Antimicrobial Effectiveness of Endodontic Irrigants Against E. Faecalis: An In vitro Study

ABSTRACT

Aim:

This in vitro study compared the antimicrobial efficacy of 5.25% sodium hypochlorite, 10% citric acid, and 3% hydrogen peroxide against Enterococcus faecalis (E. faecalis), using normal saline as a control.

Method:

E. faecalis isolates were exposed to different irrigants, with bacterial colony counts measured at various intervals using the spread plate method.

Results:

Sodium hypochlorite and citric acid effectively inhibited E. faecalis, while hydrogen peroxide was less effective. Sodium hypochlorite dissolved tissues and acted germicidally; citric acid disrupted bacterial structures through low pH. Hydrogen peroxide’s effect was due to free radical formation and oxygen release.

Conclusion:

Both 5.25% sodium hypochlorite and 10% citric acid showed equal efficacy against E. faecalis, while hydrogen peroxide was less effective. Further research should explore clinical applications and polymicrobial biofilms.

INTRODUCTION

Bacteria are the primary cause of necrotic dental pulps, periapical pathology, and complications following root canal therapy. Successful treatment requires removing bacteria from the pulpal tissue and root canals through a combination of instrumentation, obturation, and disinfection.[1] While mechanical instrumentation is key to reducing bacteria, achieving bacteria-free root canals remains challenging.[2] Studies show that saline irrigation and mechanical preparation alone are often inadequate.[3,4,5] As a result, antibacterial irrigating solutions like sodium hypochlorite (NaOCl), cetrimide, citric acid, chlorhexidine, hydrogen peroxide (H2O2), and EDTA have gained attention, with NaOCl being the most commonly used.[6] Despite these efforts, certain bacteria, notably staphylococci and enterococci, can persist. E. faecalis, a Gram-positive facultative anaerobe, is frequently found in cases of failed endodontic treatment.[7] The persistence of these bacteria suggests that the choice of irrigant is still largely empirical. This study evaluates the comparative antibacterial efficacy of different endodontic irrigants against E. faecalis.

METHODOLOGY

Study Type: Observational cross-sectional Invitro study.

Study Period: Two months (April and May 2023).

Study Settings: Department of Microbiology and Department of Dentistry, AIIMS Kalyani.

Study Isolate: E. faecalis clinical isolates.

Study Reagents: Three root canal irrigants i.e., 5.25% NaOCl, 10% citric acid, and 3% H₂O₂ were used. 0.9% normal saline was used as a control.

The clinical isolate of E. faecalis was identified using the Vitek2 compact system [Biomerieux, USA]. The efficacy of root canal irrigants was studied using the surface viable count method by spread plate method. E. faecalis broth was prepared with a known 0.5 McF turbidity using peptone water. This contains around 1.5 × 10⁸ colony-forming units (CFU)/ml of E. faecalis. Serial ten-fold dilution of irrigant solutions was done using normal saline as a diluent. The dilutions ranged from 1/10 to 1/10000. Each dilution of irrigant solutions was inoculated with 0.01 ml of 0.5 McF of E. faecalis broth. However, 0.9% normal saline was used as a control. Using a calibrated micropipette, 0.1 ml of inoculum from each dilution of respective irrigant solutions was poured over the designated quadrant of one dried nutrient agar plate and spread evenly using a Nichrome loop. Inoculum from each dilution of respective irrigant solutions was cultured at time intervals of 0 min, 5 min, 15 min, 30 min, and 24 hours [Figures 1 and 2]. Inoculated nutrient agar plates were incubated for 18–20 hours in ambient air aerobic conditions at 35 ± 2°C. The next day, cultured agar plates were read, and the viable count was calculated as the average CFUs per designated dilutions of irrigant solutions. The average CFUs for each dilution were compared at the mentioned time intervals. The same method of culture was followed for each irrigant solution. The whole experiment was performed three times using the same isolate, irrigant solutions, and methodology.

Figure 1.

Initial Bacterial Growth (at 0 minute)

Figure 2.

Irrigants Effect On Bacterial Growth After Overnight Incubation (At Time Interval of 5 minutes, 15 minutes, 30 minutes, 24 hours). Top row to bottom: 5.25% Sodium hypochlorite, Normal saline (control), 10% Citric acid, 3% hydrogen peroxide A) 1/10 dilution, B) 1/100 dilution, C) 1/1000 dilution, D) 1/10000 dilution

OBSERVATIONS AND RESULTS

Results were analyzed graphically, taking “time interval” in the X-axis and “dilutions of solutions” in the Y-axis [Figure 3].

