018; Lucio-Sauceda et al. 2019). In addition, Gram-negative and Gram-positive bacteria have distinct biofilm-regulating signaling molecules (Papenfort and Bassler 2016). Neutral electrolyzed water decreased the expression of quorum-sensing genes (and hence biofilm formation) in the Gram-negative bacteria Helicobacter pylori (Lucio-Sauceda et al. 2019). These also explains the lower or nonexistent abundance of the Gram-negative anaerobic commensal V. parvula compared to the control PBS-rinsed biofilms. Less selectivity was observed with the NaOCl rinse, as both subcommunities were similarly affected and reduced in numbers. The damage caused by NaOCl to both Gram-negative and Gram-positive cells affects both aggregation and recolonization, unlike EOS, which affected periopathogens significantly more.

In accordance with the microbiological data, dysbiotic biofilms rinsed with EOS and NaOCl had fewer metabolic acids and a simpler metabolic network. The intricate interactions and metabolic pathways within the oral community are influenced by a variety of factors, including the microbial load and composition (Verspecht et al. 2021). Shifts in the microbial community composition can then distinguish distinct metabolic fingerprints. Less microbial load and a significant ecological shift toward commensal streptococci dominance led to the prevalence of homeostasis-associated metabolic acids, with an increase in lactate and succinate production. Due to the restoration of biofilm homeostasis, butyric, propionic, formic, and acetic acid production decreased (Verspecht et al. 2021). Despite having fewer total metabolites, both EOS- and NaOCl-rinsed biofilms had higher metabolic activity per bacterial cell. However, the increase was more pronounced in NaOCl-rinsed biofilms.

Despite the homeostatic microbial ecology and low microbial load, NaOCl- but not EOS-rinsed symbiotic biofilms increased IL-8 expression in the human oral keratinocytes more than PBS-rinsed ones. The proinflammatory cytokine IL-8 has been associated with immunopathology. The release of IL-8 by gingival epithelial cells induces neutrophil chemotaxis, thereby enhancing the inflammatory drive of oral diseases (Belibasakis et al. 2013). The accumulation of reactive oxygen species and oxidative damage products after antimicrobial rinsing promotes proinflammatory mediator release. Moreover, associated changes in biofilm phenotype are highlighted by the varying inflammatory responses toward rinsed biofilms with similar microbial composition. High chlorine stress induced by NaOCl rinsing can increase the secretion of virulence proteins and the acquisition of antibiotic resistance genes (da Cruz Nizer et al. 2020). Thus, despite homeostatic ecology, the biofilm’s transcriptomic profile shifted toward a more pathogenic behavior, which could potentially trigger a host-immune response mechanism, increasing IL-8 levels. However, further analysis of rinsed biofilms should be conducted using transcriptomics.

In addition to these findings, the biocompatibility of EOS was investigated through a cytotoxicity assay using human keratinocytes. Cytotoxicity is a persistent issue with different oral antiseptics like chlorhexidine-, cetylpyridinium chloride-, and NaOCl-based antiseptics and alcohol-containing ones (Aubut et al. 2010; De Oliveira et al. 2018; Liu et al. 2018). Interestingly, investigating the impact of EOS on HOK viability did not reveal cytotoxicity. On the other hand, despite its satisfactory microbiological outcome and current clinical uses, NaOCl showed cytotoxicity up to 1:8 dilutions. Similarly, at the effective antimicrobial concentrations, no cytotoxicity was reported for electrolyzed water, either in vitro for human gingival fibroblasts and HOKs (Song et al. 2019; Aherne et al. 2022) or in vivo for mice (Morita et al. 2011). According to Morita and colleagues, electrolyzed water did not harm periodontal or gastrointestinal tissue when administered orally (Morita et al. 2011), which supports EOS’s safety for clinical use as a mouthrinse adjuvant to maintenance or nonsurgical periodontal treatments. In addition, the inflammatory response of HOK to EOS was favorable, with no upregulation of IL-8 production compared to control naive or PBS-exposed HOKs. Moreover, HOCl, the antimicrobial component of EOS, has demonstrated anti-inflammatory and tissue protection properties (Kim and Cha 2014; Castillo et al. 2015).

In conclusion, all the findings propose potential application of EOS as a mouthrinse, not only for reducing periodontal dysbiosis but also for maintaining oral hygiene in healthy subjects. Additional investigations using more complex ex vivo microbial communities, as well as additional clinical assessments, are needed to confirm the applicability of EOS.

