Antibacterial Activity of Calcium Hydroxide and Zinc Oxide Combined with Several Solutions against Enterococcus faecalis Growth

Abstract

Objective This study aims to compare the antimicrobial activity of calcium hydroxide (CaOH) and zinc oxide (ZnO) when incorporated with other solutions such as 2% chlorhexidine (CHX), 2.5% sodium hypochlorite (NaOCl), 1% povidone-iodine (PVP-I), and sterilized distilled water (ddH₂O) against Enterococcus faecalis.

Materials and Methods The materials were prepared by mixing CaOH and ZnO with other solutions (CHX, PVP-I, NaOCl, and ddH₂O) separately. The antibacterial activity of CaOH and ZnO mixtures against E. faecalis was done by using disk diffusion assay (DDA). Twofold serial dilutions of the mixtures were used against E. faecalis to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. Biofilm inhibition of E. faecalis had been measured by using crystal violet assay.

Statistical Analysis The quantitative data of this study had been analyzed by using two-way analysis of variance with software SPSS version 27. The result is considered as significant if the value of analysis is p-value less than 0.05.

Results From the DDA results, the lowest zone of inhibition toward E. faecalis was CaOH-PVP-I (6.00 ± 0.00 mm), while the highest zone of inhibition toward E. faecalis was CaOH-CHX (22.73 ± 0.02 mm). Besides that, ZnO-PVP-I showed the lowest zone of inhibition (16.50 ± 0.06 mm), while ZnO-CHX showed the highest zone of inhibition (18.30 ± 0.08 mm) against E. faecalis. The MIC and MBC values of CaOH-CHX and ZnO-CHX were 0.78 and 6.25 mg/mL, respectively. In biofilm assay, CaOH-CHX and ZnO-CHX were reduced biofilm formation of E. Faecalis.

Conclusion Both CaOH-CHX and ZnO-CHX showed the highest antimicrobial activities toward E. faecalis. CaOH and ZnO alone showed no antimicrobial activities against E. faecalis.

Keywords

calcium hydroxide - zinc oxide - Enterococcus faecalis

Introduction

Failure in root canal treatment can be due to various factors. One of the prime factors is the persistence of bacteria inside the root canal. A previous study reported that the most isolated pathogenic bacteria present in the root canal system responsible for endodontic treatment failure is Enterococcus faecalis.[1] [2] E. faecalis is a coccus-shaped, Gram-positive bacteria and facultative anaerobe. E. faecalis can resist the harsh environment inside the root canal and adapt to it.[3] Zhang et al reported that E. faecalis has a higher prevalence in persistent endodontic infection compared with untreated intra articular infection.[4]

In dental clinic settings, calcium hydroxide (CaOH) is widely used as a medicament during root canal treatment. Ba-Hattab et al mentioned that CaOH is the most popular choice for endodontic therapy due to its ideal properties toward the root canal.[5] CaOH has high pH that contributes to the alkaline environment, inducing the formation of reparative dentine and lowering the bacterial loads for the affected dentin.[6]

OdontoPASTE is a commercially available zinc oxide (ZnO)-based paste. Its usage is to temporarily obturate root canals and is extremely successful in reducing bacterial contamination during endodontic therapy.[7] Pain after endodontic operations is frequently caused by inflammation of the pulp and periapical tissues. OdontoPASTE that has anti-inflammatory properties with clindamycin hydrochloride (a broad-spectrum antibiotic) and triamcinolone acetonide (a steroid-based anti-inflammatory medication) that act through direct contact with the inflamed tissue and help to alleviate this discomfort.

However, studies stated that CaOH and ZnO are unable to totally eradicate bacteria inside the root canal such as E. faecalis and only exhibited a minor antimicrobial property.[8] [9] According to Djearamane et al ZnO was resistant to E. faecalis growth.[10] Adding to that, E. faecalis colonies in dentinal tubules that have size up to 300 μm cause it to be hard to eliminate by the intracanal medicaments. Adl et al reported that CaOH does not remove the bacteria in the dentinal tubules effectively as it is unable to penetrate it.[11] This finding was supported by Dewi et al reported E. faecalis colonized in dentinal tubules was not affected and remained after being treated with CaOH.[12] Hence, even with the use of medicaments that possess antibacterial properties, it is hard to eradicate E. faecalis from the root canal. E. faecalis also has resistance toward CaOH by the formation of biofilm alongside surviving in alkaline conditions up to a pH of 11.5 for more than 10 days.[13] To overcome the problem, the combination of CaOH and ZnO with other solutions had been studied.

