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Viability and Effects on Bacterial Proteins by Oral Rinses with Hypochlorous Acid as Active Ingredient

Abstract:

This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients.

Key Words:
hypochlorous acid; chlorhexidine; bacterial viability; proteins; Porphyromonas gingivalis; Aggregatibacter actinomycetemcomitans; Streptococcus mutans.

Resumo:

Este estudo investigou o efeito de enxaguantes à base de ácido hipocloroso (HOCl) e clorexidina (CHX) sobre a viabilidade bacteriana de S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae e E. cloacae. O percentual de bactérias sobreviventes foi testado pelo método de fluorescência utilizando Live/Dead kit(r) e BacLight (Molecular Probes(r)), fazendo comparação entre os grupos com os testes de Kruskal-Wallis e U Mann-Whitney e correção de Bonferroni (p<0,012). O efeito de HOCl e CHX sobre P. gingivalis e S. mutans foi determinado por SDS-PAGE. O CHX mostrou eficácia superior ao HOCl contra S. mutans, A. israelii, E. corrodens e E. cloacae (p<0,001), ao passo que P. gingivalis, A. actinomycetemcomitans, C. rectus e K. oxytoca foram melhores que o CHX para o HOCl (p=0,001). O K. pneumoniae teve efeito similar para o CHX e para o HOCl. As proteínas de P. gingivalis e S. mutans foram afetadas de modo semelhante por CHX e HOCl. O HOCl reduziu a viabilidade bacterial, especialmente nas bactérias periodontopáticas, o que pode recomendar o uso no controle do biofilme subgingival em pacientes periodentais.

Introduction

Dental plaque is the most studied biofilm and it is the most common form of bacterial growth in the oral cavity 11. Palmer RJ. Oral bacterial biofilms - history in progress. Microbiology2009;155: 2113-2114.. Many substances with antimicrobial effects have been developed and evaluated for the control of dental biofilm and gingivitis 22. Teles RP, Teles FR. Antimicrobial agents used in the control of periodontal biofilms: effective adjuncts to mechanical plaque control? Braz Oral Res 2009;23Suppl1:39-48.. Chlorhexidine (CHX) has been extensively studied in the inhibition of dental plaque, gingivitis reduction and is widely used in full-mouth disinfection protocols 33. Van Strydonck DA, Slot DE, Van der Velden U, Van der Weijden F. Effect of a chlorhexidine mouth rinse on plaque, gingival inflammation and staining in gingivitis patients: a systematic review. J. Clin. Periodontol 2012;39:1042-1055.. However, CHX does not seem to be more effective than an essential-oil mouthwash to reduce gingivitis in long-term studies and its main action is the reduction of dental plaque 44. Van Leeuwen MP, Slot DE, Van der Weijden GA. Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review. J. Periodontol 2011;82:174-194. 55. Neely AL. Essential oil mouth wash (EOMW) may be equivalent to chlorhexidine (CHX) for long-term control of gingival inflammation but CHX appears to perform better than EOMW in plaque control. J Evid Based Dent Pract 2012;12:69-72..

There is a great interest in the development of new molecules with antimicrobial effect against oral microorganisms with a significant control of dental caries and periodontal disease. Hypochlorous acid (HOCl) is a non-antibiotic antimicrobial solution developed during the First World War by the dilution and acidification of sodium hypochlorite (Dakin solution) for the treatment of infected wounds 66. Levine JM. Dakin's solution: past, present, and future. Adv Skin Wound Care 2013;26:410-414.. HOCl is part of a group of small molecules known as reactive oxygen species (ROS) synthesized by immune system cells during phagocytosis of antigens 77. Green JN, Kettle AJ, Winterbourn CC. Protein chlorination in neutrophil phagosomes and correlation with bacterial killing. Free Radic Biol Med 2014;77:49-56.. The molecule has demonstrated a broad antimicrobial spectrum for the inhibition of multiple Gram positive and Gram negative microorganisms, without causing side effects such as irritation of the mucosa or extrinsic pigmentation on the tooth surface or restorations. HOCl also has an important anti-inflammatory and proliferative activity (88. 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;11;6:e5. 99. 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;278:28403-28409. 1010. Kim C, Cha YN. Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects. Amino Acids 2014;46:89-100.).

