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 Table of Contents  
Year : 2018  |  Volume : 8  |  Issue : 1  |  Page : 19-23

Efficacy of ozonated water, 2% chlorhexidine and 5.25% sodium hypochlorite on five microorganisms of endodontic infection: In vitro study

1 Department of Conservative Dentistry, Krishnadevaraya College of Dental Sciences, Bengaluru, Karnataka, India
2 Department of Prosthodontics, Krishnadevaraya College of Dental Sciences, Bengaluru, Karnataka, India
3 Department of Microbiology, Krishnadevaraya College of Dental Sciences, Bengaluru, Karnataka, India

Date of Web Publication5-Jan-2018

Correspondence Address:
D Savitri
Flat 505, Venkatadri Towers, Prabhata Nagar, Chaitanyapuri, Dilsukhnagar, Hyderabad - 500 060
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/AIHB.AIHB_45_16

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Introduction: The aim of this study was to assess the antimicrobial efficacy of ozonated water (4 mg/l), 2% chlorhexidine solution, 5.25% sodium hypochlorite solution on five common endodontic microorganisms. Methods: The organisms chosen in the study were Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, Candida albicans, Kocuria rhizophila. Agar well diffusion test direct contact test were used as methods to assess antimicrobial effectiveness. In agar well diffusion test, the maximum zone of inhibition formed around the well in an agar plate after incubation of test materials against each microorganism for 24 h and 48 h were measured. In direct contact test, the colonies of E. faecalis formed on agar plates with each test material were calculated. Results: The results showed that 2% chlorhexidine showed highest zone size and minimum colony forming units indicating its highest potency and ozonated water was showed the least efficacy with a significant difference between both groups. The colony forming units showed an increase in number when ozonated water was used against E. faecalis

Keywords: 2% chlorhexidine, 5.25% sodium hypochlorite, agar well diffusion test, antimicrobial efficacy, Candida albicans, colony forming units, Enterococcus faecalis, irrigation, Kocuria rhizophila, ozonated water, Staphylococcus aureus, Streptococcus mutans

How to cite this article:
Savitri D, Shetty S, Sharath Chandra S, Jayalakshmi K, Gowda M, Rai N, Selvan A, Reddy S. Efficacy of ozonated water, 2% chlorhexidine and 5.25% sodium hypochlorite on five microorganisms of endodontic infection: In vitro study. Adv Hum Biol 2018;8:19-23

How to cite this URL:
Savitri D, Shetty S, Sharath Chandra S, Jayalakshmi K, Gowda M, Rai N, Selvan A, Reddy S. Efficacy of ozonated water, 2% chlorhexidine and 5.25% sodium hypochlorite on five microorganisms of endodontic infection: In vitro study. Adv Hum Biol [serial online] 2018 [cited 2023 Feb 9];8:19-23. Available from: https://www.aihbonline.com/text.asp?2018/8/1/19/222246

  Introduction Top

Endodontic therapy is accomplished with the reduction or complete removal of the microorganisms from the root canal system.[1] Instrumentation alone cannot result in complete elimination of microbes as the root canal system is complex.[2] It is recommended that the intracanal irrigants or medicaments have potent antimicrobial property, dissolve organic tissues and debride the root canal system and be nontoxic to the periapical tissues.

Microorganisms of the infected root canals consist complex flora such as cocci, rods, spirochaetes, including fungi. Some of the organisms found commonly in endodontic infections include Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, Actinomyces, Candida, Treponema, Porphyromonas, Kocuria rhizophila and Prevotella.[3],[4] E. faecalis is noticed from the root canals of teeth with treatment failure. Candida albicans is also commonly found fungus in endodontic infections.[5] K. rhizophila is one of the commonly found bacterial strain in endodontic infection.[3] One possible agent of interest for dealing with persistent organisms is ozone. The bactericidal and virucidal properties of ozone are well recognised[6],[7] and have shown to reduce the level of viable E. faecalis in dentinal tubules.[8] Various studies have been done to establish the role of ozone in dentistry. Studies have shown the effectiveness of ozonated water in killing various oral microorganisms and including oral C. albicans.[9] Ozone is a possible alternative antimicrobial agent in dentistry without the development of drug resistance.[10] Chlorhexidine digluconate is an antimicrobial agent effective against a broad range of microorganisms. At higher concentration (2%), CHX acts as bactericidal agent. It acts by cytoplasmic precipatation and ultimately death of the cell occurs.[11] Sodium hypochlorite has both oxidising and hydrolysing properties.[12] Concentrations varying from 1% to 5.25% of sodium hypochlorite is now accepted widely for endodontic irrigation.

