ABSTRACT

Objective:

To evaluate the antibacterial effect of electrolytically generated hypochlorous acid on Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis.

Material and Methods:

In this in vitro experiment, the effect of hypochlorous acid (HOCl) on the strains S. gordonii, F. nucleatum, and P. gingivalis was evaluated using 4% sodium hypochlorite, 0.12% chlorhexidine, and distilled water as controls. The four groups were placed on each plate, and each group was replicated five times. The agar diffusion method by zones measurement was used. The data were processed with SPSS using the Kruskal-Wallis test and multiple comparison tests.

Results:

Hypochlorous acid showed an average inhibition halo of 9.28 mm on S. gordonii. 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 and P. gingivalis.

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.

Keywords:
Microbiology; Anti-Bacterial Agents; Hypochlorous Acid; Sodium Hypochlorite

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 [¹1 Takahashi N. Oral microbiome metabolism: From "who are they?" to "what are they doing?" J Dent Res 2015; 94(12):1628-37. https://doi.org/10.1177/0022034515606045
https://doi.org/10.1177/0022034515606045... , ²2 Rodr√≠guez JAL, Casana STV. Effectiveness of chlorhexidine and essential oils associated with scaling and root planing in the treatment of chronic periodontitis. Rev Cienc Salud 2020; 18(3):1-11.]. 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 [³3 Amaranto REB, Millones-Gómez P. Efectividad analgésica del clonixinato de lisina asociado con el paracetamol en el tratamiento posoperatorio de exodoncias. Rev Cienc Salud 2019; 17(2):321-33. https://doi.org/10.12804/revistas.urosario.edu.co/revsalud/a.7943 [In Spanish].
https://doi.org/10.12804/revistas.urosar... ]. 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 [⁴4 Muras A, Otero-Casal P, Blanc V, Otero A. Acyl homoserine lactone-mediated quorum sensing in the oral cavity: a paradigm revisited. Sci Rep 2020; 10(1):9800. https://doi.org/10.1038/s41598-020-66704-4
https://doi.org/10.1038/s41598-020-66704... ].

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 [⁴4 Muras A, Otero-Casal P, Blanc V, Otero A. Acyl homoserine lactone-mediated quorum sensing in the oral cavity: a paradigm revisited. Sci Rep 2020; 10(1):9800. https://doi.org/10.1038/s41598-020-66704-4
https://doi.org/10.1038/s41598-020-66704... ].

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 [⁵5 Gray MJ, Wholey WY, Jakob U. Bacterial responses to reactive chlorine species. Annu Rev Microbiol 2013; 67:141-60. https://doi.org/10.1146/annurev-micro-102912-142520
https://doi.org/10.1146/annurev-micro-10... ]. 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 [⁶6 Wang L, Bassiri M, Najafi R, Najafi K, Yang J, Khosrovi B, et al. Hypochlorous acid as a potential wound care agent: part I. Stabilized hypochlorous acid: a component of the inorganic armamentarium of innate immunity. J Burns Wounds 2007; 6:e5.].

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 [⁷7 Selkon JB, Cherry GW, Wilson JM, Hughes MA. Evaluation of hypochlorous acid washes in the treatment of chronic venous leg ulcers. J Wound Care 2006; 15(1):33-7. https://doi.org/10.12968/jowc.2006.15.1.26861
https://doi.org/10.12968/jowc.2006.15.1.... ]. Regulation of the normal flora contributes to periodontal health, and HOCl appears to have the ability to attack gram-negative pathogens during periodontitis [⁸8 Sam CH, Lu HK. The role of hypochlorous acid as one of the reactive oxygen species in periodontal disease. J Dent Sci 2009; 4(2):45-54. https://doi.org/10.1016/S1991-7902(09)60008-8
https://doi.org/10.1016/S1991-7902(09)60... ]. 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.

Material and Methods

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.

200 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 200 ppm chlorine in HOCl. [⁹9 Hakim H, Thammakarn C, Suguro A, Ishida Y, Kawamura A, Tamura M, et al. Evaluation of sprayed hypochlorous acid solutions for their virucidal activity against avian influenza virus through in vitro experiments. J Vet Med Sci 2015; 77(2):211-5. https://doi.org/10.1292/jvms.14-0413
https://doi.org/10.1292/jvms.14-0413... ].