The purpose of this study was to evaluate the antimicrobial activity of hypochlorous acid rinses on oral bacteria (Gram positive and Gram negative) and study its effects on proteins of two of the major opportunistic and pathogenic oral microorganisms: Porphyromonas gingivalis and Streptococcus mutans.

Material and Methods

HOCl Preparation

A special formulation of HOCl for oral health was developed according to formulations and technology patented by Aquilabs S.A. (US patent: US2009/0258083A1) 1111. Method of producing and applications of composition of hypochlorous acid. Available in: Available in: http://www.wipo.int/portal/en/ .s.ds.d
http://www.wipo.int/portal/en/...
. The solutions were obtained with a concentration of available chlorine as HOCl of 250 ppm (0.025%) and 500 ppm (0.05%) according to NTC 1847 with an ORP (oxide reduction potential) of 950-1100 MV and a conductivity of 25.3 ds/m, at a density of 1.01 g/mL at pH 5.8±0.2. HOCl solution was subjected to an accelerated stability test to ensure a stable solution for more than 12 months before losing 10% of its active ingredient.

Antimicrobial Mechanisms

The viability for Streptococcus mutans ATCC 25175, Actinomyces israelii ATCC 12012, Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 29523, Eikenella corrodens ATCC 23834, Campylobacter rectus ATCC 33238, Klebsiella oxytoca ATCC 43086, Klebsiella pneumoniae ATCC 700603, Enterobacter cloacae ATCC 13047 was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)).

Bacterial Culture and Inoculum Standardization

ATTC strains of P. gingivalis, A. israelii, E. corrodens and C. rectus were grown in supplemented brucella agar (0.3% Bacto agar, 0.2% yeast extract, 5% defibrinated sheep blood, 0.2% hemolyzed blood, 0.0005%, hemin, 0.00005% and menadione) and incubated at 37 °C for 4 days in anaerobic conditions (Anaerogen, Oxoid, Hampshire, UK). A. actinomycetemcomitans was seeded in agar Dentaid 1 and incubated at 37 °C for 72 h with 5-10% CO2. S. mutans was grown in blood agar (Blood Agar Base with 5% sheep blood) and incubated at 37 °C for 24 h with 5-10% CO2. K. oxytoca, K. pneumoniae and E. cloacae were plated on BHI agar (Brain Heart Infusion) and incubated for 24-48 h at 37 °C in aerobic atmosphere. Subsequently, inocula were performed in BHI broth (Brain Heart Infusion) and were quantified by spectrophotometry for specific optical densities (OD) of 1 x 108 cells/mL or higher according to Time Kill Assay protocol 1212. Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure 1. International Standards Worldwide 2004;E:2315-03.. After obtaining the expected OD base 10, serial dilutions were plated on appropriate agar for each type of microorganism and incubated under the conditions referred above, to confirm count colony forming units (CFU). Assays were made in triplicate and the results are shown as mean and standard deviation (Table 1).

Table 1
Standardized bacterial inoculum

Bacterial Viability Test

To evaluate the number of live and dead bacteria after exposure to different concentrations of HOCl and CHX 0.2%, the inocula of all previously described bacteria were adjusted to a 1 x 108 bacteria/mL concentration and cultured in BHI broth. 230 μL of each bacteria in culture were exposed by for 30 s to 700 μL of HOCl at 250 ppm and 500 ppm concentrations at pH=5.8. Reactions were neutralized with 70 μL of sodium thiosulfate 0.10 N and the mixture (HOCl + bacteria + neutralizer), and centrifuged at 14,000 rpm during 5 min. The pellet was re-suspended in 50 μL of the supernatant and the excess was discarded. 0.15 μL of the viability mixture (Live/Dead(r) BacLight Bacterial Viability) was added to the 50 μL pellet. The mixture contains two dyes: SYTO 9 3.34 mM and propidium iodide 20 mM. Samples were incubated in low-light conditions for 15 min. The dyes were used to differentiate the bacteria with intact membrane (green fluorescence) from the bacteria with abnormal membrane (red fluorescence). Positive control with CHX 0.2% and negative control without treatment were used in the same experimental conditions. The laminas were preincubated with 2 μL of formaldehyde at 2%, in order to immobilize the bacteria. Images were observed in a fluorescence microscope (Axio-Imager A2; Zeiss, Jena, Germany) in greater magnification and digitized using the AxioVision LE 4.8 software (Zeiss Microscopy). Quantification was determined by the number of pixels for live and dead bacteria using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and data were expressed as percent of viability.

