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. 2026 Apr 9;16:16187. doi: 10.1038/s41598-026-46204-7

Evaluation of bacterial reduction by contemporary irrigation activation methods against Enterococcus faecalis biofilm in root canals

Tuğçe Özdemir Sakin 1, Fatoş Albayrak 2, Tutku Tunç 3,✉
PMCID: PMC13201574  PMID: 41957515

Abstract

The aim of this study was to evaluate the bacterial reduction achieved under in vitro conditions by different irrigation activation methods applied with sterile physiological saline solution in an Enterococcus faecalis root canal biofilm model. Seventy single-rooted and single-canal mandibular premolar teeth were divided into seven groups (n = 10): positive and negative control groups and five experimental groups (Standard needle irrigation-SNI, EDDY, passive ultrasonic irrigation-PUI, XP-Endo Finisher, Erbium: Yttrium-Aluminum-Garnet-Er: YAG laser). On the 21st day of biofilm formation, initial samples (S1) were obtained; they were cultured on blood agar, quantified as CFU/mL, and analyzed using scanning electron microscopy. Following irrigation activation, second samples (S2) were obtained and evaluated using the same procedure. The data were analyzed using SPSS 23 software. Since the assumptions of parametric tests were not met, the Kruskal–Wallis and Bonferroni-adjusted Mann–Whitney U tests were used for intergroup comparisons, and the Wilcoxon test was used for pre- and post-procedural comparisons (p < 0.05). No significant difference was found among the groups in terms of S1 values (p = 0.272). In all experimental groups, a significant reduction in bacterial counts was detected as a result of the comparison of S1 and S2 values (p < 0.005). A significant difference was determined among the groups with respect to S2 values (p < 0.001). The EDDY group exhibited the lowest mean value and was found to be significantly different from all other groups (p < 0.001). No significant difference was observed among the PUI, XP-Endo Finisher, and Er: YAG laser groups (p > 0.001); however, each of these groups demonstrated significantly lower bacterial counts compared to the SNI group (p < 0.001). In conclusion, all evaluated methods provided a significant reduction in bacterial counts; however, EDDY was determined to be the most effective method. Further studies are required to evaluate its clinical effectiveness.

Keywords: EDDY, Enterococcus faecalis, Er:YAG laser, Passive ultrasonic, XP-Endo finisher

Subject terms: Diseases, Health care, Medical research, Microbiology

Introduction

The primary goal of root canal treatment is to effectively shape the root canal system mechanically, disinfect it, and then fill it in a leak-proof manner1,2. When root canal disinfection is insufficient, microorganisms remaining in the root canal system can multiply under favorable conditions after endodontic treatment, reaching virulence levels capable of causing disease and leading to treatment failure2.

The bacterial species Enterococcus faecalis (E. faecalis) has been frequently isolated in secondary and persistent infections2,3. E. faecalis is 0.5-1 microns in diameter, allowing it to easily pass through dentinal tubules. E. faecalis is frequently encountered in failed root canal treatments due to its strong resistance mechanism and biofilm formation capacity4.

One of the most critical stages determining the success of endodontic treatment is the effective chemomechanical preparation of the root canal system. Adequate disinfection cannot be achieved in areas inaccessible to mechanical preparation, such as accessory canals, isthmuses, lateral canals, and anastomoses. Additionally, the smear layer formed during preparation further complicates chemical debridement and hinders complete cleaning of the root canal system5.

Biofilm formation provides optimal conditions for microbial cells to undergo mutations, thereby enhancing their long-term survival capacity. Due to the polymeric matrix constituting the biofilm structure, antimicrobial agents cannot fully penetrate the biofilm, which is thought to increase bacterial resistance to antimicrobials6,7. Therefore, irrigation solutions and activation techniques play a crucial role in overcoming increased bacterial resistance.

Sodium hypochlorite (NaOCl) is the most commonly used irrigation solution for the elimination of microorganisms. Its ability to dissolve both necrotic and vital tissues provides broad-spectrum antimicrobial activity8. Another irrigation solution is physiological saline, which has an isotonic structure at a concentration of 0.9%. Physiological serum has a very low antibacterial effect compared to NaOCl and chlorhexidine9. Physiological serum solution lacks the ability to dissolve necrotic tissue10. Its capacity to clean root canals is very low. It can also remove the smear layer in very small amounts11–14.