The aim of the present study was to compare in vitro the antimicrobial efficacy of ozonated water, 2% chlorhexidine and 5.25% sodium hypochlorite on E. faecalis, S. mutans, S. aureus, K. rhizophila and C. albicans. It was also purposed to assess the efficacy of these irrigating agents at different time intervals.

  Methodology Top

The antibacterial efficacy was evaluated using both agar well diffusion test and direct contact test.

The study used E. faecalis, S. mutans, S. aureus, K. rhizophila strains cultured on blood agar and C. albicans cultured on Sabouraud dextrose agar.

Agar well-diffusion test: Preparation of inoculum

The cultures were inoculated into sterile brain heart infusion (BHI) broth and sabouraud dextrose broth for bacterial culture and C. albicans respectively and incubated at 37°C until it achieved or exceeded the turbidity of 0.5 Mcfarland standard. The turbidity was adjusted with normal isotonic saline which would result in a suspension approximately containing 1.5 × 108 CFU/ml.

Inoculation of agar plates

Under aseptic conditions, 25 ml of BHI agar/standard deviation agar (Himedia, Mumbai) was prepared, and dispensed into conical flasks and sterilised by autoclaving at 15 pounds of pressure for 15 min.

The sterile media are allowed to cool, and at the temperature, at around 45°C, 50 μl of the opacity adjusted cultures were added, mixed well and poured into sterile Petri plates (90 mm diameter, TARSON). The media was allowed to set and dried at 37°C in incubator for 15 min.

Four equidistant points were chosen to cut the wells to receive the test materials and labeled [Figure 1]. The template was placed over the chosen areas. Four wells were cut in the agar medium using sterile templates of the diameter of 5 mm. 100 μl of antimicrobials were inoculated into the 4 wells using sterile micro tips.

The plates were preserved for 1 h at room temperature, and then incubation was done at 37°C for 24 h. E. faecalis and S. mutans were incubated in a candle extinction jar and other microbes incubated under aerobic conditions. Microbial inhibitory zones formed around wells containing the test materials was measured and recorded after 24 h and 48 h of incubation.

The inhibitory zone was calculated as the diameter of the zone of inhibition of growth of mocrobes around the well-using vernier calipers.

All the investigations were performed under aseptic conditions and in three replicates were performed.

The potency of the ozonated water in comparison with the 2% chlorhexidine and 5.25% sodium hypochlorite on the E. faecalis was also evaluated by the direct contact test.

Direct contact test

In a sterile Eppendorf tube, 50 μl of antimicrobial agent was added and 50 μl of E. faecalis culture was adjusted to 0.5 McFarland (1.5 × 108 CFU/ml).

After intervals of 2 min, 10 min, 20 min, 30 min and 1 h, 250 μl of sterile BHI broth was added and mixed well. 50 μl was transferred on sterile BHI agar.

  Results Top

The analysis of variance (ANOVA) has been used in this study to evaluate the significance of study parameters between groups. Descriptive and inferential statistical analysis has been performed in the present study.

Statistical software

The statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1, Medcalc 9.0.1, Systat 12.0 and R environment ver.2.11.1(IBM, USA) were used for the analysis of the data.

The mean values of the inhibitory zones were then calculated for all the groups at 24 h, and 48 h and data were tabulated. Each plate was triplicated, and average zone size was calculated. The average zone sizes for each organism against different irrigants at 24 h were tabulated in [Table 1] and at 48 h were tabulated in [Table 2]. The values were then statistically analysed using ANOVA and paired t-test.
Table 1: Comparison of zone size (mm) of four endodontic irrigants on five different organisms at 24 h

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Table 2: Comparison of zone size (mm) of four endodontic irrigants on five different microorganisms at 48 h