Evaluation of the Protein Integrity

P. gingivalis ATCC 33277 and S. mutans ATCC 25175 were cultured as described above. A suspension of each bacterium was made in BHI broth and standardized to a concentration of 1 x108 bacteria/mL and adjusted spectrophotometrically as above described. Bacterial suspensions were exposed to HOCl at 250 ppm and 500 ppm at pH=5.8. CHX 0.2% was used as positive control and untreated inoculum as negative control. The cell pellet was washed in PBS (Phosphate Buffer Saline) and re-suspended in 2 mL of lysis buffer (50 mM Tris pH=7.5, 50 mM NaCl, 5% glycerol). For P. gingivalis, bacterial inhibitor and proteinase K (10 mg/mL) was added and incubated for 15 min at 65 °C. For S. mutans, buffer plus bacterial protease inhibitor and 20 mg/mL of lysozyme was added and incubated at 37 °C overnight. The samples ere sonicated on ice in a Sonics Vibra-Cell VCX 130 ultrasonic processor (Sonics & Materials, Inc. Newtown, CT, USA) with 40% amplitude, totalizing 7 cycles (30 s on, 15 s off). The extracts were stored at -20 °C.

The protein concentration was determined using the bicinchoninic acid reagent (BCA) to quantify the outer membrane protein by a colorimetric method 1313. Smith PK, Krohn RI, Hermanson G, Mallia A, Gartner FH, Provenzano M, et al.. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76-85. and determining the concentration of the protein according to the manufacturer's instructions (Thermo Specific).

The integrity of the proteins was evaluated using electrophoresis in polyacrylamide gel in 10% SDS (sodium dodecyl sulfate) and a standard pattern of known molecular weight was used to determine the molecular weights of the different bacterial proteins. Separating gels (12% acrylamide/bisacrylamide) were used and the samples diluted in Laemmli buffer in each well with the same protein concentration (20 µg/µL) to evaluate the differences in the electrophoretic profile of the bacteria treated with the different HOCl concentrations. A run buffer with 3% Tris base, 14% glycine, 1% SDS, pH=8.3 was used and the gels were run for 80 min at 100 V. Past the runtime each gel was stained using Silver Stain Kit Pierce(r) (Thermo Fisher Scientific, Inc., Waltham, MA, USA.), following the manufacturer's recommendations.

Statistical Analysis

The average of live bacteria for each experiment was calculated as percentage. The viability reduction was calculated as the difference of living bacteria between the untreated controls per experiment. Descriptive analysis of the means and standard deviations were made. Viability percentages between different rinses were compared through the Kruskal Wallis and U Mann-Whitney test with Bonferroni correction. A pvalue of <0.012 was established for differences between groups.

Results

Inoculum Standardization

Table 1 shows the average and standard deviation of CFU counts for reference strains used in the standardization phase of the bacterial inoculum, required to perform the tests at a concentration of 1x108 bacteria/mL or higher according to the recommendations of the ASTM E2315-03 for the Time Kill Method for antimicrobial agents 1212. Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure 1. International Standards Worldwide 2004;E:2315-03..

