Journal of Clinical Pediatric Dentistry. 2025; 49(4): 148-153. doi: 10.22514/jocpd.2025.085
Original Research

Antimicrobial activity of different irrigation solutions on Enterococcus faecalis in the root canal of the primary teeth—an in vitro comparative study

Peris Celikel1, Banu Ceyhan1, Murat Buyuksefil1, Anna Levchenko2, Fatih Sengul1,*,, Sera Simsek Derelioglu1, Mehmet Cemal Adiguzel2

1Faculty of Dentistry, Ataturk University, 25240 Erzurum, Turkey

2Department of Microbiology, Faculty of Veterinary Medicine, Ataturk University, 25240 Erzurum, Turkey

*Corresponding Author(s):fsengul@atauni.edu.tr (Fatih Sengul)

History Submitted: 09 August 2024 | Accepted: 28 October 2024 | Published: 03 July 2025
Copyright:  ©2025  The Author(s). Published by MRE Press.
This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

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Abstract

Background: Enterococcus faecalis is a key pathogen in persistent endodontic infections, particularly in primary teeth with complex root canal anatomy. This study aimed to compare the antimicrobial efficacy of five irrigation solutions against E. faecalis. Methods: Seventy-five extracted primary teeth were prepared and inoculated with E. faecalis (3 × 109 CFU/mL) then divided into five groups (n = 15): 2.5% sodium hypochlorite (NaOCl), 5% ethylenediaminetetraacetic acid (EDTA), 9% etidronic acid, 17% glycolic acid, and 0.9% physiological saline (NaCl). Root canals were irrigated, and bacterial samples were collected at 30 minutes, 24 hours, and 72 hours. Bacterial counts were analyzed using ANOVA (p < 0.05). Results: NaOCl, etidronic acid, and glycolic acid significantly reduced E. faecalis counts within 30 minutes. EDTA showed no immediate effect but inhibited bacterial growth by 24 hours. NaCl caused a gradual reduction in bacterial load over time (p = 0.02). Conclusions: Etidronic and glycolic acids were effective alternatives to NaOCl in disinfecting primary tooth root canals. These agents may offer biocompatible options in pediatric endodontics, but further in vivo studies are warranted.

Keywords:Enterococcus faecalis;Etidronic acid;Glycolic acid;Root canal irrigation;Sodium hypochlorite
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Cite this article

Peris Celikel, Banu Ceyhan, Murat Buyuksefil, Anna Levchenko, Fatih Sengul, Sera Simsek Derelioglu, Mehmet Cemal Adiguzel. Antimicrobial activity of different irrigation solutions on Enterococcus faecalis in the root canal of the primary teeth—an in vitro comparative study. Journal of Clinical Pediatric Dentistry. 2025; 49(4): 148-153. doi: 10.22514/jocpd.2025.085

1. Introduction

Microorganisms play a significant role in pulpal and periradicular diseases [1]. The main reason for the failure of endodontic treatment is the inability to remove bacteria such as Enterococcus faecalis (E. faecalis), Streptococcus mutans (S. Mutans), and Candida albicans from the root canal system [2]. E. faecalis is one of the most important bacteria in recurrent root canal infection and is among the most resistant species to treatment. Several key factors, including the ability to survive on its own, penetrate deeply into the tiny tubules in the dentin, withstand high pH levels, and in low nutrients, contribute to E. faecalis’s resistance to chemomechanical irrigation in the root canal [3].

The studies suggest that the root canal systems of primary teeth are more complex compared to permanent teeth [4]. Primary teeth with branching and microchannels may make it more challenging to eradicate bacteria especially E. faecalis from the root canals during treatment, demanding more complex procedures [5]. In addition to mechanical instrumentation, various intra-canal irrigants such as physiological saline (NaCl), sodium hypochlorite (NaOCl), chlorhexidine gluconate (CHX), and ethylenediaminetetraacetic acid (EDTA) are used for bacterial elimination during root canal treatment of primary teeth. Furthermore, newly developed irrigation protocols and solutions in endodontics are promising in effectively removing bacteria from root canals [6].

