Journal of South Asian Association of Pediatric Dentistry
Volume 6 | Issue 2 | Year 2023

Comparative Evaluation of Antimicrobial Efficacy of Etidronic Acid and Propolis against E. faecalis in Primary Teeth: An In Vivo Study

Veena Arali1https://orcid.org/0000-0002-6614-3405, Atluri N Supraja2https://orcid.org/0000-0002-2298-0965, Harika Rapala3https://orcid.org/0000-0002-9043-9783, Charan T Vemagiri4https://orcid.org/0000-0001-9940-802X, Mounika Kallakuri5https://orcid.org/0000-0002-4747-4348, Gannamani LGSP Kumar6https://orcid.org/0000-0003-2589-420X

1-6Department of Pediatric & Preventive Dentistry, GSL Dental College and Hospital, Rajahmundry, Andhra Pradesh, India

Corresponding Author: Veena Arali, Department of Pediatric & Preventive Dentistry, GSL Dental College and Hospital, Rajahmundry, Andhra Pradesh, India, Phone: +91 7760748787, e-mail: pedo.veena@gmail.com

Received on: 19 March 2023; Accepted on: 19 April 2023; Published on: 23 August 2023


Aim and background: The study aimed to assess the antimicrobial efficacy of 11% alcoholic extract of propolis and 9% etidronic acid against Enterococcus faecalis (E. faecalis) in endodontically treated necrotized primary teeth.

Methods and materials: A total of 30 necrotic primary molars, either maxillary or mandibular teeth in children of age 3–8 years, were randomly included, of these 15 teeth were irrigated with 11% alcoholic extract of propolis (group I) and 15 teeth with 9% etidronic acid (group II); two microbiological samples with sterile paper points were taken from within the canal in both groups, first sample after the access opening and before first irrigation (S1), and another sample after final irrigation and debridement of infection, before obturating the tooth (S2). All samples (S1 and S2) were evaluated for the presence of E. faecalis by colony-forming units per milliliter (CFU/mL).

Results: In group I, a statistically significant decline in CFU/mL (p < 0.05) was observed in samples before and after irrigation. The same results were obtained with group II, with a statistically significant decline of CFU/mL (p < 0.05). Among both groups, after irrigation, a statistically significant decline was noticed in support of 9% etidronic acid.

Conclusions: The 9% etidronic acid exhibited a greater decrease of CFU/mL against E. faecalis than that observed with an 11% alcoholic extract of propolis. Hence, etidronic acid can be proposed as an alternative irrigant for necrotic primary teeth.

How to cite this article: Arali V, Supraja AN, Rapala H, et al. Comparative Evaluation of Antimicrobial Efficacy of Etidronic Acid and Propolis against E. faecalis in Primary Teeth: An In Vivo Study. J South Asian Assoc Pediatr Dent 2023;6(1):51-55.

Source of support: Nil

Conflict of interest: None

Keywords: Enterococcus faecalis, Etidronic acid, Propolis.


The maintenance of primary teeth is necessary for the harmonious development of occlusion, maintenance of arch length, optimal function of chewing and speech, and maintenance of a healthy oral environment.1 Pulp therapy is required to sustain the deciduous teeth in the arch and to preserve the normal development of its succedaneous teeth.2 Pulpectomy is thought to be a complicated task due to peculiar internal geometry with additional characteristics like furcational connections and horizontal anastomoses that are unique to primary teeth.3 Success of endodontic therapy depends on various factors, like appropriate diagnosis, thorough cleaning, a proper disinfection protocol with the aid of intracanal medicaments and irrigation solutions, then obturation of root canal followed by final restoration.4 Primary teeth contain lateral canals, apical ramifications, and dentinal tubules, which remain uninstrumented when mechanical preparation is used alone. In order to completely debride and disinfect the root canal, irrigation and instruments work in tandem.5

During clinical practice, various intracanal irrigants have been used for primary teeth, like sodium hypochlorite (NaOCl), chlorhexidine gluconate (CHX), ethylenediaminetetraacetic acid (EDTA), citric acid, mixture of tetracycline, acid and detergent (MTAD), hydrogen peroxide, etc. The ideal irrigating solution for pulpectomies in primary teeth remains an issue. Hence, it is a task for pediatric dentists to select the appropriate irrigating solution during pulp therapy.6 Many studies on the effect of irrigants on Enterococcus faecalis (E. faecalis) were done on permanent teeth, but there is insufficient data on primary teeth.

