An In Vitro Comparison of the Effect of Wound Irrigating Solution (0.01% Hypochlorous Acid) and 2.5% Sodium Hypochlorite Against Enterococcus Faecalis.

Aim: The aim of this in vitro study was to evaluate and compare the antimicrobial activity of normal saline, 2.5% sodium hypochlorite, and 0.01% hypochlorous acid against Enterococcus faecalis. Materials and
Methods: Antimicrobial activity was assayed by Kirby-Bauer cup-well agar diffusion method. Three cup-wells were bored in each petriplate and were filled with 50 μl of each of the respective test agents. Culture plates were placed in an incubator at 37°C and after 24 hours, the size of the magnification inhibition zone was quantified with a caliper and recorded in respective tables. The experiment was reiterated ten times for each reagent, and the diameter of the magnification inhibition zones caused by the irrigation solutions was recorded. The antimicrobial activity of the three test solutions was determined.
Results: The antibacterial property of the three groups was compared by utilizing ANOVA test. The association was found to be statistically significant (P = 0.000). The antibacterial property between 2.5% NaOCl and 0.01% HOCl was further compared with independent t test. This association was however not statistically significant (P = 0.02). Copyright:

INTRODUCTION

Endodontic ailment is brought about by polymicrobial infections.[1] Microbes are available in all parts of the root canal system, including fins and anastomoses, and might be found at different profundities of up to 300 μm inside the dentinal tubules from the pulpal end.[2]

The morphology of root channel structure is multifaceted and this favors development of microbes as biofilms.[3]

The literature has demonstrated that microorganisms are real contributors to the start and propagation of pulpal and periapical diseases.[456]

Enterococci now rank among the main three nosocomial bacterial pathogens, and strains impervious to, as of now, accessible anti-microbials posture genuine restorative troubles. Up to 90% of enterococcal diseases in people are brought on by Enterococcus faecalis. It is the most secluded or recognized species of oral diseases, including marginal periodontitis, infected root canals, and periradicular abscesses.[7]

E. faecalis are gram-positive cocci and facultative anaerobes.[8] It is known to be a resistant species which can withstand certain substance specialists, including a few anti-infection agents which would be exceedingly dangerous to other organisms.[9] They are ordinary intestinal living beings and may occupy the oral depression and gingival sulcus. When this bacterium is available in little numbers, it is effortlessly wiped out; yet in the event that it is in extensive numbers, it is difficult to eradicate.[8]

The utilization of irrigating solutions is a vital piece of powerful chemo-mechanical preparation. It improves bacterial elimination and encourages expulsion of necrotic tissue and dentine chips from the root canal; consequently, irrigants avoid pressing of contaminated tissue apically in the root canal and into the periapical region.[10]

To boost the expulsion of microorganisms, the shaping and mechanical extension of a root canal must be joined by overflowing irrigation.[11]

The utilization of chemical solutions are required with a specific goal to clean the root canal system and wipe out as many microorganisms as possible.[12]

Sodium hypochlorite (NaOCl) has broadly been acknowledged as a root canal irrigant since its initially reported use by Walker in 1936.[13] Known as the most contemplated specialist, sodium hypochlorite (NaOCl) has turned into the gold standard for the irrigation of root canal space in view of the clinical efficacy in endodontic treatment. It has dissolvable action for both necrotic and vital tissues. In any case, there are still some worries about its toxic effects, terrible stench and taste, destructive potential, and unfavorably susceptible reactions.[14]

In vivo, HOCl is produced intracellularly in abundance because of phagocytosis of pathogens by neutrophils, and assumes a critical part in the decimation of pathogens.[15]

The center of infection is the root canal and the dentinal tubules, which are difficult to reach to the components of the host defence system. Subsequently, treatment or preventive techniques ought to principally incorporate local as opposed to systemic means.[7]

The perfect irrigant ought to have an antimicrobial activity, low toxicity, and great biocompatibility to oral tissues. HOCl is biocompatible, antimicrobial against a wide scope of microorganisms,[11] non-cytotoxic, and non-irritating.[16]

The antimicrobial activity of hypochlorous acid against E. faecalis has not been determined.

Thus, hypochlorous acid was selected in the present study to determine its anti-bacterial efficacy against E. faecalis.

MATERIALS AND METHODS

The present in vitro study has been carried out in the Department of Pedodontics and Preventive Dentistry, Rajah Muthiah Dental College & Hospital, in association with the Department of Chemistry and Department of Microbiology, Annamalai University to compare the anti-bacterial effect of 2.5% sodium hypochlorite and 0.01% hypochlorous acid with saline being used as a control group against E. faecalis.

Preparation of 2.5% sodium hypochlorite

Reagent-grade 5% NaOCl (Spectra Chemicals) was weakened with distilled water to acquire 2.5% NaOCl concentration.