Thanks to its properties such as disinfection, excellent organic tissue solvent, and anti-inflammatory action, NaOCl is the main irrigant used in endodontic treatment [7]. On the other hand, it has significant drawbacks, including a toxic property, bad smell/taste, and inability to completely remove smear layers and microorganisms, emphasizing the necessity to find new alternatives [8]. EDTA solution, which has an antimicrobial effect in removing inorganic residues such as smear layer, is also used as canal irrigant in primary teeth [9]. Etidronic acid, also known as etidronate or 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), is a chelator alternative to EDTA and citric acid and is used in combination with NaOCl [10]. Glycolic acid, which is colorless, odorless and biocompatible monomer with high solubility in water, are in the alpha hydroxy acids group containing citric acid [11, 12]. Glycolic acid offers optimal abrasion on tooth surfaces thanks to its low molecular weight and organic structure, so it has been proposed to be used as an enamel and dentin surface abrasive by replacing phosphoric acid in recent years [13]. Glycolic acid, which is readily biodegradable [14]. It has been reported to effectively remove the smear layer from root canal dentin [15].

There are studies in the literature assessing the antimicrobial efficacy of different irrigation solutions against E. faecalis in primary teeth [16, 17, 18, 19]. However, to the best of our knowledge, there is currently no report assessing the antibacterial activity of glycolic acid as an irrigation agent for the treatment of root canals in primary teeth. This study was aimed to assess the effectiveness of NaCl, NaOCl, EDTA, etidronic acid, and glycolic acid on E. faecalis to fill this gap in the literature. The null hypothesis of the present study is that there is no difference in the antimicrobial activity of the irrigation solutions used on E. faecalis.

2. Material and methods

The Local Ethics Committee of Atatürk University Faculty of Medicine (7/61, 26 October 2023) approved our study’s protocol as it complies with local legislation and the principles of the Declaration of Helsinki. All parents participated in the current study signed the informed consent form.

2.1 Teeth preparation

The sample size was calculated according to a previous similar study [20]. The significant level was set at 0.05, and the statistical power of the study was set at 95%. It was estimated that 75 teeth (15 per group) were required to demonstrate an effect size (0.53).

The current ex vivo study included 75 primary central incisors or primary second molar palatal roots that were recently extracted due to infection or extensive decay. Teeth with resorption of more than 2/3 of the original root length and any signs of cracks or grooves in the root were excluded. The tissue residues on the teeth were removed using a Cavitron device (Cavitron® Jet SPSTM, Dentsply International Inc., York, PA, USA). Teeth were disinfected in the 2.5% sodium hypochlorite solution for 2 hours, then stored in 70% ethanol until use [21].

The dental crowns were removed with a diamond disk (Dentsply, Maillefer, Baillaigues, Switzerland), resulting in a root length of 10 mm. The working length was then determined to be 1 mm shorter than this root length. Root canals were prepared using a traditional technique with 40 K-File (Dentsply, Maillefer, USA) sizes 15 through 40 [22]. During the cleaning and shaping process, 1 mL of sterile distilled water was used after each instrument size. The canals were irrigated for 5 minutes each with 17% EDTA and 2.5% sodium hypochlorite to remove the smear layer. A final irrigation was then performed with 1 mL of 0.9% NaCl for one minute [23]. After sealing the apices with flowable composite (3 M flow Ultimate, MN, USA), the external surfaces of the tooth roots were meticulously sealed using cyanoacrylate adhesive to prevent any inadvertent leakage of the bacterial suspension. The teeth were then placed in acrylic resin (Bosworth, Neocryl, USA) blocks for easier handling during the experiment. Finally, the samples were sterilized in an autoclave at 121 °C for 15 minutes [24].

2.2 Bacterial strain and culture conditions

Frozen glycerol stocks of strain E. faecalis American Type Culture Collection (ATCC) 29212 in the Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Ataturk University (Erzurum, Türkiye) were streaked on Mueller Hinton (MH) agar plates and incubated at 37 °C for about 24 hours under aerobic condition. A fresh culture of E. faecalis was obtained after a sub-passage on MH agar plates for approximately 20 hours of incubation under the same conditions as described above [25]. After the incubation, bacterial strain was then diluted to 3 × 109 colony forming units per milliliter (CFU/mL) in sterile NaCl [26].

