Antibacterial Activity of Various Intracanal Medicament against Enterococcus faecalis, Streptococcus mutans and Staphylococcus aureus: An In vitro Study.

AIM: The aim of this study was to evaluate and compare the antimicrobial efficacy of triple antibiotic paste (TAP), calcium hydroxide, and Odontopaste which are used as intracanal medicaments against Enterococcus faecalis, Streptococcus mutants, and Staphylococcus aureus.
MATERIALS AND METHODS: Antibiotic sensitivity testing was done using agar well diffusion method of the following intracanal medicaments: Group 1: TAP (Ciprofloxacin, metronidazole, and doxycycline mixed in a ratio of 1:1:1; Group 2: Calcium hydroxide paste; Group 3: Odontopaste; Group 4: Sterile saline (0.9%) as a negative control. Each medicament was tested against E. faecalis, S. mutants, and S. aureus. The maximum zone of inhibition was noted by diameter size (mm) and analyzed with one-way ANOVA.
RESULTS: TAP showed the highest zone of inhibition. There is no significant difference between Odontopaste and calcium hydroxide against E. faecalis and S. aureus. There is a significant difference between TAP, Odontopaste, and calcium hydroxide against S. mutants. The order of the highest zone of inhibition is TAP > Odontopaste>Calciumhydroxide.
CONCLUSION: With in the limitations of the study, TAP had better antibacterial activity against E. faecalis, S. mutants, and S. aureus followed by odontopaste and calcium hydroxide, Hence, Odontopaste can be used as an alternative for calcium hydroxide in the infected root canal. Copyright:

INTRODUCTION

The primary etiological factors for pulpal necrosis and periapical lesions are considered to be bacteria and their by-products. The ultimate goal of treating the infected root canal is to eliminate microorganisms and their by-products before final restoration.[1] A favorable treatment result of endodontically treated teeth presenting with apical periodontitis depends on the efficient disinfection of the infected root canal system.[2] Chemo-mechanical shaping and cleaning will effectively decrease the microbes but does not completely eradicate them.[3] Various factors which may be responsible for the secondary endodontic infection are intraradicular and extraradicular infection, formation of a cyst containing cholesterol crystals, and foreign body reaction.[4] Studies show that the bacterial cells and anaerobic microorganisms increase over time when root canals remain infected.[5]

Oxygen consumption and a reduced oxidation-reduction potential is the reason for the decrease of aerobic microbes and increase of anaerobic microbes.[6] The frequently found resistant microorganisms in the root canal system environment are Enterococcus faecalis and Candida albicans.[7] The most prevalent microorganism isolated from root canals of previously root-filled teeth with chronic apical periodontitis is E. faecalis.[8] Therefore, the eradication of this facultative anaerobe requires an effective antimicrobial treatment regime.[9]

Intracanal medicaments are primarily used as an antimicrobial agent to eliminate residual bacteria after chemomechanical preparation of the infected root canal. They are used to eliminate remaining microbes present in the canal, to dissolve tissue, to form a barrier against microleakage, and to reduce seepage of fluids from the periapical region into the root canal system.[10]

Various materials were used as intracanal medicament over the years. Calcium hydroxide is the extensively used medicament due to the alkaline nature; it does not eliminate certain facultative anaerobes such as E. faecalis.[11] In the view of finding newer medicaments for eliminating resistant bacteria, approaches for the treatment of periradicular lesion, TAP has been used as an intracanal medicament for disinfecting the root canals, which is a mixture of metronidazole, ciprofloxacin, and minocycline in the ratio of 1:1:1.

Odontopaste is a zinc oxide-based root canal medicament consisting of 5% clindamycin hydrochloride (50 mg), 1% triamcinolone acetonide (10 mg), and approximately 0.5%–1% of calcium hydroxide. It has proven effectiveness against a broad spectrum of microorganisms associated with endodontic infections.[12]

This study aims to evaluate the antibacterial activity by measuring the maximum zone of inhibition between odontopaste, triple antibiotic paste (TAP), and calcium hydroxide as intracanal medicaments used in endodontic therapy against E. faecalis, Streptococcus mutants, and Staphylococcus aureus.

MATERIALS AND METHODS

The study design was an in vitro microbiological analysis to compare the antibacterial efficacy of three intracanal medicaments against E. faecalis American Type Culture Collection (ATCC) 35550, S. aureus ATCC 12598, and Streptococcus mutans ATCC 25175.