Propolis, a natural product, has drawn more attention because of its antimicrobial activity against various pathogenic microorganisms7 which consists of resin and balsams (50–60%), pollen (5–10%), and other ingredients like amino acids, minerals, vitamins A and B complex, as well as bioflavonoids (vitamin P), phenols, and aromatic compounds, which are highly active biochemical substances.8 It has also been used as a pulp capping agent, storage media for avulsed teeth, prevention of caries, and dentine hypersensitivity,9 but its use as root canal irrigant is yet to be explored in deciduous teeth.

Sodium hypochlorite (NaOCl) is the most commonly used irrigating solution; its drawbacks are unpleasant to taste and toxicity when infused into periradicular tissues.10 Chelating agents have been introduced into endodontics as final rinses in irrigation protocols which aid in the preparation of narrow, calcified root canals. The adjunctive use of chelators like EDTA, citric acid, etidronic acid, etc., have antimicrobial properties and can eliminate E. faecalis. Etidronic acid, also known as etidronate or 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP), is a chelator which is an alternative to EDTA or citric acid and is used along with NaOCl.11

As propolis and etidronic acid are antimicrobial and are used for various other treatment procedures in dentistry, this study aims to assess the antimicrobial efficacy against E. faecalis. As etidronic acid is a chelator, the other objective was to evaluate its efficacy in cleaning the root canals as a substitute for NaOCl.


The sample size for the present study was estimated using G*Power software. The sample size estimation was performed at 5% α-error, with an effect size of 65%, and the power of the study at 80%, demonstrating that a minimum of 30 samples were needed for the present study. So, each group consists of 15 samples each.

The study was performed in the Department of Pediatric and Preventive Dentistry after the approval from the Ethics committee of the institution and registered with the designation CTRI/2020/08/027096 in the Clinical Trials Registry of India. A total of 30 teeth from the patients requiring pulpectomy of 3–8 years old are included; after explaining the objective of the study, a written informed consent was signed by the parents of the children.

Inclusion Criteria12

  • Children in good general health.

  • Deciduous molars with an abscess, or sinus tract, having at least one necrotic primary canal.

  • Appearance of radiolucency in furcation area or periapical region.

  • There is still at least two-thirds of the root remaining.

  • A tooth structure strong enough to support a rubber dam.

Exclusion Criteria12

  • Individuals who had taken antibiotics within the previous 2 weeks of the sampling or who had any type of systemic impairment.

  • Patients who had pathological root resorption, perforated pulpal floor, nonrestorable teeth, or excessive mobility.

Preclinical Laboratory Procedure

  • Preparation of 9% etidronic acid: According to the manufacturer’s instructions, two capsules of weight 0.246 gm are dissolved in 10 mL of 3% NaOCl to produce 9% etidronic acid.

  • Preparation of 11% propolis: Dilute 33% commercially available alcoholic extract with warm saline in 2:1 proportion to produce 11% propolis.


A total of 30 pulpectomies were performed in necrotic primary molars using the following irrigants:

  • Group I (n = 15): 11% propolis.

  • Group II (n = 15): 9% etidronic acid.

Subjects were identified based on the inclusion and exclusion criteria. The patient’s demographic details, including name, age, and sex, were taken prior to the procedure. Treatment was done in a single visit, and periapical radiographs of teeth were taken.