Various systems and devices that enhance the activation of irrigation solutions are used to achieve more effective disinfection in root canal treatment. The oldest irrigation method, dating back to the past, is the standart needle irrigation (SNI) method. The irrigation solution is distributed homogeneously by moving the needle up and down15. There are open-ended and closed-ended types. Open-ended needles should be used 2–3 mm behind the working length because they direct the solution directly to the apical region. This helps prevent the solution from overflowing from the apical foramen due to excessive pressure. Closed-ended needles can be placed 1 mm behind the working length because they open sideways16. EDDY (VDW, Munich, Germany) is a sonic system that operates at a frequency of 6000 Hz within the root canal. Compared to other sonic activation devices that operate on a similar working principle, EDDY has been found to have a greater effect on debris removal and smear layer removal because it operates at a higher frequency17. Urban et al.18 examined the effectiveness of SNI, EA, EDDY, and passive ultrasonic irrigation (PUI) in removing the smear layer using a scanning electron microscope (SEM). In PUI, ultrasonic tips operate at transverse vibrations at frequencies of 25–30 kHz19. It is one of the activation methods that provides effective disinfection in root canals. It performs irrigation activation without instrumentation in root canals. The increase in heat generated during PUI enhances the tissue-dissolving properties of the solution, enabling more effective disinfection of areas that are difficult to reach mechanically20. The XP-Endo Finisher (FKG Dentaire; La Chaux-de-Fonds, Switzerland) is a contemporary endodontic file system developed to enhance the activation of irrigation solutions following root canal preparation and to be applied during the disinfection protocol21. Owing to its curved design, it can be applied in conjunction with irrigation solutions to facilitate improved contact of irrigants with areas that are difficult to access and clean using conventional root canal instruments, without altering the original canal morphology22. Lasers are also used to effectively eliminate bacteria in root canals. Air bubbles created by laser beams expand in the irrigation solution and burst, accelerating the movement of the fluid. The pressure waves and secondary cavitation formed during this process ensure effective activation of the solution23. Erbium: Yttrium-Aluminum-Garnet (Er: YAG) lasers are widely used due to their high absorption in water and high affinity for hydroxyapatite. Therefore, it is generally the preferred type of laser for the activation of irrigation solutions24.

Considering the issues mentioned above, our study aims to evaluate in vitro the effect of different irrigation activation methods applied with sterile physiological saline solution on bacterial reduction in E. faecalis biofilm within root canals. The null hypothesis of our study is that there is no significant difference among the five different irrigation activation methods in terms of bacterial reduction in E. faecalis biofilm.

Materials and methods

This study was approved by the Sivas Cumhuriyet University Non-Interventional Clinical Research Ethics Committee on March 21, 2024, with decision number 2024-03/53. All methods were carried out in accordance with the principles of the Declaration of Helsinki (2008). Written informed consent was obtained from all patients prior to their inclusion in the study.

Tooth selection

The study used 70 human permanent mandibular premolar teeth extracted for periodontal, orthodontic, or prosthetic reasons. Soft and hard tissue remnants were cleaned with periodontal curettes without damaging the tooth root. The cleaned teeth were stored in distilled water until use. Preoperative radiographs were taken in the mesiodistal and buccolingual directions to confirm that the teeth had a single root and a single canal.

Inclusion criteria for teeth used in the study:

  • Must be single-rooted and single-canal.

  • Root development must be complete.

  • The first file stuck in the root canal must not be larger than a size 15 K-type file.

  • There should be no restoration or root canal treatment on the tooth.

  • There should be no calcification or resorption in the root canals.

  • There should be no fractures, caries, or structural defects on the root surface.

Teeth that did not meet these criteria were excluded from the study.

Preparation of samples

To ensure standardization of the selected teeth, their crowns were cut to a length of 15 mm using a sterile diamond fissure bur attached to a high-speed motor under water cooling. After checking the apical opening of the teeth with size 10 K-type hand files (Dentsply Maillefer, Ballaigues, Switzerland) placed in the root canal, the working length was determined 1 mm behind the apical foramen. After determining the working length, the root canals were shaped with a One Curve mini (Micro-Mega SA, Besancon Cedex, France) Ni-Ti rotary file system with a 25/0.06 taper. In all irrigation procedures, a 30-G side-vented irrigation needle (RaitO, Hong Kong, China) was used. Before initiating preparation, the root canals were irrigated with 2 mL of 5% NaOCl solution (Saver®, Prime Dental Products PVT Ltd., Maharashtra, India). The apical region of the root canal was reached using a size 15 K-type file (Dentsply Maillefer, Ballaigues, Switzerland). Subsequently, irrigation was performed with 2 mL of 5% NaOCl solution. Using the 25/0.06 One Curve Mini file system, instrumentation was continued to the working length with irrigation using 4 mL of 5% NaOCl solution. After reaching the working length, the root canals were irrigated with an additional 2 mL of 5% NaOCl solution. A total of 10 mL of 5% NaOCl solution was used.