Preparation of 0.01% hypochlorous acid

Hypochlorous acid was set up in 154 mM NaCl by acidifying reagent-grade NaOCl to the pH range of 3.5 to 4.0 with dilute HCl. A pH meter was utilized to precisely quantify the last pH values. The concentration of active total chlorine species in solution communicated as [HOCl] T=[HOCl]+[Cl2]+[Cl-3]+[OCl-] in 0.9% saline was controlled by changing all of the dynamic chlorine species to OCl- with 0.1 M NaOH and measuring the concentration of OCl-. The concentration of OCl- was resolved spectro-photometrically at 292 nm (ε = 362 M-1 cm-1) with a Systronics Digital Spectrophotometer 105. This strategy for hypochlorous acid preparation was as per Wang L et al.[15]

Microbiological procedures

E. faecalis strains (ATCC 49532) were collected from the Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu.

This in vitro experimental study assessed and looked at the antimicrobial efficacy of hypochlorous acid with sodium hypochlorite on E. faecalis. Bile esculin agar medium was utilized for the culture of E. faecalis. A 0.5 McFarland suspension was set up from the bacterial species and was affirmed with spectrophotometer. At 0.5 McFarland, number of microorganisms is equivalent to 1.5 × 108. The bacterial suspension was swabbed to the whole surface of the bile esculin agar culture plate using a sterile cotton swab.

Antimicrobial action was measured by the Kirby–Bauer cup-well agar diffusion method.[8] Three cup-wells were bored into each petriplate and were loaded with 50 μl[1718] of each of the separate test solutions. Culture plates were loaded in an incubator at 37°C and following 24 hours, the size of the growth inhibition zone was measured with a caliper and recorded in the corresponding tables. E. faecalis causes a dark staining on bile esculin agar medium because of sedimentation of iron.[19] The examination was rehashed ten times for every reagent and the distance across the development restraint zones brought about by irrigation solutions was recorded.

The zones of inhibition were measured with a caliper in mm. The values obtained were compared using ANOVA and independent t test using SPSS software.

P value of < 0.01 was considered as statistically significant.

RESULTS

The antimicrobial activity of the three test solutions was determined. This was found to be 0 mm, 18.20 mm and 15.40 mm for saline, 2.5% NaOCl and 0.01% HOCl respectively [Table 1 and Graph 1]. The antibacterial property of the three groups was compared using ANOVA. The association was found to be statistically significant (P = 0.000) [Table 1].

Table 1

Zone of inhibition of saline vs 2.5% NaOCl vs 0.01% HOCl

GR	AGENT	n	MEAN (mm)	RANGE (mm)	MEAN±SD (mm)	ANOVA
						F	P
I	Saline	10	0	0	0.00	1752	0.000
II	NaOCl	10	18.20	17-20	18.20±0.92
III	HOCl	10	15.40	14-17	15.40±1.08

Graph 1

Zone of inhibition of saline vs 2.5% NaOCL vs 0.01% HOCl

The antibacterial property between 2.5% NaOCl and 0.01% HOCl was further compared with independent t test. This association was however not statistically significant (P = 0.02) [Table 2].

Table 2

Inhibition zone exhibited by 0.01% HOCl, 2.5% NaOCl and saline

T	P
2.236	0.02

DISCUSSION

E. faecalis is viewed as the most noteworthy species, both regarding recurrence of segregation from sites of disease and in the exchange of antibiotic resistance.[20] The most elevated frequencies of seclusion of E. faecalis have been accounted for by Peciuliene et al.[21] and Pinheiro et al.[22] at 64% and 53% of the culture-positive teeth, respectively.

E. faecalis was accounted as treatment-safe microscopic organisms in root canals.[23] In both short-term and long-term incubation periods, E. faecalis has appeared to colonize sedated root canals with conceivable biofilm formation. This is the motivation behind why E. faecalis is regularly utilized as a part of studies with respect to the effectiveness of the endodontic irrigants in cleaning the root canal system.[24]

It was Fleming who reported the inhibitory impact of what in the long run came to be known as penicillin on solid media by observing an area of growth inhibition of Staphylococcal colonies adjoining a Penicillium contaminant on an agar plate. This, in any case, was not the principal portrayal of what was in the long run termed as agar diffusion, as this was initially used by Beijerinck in 1889 for contemplating the impact of various auxins on bacterial growth. Notwithstanding the revelation of penicillin, Fleming made two further critical commitments to the field of antimicrobial susceptibility testing in the 1920s. In 1924, he presented the utilization of the ditch plate strategy for assessing antimicrobial characteristics of antiseptic solutions. Rosy modified this system by cutting wells into the agar and filling the wells with antiseptic solutions.[25]

In a study directed by C Chuard and L B Reller,[26] it was observed that the bile esculin test functions admirably to quickly isolate Enterococci and group D Streptococci from non-group D viridans Streptococci, requiring little to no effort and with great sensitivity (99%) and specificity (97%), if it is performed on agar slants containing 40% bile, done with an standardized inoculum (10 ml of a 0.5 McFarland standard bacterial suspension), and read at 24 hours. Therefore, in the present study, bile esculin agar medium was utilized to culture E. faecalis.