Bacterial Viability Assessment

Viability percentages for Gram positive observed in Fig.1A. The viability of S. mutans and A. israelii was significantly affected by CHX to 0.2% and HOCl at 500 ppm when compared with the untreated control and with HOCl at 250 ppm (p=0.001). However, 0.2% CHX showed a higher efficacy against these microorganisms when compared with all groups (p<0.001). HOCl 250 ppm showed greater effect than the control for A. israelii (p=0.0032), but did not show differences against S. mutans (p= 0.64) (Fig. 1). 500 ppm HOCl showed to be the most effective substance reducing the viability of Gram negative organisms associated with periodontal disease. P. gingivalis, A. actinomycetemcomitans and C. rectus showed significant differences for HOCl at 500 ppm in all evaluated groups including CHX 0.2% (p=0.001). CHX 0.2% showed better effectiveness in E. corrodens compared to all groups (p<0.001). However, HOCl to 250 ppm and 500 ppm were more effective than the control group for this microorganism (p<0.001) Figure 1B. Viability of P. gingivalis after treatments is observed in Figure 2.

Figure 1
Percentage of bacterial viability after 30 seconds treatment with CHX, HOCl de 250 ppm and 500 ppm. A: Gram positive bacteria: S. mutans ATCC 25175 and A. israelii ATCC 12012. B. Gram negative bacteria associated to periodontal disease: P. gingivalis ATCC 33277, A. actinomycetemcomitans ATCC 29523, C. rectus ATCC 33238 and E. corrodens ATCC 23834. C. Enteric rods: E. cloacae ATCC 13047, K. oxytoca ATCC 43086 and K. pneumoniae ATCC 700603. p-values come from t-test or Kruskal Wallis y U Mann-Whitney (p≤0,012). a: p-value for differences with untreated; b: p-value for differences with chlorhexidine; c: p-value for differences with HOCl 250 ppm; d: p-value for differences with HOCl 500 ppm.

Figure 2
Bacterial viability of P. gingivalis ATCC 33277. Green fluorescent bacteria are alive and red fluorescing bacteria are dead. A: untreated; B: 0.2% chlorhexidine; C: HOCl 250 ppm, pH 5.8 and D: HOCl 500 ppm, pH 5.8.

The enteric rods commonly found as contaminants in samples of saliva and subgingival plaque were affected by the solutions in study. K. pneumoniae showed a similar reduction to 0.2% CHX and the two concentrations of HOCl (p>0.05). K. oxytoca showed reduced viability for HOCl 500 ppm for all evaluated groups (p<0.001). CHX 0.2% showed a better efficacy against K. oxytoca than HOCl at 250 ppm and the control group (p<0.001). E. cloacae showed a significant decrease in bacterial viability for 0.2% CHX compared to all evaluated groups (Fig. 1C).

Evaluation of the Protein Integrity

After the treatment with HOCl at 500 ppm, 250 ppm and CHX there was an important change in the banding pattern in bacterial proteins for the reference strains of P. gingivalis and S. mutans. A disappearance of a large part of the bands is observable compared to the untreated control.

For P. gingivalis some of the low molecular weight proteins are conserved, on the other side the high molecular weight proteins lose their sharpness as if the proteins concentrations decreased after the treatment with HOCl. After treatment with CHX thin proteic profile is observed. However the protein concentration seems to be affected, suggesting some kind of protein alterations, but less than with HOCl. In S mutans is more evident the disappearance of the bands with high molecular weight and a band of approximately 28 kD is conserved for all treatments. A band of 70 kD for the HOCl at 250 ppm is conserved but in minor concentration. All experiments were made by triplicate and presented the same behavior (Fig. 3).

Figure 3
Total protein electrophoretic profile of P. gingivalis ATCC 33277 (A) and S. mutans ATCC 25175 (B). Lanes: 1 molecular weight marker; 2. Treatment with HOCl 250ppm; 3. Treatment with HOCl 500ppm; 4. Treatment with CHX; 5. Without treatment.