Hence, this study aims to evaluate the antibacterial activity of CaOH and ZnO combined with other solutions such as 2% CHX, 2.5% sodium hypochlorite NaOCL, and PVP-I toward E. faecalis.

Materials and Methods

Methodology

This experimental study was conducted to demonstrate the antimicrobial activity of materials used in endodontic treatment; CaOH paste and ZnO paste mixed with other solutions toward E. faecalis. [Fig. 1] is the workflow of the study.

Fig. 1 Workflow of this study to determine antibacterial activities of CaOH and ZnOH combined with several solutions against Enterococcus faecalis. CaOH, calcium hydroxide; CHX, chlorhexidine; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NaOCL, 2.5% sodium hypochlorite; PVP-I, 1% povidone-iodine; ZnOH, zinc hydroxide.

Preparation of Materials

Calcicur that is CaOH paste (Voco dental, Indian Land, United States) and OdontoPASTE that is ZnO paste (Voco dental, Indian Land, United States) were prepared in two different concentrations (50 and 100 mg/mL) and mixed with four different solutions; 0.12% chlorhexidine (CHX) (Oradex, Damansara, Malaysia), 3% sodium hypochlorite (NaOCl), (Coltene, Switzerland) 1% povidone-iodine (PVP-I) (Betadine, Stamford, United States), and sterilized distilled water (negative control).

Microorganisms and Culture Conditions

Enterococcus faecalis (ATCC 29212) bacteria was obtained from the laboratory of microbiology at the Faculty of Dentistry Universiti Sains Islam Malaysia. The bacteria were cultured in Tryptic Soy Broth (Oxoid, Thermo Fisher Scientific, United Kingdom, England) and incubated at 37°C for 24 hours under aerobic conditions.[14]

Preparation of Media Plates for the Antimicrobial Sensitivity Testing

Mueller Hinton agar (MHA) (Oxoid, Thermo Fisher Scientific, United Kingdom, England) and Mueller Hinton broth (MHB) (Oxoid, Thermo Fisher Scientific, United Kingdom, England) were used in antimicrobial sensitivity testing. All media were prepared according to the manufacturer's instructions and sterilized by autoclaving at 121°C for 3 hours. All plates are labeled according to the treatment for each section.

Disk Diffusion Agar Test

Antibacterial activity was determined by using the disk diffusion method with modification.[15] The modification of this method was on concentration of the samples and type of bacteria. Overnight cultures of E. faecalis (ATCC 29212) were adjusted to 0.5 McFarland standards (1.5 × 10⁸ colony forming unit [CFU]/mL; Sigma-Aldrich, St. Lous, United States). Then, the bacteria were cultured onto MHA using sterile cotton swabs. Paper disks were impregnated with 50 and 100 mg/mL of CaOH-CHX, CaOH-NaOCl, CaOH-PVP-I, CaOH-ddH₂O, ZnO-CHX, ZnO-NaOCl, ZnO-PVP-I, and ZnO-ddH₂O, respectively. Zones of inhibition were measured from the diameter of the disks to the circumference of the inhibition zone after incubating at 37°C for 24 hours, aerobically.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

E. faecalis was subcultured in the MHB and incubated at 37°C overnight. Gram staining was employed to ensure no contamination in the colony. The MIC was performed using the twofold serial dilution method in a 96-well plate (Sigma-Aldrich, St. Lous, United States). Approximately 100 μL of E. faecalis (1.5 × 10⁸ CFU/mL) were added into 100 μL of various combination solutions in 96-well. The details of the combination solutions were shown below:

• a) CaOH

  • Column A: Sterilized MHB only

  • Column B: CaOH-ddH₂0 (50–0.39 mg/mL)

  • Column C: CaOH-CHX (50–0.39 mg/mL)

