Introduction

Endodontic therapy aims to eliminate microorganisms and their by-products from the

root canal system. Although mechanical instrumentation is one of the main steps in root canal treatment, it cannot achieve complete removal of microorganisms [1]. Enterococcus faecalis is a Gram-positive facultative anaerobe that is thought to be one of the most resistant species in infected root canals and is frequently linked to failure of endodontic therapy. Difficulty in its removal may be related to inherent antimicrobial resistance, deep penetration into dentinal tubules and biofilm formation [2]. Owing to complex root canal anatomy, antimicrobial irrigation solutions are critical during cleaning and shaping. They can penetrate dentinal tubules, reach canal ramifications and other difficult areas, and therefore act as an adjunct to mechanical instrumentation [3]. MTAD (mixture of tetracycline isomer, citric acid and detergent) is a comparatively newer irrigating solution used as a final rinse for smear-layer removal and antimicrobial action against resistant microorganisms such as E. faecalis [4]. Sodium hypochlorite is widely used because of its antimicrobial activity and ability to dissolve organic matter. Chlorhexidine is used at concentrations of 0.2% to 2%, with 2% commonly preferred for its antimicrobial effect and substantivity. Acetic acid has been studied as a mild chelating and antimicrobial solution [5].

Aims and Objectives

The aim of this study was to evaluate the antimicrobial efficiency of MTAD, 2% chlorhexidine, 5% acetic acid and 5% sodium hypochlorite against E. faecalis.

Objectives:

• To evaluate the antimicrobial efficiency of MTAD against E. faecalis.
• To evaluate the antimicrobial efficiency of 2% chlorhexidine against E. faecalis.
• To evaluate the antimicrobial efficiency of 5% acetic acid against E. faecalis.
• To evaluate the antimicrobial efficiency of 5% sodium hypochlorite against E. faecalis.
• To compare which irrigating solution provides the highest antibacterial effect against E. faecalis.

Materials

Study Sample

The present in-vitro analysis compared the antimicrobial efficacy of four irrigating solutions against E. faecalis in root-canal biofilm. Sixty-five freshly extracted human mandibular premolars with fully developed root apices were collected after ethical approval. The teeth were cleaned of calculus and soft-tissue debris and disinfected with 0.5% sodium hypochlorite for 24 h followed by 0.9% normal saline until instrumentation.

Inclusion Criteria

• Mandibular premolars with straight roots Single canal
• Non-carious tooth
• Completely formed apex

Exclusion Criteria

• Mutilated teeth
• Fractured teeth
• Teeth with root resorption
• Teeth with more than one canal

Armamentarium and Materials

• Airotor handpiece (Waldent) and carbide disc for
• K-file (Waldent), Endoblock, side-vented needle, 5.25% sodium hypochlorite, 17% EDTA (Waldent) and paper points.
• Autoclave, centrifugal machine, biosafety cabinet, laminar airflow, thermocol plates and glass tubes.
• 5% sheep blood agar plates, micropipette, inoculating wire and incubator.
• Digital colony counter for colony-count Irrigating Solutions
• MTAD (mixture of tetracycline isomer, citric acid and detergent)
• 2% chlorhexidine
• 5% acetic acid
• 5% sodium hypochlorite

Sample Size

The sample size was based on the expected mean difference and standard deviation in antimicrobial efficacy between groups, with a = 0.05, 80% power and a 1:1 ratio. The calculated minimum sample size was 60 specimens, corresponding to 15 samples per experimental group.

Methodology

Sixty-five freshly extracted mandibular premolars were decoronated at the cementoenamel junction to obtain a standardized root length of 14 mm. Working length was determined using a size 10 K-file, and the apices were sealed with adhesive to prevent extrusion of irrigating solutions. Working length was confirmed by subtracting 1 mm from the length of the apical foramen. Root canals were prepared by hand instrumentation up to master apical file #40. Seventeen percent EDTA and normal saline were used with disposable side-vented needles for smear layer removal. After sterilization, all specimens were transported and manipulated under aseptic conditions using sterile instruments and equipment. Prepared root specimens were autoclaved in glass tubes containing 3 mL BHI broth at 121°C for 20 min and incubated for 24 h at 37°C to confirm sterility. Pure culture of E. faecalis (ATCC 29212) grown in BHI broth for 24 h at 37°C was used to contaminate the root canals. A 10-µL inoculum was introduced into the prepared canals, and the teeth were incubated for 48 h. The teeth were randomly divided into four experimental groups of 15 samples each and one positive-control group of five samples. Experimental groups were irrigated with 5 mL of the respective solution for 5 min. The positive-control group was irrigated with 5 mL sterile normal saline for 5 min. Size 35 paper points were inserted to 1 mm short of working length for 10 s and then transferred into tubes containing 10 µL sterile normal saline and vortexed for 1 min. Samples were cultured using a standard loop technique on 5% sheep blood agar and incubated at 37°C for 48 h under aerobic conditions. Colony counts were obtained using a digital colony counter.