Finally, the irrigation protocol was completed sequentially with 10 mL of 17% EDTA (IMICRYL, Konya, Türkiye), 10 mL of 5% NaOCl, and 10 mL of distilled water. The root canals were dried with size 25 paper points (Rogin, China), and the teeth were rendered ready for further procedures. To prevent potential microbial leakage, the entire root surface and the apical foramen were coated with three layers of nail varnish (Flormar, Türkiye).

Glass vial bottles were used to ensure stabilization of the teeth during microbiological and endodontic procedures. The caps of the vials were sterilized by hydrogen peroxide sterilization (Sterrad 100 S, ASP, USA) at 40 °C for 72 min (min). The prepared teeth25,26 and glass vials were sterilized in an autoclave (Sümer International Industry and Trade Inc., Ankara, Türkiye) at 121 °C for 20 min.

Formation of the control and experimental groups

The tooth specimens included in the study were randomly assigned to groups in accordance with randomization principles using a web-based software program (www.randomizer.org).

To monitor biofilm formation, 10 teeth were designated as the positive control group, while 10 teeth were assigned to the negative control group to evaluate microbial contamination. The remaining 50 teeth were divided into five experimental groups, with 10 teeth in each group (n = 10): SNI, EDDY, PUI, XP-Endo Finisher, Er: YAG laser.

Contamination of samples

E. faecalis strain (ATCC 29212) registered in the American Type Culture Collection (ATCC) was used as the bacterium in the study. The obtained E. faecalis culture was incubated at 37 °C in a microaerophilic environment for 24 h. The McFarland turbidity value was determined as 0.5. Bacteria were taken from E. faecalis cultures prepared on blood agar using a loop, and bacterial suspensions were prepared to be 0.5 McFarland (1.5 × 108 CFU/mL). Ten µL of bacterial culture suspension was transferred into the mechanically enlarged root canal space using a 2.5 mL sterile syringe. The root canal openings were sealed with temporary filling material and sterile aluminum foil and incubated at 37 °C. This procedure was repeated every three days for 21 days, and bacterial growth was maintained in the root canals. Thus, mature biofilm formation was achieved over 21 days.

Scanning electron microscope (SEM)

Following biofilm formation, specimens in the positive control group (n = 10) were vertically sectioned into two halves using a sterile separator without disrupting the root canal system. Prior to imaging, the specimens were fixed in 4% glutaraldehyde at 4 °C for 12 h and subsequently dehydrated at room temperature in ascending concentrations of ethanol. Thereafter, the specimens were gold-coated using a sputter coating device (Q150R ES, Quorum Technologies, UK) and analyzed by SEM on day 21.

Additionally, 10 specimens that were not contaminated with bacteria and received sterile 1% glucose tryptic soy broth were used as a negative control group to verify the presence of external bacterial contamination and were analyzed by SEM.

Experimental groups and protocol

The root canals were first irrigated with 1 mL of sterile 0.9% saline solution. Subsequently, three sterile size 25 paper points were placed at the working length for 1 min each and transferred into Eppendorf tubes labeled with the corresponding tooth numbers. The tubes containing 1 mL of BHI were vortexed for 1 min. From each tube, 25 µL was plated onto bipartite blood agar plates using the spread plate technique and incubated at 37 °C for 24 h. The resulting colonies were counted, calculated as CFU/mL, and recorded as S1 values.

A water bath (Nüve BM 402, Belgium) was used in our study to achieve a body temperature of 37 °C and ensure optimal working conditions. The glass vials containing the teeth were placed in the water bath. Sterile physiological saline, used as the irrigation solution, was heated to 37 °C. The irrigation activation protocol was performed in the hot water bath in all groups, ensuring standardization.

Standard needle irrigation (SNI) (n = 10)

A 30-G side-vented irrigation needle placed 1 mm shorter than the working length was used. Irrigation was applied to the root canals with 2 mL of 0.9% sterile physiological saline solution using up and down movements for 30 s (s), followed by a 30 s interval. This cycle was repeated three times.

