Discussion

There is an agreement on the effect of various antiplaque substances to reduce gingivitis in long term studies ⁴. 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 ⁵. 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 ^14 15.

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 ¹⁶. 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 ¹⁰.

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 ^17 18. 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 ^19 20. 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 ²¹.

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 ²². 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 ^23 24.

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 ²⁵. 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 ⁷. 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 ¹¹. 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.