2.3 Study design

In the irrigation of each root, 5 mL of each of the five different solutions (NaOCl (260624269, Microvem, Altun Medical, Istanbul, Türkiye), EDTA (160120-1, Saver®, Prime Dental Products PVT ltd., Maharashtra, India), etidronic acid (MKCK5737, Sigma Aldrich, St Louis, MO, USA), glycolic acid (03983, Sigma Aldrich, St Louis, MO, USA), and NaCl (24474599, OSEL, Istanbul, Türkiye)) was used at the concentrations recommended for primary teeth [15, 16, 18, 27, 28]. A total of 75 primary teeth roots was randomly divided to five groups (four experimental and one control; n = 15 teeth per group), as indicated in Table 1.

Table 1.Solutions used for the irrigation of the teeth root after E. faecalis inoculation in the current study.
GroupsType of solutionConcentrationnSolution volume
Group 1Sodium hypochlorite (NaOCl)2.5%155 mL
Group 2Ethylenediaminetetraacetic acid (EDTA)5%155 mL
Group 3Etidronic Acid9%155 mL
Group 4Glycolic Acid17%155 mL
Group 5 (Control)Physiological saline (NaCl)0.9%155 mL

A 20 µL aliquot of the suspension, with a concentration of 3 × 109 bacteria counting number (CFU/mL, was inoculated into the tooth root. The teeth were then incubated for allowing inoculation of bacteria into the teeth root at 37 °C for 30 minutes. While holding the tooth with sterile forceps, final irrigation was performed for each experimental group using 5 mL of the designated solution. The root canals were then dried using a paper point.

After 30 minutes, the root canal was irrigated with 100 µL of NaCl, and the fluid in the canal was collected for bacterial counting using a sterile pipette tip (Dragonlab, Japan). The sampled teeth were incubated at 37 °C. Similarly, sample collection and bacterial counting procedures were meticulously performed at 24 and 72 hours [28].

For the enumeration of E. faecalis, a volume of 100 µL of the irrigated liquid was used. Subsequently, this volume was combined with 900 µL of physiological saline solution, and a serial dilution was performed. Aliquots of 100 µL from the appropriate dilutions were cultured on MH agar plates. These plates were then incubated at 37 °C for a duration of 24 hours. Following incubation, a random colony was meticulously selected and subsequently confirmed to be E. faecalis through routine biochemical tests, including Gram staining, catalase testing, oxidase testing, and Lancefield serogroup classification. The resulting bacterial count was expressed in CFU/mL [28].

2.4 Statistical analysis

Analyses were performed in SPSS version 25 (IBM Corp, Armonk, NY, USA) program. Levene’s test was performed to assess the homogeneity of the time-dependent bacterial counts obtained in the current study. One-Way analysis of variance (ANOVA) was used, followed by post-hoc Tukey’s or Dunnett’s T3 tests, depending on the data distribution. A p < 0.05 value was considered statistically significant.

3. Results

The five different irrigation solutions were applied to the tooth roots to reduce the E. faecalis load (Fig. 1). NaOCl, etidronic acid, and glycolic acid showed significant bacterial inhibition within the first 30 minutes of irrigation. Surprisingly, 5% EDTA solution permitted bacterial growth during the same period (p = 0.01). However, as demonstrated in Fig. 1, all four solutions (NaOCl, EDTA, etidronic and glycolic acid) successfully inhibited E. faecalis growth at both the 24-hour and 72-hour intervals. Statistical analysis revealed varying degrees of effectiveness among these substances compared to control groups (p < 0.001). The use of physiological saline solution in the control group resulted in a gradual reduction in bacterial load over each timeline (p = 0.02). These results emphasize the importance of selecting appropriate root canal irrigants to achieve effective microbial control in endodontic procedures.