Irrigation activation with EDDY (n = 10)

A 30-G side-vented irrigation needle placed 1 mm short of the working length was used. Irrigation was applied to the root canals with 2 mL of 0.9% sterile physiological saline solution for 30 s. A non-cutting polymer tip (VDW, Munich, Germany) with dimensions of 25/0.04, operating at a frequency of 6000 Hz, was placed 1 mm short of the working length into the root canals and activated with 30 s of up-and-down movements. This cycle was repeated three times.

Passive ultrasonic irrigation activation (PUI) (n = 10)

A 30-G side-vented irrigation needle placed 1 mm short of the working length was used. Irrigation was applied to the root canals with 2 mL of 0.9% sterile physiological saline solution for 30 s. An ultrasonic device (Ultra X, Eighteeth, Changzhou, China) with passively used tips of size 20/0.02 was placed into the root canals 1 mm shorter than the working length and activated for 30 s. This procedure was performed with 2–3 mm strokes in the up and down directions without contacting the root canal wall. This cycle was repeated three times.

Irrigation activation with XP-endo finisher (n = 10)

A 30-G side-vented irrigation needle placed 1 mm short of the working length was used. Irrigation was applied to the root canals with 2 mL of 0.9% sterile physiological saline solution for 30 s. An XP-Endo Finisher file (FKG Dentaire; La Chaux-de-Fonds, Switzerland) with an ISO 25 apical diameter and 0% taper was set at 800 rpm and 1 Ncm torque and placed into the root canals 1 mm short of the working length. Activation was performed with 30 s of up-and-down movements. This cycle was repeated three times.

Irrigation activation with Er: YAG Laser (n = 10)

A 30-G side-vented irrigation needle placed 1 mm short of the working length was used. Irrigation was applied to the root canals with 2 mL of 0.9% sterile physiological saline solution using 30 s of up and down movements. While the irrigation solution was being applied, an Er: YAG laser device (Smart 2940D, DEKA, Calenzano, Italy) with a wavelength of 2940 nm was used simultaneously. The conical fiber optic tip of the Er: YAG laser was placed in the coronal reservoir27, and pulsed irradiation (Power output 2.0 W, 20 Hz, 100 mJ) was performed to activate the root canals. After the irrigation activation process was completed, a 30-s interval was given. This cycle was repeated three times.

Final irrigation protocol

A total of 6 mL of sterile physiological saline solution was used in all groups during the irrigation activation procedures performed in three cycles. Finally, the root canals were irrigated with 2 mL of physiological saline for 30 s. A total of 120 s of irrigation and a 90-s activation/rest period were applied in the root canals.

After completion of the irrigation activation procedures, three sterile size 25 paper points were placed at the working length for 1 min each and transferred into Eppendorf tubes labeled with the corresponding tooth numbers. The tubes containing 1 mL of Brain Heart Infusion Broth (BHI; Acumedia Manufactures, Inc., Lansing, Michigan, USA) were vortexed for 1 min. From each tube, 25 µL was plated onto the second half of bipartite blood agar plates using the spread plate technique and incubated at 37 °C for 24 h. The resulting colonies were counted, calculated as CFU/mL, and recorded as S2 values.

To visually confirm the elimination of bacteria following the application of irrigation activation protocols, SEM examination was performed on 2 randomly selected samples from each experimental group. In this context, a total of 10 samples were prepared for SEM, and randomly selected areas along the root canal lumen of each sample were imaged. SEM evaluation was not intended for quantitative analysis but was conducted for qualitative and illustrative purposes.

Statistical analysis

The effect size was assumed to be f = 0.50 based on values reported in a similar study28. The sample size was calculated using a power analysis performed with G*Power software (version 3.1.9.7). A one-way ANOVA test was assumed for the comparison of seven groups, with the significance level set at α = 0.05 and the statistical power (1 − β) at 0.80. Under these parameters, the minimum required total sample size was calculated as 63, corresponding to at least 9 specimens per group. To account for potential data loss, 10 specimens were included in each group, resulting in a total of 70 samples. With this sample size, the achieved statistical power was 0.816. Accordingly, the study was designed with sufficient power to detect statistically significant differences among the groups.

