Antibacterial Effect of Hypochlorous Acid on Bacteria Associated with the Formation of Periodontal Biofilms: An in vitro Pilot Study

periodontal biofilms


Introduction
The control of microorganisms and biofilms is one of the measures used to prevent common oral diseases, such as caries and periodontitis, which can only be achieved by mechanical methods such as tooth brushing and flossing.Mouthwashes are widely used and have relatively complicated formulas, and most of them contain antimicrobial agents such as chlorhexidine (CHX), triclosan, cetylpyridinium chloride, chlorine dioxide, and cationic peptides [1,2].Although these substances have marked antibacterial effects, oral rinses that do not alter the normal oral ecosystem but can significantly reduce biofilms are preferred for daily use.
Periodontal disease is mainly associated with the formation of bacterial biofilms.The main periodontal pathogens are gram-negative and anaerobic bacteria, some of which are highly proteolytic and cause bad breath [3].Although selective antibacterial agents against these bacteria are not available, an overall reduction in the number of these bacteria will contribute to the control of periodontal disease [4].
Although the role of S. gordonii in the formation of subgingival biofilms is not defined, it has been shown in in vitro studies that when P. gingivalis depends on signals produced by S. gordonii to form mixed biofilms with P. gingivalis [4].
Hypochlorous acid is the active component of sodium hypochlorite without its adverse effects; thus, it could be considered a potent antiplaque for use in oral cavity as it has been shown to have a high antimicrobial effect [5].HOCl has been shown to have a broad-spectrum antimicrobial effect at concentrations ranging from 0.1 to 2.8 mg/ml over a 2-minute exposure period.This microbicidal activity, although more effective for bacterial forms than spores and fungi, encompasses clinically relevant microorganisms such as Gram-negative and Gram-positive bacteria, parasites, and fungi [6].
In recent years, interest has increased in new high-potency molecules with antiplaque effects and bioequivalence with CHX but fewer adverse effects.Hypochlorous acid (HOCl) has been proposed as an antiplaque agent and as an agent for the healing of wounds in the oral mucosa due to its low toxicity, proven antimicrobial effectiveness, anti-inflammatory and cell proliferation-inducing effects, and history of use as a topical substance for wound disinfection in medicine [7].Regulation of the normal flora contributes to periodontal health, and HOCl appears to have the ability to attack gram-negative pathogens during periodontitis [8].However, the lack of studies necessitates further investigation of the effect of HOCl on oral microorganisms, especially those that form biofilms associated with highly prevalent diseases such as periodontitis.
The objective of this study was to evaluate the antibacterial effect of electrolytically generated HOCl ('electrolyzed water') on three of the main microorganisms associated with the formation of periodontal biofilms: Streptococcus gordonii ATCC 51656, Fusobacterium nucleatum ATCC 10953, and Porphyromonas gingivalis ATCC 33277.

Study Design
This research was an in vitro experimental study carried out in the Bacteriology Laboratory of the College of Sciences at the Universidad Peruana Cayetano Heredia (Cayetano Heredia University), Lima, Peru.

ppm Hypochlorous Acid Preparation
To obtain the HOCl, the EcoloxTech 240 System (EWCO, Miami Beach, FL, USA) was used.One liter of distilled water and 1 g of sodium chloride plus acetic acid were added (calibration at pH 0.7), which yielded

Antibacterial Susceptibility Tests
To evaluate antibacterial effects, plates containing brain heart infusion (BHI) agar for S. gordonii, BHI supplemented with 5% sheep blood plus menadione and vitamin K for F. nucleatum, and BHI agar supplemented with horse blood plus menadione and vitamin K for P. gingivalis were monitored for 24 hours to confirm sterility [10,11].
The strains were cultured in BHI broth for 24 hours, and then, the turbidity was calibrated to 0.5 on the McFarland scale by using a swab to soak up the previously prepared inoculum and then streaking the surface of the agar four times.Next, the agar was allowed to rest for 5 minutes, and then, 6-mm filter paper discs (Whatman 3, Danaher Corporation, Washington, D.C., USA) impregnated with 10 µl of 200 ppm HOCl, 10 µl of 4% NaClO, 10 µl of 0.12% CHX, and 10 µl of distilled water were placed on the plate.Then, all plates were incubated at 37 °C for 48 hours under anaerobic conditions [11].This procedure was repeated five times.
After 48 hours of incubation, the plates were examined, and the zones of inhibition were measured in millimeters using a calibrated Truper caliper.The four groups were placed on each plate, and each group was replicated five times.The number of plates to use (five) was determined assuming a maximum difference of 1.2 mm between the treatment means and a common standard deviation of 0.5 mm, with a type I error of 5% and power of 80%, necessary for the analysis of variance using Minitab 19 (Minitab LCC., State College, PA, USA).