Discussion

There is an agreement on the effect of various antiplaque substances to reduce gingivitis in long term studies 44. Van Leeuwen MP, Slot DE, Van der Weijden GA. Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review. J. Periodontol 2011;82:174-194.. However, the effective reduction of plaque and gingivitis in the short term is still under study. Chlorhexidine remains as the gold standard as antiplaque agent in short and long term effect 55. Neely AL. Essential oil mouth wash (EOMW) may be equivalent to chlorhexidine (CHX) for long-term control of gingival inflammation but CHX appears to perform better than EOMW in plaque control. J Evid Based Dent Pract 2012;12:69-72.. Despite their wide use, some adverse effects have discouraged its use, as tooth discoloration as it is easily mixed with the dietary chromogens, weak microbicidal activity at low concentrations and at high concentrations may produce dermatitis and desquamation of oral mucosa as well as delay in healing 1414. Jones CG. Chlorhexidine: is it still the gold standard? Periodontol2000 1997;15:55-62. 1515. Graziani F, Gabriele M, D'Aiuto F, Suvan J, Tonelli M, Cei S. Dental plaque, gingival inflammation and tooth -discolouration with different commercial formulations of 0.2% chlorhexidine rinse: a double-blind randomised controlled clinical trial. Oral Health Prev Dent 2015;13:101-111..

Hypochlorous acid rinses are proposed for plaque control and as a wound healing agent for its use in oral health, due to its low toxicity, high antimicrobial efficacy, anti-inflammatory effect, induction to cell proliferation and its background as a topical solution in the antisepsis of wounds in clinical medicine 1616. Selkon J,B, Cherry GW, Wilson JM, Hughes M A. Evaluation of hypochlorous acid washes in the treatment of chronic venous leg ulcers. J Wound Care 2006;15:33-37.. HOCl has also the ability to oxidize the amino acid taurine and induce the formation of chlorine-taurine (TauCl) which has broad spectrum antimicrobial activity. The TauCl has a significant protective effect on tissues because it can inhibit the production of inflammatory mediators and thereby contribute to the processes of tissue protection 1010. Kim C, Cha YN. Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects. Amino Acids 2014;46:89-100..

In this study, HOCl showed a significant effect on Gram positive bacteria but did not exceed the effect of chlorhexidine. Chlorhexidine has shown better effect on Gram positive microorganisms but less on Gram negative microorganisms 1717. De Rossi A, Ferreira DC, da Silva RA, de Queiroz AM, da Silva LA, Nelson-Filho P. Antimicrobial activity of toothpastes containing natural extracts, chlorhexidine or triclosan. Braz Dent J 2014;25:186-190. 1818. Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-Filho FJ. Comparative study of the antimicrobial efficacy of chlorhexidine gel, chlorhexidine solution and sodium hypochlorite as endodontic irrigants. Braz Dent J 2007;18:294-298.. The antimicrobial action of HOCl appears to be greater in Gram negative than in Gram positive possibly because Gram negative bacteria has sulfur and hem groups (rich in iron) in its membrane which causes an irreversible reaction HOCl/membrane proteins, producing structural damage, and altering cell permeability, affecting bacterial viability in Gram negative bacteria 1919. Rosen H, Klebanoff S. Oxidation of Escherichia coli iron centers by the myeloperoxidase-mediated microbicidal system. J Biol Chem 1982;257:13731-13735. 2020. Mckenna SM, Davies KJ. The inhibition of bacterial growth by hypochlorous acid. Possible role in the bactericidal activity of phagocytes. Biochem J 1988;254:685-692.. The HOCl oxidizes and/or chlorinates endotoxins and exotoxins such as lipopolysaccharides and gingipains as Rgp and Kgp neutralizing their action. In Gram positive bacteria HOCl oxidizes glycine residues present in the peptidoglycan, on the other hand chlorination reactions in this group of microorganisms differs in the action on the target 2121. Chong-Hou S, Hsein-Kun L. The role of hypochlorous acid as one of the reactive oxygen species in periodontal disease. J Dent Sci 2009;4:45-54..