It has been accounted for that around 80% of root canals can be rendered microbe-free with the utilization of an antimicrobial irrigant (Bystrom and Sundqvist 1981, Bystrom and Sundqvist 1983).[2]

According to J F Siqueira J R et al.[27] root canals irrigated with sterile saline solution were positive for bacterial growth. This is in accordance with the present study.

Taking into account the study led by Jefferson J C Marion et al.,[28] both 2.5% NaOCl and 5.25% NaOCl have comparative properties, yet the first is less cytotoxic, being more indicated for root canals endodontic treatment. Subsequently, in the present study 2.5% NaOCl was utilized as one of the testing solutions.

NaOCl has a few undesirable attributes, for example, tissue lethality at high concentrations, danger of emphysema, and unfavorably susceptible potential. In addition, NaOCl does not thoroughly clean the surfaces of the root canals. These issues indicate that NaOCl irrigation is not completely advanced and there is a need to recognize irrigants which are powerful, additionally biocompatible, to keep away from the danger of harming patients.[11] Thus, to overcome these limitations, it was decided to evaluate the efficacy of a new agent as a root canal irrigant.

Hypochlorous acid can be incorporated using one of the three techniques: Hydrolysis of chlorine gas (eq1), electrolysis of salt arrangement (eq2a and eq2b), and acidification of hypochlorite (eq3).

eq1: Cl2 + H2O → HOCl + H+ + Cl-

eq2a: 2Cl- + 2e- →Cl2

eq2b: Cl2 + H2O → HOCl + H++ Cl-

eq3: OCl- + H+ → HOCl

The confinements of utilizing (eq1) are the natural risks of taking care of chlorine and the trouble in control.

The weakness of the electrolysis strategy (eq2) is the trouble in controlling the objective concentration of solution.

Since hypochlorite is industrially accessible, the utilization of hypochlorite (eq3) is the favored technique, and is more advantageous, safe, and controllable when contrasted with the other two strategies. So, in the present study, the third strategy was favored for the preparation of hypochlorous acid (HOCl).

In the present study, the pH of the prepared HOCl solution was adjusted to 4 on the grounds that at a higher pH, hypochlorite particle (OCl−) is formed, while at lower pH, the solution exists as a blend of chlorine (Cl2) in solution, chlorine gas in the headspace, and HOCl.

To keep the solution stable and keep up its desired action, the pH of the solution ought to stay somewhere around 3.5 and 5, and the solution ought to be put away in a firmly sealed container.

Stable HOCl is set up by addition of NaOCl to a solution of sodium chloride in sterile water, followed by the addition of a solution of hydrochloric acid to form the active segment, HOCl. Stabilized HOCl is a dilute solution of HOCl in 150 mM (0.9%) sodium chloride at an un-buffered pH of 3.5. This is as per the preparation done by Wang L et al.[15]

The cytotoxicity test led by Dmitri Debabov et al.[30] utilizing L929 (ATCC CCL-1TM) mouse fibroblast cells demonstrated that the viable cell count was 73.77% ± 7.32% with 0.01% HOCl when contrasted with the untreated control. Then again, when the 0.02% hypochlorous acid was tested, the viable cell count was 2.44% ± 0.24% when contrasted with the untreated control. In this manner, in this cytotoxicity examination, 0.01% HOCl was non-cytotoxic, though 0.02% hypochlorous acid was cytotoxic. Hence, in the present study, a concentration of 0.01% HOCl was chosen as another testing solution against E. faecalis.

In the present study, no zone of inhibition was produced for saline wells. This is in accordance with a study conducted by Uday Kamath et al.[18] who found that there was no zone of inhibition shown by normal saline.

The mean zone of inhibition for sodium hypochlorite was found to be 18.20 ± 0.92 mm in the present study [Table 1, Figure 1]. This was in accordance with the study conducted by Sandeep Dubey et al.[17]

Figure 1

Effect of saline, 0.01% Hypochlorous acid and 2.5% sodium hypochlorite against E.faecalis

There have been no studies to test the zone of inhibition produced by HOCl against E. faecalis. In the present study, this zone of inhibition was determined to be 15.40 ± 1.08 mm.

It is recommended that further studies be carried out to determine the toxicity level and other properties of hypochlorous acid for its effective use as an endodontic irrigation solution.

CONCLUSION

Within the limitations of present study, it was found that 0.01% HOCl and 2.5% NaOCl exhibits an almost similar anti-bacterial effect against E. faecalis. Also prepared HOCl resembles the HOCl molecule made by neutrophils during oxidative burst (a natural defense process against invading microorganisms), and hence it could be used effectively as a root canal irrigant with further research, mimicking a more accurate in vivo model.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.