Statistical Analysis
The data were processed with SPSS version 26 (IBM Corp., Armonk, NY, USA) using the nonparametric Kruskal-Wallis test and multiple comparison tests to compare the antimicrobial susceptibility to HOCl, including three controls, based on the diameter ranges of the zones of inhibition.The Kruskal-Wallis test was adopted as an alternative to the analysis of variance due to the evident non-normality and heterogeneity of the errors in analysis of variance.A p-value of 0.05 was considered statistically significant.

Results
Measurement of the zones of inhibition in the five plates at 48 hours of incubation allowed us to perform the comparisons provided in Table 1 for each of the oral bacteria under study.0 ± 0 a 0 ± 0 a 0 ± 0 a ANOVA: P-value 0.000 0.000 0.000 Levene test for homogeneity of variances: P-value 0.032 0.008 0.000 Kruskal-Wallis, multiple comparisons (a,b,c)  0.000 0.000 0.000 HOCl showed an effect on S. gordonii (Figure 1A).As expected with distilled water, no zone of inhibition was noted for any of the bacteria, nor were zones of inhibition observed with HOCl for F. nucleatum ATCC (Figure 1B) 10953 and P. gingivalis ATCC (Figure 1C).The range-based Kruskal-Wallis test showed differences in the zones of inhibition for the three oral bacteria S. gordonii ATCC 51656 (p=0.000),F. nucleatum ATCC 10953 (p=0.001), and P. gingivalis ATCC (p=0.000).The 0.12% CHX treatment showed greater control of the growth of S. gordonii ATCC 51656 (15.20 ± 0.66 mm.) and P. gingivalis ATCC (15.52 ± 0.43 mm.), but the effect was not significantly different from that of the NaClO treatment.In contrast, NaClO showed greater control of F. nucleatum ATCC 10953 (9.08 ± 4.53), but the effect was again not significantly different from that of 0.12% CHX.

Discussion
This study demonstrated that the 0.12% CHX and 4% NaClO groups showed bacterial reductions for the three bacteria evaluated.However, HOCl at 200 ppm was effective against only S. gordonii, a bacterium considered a primary colonizer in oral biofilms and responsible for biofilm adherence to surfaces [12,13].Sarduy-Bermúdez and González Díaz [14] mentioned that these initial colonizers adhere to the film through specific molecules, called adhesins, that are present on the bacterial surface and interact with receptors in the dental film.
HOCl appears to have a greater effect on bacteria that favor adherence than on late colonizers that constitute the biofilm.
Hypochlorous acid has uses in many industries, ranging from applications in agriculture and restaurants to medical care, including in the care and disinfection of chronic wounds [15,16].In addition to its use as a liquid disinfectant, nebulization with hypochlorous vapor has been shown to have virucidal effects against several viruses and bacteria [17].Numerous studies have confirmed that HOCl has efficacy in many clinical fields.In ophthalmology, HOCl in saline solution at a concentration of 100 ppm proved to be effective in decreasing the periocular bacterial load, reducing the staphylococcal load by 99% [18]; as a surface disinfectant, HOCl is effective at a concentration of 1000 ppm [19], and as a hand antiseptic, it has efficacy at a concentration between 100 and 200 ppm [20].
As a mouth rinse, HOCl has not been shown to have a systemic effect and appears to be safe for use in humans [21].In other studies, HOCl has shown a broad antimicrobial spectrum for the inhibition of multiple microorganisms [22,23].Castillo et al. [24] conducted an extensive investigation of the antimicrobial properties of a 0.050% and 0.0250% HOCl mouthwash and determined that HOCl was more effective than CHX (0.2%) against P. gingivalis, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Klebsiella oxytoca.
In the present study, of the three bacteria evaluated, only S. gordonii showed an antibacterial effect.
However, these results may not be very encouraging with respect to some antecedents [25,26].This difference may be due to the different concentrations used, with different pH h in previous studies, the equipment used and even the inputs and volumes used in their preparation.Even so, it constitutes a starting point in a series of steps that must be rigorously followed to reach conclusions on its safety and efficacy.

Conclusion
Hypochlorous acid showed antimicrobial properties against only S. gordonii and was less effective than 4% sodium hypochlorite and 0.12% chlorhexidine, although no significant differences were found between the latter.