Different authors have controversial uses of microbiological culture methods to evaluate the efficacy of antimicrobials. The bacterial viability assessment with specific methods such as fluorochromes or epifluorescence has been proposed for evaluation of antimicrobials 2222. Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T. Assessment and interpretation of bacterial viability by using the LIVE ⁄DEAD Baclight Kit in combination with flow cytometry. Appl Environ Microbiol 2007;73:3283-3290.. In the present study a method of epifluorescence was used for the evaluation of the bacterial viability similar to the reported by other studies that have evaluated the substantivity of antiplaque substances 2323. García-Caballero L, Carmona IT, González MC, Posse JL, Taboada JL, Dios PD. Evaluation of the substantivity in saliva of different forms of application of chlorhexidine. Quintessence Int 2009;40:141-144. 2424. Herrera D, Roldan S, Santacruz I, Santos S, Masdevall M, Sanz M: Differences in antimicrobial activity of four commercial 0.12% chlorhexidine mouth rinse formulations: an in vitro contact test and salivary bacterial counts study. J Clin Periodontol 2003;30:307-314..

Proteins of P. gingivalis and S. mutans were affected after 30 s of treatment with the test solutions. For P. gingivalis, after HOCl and CHX treatments it is observed a similar reduction of the protein concentrations in many bands when is compared with the untreated control. In S. mutans there is an elimination of almost all the proteins for the different treatments when compared with the untreated control. Cheung et al., in 2011 have shown changes in the proteic profile in Bacillus subtilis and E. coli after treatment with CHX, suggesting that the mechanism of action of CHX is related with an alteration of lipidic stability of the cell membrane 2525. Cheung HY1, Wong MM, Cheung SH, Liang LY, Lam YW, Chiu SK. Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli. PLoS One 2012;7:e36659.. It is not clear which is the action of HOCl on bacteria, however oxidation and chlorination of amino groups in some functional and structural proteins in Gram positive and Gram negative bacteria is suggested 77. Green JN, Kettle AJ, Winterbourn CC. Protein chlorination in neutrophil phagosomes and correlation with bacterial killing. Free Radic Biol Med 2014;77:49-56.. In further it is important to identify the proteins affected by HOCl action and thus explain how this antimicrobial molecule affects the bacterial viability and why in some Gram negative bacteria as E. cloacae does not have an important reduction in the viability.

The formulation of HOCl has been stabilized in Colombia and patented as an substance with antimicrobial effects for medical applications such as the treatment of chronic and non-healing wounds 1111. Method of producing and applications of composition of hypochlorous acid. Available in: Available in: http://www.wipo.int/portal/en/ .s.ds.d
http://www.wipo.int/portal/en/...
. Findings of this study could support future research of HOCl as antimicrobial and antiplaque agent in dentistry.

HOCl showed better effects on bacterial viability than CHX in Gram negative microorganism specially in P. gingivalis, A. actinomycetemcomitans and C. rectus. HOCl could have a significant effect on periodontophatic bacteria that could colonize and aggregates as dental biofilm.

Acknowledgements

The authors thanks to the Colombian Department for Science, Technology and Innovation (COLCIENCIAS) as the sponsor of this project through the Grant No 130850227678 To Dr. Marcela Buitrago for it assistance in the development of the SDS-Page.