The medicaments tested were as follows:

Group 1: TAP (1:1:1 mixture of ciprofloxacin, metronidazole, and doxycycline with sterile saline)

Group 2: Odontopaste (Australian Dental Manufacturer OP8G)

Group 3: Calcium hydroxide (RC Cal)

Group 4: Sterile saline (0.9%) (negative control).

The methodology performed in this study is the agar well diffusion method.

Sample preparation

Odontopaste and calcium hydroxide were mixed with saline separately and vortexed for maximum dissolution. Ciprofloxacin, metronidazole, and doxycycline were mixed in a ratio of 1:1:1, and sterile saline individually used as a negative control.

Procedure

Luria Bertani Agar plates were inoculated with the test organism. The plates were evenly spread out. Then, wells were prepared in the plates with a cork borer. Each well was loaded with 50 μl. The plates were incubated at 37°C for 24 h. The development of the inhibition zone around the well was measured (diameter) and recorded in millimeters using Vernier calipers and then mean values were calculated.

Statistical analysis

Data collected by experiments were statistically analyzed using SPSS version 16.0. Mean and standard deviation were calculated. One-way ANOVA was used for multiple group comparisons, followed by Turkey post hoc for group-wise comparisons. Value of P < 0.05 was considered statistically significant.

RESULTS

Antimicrobial activity was seen against all the three test microorganisms by all the medicaments after incubation at 37°C for 24 h. Zone of inhibition was recorded [Figure 1a–c].

Figure 1

Zone of inhibition of the medicaments against (a) Enterococcus faecalis, (b) Streptococcus mutans and (c) Staphylococcus aureus

Table 1 depicts the comparison of the zone of inhibition of different groups against E. faecalis, S. aureus , and S. mutans. Zone of inhibition against E. faecalis was highest in TAP (44.750.95) followed by Odontopaste (18.5 ± 0.57), RC Cal (17.0 ± 1.15), and saline. Zone of inhibition against S. aureus was highest in TAP (43.51.73) followed by Odontopaste (18.25 ± 1.25), RC Cal (17.5 ± 1.29), and saline. Zone of inhibition against S. mutans was highest in TAP (46.751.5) followed by Odontopaste (30.75 ± 1.5), RC Cal (22.5 ± 1.29), and saline (16.0 ± 3.36). The differences were statistically significant among all the groups against E. faecalis, S. aureus, and S. mutans [Table 1].

Table 1

Comparison of the zone of inhibition of different groups against Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans

Microorganism	Group	Mean±SD	F	P
Enterococcus faecalis	TAP	44.75±0.95	2113.645	0.000
	Odontopaste	18.50±0.57
	RC Cal	17.00±1.15
	Sterile saline	0.0000±0.00000
	Total	20.06±16.53
Staphylococcus aureus	TAP	43.5±1.73	820.413	0.000
	Odontopaste	18.25±1.25
	RC Cal	17.50±1.29
	Sterile saline	0.0000±0.00000
	Total	19.81±16.05
Streptococcus mutans	TAP	46.75±1.50	116.33	0.000
	Odontopaste	30.75±1.50
	RC Cal	22.50±1.29
	Sterile saline	16.00±3.36
	Total	29.00±12.02

SD: Standard deviation

Pairwise comparison within different groups against E. faecalis shows a statistically significant difference between RC Cal and TAP (Mean difference-27.75) and Odontopaste and TAP (Mean difference-26.25). Pairwise comparison within different groups against S. aureus shows a statistically significant difference between RC Cal and TAP (Mean difference-26) and Odontopaste and TAP (Mean difference-25.25). Pairwise comparison within different groups against S. mutans shows a statistically significant difference between saline and RC Cal (Mean difference-6.50); saline and Odontopaste (Mean difference-14.75); saline and TAP (Mean difference-30.75); RC Cal and Odontopaste (Mean difference-8.25); RC Cal and TAP (Mean difference-24.25); and Odontopaste and TAP (Mean difference-16) [Table 2].