  • Column D: CaOH -PVP-I (50–0.39 mg/mL)

  • Column E: CaOH-NaOCl (50–0.39 mg/mL)

  • Column F: E. faecalis only

• b) ZnO

  • Column A: Sterilized MHB only

  • Column B: ZnO-ddH₂O (50–0.39 mg/mL)

  • Column C: ZnO-CHX (50–0.39 mg/mL)

  • Column D: ZnO-PVP-I (50–0.39 mg/mL)

  • Column E: ZnO-NaOCl (50–0.39 mg/mL)

  • Column F: E. faecalis only

After 24 hours of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich, St. Lous, United States) was added to all wells (10 µL per well). The color changes were observed after incubation at 37°C for 1 to 4 hours. The MBC was determined by directly plating 10 µL of an aliquot from wells that displayed no growth in MIC wells onto MHA. After incubating overnight at 37°C, the MBC value was identified. MBC of different combination solutions was defined as the lowest concentration that kills E. faecalis.

Anti-biofilm Assay

Anti-biofilm assay was done by following Hanafiah et al.[15] Briefly, 100 μL of mixtures at MIC was added into 100 μL of E. faecalis (1.5 × 10⁸ CFU/mL). E. faecalis treated with CaOH-ddH₂0 and ZnO-ddH₂O acted as negative control, respectively. After inoculation, all the samples were incubated in an incubator at 37°C for 24 hours. Biofilm formation was determined by crystal violet assay. The culture medium was then decanted, and the plates were gently washed twice with 200 μL of sterile distilled water to remove the planktonic and loosely bound cells. The adherent bacteria were stained with 50 μL of 0.1% crystal violet for 15 minutes. After rinsing twice with 200 μL of water, the bound dye was extracted from the stained cells using 200 μL of 99% ethanol. Biofilm formation was then quantified by measuring the absorbance of the solution at 600 nm in a microplate reader.

Results

Disk Diffusion Assay of CaOH and ZnO Combined with Solutions against E. faecalis

The inhibition zone of CaOH combined with several solutions against E. faecalis was shown in [Fig. 2]. The mean of the diameter inhibition zone for E. faecalis after treated with CaOH-CHX and CaOH-NaOCl was found to be significant when compared with control (CaOH-ddh2O) (p < 0.05), while there was no significant difference between PVP-I and control. CaOH-CHX shows the highest zone of inhibition compared with other groups in concentrations 50 and 100 mg/mL that were 22.26 ± 0.03 and 22.73 ± 0.02mm, respectively.

Fig. 2 The inhibition zone of CaOH combined with several solutions against Enterococcus faecalis. Values are represented as mean ± standard deviation. Values on the same group followed by asterisk differ significantly (p < 0.05). CaOH, calcium hydroxide; CHX, chlorhexidine; NaOCL, 2.5% sodium hypochlorite; PVP-I, 1% povidone-iodine; ZnO, zinc oxide.

The inhibition zone of ZnO combined with several solutions against E. faecalis was shown in [Fig. 3]. The mean of the diameter inhibition zone for E. faecalis after being treated with ZnO-CHX, ZnO-NaOCl, and ZnO-PVP-I were found to be significant, (p < 0.05) when compared with control. ZnO-CHX shows the highest zone of inhibition compared with other groups in concentrations 50 and 100 mg/mL that were 17.40 ± 0.02 and 18.30 ± 0.08 mm, respectively.

Fig. 3 The inhibition zone of ZnO combined with several solutions against Enterococcus faecalis. Values are represented as mean ± standard deviation. Values on the same group followed by asterisk differ significantly (p < 0.05). CaOH, calcium hydroxide; CHX, chlorhexidine; NaOCL, 2.5% sodium hypochlorite; PVP-I, 1% povidone-iodine; ZnO, zinc oxide.

