Microbiological evaluation of different agitation methods of irrigation during endodontic procedure – A randomized clinical trial

Abstract

Introduction:

Elimination of microorganisms from the root canal system is a prerequisite for predictable endodontic success. Among persistent pathogens, Enterococcus faecalis poses a significant challenge due to its biofilm-forming capacity and resistance to standard irrigants. This study aimed to compare the antimicrobial efficacy of three irrigation modalities – conventional syringe irrigation, sonic activation, and ultrasonic activation – in reducing E. faecalis colony-forming unit (CFU) counts in infected canals.

Methods:

Sixty-five patients scheduled for endodontic retreatment were randomly assigned to three irrigation protocols: conventional (n = 20), sonic (n = 22), and ultrasonic (n = 23) irrigation. Microbial samples were collected pre- and postirrigation. Bacterial reduction was evaluated using culture-based CFU analysis, followed by appropriate parametric and nonparametric statistical tests (α = 0.05).

Results:

Only the ultrasonic group demonstrated a statistically meaningful reduction in CFU levels (P = 0.011). Comparative analysis revealed a significant difference among the three groups, with ultrasonic activation showing superior antimicrobial effectiveness (ANOVA P = 0.011; Kruskal–Wallis P = 0.0003). Sonic activation produced inconsistent reductions, while syringe irrigation showed a negligible effect.

Conclusion:

Ultrasonically activated irrigation demonstrated superior antimicrobial efficacy over sonic and conventional techniques, supporting its use as an adjunctive strategy for improved endodontic disinfection.

INTRODUCTION

Complete eradication of intracanal microorganisms remains one of the most critical determinants of endodontic treatment success. Although contemporary instrumentation techniques efficiently shape root canals, their ability to address anatomical complexities – such as fins, lateral canals, and apical ramifications – is inherently limited. As a result, chemical irrigation remains indispensable for achieving effective disinfection.[1,2]

Clinical outcome studies have consistently shown that the microbial status of the canal at the time of obturation significantly influences treatment prognosis. Sjögren et al.[3] reported markedly higher success rates when canals were rendered culture-negative before filling. Despite this, persistent infections continue to occur, particularly in retreatment cases. Among the microorganisms implicated, Enterococcus faecalis is notable for its ability to survive nutrient deprivation, penetrate dentinal tubules, and establish biofilms that resist chemical and mechanical disruption.[4,5,6]

Traditional needle irrigation frequently fails to deliver irrigants effectively to the apical third and complex canal recesses. To overcome these limitations, activation techniques such as sonic and ultrasonic agitation have been introduced to enhance fluid dynamics within the canal system.[7,8] While laboratory studies have reported improved antimicrobial effects with activated irrigation, the majority of these investigations rely on in vitro models, limiting direct clinical extrapolation.[9,10,11]

Accordingly, the present randomized clinical trial was designed to compare the effectiveness of conventional syringe irrigation, sonic activation, and ultrasonic activation in reducing E. faecalis from clinical root canal samples.

METHODS

Sample selection

Following institutional ethical approval, 65 patients requiring endodontic retreatment of single-rooted teeth were recruited from the outpatient endodontic clinic at PGIMER, Chandigarh. Informed consent was obtained before participation.

Inclusion criteria

1. Patient with a single-rooted retreatment tooth infection

2. Chronic apical periodontitis with or without periapical pathology.

Exclusion criteria

1. Medically compromised patients

2. Teeth with open apex, root resorption, and perforation

3. Patients with systemic diseases, pregnant or lactating female patients, psychologically disturbed patients, or patients allergic to any sort of medications used in this study

4. Teeth with abnormal anatomy or calcified canals or with grade two or three mobility were all excluded.

Methodology

Clinical trial registration

The study design and protocol were prospectively registered under the identifier “CTRI/2021/09/036709” in accordance with standard research governance requirements.

Access cavity preparation

The access cavity was performed with a high-speed handpiece using a sterile round carbide bur size 3 (Dentsply, Tulsa Dental, Dentsply Maillefer, TN, USA), and flaring was done with an Endo Z bur (Dentsply).

Root canal preparation

Using rubber dam (Dental Dam, Sanctuary, UK) the tooth was isolated and after removal of gutta percha, an electronic apex locator (Root ZX, J. Morita USA, Irvine, CA, USA) was used to measure working length and was then confirmed with digital intraoral periapical radiograph (Soredex, Digora Toto sensor, Finland) to be within 0.5–1 mm short from radiographic apex. Crown-down technique was performed in instrumentation of root canal using ProTaper Next (Dentsply Maillefer, Ballaigues, Switzerland) NiTi rotary files according to the manufacturer’s instructions.