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Furthermore, a direct contact test was done on E. faecalis using all four irrigants at different time intervals as 2 min, 10 min, 20 min, 30 min and 1 h and number of colonies expressed in CFU's were calculated for individual irrigant and tabulated in [Table 3].
Table 3: Comparison of colony forming units on direct contact test of four endodontic irrigants on Enterococcus faecalis

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In [Table 1], 2% chlorhexidine group showed highest zone size of inhibition in all the given experimental microorganisms. Statistically significant difference (P < 0.001) was found in all four irrigant groups in each of the five microorganisms. A maximum zone of inhibition of 25.23 ± 0.25 mm is seen with 2% chlorhexidine group on S. aureus and the least zone of inhibition of 18.93 ± 0.40 mm is seen in S. mutans. There was no significant difference between C. albicans, E. faecalis and K. rhizophila.

5.25% Sodium hypochlorite gave the highest zone size of inhibition of 14.50 ± 0.30 mm with S. mutans followed by E. faecalis with zone of inhibition 12.40 ± 0.20 mm and least zone size of 9.53 ± 0.06 mm with S. aureus with no significant difference between S. aureus, C. albicans and K. rhizophila.

Ozonated water (4 mg/l) showed the least efficacy and antimicrobial activity against all the five test organisms compared to 2% chlorhexidine and 5.25% sodium hypochlorite. Ozonated water showed highest zone of inhibition of 12.37 ± 0.11 mm with S. mutans while no significant difference was seen among other test microorganisms.

[Table 2] shows the zone size of inhibition on five microorganisms when the agar plates were observed after an incubation period of 48 h. There was no statistically significant difference among the zone size of inhibition among 24 h and 48 h time intervals.

In [Table 3], direct contact test, 2% chlorhexidine showed gradual decrease in the colony forming units of E. faecalis from 2 min to 20 min time interval followed by constant results. With 5.25% sodium hypochlorite maximum colony forming units were observed at 2 min followed by a gradual decrease. With ozonated water (4 mg/l) there was decrease followed by an increase in colony forming units at 30 min and 60 min time interval. Control group has shown maximum number of colony forming units of E. faecalis.

  Discussion Top

Primary root canal infections consist of different types of microbes, mostly consisting of obligate anaerobes.[13] It is believed that both mechanical enlargement and copious irrigation of root canals aids is maximum removal of microorganisms as possible.[14],[15] An irrigant should impart mechanical, microbicidal properties, be biocompatible and aid in dissolving organic tissues without damaging the periradicular tissues if extrusion of irrigant occurs into the periodontium. Ozone is a very strong, unstable, oxidising bactericidal agent. Studies suggest that ozone at minimal low concentration, 0.1 ppm, inactivates bacteria along with spores.[16] It is available in atmosphere and can be produced by ozone generator. Ozone dissolves rapidly in water and also disintegrates quickly. Although ozonated water is a strong antimicrobial agent against broad range of microbes, its efficacy on bacterial biofilm and in root canal infection has not been studied in detail.[17],[18] Ozonated water was used in the present study as it has high oxidative power and antimicrobial efficacy.

Agar well diffusion test is performed in this study as the inhibitory zones evaluate the antimicrobial efficacy of individual irrigant against various microbe/s.[19] Direct contact test is a test for evaluating the antiseptic property of an irrigant, wherein the irrigant in specified quantities is mixed with a bacterial suspension. The antibacterial efficacy is assessed by culture of suspension in a nutrient medium and, the presence or absence of microbial growth is evaluated for individual microbe, each irrigant with time.[20],[21]

The results of the agar well diffusion tests showed that 5.25% sodium hypochlorite was an effective agent against the growth of all the tested individual microorganisms. 5.25% NaOCl is used in the current study as the results of the studies done previously stated that 5.25% sodium hypochlorite shows best antimicrobial efficacy in very short time.[22] Using the agar diffusion method, previous studies demonstrated that NaOCl has comparatively less antiseptic activity than 2% CHX[23],[24],[25],[26] which is in accordance with the present study. 5.25% NaOCl produced highest zone of inhibition with S. mutans (14.50 ± 0.30 mm), while, no significant difference was seen between inhibition zones of other four microorganisms which is in accordance with a study which reported elimination of S. mutans conducted by Nanjundaswamy et al.[27] These results can be explained by the proven disinfecting efficacy of NaOCl. The undissociated hypochlorous acid provides its antibacterial efficacy by oxidation on sulphydryl groups of bacterial enzymes. Thus, metabolic reactions are interrupted, and bacterial cell are killed.[28]

