|Year : 2022 | Volume
| Issue : 1 | Page : 12-16
Comparative Evaluation of Enterococcus faecalis Counts on Different Tapers of Rotary System with Different Irrigating Solutions: An In Vitro Study
Rahimath Shettybettu Hydros Shariq, Harish Kumar Shetty, Prathap Mulakkal Sreekantan Nair, Vivian Flourish D’Costa
Department of Conservative Dentistry and Endodontics, Yenepoya Dental College, Yenepoya (Deemed to be University), Deralakatte, Mangalore, India
|Date of Submission||28-Jan-2022|
|Date of Decision||24-Apr-2022|
|Date of Acceptance||25-Apr-2022|
|Date of Web Publication||05-Aug-2022|
Vivian Flourish D’Costa
Department of Conservative Dentistry and Endodontics, Yenepoya Dental College, Yenepoya (Deemed to be University), Deralakatte, Mangalore – 575018
Source of Support: None, Conflict of Interest: None
Introduction: The conservation of tooth structure and prevention of extrusion of obturating materials have been cited as primary advantages of minimal apical enlargements. The aim of this study is to compare the effects of different rotary file tapers and two irrigating solutions and a combination of two irrigating solutions on E. faecalis counts. In this study, 84 freshly extracted single rooted single canal human premolars were selected and enlarged to ISO #20 K-file. Materials and Methods: The samples were sterilized and inoculated with E. faecalis for 72 hours, divided into six experimental groups, and prepared with #30 nickel-titanium rotary files with 0.04 and 0.06 tapers. Group I was irrigated with 2 mL of 3% sodium hypochlorite, Group 2 was irrigated with 2 mL of 2% chlorhexidine (CHX), and Group 3 was irrigated with 2 mL of a combination of 3% sodium hypochlorite, saline, and 2% CHX irrigation after each file. Cleaning efficacy was evaluated in terms of the reduction of colony forming units. Comparison of the E. faecalis among the groups at 0.04 taper was performed using the Kruskal-Wallis test after establishing normality of variance using the Shapiro-Wilk test. A comparison of the E. faecalis between 0.04 and 0.06 taper within the group was performed using the Mann-Whitney test. Results: Group 1 (3% sodium hypochlorite) and Group 3 (3% sodium hypochlorite + saline + 2% chlorhexidine gluconate) showed no statistically significant difference between the subgroups (0.04 and 0.06 taper), whereas statistically significant difference with E. faecalis was seen with respect to Group 2 between 0.04 and 0.06 taper (2% chlorhexidine gluconate). Conclusion: The study proved the use of sodium hypochlorite, saline, and CHX in combination at both 0.04 taper and 0.06 taper gave better results with no significant change.
Keywords: Chlorhexidine gluconate, E. faecalis, irrigating solutions, nickel-titanium rotary files, sodium hypochlorite
|How to cite this article:|
Shariq RS, Shetty HK, Nair PM, D’Costa VF. Comparative Evaluation of Enterococcus faecalis Counts on Different Tapers of Rotary System with Different Irrigating Solutions: An In Vitro Study. J Orofac Sci 2022;14:12-6
|How to cite this URL:|
Shariq RS, Shetty HK, Nair PM, D’Costa VF. Comparative Evaluation of Enterococcus faecalis Counts on Different Tapers of Rotary System with Different Irrigating Solutions: An In Vitro Study. J Orofac Sci [serial online] 2022 [cited 2022 Aug 7];14:12-6. Available from: https://www.jofs.in/text.asp?2022/14/1/12/353473
| Introduction|| |
Apical periodontitis is an inflammatory condition of the periapical tissues caused by a microbial infection within the root canal system considered to be a direct consequence of dental caries. The dominance of E. faecalis in root canal failures plays a pivotal role in its management. It lives as a single organism or in conjugation with other oral flora. It enters into the root canal system during the treatment, intervals between appointments, or even posttreatment completion. Consequently, it is important to consider treatment regimens intended at eradicating or avoiding the passage of E. faecalis caused infection during each of these phases. Hence, the apical portion must be prepared for a larger instrument to facilitate the penetration of antimicrobials to eliminate intratubular bacteria.
The ultimate goal of root canal treatment is the disinfection of the root canal space by chemo-mechanical means. Root canal irrigants play a critical role during the process as it reduces the friction between cutting instrument and dentine, which in turn enhances the cutting efficiency of files, dissolves tissues, and disinfects the canal space that was not touched by mechanical instrumentation.
The use of nickel-titanium (NiTi) rotary instruments has revolutionized mechanical instrumentation and minimized iatrogenic errors such as ledging, zipping, canal transportation, and/or apical blockage. Irrigants and intracanal medicaments form a necessary adjunct that increases the antimicrobial effect of mechanical cleansing and thus increases overall clinical efficacy. Several irrigating solutions are used against planktonic bacteria; however, only sodium hypochlorite (NaOCl) and chlorhexidine (CHX) showed high antimicrobial activity against both planktonic and biofilm bacteria.
Therefore, the present study is aimed at comparing the effects of different rotary file tapers and two irrigating solutions and the combination of the two irrigating solutions on the E. faecalis counts.
| Materials and Methods|| |
Ethical approval for this study (Protocol No: YEC2/187) was provided by the Yenepoya Ethics Committee 2, Yenepoya (Deemed to be University), Mangalore on November 20, 2019.