The data obtained from the study were transferred to and analyzed using IBM SPSS Statistics Version 23 (Statistical Package for the Social Sciences, IBM SPSS®, Chicago, USA). Since the assumptions of parametric tests were not met in the evaluation of the data, the Kruskal-Wallis test was used for the comparison of more than two independent groups, and the Mann-Whitney U test with Bonferroni correction was used for multiple comparisons. For the comparison of pre-procedural and post-procedural bacterial samples obtained from the same specimens, the Wilcoxon test was used. The data were presented in tables as arithmetic mean and standard deviation values, and the level of statistical significance was accepted as 0.05.

Results

SEM analysis

A positive control group was included to verify the growth and biofilm formation of E. faecalis in the experimental teeth. SEM analysis confirmed the proliferation of E. faecalis and demonstrated its characteristic biofilm colonization pattern on the root canal dentin surfaces (Fig. 1).

Fig. 1.

Fig. 1

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Representative SEM images of the positive control group demonstrating E. faecalis growth and biofilm formation. A: SEM image at 5000× magnification showing the coronal third of the root canal in the positive control group, B: SEM image at 5000× magnification showing the middle third of the root canal in the positive control group, C: SEM image at 5000× magnification showing the apical third of the root canal in the positive control group.

A negative control group was included to verify the absence of bacterial contamination. SEM analysis confirmed that no bacterial colonization or biofilm formation was detected on the root canal dentin surfaces (Fig. 2).

Fig. 2.

Fig. 2

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Representative SEM images of the negative control group confirming the absence of bacterial contamination. D: SEM image at 5000× magnification showing the coronal third of the root canal in the negative control group, E: SEM image at 5000× magnification showing the middle third of the root canal in the negative control group, F: SEM image at 5000× magnification showing the apical third of the root canal in the negative control group.

After applying irrigation activation methods, the remaining bacteria were visually evaluated by SEM examination (Fig. 3).

Fig. 3.

Fig. 3

Fig. 3

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Representative SEM images of residual bacteria after different irrigation activation methods. a1: SEM image at 5000× magnification showing the coronal third in the SNI group, a2: SEM image at 5000× magnification showing the middle third in the SNI group, a3: SEM image at 5000× magnification showing the apical third in the SNI group, b1: SEM image at 5000× magnification showing the coronal third in the EDDY group, b2: SEM image at 5000× magnification showing the middle third in the EDDY group, b3: SEM image at 5000× magnification showing the apical third in the EDDY group, c1: SEM image at 5000× magnification showing the coronal third in the PUI group, c2: SEM image at 5000× magnification showing the middle third in the PUI group, c3: SEM image at 5000× magnification showing the apical third in the PUI group, d1: SEM image at 5000× magnification showing the coronal third in the XP-Endo Finisher group, d2: SEM image at 5000× magnification showing the middle third in the XP-Endo Finisher group, d3: SEM image at 5000× magnification showing the apical third in the XP-Endo Finisher group, e1: SEM image at 5000× magnification showing the coronal third in the Er: YAG laser group, e2: SEM image at 5000× magnification showing the middle third in the Er: YAG laser group, e3: SEM image at 5000× magnification showing the apical third in the Er: YAG laser group.

Analysis of Bacterial Reduction in Experimental Groups

Descriptive statistics of bacterial counts (log CFUs/mL) determined at S1 and S2, along with bacterial load reduction percentages for all experimental groups, are presented in Table 1; Fig. 4.

Table 1.

Descriptive statistics (mean ± SD) of log CFU/mL values for E. faecalis biofilm and bacterial reduction rates according to the applied irrigation activation methods.

Groups S1 S2 p value Bacterial Load Reduction Percentage (%)
Mean ± SD Mean ± SD Mean ± SD
SNI 8.15 ± 0.03 X 4.52 ± 0.83 T 0.005* 44.54 ± 10.20 A
EDDY 8.18 ± 0.04 X 3.03 ± 0.51 Y 0.005* 63.00 ± 6.32 B
PUI 8.17 ± 0.04 X 3.68 ± 0.29 Z 0.005* 55.01 ± 3.57 C
XP-Endo Finisher 8.17 ± 0.03 X 3.63 ± 0.77 Z 0.005* 55.59 ± 9.34 C
Er: YAG laser 8.14 ± 0.05 X 3.81 ± 0.47 Z 0.005* 53.22 ± 5.76 C
p value 0.272 < 0.001* < 0.001*
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SNI: Standard needle irrigation, PUI: Passive ultrasonic irrigation, Er: YAG laser: Erbium: Yttrium-Aluminum-Garnet. SD: standard deviation. Different uppercase letters indicate statistically significant differences between the groups (p < 0.05). Letters A–B indicate differences between columns, whereas letters X-Y-Z indicate differences between rows.