Author Contributions

N. Zayed, H. Munjaković, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; M. Kübra Aktan, A. Braem, F. Pamuk, M. Saghi, W. Van Holm, contributed to data acquisition, critically revised the manuscript; K. Simoens, contributed to data acquisition and analysis, critically revised the manuscript; K. Bernaerts, contributed to data acquisition, analysis, and interpretation, critically revised the manuscript; N. Boon, contributed to conception, data analysis and interpretation, critically revised the manuscript; A. Fidler, R. Gašperšič, contributed to conception and design, data interpretation, critically revised the manuscript; W. Teughels, contributed to conception and design, data analysis and interpretation, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

sj-docx-1-jdr-10.1177_00220345231216660 – Supplemental material for Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro

Supplemental material, sj-docx-1-jdr-10.1177_00220345231216660 for Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro by N. Zayed, H. Munjaković, M.K. Aktan, K. Simoens, K. Bernaerts, N. Boon, A. Braem, F. Pamuk, M. Saghi, W. Van Holm, A. Fidler, R. Gašperšič and W. Teughels in Journal of Dental Research

Footnotes

Acknowledgements

We thank Dr. Martine Pauwels (laboratory of oral biology, KU Leuven) for her help with the v-qPCR.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by grants from the Fund for Scientific Research Belgium (FWO G0B2719N), KU Leuven (C24/17/086), and the Ministry of Higher Education, Science and Innovation, Republic of Slovenia (Grant P3-0293).

ORCID iDs

N. Zayed

H. Munjaković

K. Simoens

A supplemental appendix to this article is available online.

References

Aherne

Ortiz

Fazli

Davies

. 2022. Effects of stabilized hypochlorous acid on oral biofilm bacteria. BMC Oral Health. 22(1):1–12.

Al-Radha

ASD

Pal

Pettemerides

Jenkinson

. 2012. Molecular analysis of microbiota associated with peri-implant diseases. J Dent. 40(11):989–998.

Aubut

Pommel

Verhille

Orsière

Garcia

About

Camps

. 2010. Biological properties of a neutralized 2.5% sodium hypochlorite solution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 109(2):e120–e125.

Belibasakis

Thurnheer

Bostanci

. 2013. Interleukin-8 responses of multi-layer gingival epithelia to subgingival biofilms: role of the “red complex” species. PLoS One. 8(12):e81581.

Brookes

Bescos

Belfield

Ali

Roberts

. 2020. Current uses of chlorhexidine for management of oral disease: a narrative review. J Dent. 103:103497.

Cárdenas

Campos-Bijit

Di Francesco

Schwarz

Cafferata

Vernal

. 2022. Electrolyzed water for the microbiologic control in the pandemic dental setting: a systematic review. BMC Oral Health. 22(1):1–43.

Castillo

Delgadillo

Neuta

Jola

Calderón

Lafaurie

. 2015. Viability and effects on bacterial proteins by oral rinses with hypochlorous acid as active ingredient. Braz Dent J. 26:519–524.

Chen

B-K

Wang

C-K

. 2022. Electrolyzed water and its pharmacological activities: a mini-review. Molecules. 27(4):1222.

Cheng

Tian

Zhao

Liu

. 2016. Bactericidal effect of strong acid electrolyzed water against flow enterococcus faecalis biofilms. J Endod. 42(7):1120–1125.

10.

Christensen

McNeal

Eleazer

. 2008. Effect of lowering the pH of sodium hypochlorite on dissolving tissue in vitro. J Endod. 34(4):449–452.

11.

da Cruz Nizer

Inkovskiy

Overhage

. 2020. Surviving reactive chlorine stress: responses of gram-negative bacteria to hypochlorous acid. Microorganisms. 8(8):1220.

12.

De Oliveira

Belato

De Oliveira

Jorge

AOC

Camargo

SEA

De Oliveira

. 2018. Mouthwashes: an in vitro study of their action on microbial biofilms and cytotoxicity to gingival fibroblasts. Gen Dent. 66(2):28–34.

13.

Dube

Jain

. 2018. Electrolyzed saline . . . an alternative to sodium hypochlorite for root canal irrigation. Clujul Medical. 91(3):322.

14.

Farah

RfI

Al-Haj Ali

. 2021. Electrolyzed water generated on-site as a promising disinfectant in the dental office during the Covid-19 pandemic. Front Public Health. 9:629142.

15.

Galván

Gonzalez

Cohen

Alonaizan

Chen

CTL

Rich

Slots

. 2014. Periodontal effects of 0.25% sodium hypochlorite twice-weekly oral rinse. A pilot study. J Periodontal Res. 49(6):696–702.

16.

Herrero

Slomka

Boon

Bernaerts

Hernandez-Sanabria

Quirynen

Teughels

. 2016. Dysbiosis by neutralizing commensal mediated inhibition of pathobionts. Sci Rep. 6(1):1–10.

17.

Huang

Hung

Hsu

Huang

Hwang

. 2008. Kangen water proof book table of contents. Food Control. 19(4):329–345.

18.

Kamagate

Kone

Coulibaly

Ahnoux

. 2005. Subgingival irrigation combined with scaling and root planing. Results of a study with chlorhexidine and sodium hypochlorite. Odontostomatol Trop. 28(109):28–32. in French.

19.

Kim

Cha

Y-N

. 2014. Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects. Amino Acids. 46(1):89–100.

20.