Strains Used

The strains used were S. gordonii ATCC 51656, F. nucleatum ATCC 10953, and P. gingivalis ATCC 33277.

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 [¹⁰10 Millones-Gómez PA, Maurtua-Torres D, Bacilio-Amaranto R, Calla-Poma RD, Requena-Mendizabal MF, Valderrama-Negron AC, et al. Antimicrobial activity and antiadherent effect of peruvian Psidium guajava (Guava) leaves on a cariogenic biofilm model. J Contemp Dent Pract 2020; 21(7):733-40. https://doi.org/10.5005/jp-journals-10024-2893
https://doi.org/10.5005/jp-journals-1002... , ¹¹11 Millones Gómez PA, Tay Chu Jon LY, Maurtua Torres DJ, Bacilio Amaranto RE, Collantes Díaz IE, Medina CAM. Antibacterial, antibiofilm, and cytotoxic activities and chemical compositions of Peruvian propolis in an in vitro oral biofilm. F1000 Res 202; 10:1093. https://doi.org/10.12688/f1000research.73602.1
https://doi.org/10.12688/f1000research.7... ].

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 [¹¹11 Millones Gómez PA, Tay Chu Jon LY, Maurtua Torres DJ, Bacilio Amaranto RE, Collantes Díaz IE, Medina CAM. Antibacterial, antibiofilm, and cytotoxic activities and chemical compositions of Peruvian propolis in an in vitro oral biofilm. F1000 Res 202; 10:1093. https://doi.org/10.12688/f1000research.73602.1
https://doi.org/10.12688/f1000research.7... ]. This procedure was repeated five times.

Four groups were formed: HOCl, 4% sodium hypochlorite (NaClO), 0.12% CHX, and distilled water. 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.

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).

Figure 1
Agar plates with hypochlorous acid, 0.12% chlorhexidine, distilled water, and 4% sodium hypochlorite on S. gordonii (A), F. nucleatum (B), and P. gingivalis (C)

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=0.00l), 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% NaCIO 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 [¹²12 Kriswandini IL, Diyatri I, Tantiana, Nuraini P, Berniyanti T, Putri IA, et al. The forming of bacteria biofilm from Streptococcus mutans and Aggregatibacter actinomycetemcomitans as a marker for early detection in dental caries and periodontitis. Infect Dis Rep 2020; 12(Suppl 1):8722. https://doi.org/10.4081/idr.2020.8722
https://doi.org/10.4081/idr.2020.8722... , ¹³13 Millones-G√≥mez PA, Amaranto REB, Torres DJM, Calla-Poma RD, Requena-Mendizabal MF, Alvino-Vales MI, et al. Identification of proteins associated with the formation of oral biofilms. Pesqui Bras Odontopediatria Clin Integr 2021; 21:e0128. https://doi.org/10.1590/pboci.2021.084
https://doi.org/10.1590/pboci.2021.084... ]. Sarduy-Bermúdez and González Díaz [¹⁴14 Sarduy Bermúdez L, González Díaz ME. Biofilm: a new conception of dentobacterial plaque. Medicentro 2016; 20(3):167-75.] 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 [¹⁵15 Ishihara M, Murakami K, Fukuda K, Nakamura S, Kuwabara M, Hattori H, et al. Stability of weakly acidic hypochlorous acid solution with microbicidal activity. Biocontrol Sci 2017; 22(4):223-7. https://doi.org/10.4265/bio.22.223
https://doi.org/10.4265/bio.22.223... , ¹⁶16 Veasey S, Muriana PM. Evaluation of electrolytically-generated hypochlorous acid ('electrolyzed water') for sanitation of meat and meat-contact surfaces. Foods 2016; 5(2):42. https://doi.org/10.3390/foods5020042
https://doi.org/10.3390/foods5020042... ]. 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 [¹⁷17 Green JN, Kettle AJ, Winterbourn CC. Protein chlorination in neutrophil phagosomes and correlation with bacterial killing. Free Radic Biol Med 2014; 77:49-56. https://doi.org/10.1016/j.freeradbiomed.2014.08.013
https://doi.org/10.1016/j.freeradbiomed.... ] 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% [¹⁸18 Stroman DW, Mintun K, Epstein AB, Brimer CM, Patel CR, Branch JD, et al. Reduction in bacterial load using hypochlorous acid hygiene solution on ocular skin. Clin Ophthalmol 2017; 11:707-14. https://doi.org/10.2147/opth.s132851
https://doi.org/10.2147/opth.s132851... ]; as a surface disinfectant, HOCl is effective at a concentration of 1000 ppm [¹⁹19 Overholt B, Reynolds K, Wheeler D. 1151. A safer, more effective method for cleaning and disinfecting gi endoscopic procedure rooms. Open Forum Infect Dis 2018; 5(Suppl 1):S346. https://doi.org/10.1093/ofid/ofy210.984
https://doi.org/10.1093/ofid/ofy210.984... ], and as a hand antiseptic, it has efficacy at a concentration between 100 and 200 ppm [²⁰20 Wolfe MK, Gallandat K, Daniels K, Desmarais AM, Scheinman P, Lantagne D. Handwashing and Ebola virus disease outbreaks: A randomized comparison of soap, hand sanitizer, and 0.05% chlorine solutions on the inactivation and removal of model organisms Phi6 and E. coli from hands and persistence in rinse water. PLoS One 2017; 12(2):e0172734. https://doi.org/10.1371/journal.pone.0172734
https://doi.org/10.1371/journal.pone.017... ].