Fig. 4.

Fig. 4

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Bar graph illustrating the mean percentage reduction in bacterial load (%) among the experimental groups. Distinct uppercase letters denote statistically significant differences (p < 0.001).

No statistically significant difference was found among the experimental groups with respect to the S1 values of E. faecalis counts (p = 0,272). In contrast, a statistically significant difference was observed between the groups when the S2 values were analyzed (p < 0.001). Furthermore, comparison of S1 and S2 measurements within all experimental groups revealed a statistically significant reduction in bacterial counts (p < 0.005).

With respect to the S2 values, a statistically significant difference was detected among the groups (p < 0.001). The EDDY group (3.03 ± 0.51; Y) exhibited the lowest mean value and was significantly different from all other groups. No statistically significant difference was observed among the PUI (3.68 ± 0.29; Z), XP-Endo Finisher (3.63 ± 0.77; Z), and Er: YAG laser (3.81 ± 0.47; Z) groups (p > 0.001); however, each of these groups demonstrated significantly lower bacterial counts compared with the SNI group (4.52 ± 0.83; T). A statistically significant difference was also detected among the groups in terms of bacterial load reduction percentages (p < 0.001). The highest reduction was observed in the EDDY group (63.00 ± 6.32%; B), which was significantly different from all other groups. The PUI (55.01 ± 3.57%; C), XP-Endo Finisher (55.59 ± 9.34%; C), and Er: YAG laser (53.22 ± 5.76%; C) groups showed comparable results to one another; however (p > 0.001), all three groups exhibited significantly greater bacterial reduction compared with the SNI group (44.54 ± 10.20%; A).

Discussion

Microorganisms in the root canal system cannot be eliminated from root canals using mechanical methods alone29. When microorganisms cannot be removed mechanically, adequate microbial cleaning cannot be achieved in the root canals, which may lead to failure of endodontic treatment30. Therefore, it is important to ensure disinfection with irrigation solutions in areas that cannot be reached mechanically. There are studies evaluating various methods that activate irrigation solutions and aim to increase disinfection efficacy31–33. This study aims to evaluate in vitro the bacterial reduction effect of SNI, EDDY, PUI, XP-Endo Finisher, and Er: YAG laser activation methods applied with sterile physiological saline on E. faecalis biofilm in root canals. In our study, significant differences were observed among the five different irrigation activation methods compared for their bacterial reduction efficacy against E. faecalis biofilm, and based on these findings, the null hypothesis of the study was rejected.

Although various microorganisms may be involved in root canal infections, E. faecalis is the most frequently isolated bacterium in cases of endodontic treatment failure. It is a Gram-positive facultative anaerobic bacterium capable of penetrating dentinal tubules up to 1000 μm34. It possesses adhesion factors and gelatinase activity that contribute to the formation of highly resistant biofilms. These properties enhance biofilm stability and increase resistance against antimicrobial agents, allowing E. faecalis to survive for prolonged periods within root canals35–37. For these reasons, the E. faecalis ATCC 29,212 strain commonly used in the literature38–40 was selected in the present study. However, root canal infections in clinical conditions are typically polymicrobial41. Therefore, the use of a monospecies biofilm model may be considered a limitation of this study, and the findings should be interpreted within this context.

There is no clear consensus in the literature regarding the incubation period required for E. faecalis biofilm formation. The time required for biofilm development ranges from 24 h to 4 weeks42–44. In our study, in order to obtain a mature biofilm, a fresh E. faecalis suspension was injected into the root canals every three days and maintained for 21 days45. Furthermore, the use of a single-species biofilm model allowed standardization of experimental conditions and reproducibility of bacterial quantification.

Yurdagül et al.46 emphasized that, in order to more clearly evaluate the efficacy of activation methods, the antibacterial effect of the irrigating solutions should be eliminated. Physiological saline is known to lack the ability to dissolve necrotic tissue, to remove the smear layer only to a limited extent, and to provide no substantial antibacterial activity when used alone10,12–14. In contrast, the literature consistently reports that NaOCl demonstrates significantly greater antibacterial efficacy in root canal disinfection compared with physiological saline9,47. These findings highlight the critical role of the irrigating solution in bacterial elimination. In the present study, sterile physiological saline was deliberately used to better isolate and evaluate the mechanical effects of the activation techniques. However, in clinical practice, NaOCl and/or chelating agents are routinely preferred. Therefore, the findings of this study should not be directly generalized to clinical conditions and should be interpreted within the context of this limitation.