Observation and Results

The E. faecalis colony count of four groups was summarized in Mean ± SD, median, sum of ranks and mean sum of ranks.(Table 1) The E. faecalis colony count of Group I, Group II, Group III and Group IV ranged from  0-8,  20-40,  30-55  and  10-25  (CFU  1/ml)

respectively with mean (± SD) 0.53 ± 2.07, 30.60 ± 6.33, 39.40 ± 8.38 and 16.00 ± 5.28 (CFU 1/ml)

respectively and median 0, 30, 41 and 15 (CFU 1/ml) respectively. The mean (or median) of E. faecalis colony count of Group III was the maximum followed by Group II, Group IV and Group I, the minimum (Group I < Group IV < Group II < Group III).

Randomised controlled clinical trials have assessed the role of probiotics as an adjunct to mechanical debridement in the treatment of peri-implant mucositis. One notable study found that the addition of Lactobacillus reuteri lozenges led to significant reductions in bleeding on probing and peri-implant inflammation compared to a placebo [6]. Subsequent clinical investigations reported improvements in plaque index, bleeding on probing, and peri-implant probing depths following short-term probiotic supplementation, suggesting a notable enhancement in the resolution of soft tissue inflammation [7].

Discussion

This study evaluated whether MTAD offers superior antimicrobial efficacy compared with conventional irrigants against a resistant, biofilm-forming organism such as E. faecalis [6]. At an exposure time of 5 min, antimicrobial activity decreased in the order Group I > Group IV > Group II > Group III > Group V. The superior antimicrobial performance of MTAD observed in the present study may be attributed to its combination of doxycycline/tetracycline isomer, citric acid and detergent, which facilitates penetration and biofilm disruption [7]. 5% sodium hypochlorite demonstrated the second-highest antimicrobial efficacy after MTAD [8]. Sodium hypochlorite is widely used because of its antimicrobial activity, tissue dissolving capacity and ability to disrupt organic biofilms [9]. 2% chlorhexidine showed moderate antimicrobial efficacy and performed better than 5% acetic acid but was inferior to MTAD and sodium hypochlorite [10]. Chlorhexidine has broad spectrum antimicrobial activity and substantivity, although it lacks tissue-dissolving capability [11]. Acetic acid showed the least antimicrobial efficacy among the experimental groups. Although it has mild chelating properties and may contribute to smear-layer removal, its antibacterial effect appeared insufficient for complete root-canal disinfection under the conditions of the present study [12]. The findings support the importance of combining mechanical preparation with effective chemical irrigation. The descending order of antimicrobial efficacy was MTAD > 5% sodium hypochlorite > 2% chlorhexidine > 5% acetic acid > positive control [13].

Conclusion

MTAD (Group I) was the most effective antimicrobial agent among the experimental groups. 5% sodium hypochlorite (Group IV) was less efficacious than MTAD but more effective than 2% chlorhexidine and 5% acetic acid. 2% chlorhexidine (Group II) was less effective than 5% sodium hypochlorite but more effective than 5% acetic acid. 5% acetic acid was the least effective experimental irrigant but demonstrated antibacterial activity against E. faecalis. The positive-control group showed no antimicrobial activity because no antimicrobial irrigant was used. Overall antimicrobial action was Group I > Group IV > Group II > Group III > Group V. None of the tested irrigating solutions completely eradicated E. faecalis from the root canal.

References

5. Bystrom LH, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol. 1983;55 [3]:307-12.
6. Torabinejad M, Shabahang S, Aprecio R, Kettering S. The antimicrobial effect of MTAD: An in vitro investigation. J Endod. 2003;29 [6]:400-403.
7. Torabinejad M, Shabahang S. A new solution for the removal of smear layer. J Endod. 2002;28 [3]:170-5.
8. Sundqvist G, et Ecology of the root canal flora. J Endod. 1992;18 [9]:427-30.
9. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J. 2009;42 [4]:288-302.
10. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J 2003;29 [4]:233-9. 4
11. Zehnder M, Haapasalo M, Endal U, Zandi H, Coil JM. Eradication of endodontic infection by instrumentation and irrigation solutions. Endod 2005;10 [1]:77-102.
12. Zhang R, Zhang K, Yang D, Jiang L. Antibacterial activity of chlorhexidine against Enterococcus faecalis biofilm in vitro. Sci Rep. 2015;5:12944.
13. Siqueira JF Jr, et al. Etiology of root canal treatment failure. J Endod. 2001;27 [1]:1-5.
14. Siqueira JF Jr, Rôças IN. Clinical implications of microbial diversity in endodontic infections. J Endod. 2009;35 [4]:465-73.
15. Pashley DH, Torabinejad M. The smear layer in endodontics: A review. J Endod. 1997;23 [2]:69-76.
16. Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al. A new solution for the removal of the smear layer. J Endod. 2003;29 [3]:170-
17. Nara A, Dhanu KS, Chandra RV, Anandakrishna
18. Comparative evaluation of antimicrobial efficacy of MTAD, 3% NaOCl and propolis against Enterococcus faecalis. J Clin Diagn Res. 2016;10 [5]:80-83.

Tables

Table 1- This table shows the summary statistics of E. faecalis colony count (CFU 1/ml) of four groups

Table 2. This table shows the comparison of the mean sum of ranks of E. faecalis colony count (CFU 1/ml) between groups by z-test