MIC and MBC of CaOH and ZnO Combined with Solutions against E. faecalis

The MIC and MBC values of CaOH and ZnO combined with solutions against E. faecalis were shown in [Table 1]. The MIC and MBC values of CaOH-CHX and ZnO-CHX against E. faecalis were 0.78 and 6.25 mg/mL, respectively. However, the MIC and MBC values of CaOH-PVP-I and CaOH-ddH₂O were more than 50 mg/mL, respectively. Meanwhile, the MIC and MBC values of ZnO-PVP-I and ZnO-NaOCL were 6.25 mg/mL and more than 50 mg/mL, respectively. The MIC and MBC values of ZnO-ddH₂O were 3.13 and more than 50 mg/mL, respectively. From the analysis, CHX solutions enhanced the antibacterial activity of CaOH and ZnO against E. faecalis.

Table 1 The MIC and MBC values of CaOH and ZnO combined with solutions against Enterococcus faecali s

Samples	MIC (mg/mL)	MBC (mg/mL)
CaOH-CHX	0.78	6.25
CaOH-NaOCL	3.13	6.25
CaOH-PVP-I	>50	>50
CaOH-ddH2O	>50	>50
ZnO-CHX	0.78	6.25
ZnO-PVP-I	6.25	>50
ZnO-NaOCL	6.25	>50
ZnO-ddH2O	3.13	>50

Abbreviations: CaOH, calcium hydroxide; CHX, chlorhexidine; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NaOCL, 2.5% sodium hypochlorite; PVP-I, 1% povidone-iodine; ZnO, zinc oxide.

CHX was presented as an effective solution that can enhance the antimicrobial activity of CaOH and ZnO toward E. faecalis.

Anti-Biofilm Activity

[Fig. 4] showed significant (p < 0.05) anti-biofilm activity of mixtures at MIC concentrations against E. faecalis. Biofilm formation of E. faecalis reduced significantly, when it was treated with CaOH-CHX and ZnO-CHX compared with control, respectively. Treatment with CaOH-CHX and ZnO-CHX inhibited 70 and 75% biofilm formation of E. faecalis, respectively. CaOH-PVP and CaOH-NaOCL did not inhibit biofilm of E. faecalis at MIC concentration. Meanwhile, ZnO-PVP and ZnO-NaOCL inhibited 20 to 25% of biofilm formation of E. faecalis when compared with control, respectively.

Fig. 4 Anti-biofilm activity of calcium hydroxide (CaOH) and zinc oxide (ZnO) combined with other solution against Enterococcus faecalis. In this analysis, CaOH-CHX, ZnO-CHX, ZnO-PVP-I, and ZnO-NaOCL were significantly reduced biofilm of E. faecalis at minimum inhibitory concentration (p < 0.05). CHX, chlorhexidine; NaOCL, 2.5% sodium hypochlorite; PVP-I, 1% povidone-iodine.

Discussions

E. faecalis is a significant pathogen in the oral mucosa.[16] [17] It is thought to have a key part in the etiology of post-treatment apical periodontitis because it is more prevalent in secondary endodontic infections than original infections. E. faecalis may enter the root-filled channel through coronal leakage during or after root-canal therapy as secondary invaders.[18] E. faecalis is resistant to several antibiotics and antimicrobial compounds, such as chloramphenicol, tetracyclines, macrolides, and clindamycin,[17] [19] making it difficult to eliminate. Furthermore, several irrigants used in root canal therapy showed little or no bactericidal action against E. faecalis.

The study on antibacterial activity of CaOH and ZnO combined with several solutions has proven that CaOH-CHX showed the most effective combination as antibacterial agent against E. faecalis when compared with other mixtures. Meanwhile, a combination of CaOH-PVP-I displayed the lowest antibacterial agents against E. faecalis. CHX is a synthetic antimicrobial agent that is widely used in antiplaque and antigingivitis formulations for the treatment of periodontitis due to its wide-spectrum action against both Gram-negative and Gram-positive bacteria, as well as in other applications against yeasts, fungi, and some viruses.[20] Some studies have failed to show any benefits in incorporating CHX that is in contrast to our findings. The reasons are the methods and materials used, microbiological assessments, time periods of experiments, strains and concentrations of E. faecalis, methods of bacterial inoculation, and depths of sampling.[21]