Table 2

Pairwise comparison within different groups against Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans

Dependent variable	Group (I)	Group (J)	Mean difference (I-J)	Significant
Enterococcus faecalis	RC Cal	Odontopaste	-1.50	0.088
		TAP	-27.75*	0.000
	Odontopaste	TAP	-26.25*	0.000
Staphylococcus aureus	RC Cal	Odontopaste	-0.75	0.830
		TAP	-26.00*	0.000
	Odontopaste	TAP	-25.25*	0.000
Streptococcus mutans	Saline	RC Cal	-6.50*	0.004
		Odontopaste	-14.75*	0.000
		TAP	-30.75*	0.000
	RC Cal	Odontopaste	-8.25*	0.001
		TAP	-24.25*	0.000
	Odontopaste	TAP	-16.00*	0.000

* - significant with p value less than 0.05

DISCUSSION

E. faecalis has been proven to survive in a harsh environment and has resistance to several medicaments. It has been hypothesized that in periapical infections involving E. faecalis, tissue damage is caused primarily by the reaction of the host to the bacteria rather than direct damage from the bacterial end products.[13] The virulence factor of E. faecalis is due to the capacity of the cells to occupy the dentinal tubules and adhere to the collagen,[14] it also co-aggregates with other organisms and aids in biofilm formation.

S. mutans and S. aureus were also selected as a test organism in this study due to its relevance to cause both primary and persistent endodontic infections.[15]

Odontopaste is an intracanal medicament and has a combination of 5% clindamycin hydrochloride (50 mg), 1% triamcinolone acetonide (10 mg), and 0.5%–1% of calcium hydroxide. Clindamycin hydrochloride present in Odontopaste, inhibits the peptide bond formation in the bacterial DNA and leads to cell lysis. It has been reported by Athanassiadis M et al., that E. faecalis is resistant to 4–16 μg of clindamycin. In Odontopaste, clindamycin hydrochloride has a concentration of about 50,000 μg/ml, that makes it effective against E. faecalis.[16] The advantage of local delivery is that large dose can be administered, that aids in overcoming resistance, without risk of systemic toxicity.[17]

In addition, the interaction of zinc oxide with clindamycin hydrochloride results in slow release of the antibiotic and maintains a greater concentration in the root canal for a longer time. The presence of corticosteroid (triamcinolone acetonide) decreases inflammation by inhibiting macrophages, mast cells activity, and other mediators. The antimicrobial activity of calcium hydroxide is attributed to the release of hydroxyl ions.

However, the manufacturer claims that the function of the calcium hydroxide in this medicament is to improve the consistency of the paste and not for the antibacterial property. Previously in a study, it was reported that E. faecalis acquired a greater level of resistance to clindamycin hydrochloride present in odontopaste. The possible synergistic effects of other ingredients of odontopaste and the physical properties and pH of the paste will have a more significant influence over microorganisms. Their points were based on the pharmacological aspect of clindamycin hydrochloride, without considering the other ingredients and uniqueness of its dose of application.[18]

Independently, ciprofloxacin acts against both Gram-negative and Gram-positive bacteria by inhibiting the cell division and enzyme inactivation due to its broad-spectrum of activity. Obligate anaerobes are predominantly present in the deep dentin of infected root canals, where metronidazole acts effectively by disrupting their bacterial DNA. Minocycline acts by inhibiting matrix metalloproteinase enzyme and enzyme inactivation.[18] The combination of these three antibiotics achieves higher antimicrobial action and overcomes bacterial resistance.[19] Previous studies have stated that the antibiotic mixture of ciprofloxacin, metronidazole, and minocycline has proven for favorable treatment outcomes when used as intracanal medicament.[1920] However, few demerits of using TAP have been reported include crown discoloration due to the presence of minocycline as it binds to the calcium of dentin forming insoluble complexes.[21] Hence, in this study doxycycline was used as a substitute for minocycline in combination with ciprofloxacin and metronidazole to prevent discoloration.[22] Clinically, a vehicle and consistency for TAP are not standardized by any manufacturer for application inside the root canal.[23] Calcium hydroxide can cause necrosis destroying the remaining vital tissues, and additionally, several studies have found out that calcium hydroxide was not very effective in decreasing posttreatment pain. There is more possibility that odontopaste could reduce postoperative pain as it contains steroids. Further clinical trials should be carried out to evaluate the clinical effectiveness of odontopaste such as antimicrobial efficacy, postoperative pain, and periradicular healing.

CONCLUSION

Within the limitations of this in vitro study, there is a significant difference between TAP and other medicaments against all microorganisms. There is a significant difference between Odontopaste and RC Cal against S. mutans. There is no significant difference between Odontopaste and calcium hydroxide against E. faecalis and S. aureus. Hence, Odontopaste can also be used as an alternative for calcium hydroxide against root canal microflora.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.