Microbial sampling

Microbial samples were obtained directly from the root canal of the involved tooth. Every tooth was isolated with a rubber dam. A sterile paper point was introduced to the predetermined working length and was held in place for 60 s to absorb the contents of the canal. Sampling was conducted before irrigation (S1) and immediately after completion of the irrigation protocol (S2). Samples were transferred to transport media and processed in the microbiology laboratory.

Protocol for irrigant activation

After the microbiological sampling, the samples were divided into three groups:

1. Group A (n = 20): Conventional irrigation with 2.5% NaOCl for 5 min. This constituted the control group

2. Group B (n = 22): Sonically activated irrigation according to the manufacturer’s instructions. The procedure was repeated three times. In between each activation cycle, the canal was flushed with 3 ml of irrigant using a needle

3. Group C (n = 23): Ultrasonic irrigation with Endo Ultra. The procedure was repeated three times, and in between each cycle, the canal was flushed with 3 mL irrigant using a needle.

Microbiological evaluation

Bacterial quantification was performed using the Miles and Misra technique. Serial dilutions were plated on selective media and incubated under aerobic conditions. Colony counts were expressed as colony-forming units (CFU)/mL, and organism identity was confirmed using MALDI-TOF mass spectrometry.[12]

RESULTS

This randomized clinical trial investigated the antimicrobial efficacy of three root canal irrigation protocols – conventional passive irrigation (Control), sonic agitation, and Ultrasonic agitation – in reducing the microbial load of E. faecalis. CFUs were quantified before and after irrigation for each patient. In total, 65 patients were analyzed, with 20 in the Control group, 22 in the sonic group, and 23 in the ultrasonic group. Statistical analyses included descriptive measures, paired within-group tests, and between-group comparisons. The descriptive characteristics of pre- and posttreatment CFU counts are summarized in Table 1.

Table 1.

Descriptive statistics for colony-forming unit counts in different irrigation groups

Group	n	Mean pre	Mean post	Median pre	Median post	SD pre	SD post	Mean reduction
Control	20	9.49	10.85	12.09	14.53	7.46	7.08	−1.36
Sonic	22	8.64	7.98	7.75	9.46	6.47	7.59	+0.66
Ultrasonic	23	12.30	9.52	15.22	9.23	5.88	6.02	+2.78

SD: Standard deviation

Control group

The Control group began with a mean pretreatment CFU of 9.49 (±7.46), which increased slightly posttreatment to 10.85 (±7.08). The median pretreatment CFU was 12.09, rising to 14.53 posttreatment. The mean reduction was negative (−1.36), suggesting no bacterial reduction but, paradoxically, a mild increase.

Sonic group

The Sonic group started with a mean pretreatment CFU of 8.64 (±6.47), which reduced slightly to 7.98 (±7.59) posttreatment. The median CFU values were 7.75 (pre) and 9.46 (post). Although the mean CFU reduction was + 0.66, variability was high, as reflected in the increased posttreatment standard deviation.

Ultrasonic group

The ultrasonic group displayed the highest baseline CFU levels, with a mean of 12.30 (±5.88) and a median of 15.22. Following ultrasonic agitation, the mean was reduced to 9.52 (±6.02), with a posttreatment median of 9.23. The mean reduction of 2.78 CFU was the most substantial among the three groups.

Within-group statistical comparisons

Paired t-tests were conducted within each group to evaluate whether reductions from pre- to posttreatment were statistically significant [Table 2]. In the control group, no significant difference was detected between pre- and posttreatment CFU counts (P = 0.20). In fact, several cases showed increased CFU counts following irrigation, confirming the ineffectiveness of passive irrigation in microbial elimination. In the Sonic group, although a slight mean reduction was noted, the change was not statistically significant (P = 0.40). This suggests that while sonic agitation may disrupt biofilms in some patients, it does not provide consistent or robust microbial clearance. In the Ultrasonic group, a statistically significant reduction was achieved (P = 0.011). This confirms that ultrasonic agitation was the only technique among the three to demonstrate meaningful within-group efficacy, highlighting its superiority in microbial reduction.

Table 2.

Paired t-test results (pre vs. post within groups)

Group	t-statistic	P	Significance
Control	−1.31	0.20	Not significant
Sonic	0.86	0.40	Not significant
Ultrasonic	2.77	0.011	Significant

Between-groups comparisons

To evaluate differences in effectiveness between groups, one-way ANOVA and the nonparametric Kruskal–Wallis test were applied to the CFU reductions [Table 3].

Table 3.