In the present study, 2% chlorhexidine solution exhibited highest inhibitory zones compared to 5.25% sodium hypochlorite and ozonated water (4 mg/l) on all the experimental microorganisms indicating highest efficacy. This possibly can be explained by the substantivity of 2% chlorhexidine solution. These results are similar to previous study where the growth inhibition zones produced by 2% chlorhexidine were larger than those by various concentrations of NaOCl, including 5.25%. All microbial species tested in that study were sensitive to chlorhexidine gluconate.[29]

In the present study, ozonated water (4 mg/l) showed highest zone of inhibition with S. mutans (12.37 ± 0.11 mm), while, no significant difference was seen among other microbes. In accordance with the present results, similar study showed that ozonated water (4 mg/l) applied to dental plaque for 10 s destroyed nearly all microorganisms, including S. mutans.[8] Once in the body, ozone generates ozonised compounds such as reactive oxygen species and lipid oxidation products which act as antifungal, antibacterial, antiviral and antiparasitic agents.[7] Müller et al. also explored resistance of ozone against bacteria for 60 s.[30] Complete elimination of bacteria could not be achieved in the study which is similar to the present study. Furthermore, favouring the current study results, Hubbezoglu et al. applied ozone on root canals infected with C. albicans for 300 s. Although they used longer application time, complete disinfection was not achieved.[31]

Ozonated water (4 mg/l) showed a zone of inhibition of 8.33 ± 0.29mm against K. rhizophila. The internal ability of K. rhizophila is comparatively less, and thus, it can be inactivated easily.[3] Ozonated water (4 mg/l) showed a zone of inhibition of 8.07 ± 0.11 mm against S. aureus. The oxidative potential of ozonated water distinguishes it as an efficient antimicrobial agent. Its antimicrobial action has been proven against various microbes.[32],[33]

In the present study, direct contact test was done on E. faecalis and results showed gradual decrease in colony forming units from 2 min to 1 h with 2% chlorhexidine and 5.25% sodium hypochlorite but an increase in colony forming units after 30 min with ozonated water (4 mg/l) which might have resulted because of the loss of its potency after 300 s.

The results in the present study showed the decreased efficacy of ozonated water which may be possibly explained by variation in the method of conducting the experiment, concentration and various types of irrigating solutions used, or the amount of time used to evaluate in the current study. Rapid deterioration of the ozone just after contact with organic compounds, such as culture media, which is one of its environmental disadvantages, may cause a decrease in antimicrobial effectiveness of ozonated water. The probability that the oxidative ozone reacted with some reductants in the culture media other than the test bacterila organisms. The results of the present study may not apply to in vivo conditions as the agar diffusion method has many drawbacks to apply and replicate it to clinical scenario and thus may not be reliable technique.[20] Agar diffusion method has been used regularly in various studies, but many factors, except antimicrobial activity of test materials might influence the reliability of the test.

The extrapolation of the results from work that is purely in vitro in nature must always be made with caution. The ultimately deciding clinical applicability considers in vivo and patient conditions and various clinical variables to enhance the prognosis of root canal treated teeth. In the light of the current study's results, further research should be carried out using irrigating solutions with the help of designs resembling in vivo conditions to establish their antimicrobial efficacy.

  Conclusion Top

Under the conditions of the present study and within its limitations, it can be concluded that:

  1. 2% Chlorhexidine solution is a better endodontic irrigant with maximum antimicrobial potency compared to 5.25% sodium hypochlorite and ozonated water
  2. At any given time interval, 4 mg/l ozonated water showed maximum colony forming units indicating its short span of action
  3. Agar diffusion test and direct contact test have its own drawbacks and cannot simulate the in vivo conditions which might vary the results obtained in the present study
  4. Further research is needed to investigate the availability of aqueous ozone that has more adequate properties for clinical endodontic treatments. Although the in vitro test methods are helpful in conducting and evaluating the antibacterial efficacy of various irrigants, a detailed and standardised procedure is required to enhance the reproducibility among different researchers in future.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1]

  [Table 1], [Table 2], [Table 3]


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