References

  • 1
    Palmer RJ. Oral bacterial biofilms - history in progress. Microbiology2009;155: 2113-2114.
  • 2
    Teles RP, Teles FR. Antimicrobial agents used in the control of periodontal biofilms: effective adjuncts to mechanical plaque control? Braz Oral Res 2009;23Suppl1:39-48.
  • 3
    Van Strydonck DA, Slot DE, Van der Velden U, Van der Weijden F. Effect of a chlorhexidine mouth rinse on plaque, gingival inflammation and staining in gingivitis patients: a systematic review. J. Clin. Periodontol 2012;39:1042-1055.
  • 4
    Van Leeuwen MP, Slot DE, Van der Weijden GA. Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review. J. Periodontol 2011;82:174-194.
  • 5
    Neely AL. Essential oil mouth wash (EOMW) may be equivalent to chlorhexidine (CHX) for long-term control of gingival inflammation but CHX appears to perform better than EOMW in plaque control. J Evid Based Dent Pract 2012;12:69-72.
  • 6
    Levine JM. Dakin's solution: past, present, and future. Adv Skin Wound Care 2013;26:410-414.
  • 7
    Green JN, Kettle AJ, Winterbourn CC. Protein chlorination in neutrophil phagosomes and correlation with bacterial killing. Free Radic Biol Med 2014;77:49-56.
  • 8
    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;11;6:e5.
  • 9
    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;278:28403-28409.
  • 10
    Kim C, Cha YN. Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects. Amino Acids 2014;46:89-100.
  • 11
    Method of producing and applications of composition of hypochlorous acid. Available in: Available in: http://www.wipo.int/portal/en/ s.ds.d
    » http://www.wipo.int/portal/en/
  • 12
    Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure 1. International Standards Worldwide 2004;E:2315-03.
  • 13
    Smith PK, Krohn RI, Hermanson G, Mallia A, Gartner FH, Provenzano M, et al.. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76-85.
  • 14
    Jones CG. Chlorhexidine: is it still the gold standard? Periodontol2000 1997;15:55-62.
  • 15
    Graziani F, Gabriele M, D'Aiuto F, Suvan J, Tonelli M, Cei S. Dental plaque, gingival inflammation and tooth -discolouration with different commercial formulations of 0.2% chlorhexidine rinse: a double-blind randomised controlled clinical trial. Oral Health Prev Dent 2015;13:101-111.
  • 16
    Selkon J,B, Cherry GW, Wilson JM, Hughes M A. Evaluation of hypochlorous acid washes in the treatment of chronic venous leg ulcers. J Wound Care 2006;15:33-37.
  • 17
    De Rossi A, Ferreira DC, da Silva RA, de Queiroz AM, da Silva LA, Nelson-Filho P. Antimicrobial activity of toothpastes containing natural extracts, chlorhexidine or triclosan. Braz Dent J 2014;25:186-190.
  • 18
    Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-Filho FJ. Comparative study of the antimicrobial efficacy of chlorhexidine gel, chlorhexidine solution and sodium hypochlorite as endodontic irrigants. Braz Dent J 2007;18:294-298.
  • 19
    Rosen H, Klebanoff S. Oxidation of Escherichia coli iron centers by the myeloperoxidase-mediated microbicidal system. J Biol Chem 1982;257:13731-13735.
  • 20
    Mckenna SM, Davies KJ. The inhibition of bacterial growth by hypochlorous acid. Possible role in the bactericidal activity of phagocytes. Biochem J 1988;254:685-692.
  • 21
    Chong-Hou S, Hsein-Kun L. The role of hypochlorous acid as one of the reactive oxygen species in periodontal disease. J Dent Sci 2009;4:45-54.
  • 22
    Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T. Assessment and interpretation of bacterial viability by using the LIVE ⁄DEAD Baclight Kit in combination with flow cytometry. Appl Environ Microbiol 2007;73:3283-3290.
  • 23
    García-Caballero L, Carmona IT, González MC, Posse JL, Taboada JL, Dios PD. Evaluation of the substantivity in saliva of different forms of application of chlorhexidine. Quintessence Int 2009;40:141-144.
  • 24
    Herrera D, Roldan S, Santacruz I, Santos S, Masdevall M, Sanz M: Differences in antimicrobial activity of four commercial 0.12% chlorhexidine mouth rinse formulations: an in vitro contact test and salivary bacterial counts study. J Clin Periodontol 2003;30:307-314.
  • 25
    Cheung HY1, Wong MM, Cheung SH, Liang LY, Lam YW, Chiu SK. Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli PLoS One 2012;7:e36659.

Publication Dates

  • Publication in this collection
    Oct 2015

History

  • Received
    13 Dec 2014
  • Accepted
    23 June 2015
Fundação Odontológica de Ribeirão Preto Av. do Café, S/N, 14040-904 Ribeirão Preto SP Brasil, Tel.: (55 16) 3602-3982, Fax: (55 16) 3633-0999 - Ribeirão Preto - SP - Brazil
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