The study by Alves et al.48 evaluated the elimination of E. faecalis using the PUI and XP-Endo Finisher irrigation activation methods. To simulate body temperature and ensure optimal conditions, the irrigation solution was heated to 37 °C and used, and the practical stages of the study were performed in a cabinet set to 37 °C. In the study by Carvalho et al.49 80 mandibular incisors contaminated with E. faecalis were used. The main groups of the study were divided into four groups: XP-Endo Shaper, XP-Endo Shaper + XP-Endo Finisher, Reciproc Blue, and Reciproc Blue + XP-Endo Finisher. Each group was divided into two subgroups based on the irrigation solution, namely 0.9% saline and 2.5% NaOCl, creating a total of eight experimental groups. The antibiofilm efficacy against E. faecalis was evaluated across these groups. The application stages of all experimental groups were performed in a 37 °C cabinet to mimic body temperature. In our study, the irrigation activation methods were applied in a 37 °C water bath, similar to the above studies48,49. In addition, the physiological saline solution used for root canal irrigation in our study was used at a temperature of 37 °C.

In comparing disinfection procedures in endodontic treatments, the duration of application in root canals is important50,51. In our study, to ensure standardization, all experimental groups underwent 90 s of irrigation and 90 s of activation or passive contact time. Thus, the duration of solution contact with the root canals was equal in all groups. Considering previous studies, the irrigation activation protocol in our study was performed 1 mm shorter than the working length in the SNI52, EDDY53, PUI54,55, and XP-Endo Finisher53 groups. In the Er: YAG laser group, activation was applied by placing the fiber optic tip in the coronal reservoir27.

In our study, root canal preparation was performed using the One Curve mini file system with a 25/0.06 taper. Owing to the wide apical preparation, it can be anticipated that the activated solution was able to reach the root canals, particularly the apical region, more effectively. In this context, similar to the findings reported in the sample study56, it is considered that a significant reduction in the number of E. faecalis occurred in all experimental groups after the procedure.

Al-Obaida et al.57 applied six different irrigation activation methods to teeth contaminated with E. faecalis and examined the residual bacterial loads after the procedure. The study included SNI, ultrasonic activation, EA, EDDY, positive, and negative control groups. The SNI method was found to be less successful than the other groups. Bao et al.58 evaluated the antibiofilm efficacy of SNI, PUII, and XP-Endo Finisher irrigation activation methods in their microbiology study. The findings of the study showed that the XP-Endo Finisher was more successful in bacterial elimination than PUI and SNI, while SNI was found to be the least successful method. Zeng et al.59 compared the elimination of E. faecalis bacteria from root canals after applying activation with SNI, EA, and EDDY to physiological serum and NaOCl solutions. While no significant difference was found between the EA and EDDY groups, the SNI method was found to be the least effective group. In a study by Zapata et al.60, 6% NaOCl was used in conjunction with irrigation activation methods, and the effectiveness against biofilms was examined. Fifty samples were randomly divided into five groups according to the final irrigation protocol: SNI, EA, PUI, Photon-Induced Photoacoustic Streaming (PIPS), and a control group consisting of distilled water. When the results of the study were evaluated, the most successful group was found to be the Er: YAG laser–based PIPS irrigation activation protocol. It was found to be more successful than the PUI, SNI, and EA methods, while the least successful group was the control group, followed by SNI. As a result of the findings obtained in our study, consistent with the studies mentioned above, the SNI method was found to be the least successful method in terms of antibiofilm efficacy against E. faecalis58–62. The reason why the SNI method was less successful than all other irrigation activation methods is thought to be due to the insufficient penetration of the irrigation solution into areas of the root canal system that cannot be reached by mechanical preparation. Studies in the literature also emphasize that the effectiveness of the irrigation solution is increased with the application of activation methods63,64. Furthermore, the formation of a vapor lock effect in the SNI method may have prevented the irrigation solution from reaching the apical region sufficiently. This situation can be considered a reason for the lower disinfection efficacy compared to other activation methods65.