Kim

Y-R

Nam

S-H

. 2018. Comparison of the preventive effects of slightly acidic HOCl mouthwash and CHX mouthwash for oral diseases. Biomed Res. 29(8):1718–1723.

21.

Lee

S-H

Choi

B-K

. 2006. Antibacterial effect of electrolyzed water on oral bacteria. J Microbiol. 44(4):417–422.

22.

Liao

Xiang

Cullen

Chen

Liu

Ding

. 2020. Plasma-activated water (PAW) and slightly acidic electrolyzed water (SAEW) as beef thawing media for enhancing microbiological safety. LWT. 117(4):108649.

23.

Liu

Werner

Kirsch

Zuckerman

Virk

. 2018. Cytotoxicity evaluation of chlorhexidine gluconate on human fibroblasts, myoblasts, and osteoblasts. J Bone Jt Infect. 3(4):165–172.

24.

Lucio-Sauceda

Urrutia-Baca

Gomez-Flores

La Garza-Ramos

Angélica

Tamez-Guerra

Orozco-Flores

. 2019. Antimicrobial and anti-biofilm effect of an electrolyzed superoxidized solution at neutral-pH against helicobacter pylori. Biomed Res Int. 2019:6154867. doi:10.1155/2019/6154867

25.

Morita

Nishida

Ito

. 2011. Biological toxicity of acid electrolyzed functional water: effect of oral administration on mouse digestive tract and changes in body weight. Arch Oral Biol. 56(4):359–366.

26.

Mourad

Hobro

. 2020. Developing chlorine-based antiseptic by electrolysis. Sci Total Environ. 709:136108.

27.

Ogunniyi

Dandie

Ferro

Hall

Drigo

Brunetti

Venter

Myers

Deo

Donner

. 2019. Comparative antibacterial activities of neutral electrolyzed oxidizing water and other chlorine-based sanitizers. Sci Rep. 9(1):19955.

28.

Papenfort

Bassler

. 2016. Quorum sensing signal–response systems in gram-negative bacteria. Nat Rev Microbiol. 14(9):576–588.

29.

Wang

Cai

Quan

Cui

Meng

. 2018. Effects of d-valine on periodontal or peri-implant pathogens: porphyromonas gingivalis biofilm. J Periodontol. 89(3):303–314.

30.

Qing

Akita

Kawano

Kawazu

Yoshida

Sekine

. 2006. Cleaning efficacy and dentin micro-hardness after root canal irrigation with a strong acid electrolytic water. J Endod. 32(11):1102–1106.

31.

Sauer

Thatcher

Northey

Gutierrez

. 2009. Neutral super-oxidised solutions are effective in killing P. Aeruginosa biofilms. Biofouling. 25(1):45–54.

32.

Shimada

Ito

Murai

. 2000. A comparison of the bactericidal effects and cytotoxic activity of three types of oxidizing water, prepared by electrolysis, as chemical dental plaque control agents. Int J Antimicrob Agents. 15(1):49–53.

33.

Sliepen

Van Damme

Van Essche

Loozen

Quirynen

Teughels

. 2009. Microbial interactions influence inflammatory host cell responses. J Dent Res. 88(11):1026–1030.

34.

Song

Kim

Lee

. 2019. Antimicrobial and cell viability measurement of hypochlorous acid against Streptococcus mutans and Aggregatibacter actinomycetemcomitans. J Korean Soc Integr Med. 7(2):141–151.

35.

Stroman

Mintun

Epstein

Brimer

Patel

Branch

Najafi-Tagol

. 2017. Reduction in bacterial load using hypochlorous acid hygiene solution on ocular skin. Clin Ophthalmol. 11:707–714. doi:10.2147/OPTH.S132851

36.

Takeda

Uchiumi

Matsuda

Ogawa

. 2020. Acidic electrolyzed water potently inactivates SARS-CoV-2 depending on the amount of free available chlorine contacting with the virus. Biochem Biophys Res Commun. 530(1):1–3.

37.

Verspecht

Van Holm

Boon

Bernaerts

Daep

Masters

Zayed

Quirynen

Teughels

. 2021. Potential prebiotic substrates modulate composition, metabolism, virulence and inflammatory potential of an in vitro multi-species oral biofilm. J Oral Microbiol. 13(1):1910462.

38.

Yan

Chelliah

K-h

Selvakumar

Chen

H-y

. 2022. Stability and antibiofilm efficiency of slightly acidic electrolyzed water against mixed-species of listeria monocytogenes and staphylococcus aureus. Front Microbiol. 13:865918. doi:10.3389/fmicb.2022.865918

39.

Zayed

Boon

Bernaerts

Chatzigiannidou

Van Holm

Verspecht

Teughels

. 2022. Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in-vitro oral biofilms. J Periodontal Res. 57(1):52–62.

40.

Zayed

Ghesquière

Kamarudin

Bernaerts

Boon

Braem

Van Holm

Teughels

. 2023. Oral biofilm cryotherapy as a novel ecological modulation approach. J Dent Res. 102(9):1038–1046.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.45 MB