Isolation and Operative Field Disinfection

A 2% lidocaine was used to induce the inferior alveolar nerve block to deciduous mandibular molars and infiltration to deciduous maxillary molars following antisepsis of the oral cavity. The protocol for disinfection employed was a modification of Ng et al.13 Pumice was used to clean each tooth, which was then isolated with a rubber dam. The dental crown, along with the rubber dam and clamp, were wiped with 30% H2O2, then 5.25% NaOCl for 1 minute to disinfect the operation field, followed by inactivation with 10% sodium thiosulfate. A no. 3 round carbide bur cooled in saline solution was used to remove the gross carious tissue. The cavity was disinfected, following which the roof of the pulp chamber was removed with another no. 3 round carbide bur, disinfectants were prevented by placing a sterile cotton pellet in the pulp chamber, and the orifice of the root canal was reached.

Collection of Microbiological Samples

The root canals were negotiated, then the working length was measured by preoperative radiograph. The first sample was taken from the canal by using two sterile absorbent paper points that are compatible with the root canal diameter, which was placed successively for 30 seconds each. These extracted paper points were immersed in a test tube with brain heart infusion (BHI) broth immediately, which works as a transport and growth media because of its ability to keep the sampled bacteria alive. Following sample collection, a routine pulpectomy procedure was done. The teeth were divided randomly into groups I and II and irrigated with 0.5 mL of a chosen solution between each filling. The canal was irrigated with respective irrigants of either 11% propolis or 9% etidronic acid for the final time after instrumentation and before obturation, and no intermediate irrigants were used. At this point, the second microbiological sample was collected from the same canal, using two paper points placed for 30 seconds each, and these were inserted into the BHI broth test tube. Finally, the canal was obturated using metapex obturating material, and an intraoral periapical radiograph was recorded postoperatively. The collected samples were subjected to microbiological analysis to calculate colony-forming units per milliliter (CFU/mL) against E. faecalis.

Laboratory Procedures

Samples collected were streaked on the Petri plates and placed in an anaerobic gas jar for 48 hours. When the growth of the bacteria was observed, the magenta pink-colored colonies were inoculated on the slide and the gram staining was done. We studied the samples under the microscope and counted them using the turbidimetry method and McFarland’s scale pattern based on the degree of turbidity or density exhibited, as this approach determines the CFU/mL.


Statistical analysis was performed using Statistical Package for the Social Sciences software version 20.0 and MS Excel 2013. Intragroup and intergroup comparisons were evaluated. Paired t-test was used to compare the means of before and after treatment results. An independent t-test was used to compare the means of the two groups.

A total of 30 pulpectomies were performed in children of age 3–8 years using the following irrigants:

Of these, 60 microbiological samples were obtained:

A total of 30 samples were collected from group I [15 preirrigation (Fig. 1) and 15 postirrigation (Fig. 2)] and 30 samples were collected from group II [15 preirrigation (Fig. 3) and 15 postirrigation (Fig. 4)].

Fig. 1: Figure showing growth of E. faecalis at baseline on blood agar in group I

Fig. 2: Figure showing growth of E. faecalis at final on blood agar in group I

Fig. 3: Figure showing growth of E. faecalis at baseline on blood agar in group II

Fig. 4: Figure showing growth of E. faecalis at final on blood agar in group II

S1 and S2 samples were calculated and correlated for the number of CFU/mL in both groups I and II. Under baseline conditions, the comparative statistical analysis revealed a p-value of 0.082, stating no statistical difference among groups. The difference between CFU/mL, preirrigation, and postirrigation was statistically significant in groups I and II. Finally, the experimental group was shown to have a considerable advantage over the control group (Table 1).