Neuhaus et al.33 investigated the removal of numerous microorganisms using various irrigation activation methods in their study. In the study evaluating SNI, EDDY, and PUI, EDDY was found to be the most successful group in eliminating microorganisms. Yurdagül et al.46 used four different irrigation activation methods, SNI, EDDY, PUI, and XP-Endo Finisher R, in their study on E. faecalis elimination. EDDY was found to be the most successful group in their study. In the study by Hekal et al.66 using 42 human molars, the elimination of E. faecalis was evaluated with the EDDY and XP-Endo Finisher irrigation activation methods. It was observed that EDDY removed more E. faecalis from the root canals than the XP-Endo Finisher method. Consistent with studies in the literature, the effectiveness of EDDY in bacterial elimination was found to be superior to all irrigation activation methods examined in our study. EDDY, with its polyamide tip that oscillates with 3-dimensional movement, can be advanced to the apical part of the root canal because of its flexible structure, and does not abrade the root dentin like metal tips. The polyamide tip is highly resistant to breakage, making it easy to use in curved canals67. In the irrigation activation method with EDDY, the three-dimensional movement of the polymer tip can spread the energy of the solution in all directions, whereas in the PUI activation method, the energy distribution is more linear48. Swimberghe et al.68 stated in their study that the movement of the EDDY tip was 350 μm, while that of the PUI tip was 75 μm. Due to this difference, it is thought that EDDY may contribute to more effective disinfection by spreading the irrigation solution over a wider area. At the same time, EDDY is quite effective in breaking down bacterial biofilm through the acoustic flow and cavitation it creates18,19. Since the XP-Endo Finisher performs disinfection based on physical contact, its cavitation effect is not as effective as that of EDDY69. When evaluating the differences in the working principles of irrigation activation methods, the reason why EDDY is more successful than the PUI and XP-Endo Finisher methods can be explained.

In a study conducted by Bürklein et al.70 on simulated three-dimensional printed resin blocks, it was found that the cleaning efficacy of the Er: YAG laser in root canals was more successful compared to EDDY. The findings of that study do not correspond with those of the present study. Air bubbles generated by laser beams expand and burst, accelerating the movement of the irrigation solution and thereby ensuring its effective activation23. In the aforementioned study, a laser beam directed toward the apex in the root canals was applied thanks to the thin design of the tip of the Er: YAG laser. In our study, however, the fiber optic tip used was placed in the coronal reservoir. In our study, it is thought that the disinfection efficacy was significantly reduced as a result of the strong shock wave and cavitation effect formed in the root canals decreasing toward the apex.

In a study conducted by Ballal et al.71, the effectiveness of SNI, PUI, XP-Endo Finisher, and the F-file methods in bacterial elimination was compared. XP-Endo Finisher and PUI were the most successful methods, and no significant difference was found between the two groups. Shan et al.72 compared the effectiveness of PIPS, PUI, and the Er: YAG laser methods in eliminating E. faecalis from root canals. According to the findings of the study, no significant difference was found between the PUI and Er: YAG laser groups, whereas PIPS was found to be less successful compared to the other groups.

There are limited studies in the literature that directly compare the efficacy of the XP-Endo Finisher and Er: YAG laser in eliminating E. faecalis. Most studies evaluating these two methods together focus on the removal of root canal filling materials or smear layer rather than bacterial elimination32,48. Therefore, it is thought that the findings obtained from our study will contribute to filling this gap in the literature.

Conclusions

In the present in vitro investigation, contemporary irrigation activation methods were found to be more effective than the SNI protocol in reducing E. faecalis biofilm. Among the tested approaches, EDDY produced the most pronounced decrease in bacterial counts. This outcome may be associated with the hydrodynamic and acoustic activation generated by EDDY, which could promote enhanced irrigant circulation and improved contact with canal wall surfaces. Additionally, the flexibility of its polymer tip may contribute to improved irrigant penetration into areas that are challenging to access with conventional instrumentation.

Overall, these findings indicate that modern irrigation activation strategies may improve the reduction of E. faecalis biofilm under laboratory conditions. Nevertheless, given the inherent limitations of in vitro models, further in vivo studies incorporating microbiological evaluation are necessary to clarify their potential clinical applicability.

Author contributions

Conceptualization: A. F. and ÖS. T.; data curation: A. F., ÖS. T. and T. T.; investigation: A. F., ÖS. T. and T. T.; methodology: A. F., ÖS. T. and T. T.; validation: A. F., ÖS. T. and T. T.; visualization: A. F. and ÖS. T.; writing– original draft: A. F. and ÖS. T.; writing– review & editing: A. F., ÖS. T. and T. T.

Funding

Declaration.

This study was supported by the Scientific Research Projects Coordination Unit of Sivas Cumhuriyet University (CUBAP) under project number DİŞ-2024-321.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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