As a mouth rinse, HOCl has not been shown to have a systemic effect and appears to be safe for use in humans [²¹21 Morita C, Nishida T, Ito K. Biological toxicity of acid electrolyzed functional water: effect of oral administration on mouse digestive tract and changes in body weight. Arch Oral Biol 2011; 56(4):359-66. https://doi.org/10.1016/j.archoralbio.2010.10.016
https://doi.org/10.1016/j.archoralbio.20... ]. In other studies, HOCl has shown a broad antimicrobial spectrum for the inhibition of multiple microorganisms [²²22 Fu X, Kassim SY, Parks WC, Heinecke JW. Hypochlorous acid generated by myeloperoxidase modifies adjacent tryptophan and glycine residues in the catalytic domain of matrix metalloproteinase-7 (matrilysin): an oxidative mechanism for restraining proteolytic activity during inflammation. J Biol Chem 2003; 278(31):28403-9. https://doi.org/10.1074/jbc.M304739200
https://doi.org/10.1074/jbc.M304739200... , ²³23 Wang L, Bassiri M, Najafi R, Najafi K, Yang J, Khosrovi B, et al. Hypochlorous acid as a potential wound care agent: Part I. Stabilized hypochlorous acid: a component of the inorganic armamentarium of innate immunity. J Burns Wounds 2007; 6:e5.]. Castillo et al. [²⁴24 Castillo DM, Castillo Y, Delgadillo NA, Neuta Y, Jola J, Calderón JL, et al. Viability and effects on bacterial proteins by oral rinses with hypochlorous acid as active ingredient. Braz Dent J 2015; 26(5):519-24. https://doi.org/10.1590/0103-6440201300388
https://doi.org/10.1590/0103-64402013003... ] 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 [²⁵25 Lafaurie GI, Zaror C, Díaz-Báez D, Castillo DM, De Ávila J, Trujillo TG, et al. Evaluation of substantivity of hypochlorous acid as an antiplaque agent: a randomized controlled trial. Int J Dent Hyg 2018; 16(4):527-34. https://doi.org/10.1111/idh.12342
https://doi.org/10.1111/idh.12342... , ²⁶26 Chen CJ, Chen CC, Ding SJ. Effectiveness of hypochlorous acid to reduce the biofilms on titanium alloy surfaces in vitro. Int J Mol Sci 2016; 17(7):1-5. https://doi.org/10.3390/ijms17071161
https://doi.org/10.3390/ijms17071161... ]. 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.

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

References

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