According to Nozari et al, the combination of CaOH and ZnO, especially when associated with CHX, provides a powerful antimicrobial effect against E. faecalis during root canal treatments.[22] Evans et al discovered that E. faecalis becomes susceptible to the combination of CaOH and chlorhexidine when examining the mechanism of resistance to CaOH in bovine dentin. Because of its method of action, CHX takes just a short time to exert an antibacterial effect on E. faecalis.[23] Its cationic molecule interacts with the negative charge of phosphate groups in the bacterial cell wall, causing metabolic damage and, as a result, bacterial death. This is also supported by another study that has the same result as our result where they observed that the combination of CaOH-CHX performed better than CaOH alone.[24]

OdontoPaste (ZnO) contains clindamycin hydrochloride that causes cell lysis of bacterial DNA by inhibiting the formation of peptide bonds.[25] Clindamycin hydrochloride is effective toward E. faecalis with 50,000 µg/mL concentration in OdontoPaste. In our study, the ZnO was effective against E. faecalis alone. However, when we incorporated CHX and ZnO together the results showed an increase of antibacterial activity against E. faecalis (p < 0.05). A study suggested that CHX is functional as part of the final irrigation solution in root canal infected with E. faecalis prior to obturating the canal.[26]

Povidone-iodine is often used as an alternative agent because of its increased antibacterial activity. Iodine has a strong oxidizing property resulting in disulfide linkages and effectively inhibits many root canal bacteria growth. However, in this study, CaOH-PVP-I mixture did not give any effect on E. faecalis. This may be due to basic chemical knowledge indicating that povidone-iodine is neutral when compared with CaOH.[27] Interestingly, the combination of ZnO-PVP-I has mild antibacterial activity against E. faecalis. There were no studies found that investigated the antibacterial effect of ZnO when mixed with PVP-I toward E. faecalis. Another study also investigated the combination of CaOH-PVP-I against E. faecalis. In the investigation, the combination of CaOH-PVP-I did not result in a significant reduction in E. faecalis when compared with CaOH-CHX which is equal to our result.[27]

When CaOH and ZnO combined with NaOCl, there is significant zone reduction. Besides that, the antimicrobial activity of CaOH and ZnO incorporated with NaOCl was not as good or better than other samples. Previous studies also reported that NaOCl was resistant to E. faecalis.[28] Several studies have established NaOCl's usefulness as an irrigant solution. It is widely utilized in endodontic therapy, and the concentration of 2.5% NaOCl was chosen since it is commonly used. It can preserve enough chlorine to eradicate a considerable proportion of bacteria.[24] Based on a study, their findings confirmed that when CaOH is combined with NaOCl, the antibacterial efficacy of the mixture is larger than CaOH alone.[24] This conclusion is comparable to the results of Farhad et al, who examined the antibacterial efficiency of CaOH in conjunction with H₂O, CHX, or NaOCl against E. faecalis. Their findings revealed that the antibacterial potency of CaOH can be increased by preparing it with antibacterial irrigants such as NaOCl.[29] However, we could not find any other studies that investigate the combination of ZnO with NaOCl against E. faecalis.

ZnO and CaOH alone showed antibacterial activity against E. faecalis; however, synergetic effects were shown after the materials combined with CHX. Both combinations strongly inhibit E. faecalis. The results of this study revealed that the mixtures of CaOH-CHX and ZnO-CHX have antibacterial properties. This showed that they promoted greater antibacterial activity against E. faecalis by the association of CHX. Although based on our knowledge, both CaOH and ZnO can promote antibacterial effects, the activity against E. faecalis is favored by association with CHX. Through this study, we found that research on the combination between ZnO and other intracanal medicament materials is rarely done and as we have already investigated this study by using combination of ZnO with CHX, NaOCl, and PVP-I, we acknowledged that this is the significances of our study and we recommend for others to try investigating combination of ZnO with other medicaments too so that we can do comparison of our results in the future.

Conclusion

In conclusion, this study showed that CaOH-CHX and ZnO-CHX possessed the highest effect of antibacterial activity against E. faecalis. This finding provides an insight on the use of these combination agents in endodontic treatment. These combination agents could be useful in the formulation of disinfectants and the development of intracanal medicaments for root canal treatment.

Conflicts of Interest

None declared.

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Publication History

Article published online:
26 June 2024