The bacteria counting number (CFU/mL) of E. faecalis using five different irrigation solutions for teeth root in three-time intervals. The solutions were represented with different colors on the figure. NaCl: physiological saline; NaOCl: sodium hypochlorite; EDTA: ethylenediaminetetraacetic acid.

Fig. 1.The bacteria counting number (CFU/mL) of E. faecalis using five different irrigation solutions for teeth root in three-time intervals. The solutions were represented with different colors on the figure. NaCl: physiological saline; NaOCl: sodium hypochlorite; EDTA: ethylenediaminetetraacetic acid.

4. Discussion

The excision of necrotic pulpal tissue, dentin debris, intra-canal bacteria, and the avoidance of subsequent infections are crucial in endodontic treatment [5]. Endodontic treatment of primary teeth requires some considerations. Since primary teeth have a complex root canal system due to their structure, root canal irrigation is crucial in removing microorganisms from areas where mechanical instrumentation is insufficient. However, root canal irrigation solutions currently used cannot completely remove microorganisms from the canal. Therefore, the research for an ideal irrigation solution for primary tooth root canal treatment continues [4]. Accordingly, the present study aimed to assess the effectiveness of NaCl (0.9%), EDTA (5%), NaOCl (2.5%), etidronic acid (9%), and glycolic acid (17%) in eliminating E. faecalis from primary tooth root canals. The null hypothesis was rejected; however, the results revealed that both etidronic acid and glycolic acid were promising alternatives to NaOCl, and that all three irrigants were effective.

Root canal infections are known to be associated with a wide variety of microorganisms. Studies have reported that E. faecalis is the most common species found in permanent tooth root canals and necrotic primary teeth [6, 29]. E. faecalis, an endodontic pathogen known for its resilience in challenging conditions with insufficient nutrients and its ability to survive for extended periods in treated root canals, was selected. E. faecalis is a Gram-positive facultative anaerobic cocci that can survive changes in pH and temperature [30]. The apices and root walls of all sampled teeth were sealed with cyanoacrylate adhesive to prevent contamination from the external surface of the tooth during the experiment in the current study.

Primary teeth have a more complex root canal system compared to permanent teeth in terms of canal structure, anatomical variations, and pulp morphology. This complexity result in bacteria within the root canal being irretrievable mechanical instruments alone, requiring the use of an effective antimicrobial irrigation agent [31]. Agents such as NaCl, NaOCl, EDTA and CHX are used in primary tooth root canal irrigation [6]. In this study, along with common solutions in the irrigation of primary teeth (NaCl, NaOCl, EDTA), the novel irrigation solutions, etidronic acid and glycolic acid were also used. However, CHX was not included in this study because recent findings have indicated that CHX is as cytotoxic as, or even more cytotoxic than, NaOCl and should not be used as a final irrigant [32].

NaCl solution is often used in primary teeth as an irrigant thanks to its isotonic nature, which minimizes harm to the tooth structure and surrounding tissues. While its antimicrobial efficacy is limited compared to other solutions, NaCl is effective at physically removing debris, tissue remnants, and microorganisms from the canal area [33]. Previous studies have reported that NaCl has limited antimicrobial activity [19, 34] and only removes debris from the root canal [35]. Similarly, the current study revealed that NaCl has a limited antimicrobial effect in the current study and should be used as a supportive agent with other antimicrobial agents.

While NaOCl, a common irrigation solution in root canal treatment, has antimicrobial and solvent properties, its use in primary canals is limited due to its unpleasant taste, potential for tissue damage, and cytotoxic effects [36]. EDTA is used to disinfect root canals, dissolving the inorganic part of the smear layer by chelating with calcium in dentin tissue [37]. While EDTA effectively dissolves the inorganic constituents of the smear layer in primary teeth, it also induces erosion of the dentin layer and exhibits a minimal antibacterial effect, hence limiting its usage [1, 38]. Therefore, it is recommended to use it in combination with NaOCl, which has both an antibacterial effect and effectively removes organic tissue [39]. In this study, while NaOCl has antibacterial effects against E. faecalis in the first 30 minutes, the EDTA solution was ineffective. This suggests that EDTA should not be exclusively relied upon for microbial reduction but is rather used to enhancing the efficacy of other irrigants by facilitating deeper penetration of antimicrobial solutions.