Figure 3.

Bacterial count reduction over time for different dilutions of Sodium hypochlorite 5.25%, Hydrogen peroxide 3%, Citric acid 10%, and Normal saline. Measurements taken at 0 min, 5 min, 15 min, 30 min, and 24 hours

Bacterial colony count at various dilutions and time intervals [Tables 1-4].

Table 1.

Sodium Hypochlorite 5.25%

Time	Dilutions
	1/10	1/100	1/1000	1/10000
	Bacterial count
0 min	5000	5000	5000	5000
5 min	0	0	0	0
15 min	0	0	0	0
30 min	0	0	0	0
24 hours	0	0	0	0

Table 4.

Normal Saline (Control)

Time	Dilutions
	1/10	1/100	1/1000	1/10000
	Bacterial count
0 min	5000	5000	5000	5000
5 min	5000	4000	3000	3000
15 min	5000	4000	3000	2000
30 min	5000	4000	4000	2000
24 hours	4000	3000	2000	1000

Table 2.

Citric Acid 10%

Time	Dilutions
	1/10	1/100	1/1000	1/10000
	Bacterial count
0 min	5000	5000	5000	5000
5 min	0	0	0	0
15 min	0	0	0	0
30 min	0	0	0	0
24 hours	0	0	0	0

Table 3.

3% Hydrogen Peroxide

Time	Dilutions
	1/10	1/100	1/1000	1/10000
	Bacterial count
0 min	5000	5000	5000	5000
5 min	1000	0	0	0
15 min	500	0	0	0
30 min	0	0	0	0
24 hours	0	0	0	0

DISCUSSION

E.faecalis, a Gram-positive facultative anaerobe, is often found in failed endodontic cases. It exhibits virulence factors like lytic enzymes, cytolysin, pheromones, lipoteichoic acid, and aggregation substances. These factors alter host responses, suppress lymphocyte activity, and compete with other bacteria, potentially leading to endodontic failure. E. faecalis prioritizes survival in root canals through genetic variations, dentin-binding enzymes, starvation endurance, and biofilm formation. It can withstand challenges in the root canal environment, balancing pH passively or using proton pumps, but cannot survive at pH levels above 11.5. The bacterium can persist within dentinal tubules, enduring starvation until nutrients are available. Serum from alveolar bone and the periodontal ligament facilitates its attachment to type I collagen. Biofilm formation by E. faecalis significantly increases its resistance to phagocytosis, antibodies, and antimicrobial agents. The presence of dentin components like hydroxyapatite, type-I collagen, and serum can affect the efficacy of root canal medicaments against bacteria.[8] Sodium hypochlorite, with its high pH, is effective in dissolving necrotic tissues and has germicidal properties.[9,10,11,12,13,14] It disrupts cellular metabolism, forms chloramines, and causes oxidative damage, leading to enzyme inactivation and lipid degradation. In this study, citric acid showed equal antibacterial efficacy to 5.25% sodium hypochlorite. Yamaguchi et al.[15] found citric acid effective against 12 types of bacteria in root canals which includes Staphylococcus aureus, Streptococcus sanguis, Eubacterium lentum, Lactobacillus casei, Propionibacterium avidum, Peptococcus niger, Peptostreptococcus anaerobius, Bifidobacterium bifidum, Veillonella parvula, Porphyromonas endodontalis, Actinomyces naeslundii, and Fusobacterium nucleatum, while Arias-Moliz et al.[16] reported a minimum bactericidal concentration (MBC) of 20% against E.faecalis. Citric acid reduces pH within cells, damaging DNA and proteins, leading to bacterial death. Hydrogen peroxide is broad-spectrum, producing hydroxy free radicals and oxygen, which are unfavorable for anaerobic bacteria, but was less effective against E.faecalis in this study.

CONCLUSION

5.25% sodium hypochlorite and 10% citric acid showed similar antibacterial effectiveness against E.faecalis. Further in vivo testing is needed to confirm these findings.

Limitations of the study

An in vivo clinical trial could have better demonstrated the practical effectiveness of these irrigants. Future research should focus on polymicrobial biofilms.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.