Table 1: Table showing the baseline and final mean CFU/mL of both the groups
Baseline mean p-value Final mean p-value
Group I 340173.3 0.082 1523.3 0.000
Group II 376754.6 2246.4


The success of endodontic treatment is based on the disinfection of the root canal with the help of mechanical instrumentation along with profuse irrigation to aid in the removal of necrotic pulpal tissue, dentin debris, and microorganisms.14 As primary teeth have a complex root canal system, irrigation of the root canal plays a significant role in the complete removal of microorganisms from the areas not reachable to mechanical instrumentation. Hence, the clinician should pay attention to cleaning the root canal of primary teeth because of bizarre internal geometry with accessory canals and ramifications, thereby leading to the success of pulpectomy. E. faecalis, a gram-positive facultative anaerobic microorganism, is the most predominant species found in the necrotic primary teeth.6

Although endodontic treatment was indicated in primary teeth with the absence of radiographic lesion, abscess or pulp necrosis, the necrotic primary teeth mainly consist of anaerobic gram-negative facultative bacteria, such as E. faecalis, that are resistant species and are major causes for failure. Hence, patients with the above inclusion criteria have participated in the study.

Kujumgiev et al.15 stated that the antimicrobial property of propolis is due to flavonoids and the esters of phenolic acids. Although NaOCl is a commonly used irrigating solution as it has excellent properties to dissolve organic tissue and antimicrobial property, there are some limitations because of its cytotoxicity and the potential to injure permanent tooth germs if it extrudes beyond the periapical tissue.16 In order to overcome these limitations, HEBP, a mild chelator, has been used along with NaOCl solution, termed continuous chelation. HEBP capsules should be mixed immediately prior to use with an aqueous solution of NaOCl to produce an all-in-one irrigant, and it causes the reduced activity of NaOCl after 1 hour. The main advantage of this chelating agent is that it completely eliminates the smear layer consisting of dentin debris formed during instrumentation.17

Al-Qathami and Al-Madi18 conducted a study on the antimicrobial efficacy of three endodontic irrigants of propolis, NaOCl and saline and found that the antimicrobial efficacy of propolis is the same as that of NaOCl. In our study, we found the etidronic acid that is NaOCl combined with capsules of etidronic acid found to have better antimicrobial efficacy than propolis, and hence etidronic acid can be used as an alternative irrigant to NaOCl. The efficacy was due to NaOCl mixed with etidronic acid, as it helps to penetrate deep in the root canal by removing the smear layer to kill E. faecalis.

Jaiswal et al.19 conducted an in vitro study to assess the more effective and less irritating root canal irrigant among different irrigants, and they concluded that chitosan + CHX, CHX, and propolis were as effective as NaOCl. Also, the use of natural products as irrigating solutions may be more advantageous due to the drawback’s properties of NaOCl.

Bhagwat et al.20 conducted an in vitro study to compare various irrigants on the microhardness of root dentin and showed that HEBP has the least effect, and 17% EDTA showed the maximum effect on the microhardness of dentin.

The studies available with respect to etidronic acid are in vitro studies and on permanent teeth. The studies evaluating the antimicrobial efficacy of etidronic acid and propolis against E. faecalis in deciduous teeth are sparse. Our study is the first of its kind, which evaluated in vivo antimicrobial efficacy of etidronic acid and propolis that, resulted in etidronic acid being more satisfactory in eliminating E. faecalis from the root canal of primary teeth.


Etidronic acid, in combination with NaOCl, can be used as a root canal irrigant in primary teeth due to the additive effect of removing the smear layer and penetrating deep into the root canal to kill E. faecalis. However, more clinical studies are needed to assess the efficacy of etidronic acid in necrotic primary teeth treated with pulpectomy.

Clinical Significance

  • Etidronic acid can be used to remove the inorganic debris, namely, the smear layer, produced during the instrumentation of the root canal.

  • Etidronic acid can be used as an alternate irrigant during the pulpectomy procedure in primary teeth.

  • Etidronic acid has both antimicrobial action and chelating action.


Veena Arali https://orcid.org/0000-0002-6614-3405

Atluri N Supraja https://orcid.org/0000-0002-2298-0965

Harika Rapala https://orcid.org/0000-0002-9043-9783

Charan T Vemagiri https://orcid.org/0000-0001-9940-802X

Mounika Kallakuri https://orcid.org/0000-0002-4747-4348

Gannamani LGSP Kumar https://orcid.org/0000-0003-2589-420X


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