Between-groups statistical comparisons

Test	Statistic	P	Interpretation
ANOVA	4.80	0.011	Significant
Kruskal–Wallis	16.12	0.0003	Highly significant

• ANOVA revealed a significant difference across groups (P = 0.011), confirming that at least one intervention was more effective than the others

• Kruskal–Wallis, which is more robust to nonnormality and skewness in data, also indicated highly significant differences (P = 0.0003).

Taken together, these findings strongly suggest that the Ultrasonic group outperformed both the Control and Sonic groups. The significant difference between-group effect corroborates the within-group analysis, highlighting that ultrasonic irrigation provides the most reliable microbial reduction.

Conventional irrigation alone was ineffective in significantly reducing bacterial load, with some cases showing increased CFU counts, likely due to inadequate irrigant penetration or the displacement of bacteria from deeper canal niches. Sonic agitation produced only a modest and inconsistent reduction in E. faecalis, reflecting its limited hydrodynamic efficacy. In contrast, ultrasonic irrigation achieved a statistically significant reduction (P = 0.011), with a marked decrease in CFU counts. This superior outcome is attributed to cavitation and acoustic streaming, which enhance biofilm disruption and irrigant penetration.

Collectively, these findings have important clinical implications. Passive irrigation alone is insufficient for adequate root canal disinfection and may even contribute to bacterial persistence. While sonic agitation offers a modest benefit, its inconsistent performance limits its reliability as a primary adjunctive technique. Ultrasonic agitation, on the other hand, demonstrates both statistically and clinically significant superiority in reducing microbial loads. These results strongly support the incorporation of advanced agitation techniques into contemporary endodontic practice.

DISCUSSION

In this study, we compared the antimicrobial efficacy of three irrigation modalities – conventional (control), Sonic, and ultrasonic – in reducing E. faecalis CFU counts in root canal samples across 65 patients. Notably, among the three groups, ultrasonic irrigation was the only method that achieved a statistically significant within-group reduction in CFUs (P = 0.011). Furthermore, both parametric (ANOVA, P = 0.011) and nonparametric (Kruskal–Wallis, P = 0.0003) analyses confirmed that ultrasonic irrigation outperformed both sonic and control groups.

Our findings are consistent with a growing body of literature demonstrating that ultrasonically activated irrigation (UAI) achieves superior disinfection compared to sonic or syringe methods in settings modeling E. faecalis biofilms.[7,13,14] For example, a recent in vitro study using activated sodium hypochlorite demonstrated that UAI achieved significantly greater biofilm reduction than sonic activation (23.5% vs. 7.8%, P = 0.004).[15] Similarly, ex vivo investigations have shown that both sonic and ultrasonic activation improve debris and smear layer removal, with ultrasonic methods generally performing better – especially in larger apical preparations.[16,17]

Ultrasonic devices operate at high frequencies, generating rapid fluid movement and localized shockwaves that promote irrigant penetration into fins, isthmuses, and dentinal tubules, forming the basis of their superiority.[18,19] By contrast, sonic devices, which typically operate at lower frequencies (<6 kHz), rely on large-amplitude polymer tips. Their energy output is reduced when constrained by canal walls, leading to weaker shear forces and reduced antimicrobial effectiveness.[8]

It is important to note, however, that some studies have reported conflicting findings. Some studies using low-concentration NaOCl (1%) found no significant difference between sonic, ultrasonic, and syringe irrigation in straight root canals.[20] These discrepancies might be due to differences in irrigant concentration, canal anatomy (biofilm vs. planktonic infection), or activation protocols.[21,22] Indeed, E. faecalis biofilms more closely model clinical challenges, whereas some laboratory models may overestimate efficacy.[23]

Our trial strengthens this evidence base by using actual patient-derived samples and reflecting realistic anatomical and microbial diversity. While our sonic results showed some mean reduction, the variability precluded statistical significance (P = 0.40), mirroring clinical inconsistency. This also reflects the limitation of the study, as a higher number of patient samples can statistically strengthen the external validity of the findings. Future studies, including mixed species biofilms, can be designed that may better replicate clinical conditions and hence be more significant in real time. Despite the limitations, in this study, the pronounced performance of Ultrasonic irrigation reinforces its value as a gold-standard adjunct in root canal disinfection.[16,17]

CONCLUSION

Within the limitations of this clinical study, ultrasonic irrigation demonstrated significantly greater antimicrobial efficacy against E. faecalis compared with sonic activation and conventional syringe irrigation. While sonic activation offered limited benefit, ultrasonic activation consistently achieved superior bacterial reduction. These findings support the incorporation of ultrasonic irrigation as a routine adjunct in contemporary endodontic practice to enhance root canal disinfection and improve treatment outcomes.

Ethics approval

Ethics approval was obtained with the letter number INT/IEC/2021/SPL-1213 dated August 13, 2021.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.