Etidronic acid is a biocompatible chelator that is used in combination with NaOCl as an alternative to EDTA or citric acid [16]. Organic acids such as EDTA or citric acid are not compatible with hypochlorite in terms of retaining active chlorine in solution. Upon the combined use of these two substances, the interaction between the calcium-EDTA complex and hypochlorite produces a precipitate. This precipitate can accumulate inside the root canal or at the canal orifice, preventing complete cleaning and disinfection of the canal. The antimicrobial properties of etidronic and glycolic acid were assessed in this study, driven by the side effects of EDTA and NaOCl. In contrast to EDTA, etidronic acid has been reported to have a decalcifying capacity that is compatible with NaOCl [40]. The use of etidronic acid as an irrigation solution in root canal treatment has been observed to reduce mechanical stress on rotary instruments [41]. Additionally, an in vivo study conducted on primary teeth found that 9% etidronic acid used as an irrigation solution exhibited antibacterial properties against E. faecalis [16]. A study by Supraja et al. [42] also indicated that etidronic acid may be effective in reducing bacterial load in endodontic infections.

In the literature, concentrations of etidronic acid ranging from 9% to 18% and glycolic acid from 5% to 17% have been utilized as irrigant solutions [15, 16, 27]. Glycolic acid is a biocompatible monomer of alpha hydroxy acid structure used in skin care products, dermatological treatments and tissue engineering [14]. Glycolic acid has the ability to induce collagen synthesis and fibroblast proliferation [43]. It has also been reported to exhibit similar cytotoxicity to EDTA and citric acid in cell culture and has the ability to remove the smear layer from root canal dentin. A 17% glycolic acid solution has been reported to be effective in reducing the microhardness of the superficial dentin layer and significantly facilitates biomechanical preparation under clinical conditions [15]. It has been suggested that the softening effects of glycolic acid on dentin walls may be advantageous in clinical practice due to its ability to reduce the microhardness and increase the roughness of the dentin layer [15, 44]. Gambin et al. [45] reported that glycolic acid exhibited better antibacterial properties against E. faecalis than citric acid and EDTA. The current study revealed that etidronic (9%) and glycolic acid (17%) exhibited antimicrobial activity against E. faecalis in as little as 30 minutes, suggesting they can be used as an alternative to EDTA and NaOCl.

A significant limitation of this study is its reliance on a simplified polymicrobial biofilm model, which fails to accurately replicate in vivo conditions. Conducting further studies on the efficacy of these endodontic solutions at different concentrations, using both passive and active irrigation techniques, in the presence of a more complex biofilm representative of refractory periapical infections is crucial.

5. Conclusions

NaOCl (2.5%), etidronic acid (9%) and glycolic acid (17%) were effective in eliminating E. faecalis from primary tooth root canals. Within the limits of the study, etidronic acid (9%) and glycolic acid (17%) as root canal irrigation materials for primary teeth could be used as alternatives to NaOCl, which is associated with tissue and cell toxicity, irritant effects and allergic reactions. Further research is needed to assess the effectiveness of root canal irrigants that ideally disinfect primary tooth root canals in vivo.

Availability of data and materials

The authors declare that all data supporting the findings of this study are available within the paper and any raw data can be obtained from the corresponding author upon request.

Author contributions

PC, BC—designed the study and carried them out. BC, MB, AL, FS—supervised the data collection, analyzed the data, interpreted the data. PC, SSD, MCA—prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Ethics approval and consent to participate

The protocol for our study was approved by the Atatürk University Faculty of Medicine Local Ethics Committee (7/61, 26 October 2023) as it complies with local legislation and the principles of the Declaration of Helsinki. All parents included in the current study signed the informed consent form.

Acknowledgment

Not applicable.

Funding

This research received no external funding.

Conflict of interest

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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