Eighty-four freshly extracted single rooted single canal human premolars (14 per subgroup) were selected, cleaned, and then immersed in 5.25% NaOCl solution for 30 minutes and then stored in sterile normal saline. Using a diamond disk, the tooth was sectioned at the Cemento enamel junction (CEJ) (12 mm from root tip) perpendicular to the long axis of the root. Patency of root canal was ensured using #10 and #15 K-file and then all samples were instrumented up to #20 K-file under copious distilled water irrigation up to a working length of 11 mm. The roots were then soaked first in 17% ethylenediaminetetraacetic (EDTA) acid for about 10 minutes and then in 5.25% NaOCl for 10 minutes, following which they were rinsed under sterile water. Samples were autoclaved for 20 minutes at 121°C and 15 psi. To ensure sterilization, all the samples were incubated separately in a microtube containing Brain heart infusion (BHI) for 24 hours under aerobic and aseptic conditions at 37°C, and if turbidity was seen, the process of sterilization was repeated.
In this study to create standard and controlled infection, E. faecalis species as a resistant bacterial species was used. 0.05 mL of suspension of these bacteria was injected by syringe into the canals of each sample. The access cavity was then sealed with intermediate restorative material (Cavit) and incubated at 37°C for 72 hours separately.
The samples were kept sterile and divided into three groups and six subgroups after the incubation period. The canals were prepared using NiTi rotary instruments and recapitulated with #15 K-file and irrigated with 2 mL solution. The grouping of samples is as follows:
Group I: Irrigating with 2 mL of 3% NaOCl irrigation after each file
Subgroup IA: Sequential preparation with #30 size NiTi 4% and 6% taper
Subgroup IB: Sequential preparation with #30 size NiTi 4% only
Group II: Irrigating with 2 mL of 2% CHX irrigation after each file
Subgroup IIA: Sequential preparation with #30 size NiTi 4% and 6% taper
Subgroup IIB: Sequential preparation with #30 size NiTi 4% only
Group III: Irrigating with 2 mL of a combination of 3% NaOCl, saline, and 2% CHX irrigation after each file
Subgroup IIIA: Sequential preparation with #30 size NiTi 4% and 6% taper
Sub-Group IIIB: Sequential preparation with #30 size NiTi 4% only
| Evaluation|| |
After canal preparation and final rinsing with 10 mL of distilled water, a 3 mm apical area of the root was powdered with the carbide bur. The powder was then transferred into the tubes containing sterile BHI in the same condition for all groups. After 10-fold dilutions in saline solution, aliquots of 0.1 mL were plated onto nutrient agar plates and incubated at 37°C for 48 hours. The colony forming units were counted after 24 hours.
| Results|| |
The data obtained were subjected to the “Shapiro-Wilk test” [Table 1]. Data showed nonnormal distribution; hence, nonparametric tests “Kruskal-Wallis test and Mann-Whitney” [Table 2] were applied.
|Table 2 Comparison of the E. faecalis among the groups at 0.04 taper using Kruskal-Wallis test|
Click here to view
In the present study, cleaning and shaping were done using NiTi files and the tapers used were 0.04 and 0.06. The result according to Mann-Whitney test showed that Group 1 (3% NaOCl) – (P = 0.629) and Group 3 (3% NaOCl + saline + 2% chlorhexidine gluconate) – (P = 0.31) showed no statistically significant difference between the subgroups (0.04 and 0.06 taper), whereas statistically significant difference was seen with respect to Group 2 between 0.04 and 0.06 taper (2% chlorhexidine gluconate) – (P = 0.00). Hence, according to the present study, 0.04 taper is preferred over 0.06 when NaOCl is used as an irrigant alone or in combination with CHX and saline [Table 3].
|Table 3 Comparison of the E. faecalis between 0.04 and 0.06 taper within the group using Mann-Whitney test|
Click here to view
In the present study, at both 0.04 and 0.06 taper, the median values of E. faecalis were predominantly higher in Group 2 (2% chlorhexidine gluconate) – 5245(IQR 70) followed by Group 1 (3% NaOCl) – 2299(IQR 110), and the least count of E. faecalis was seen in Group 3 (3% NaOCl + saline+ 2% chlorhexidine gluconate). Hence, according to the study, combination of NaOCl and saline and CHX gave better results against E. faecalis.
| Discussion|| |
Apical periodontitis represents a defensive reaction to primary infection in a necrotic pulp. The role of bacteria in the initiation of pulpal and periapical inflammation has been well illustrated by Kuzekanani and Moaddab. Torabinejad described primary infected root canals as untreated canals where microorganisms can access and colonize the pulpal tissue and impair its function. Microorganisms in primary infection may have been involved in the earlier stages of pulp invasion (usually via caries), or they can be latecomers that took advantage of the environmental conditions in the root canal after pulp necrosis. Apical periodontitis may also develop due to a secondary infection subsequent to endodontic treatment procedures. Posttreatment apical periodontitis is most commonly due to unsuccessful control of primary root canal infection, extrusion of root canal materials into periapical space, and/or inadequate coronal seal that allows bacterial leakage to take place.
The goal of root canal treatment is to eliminate the microorganisms from the root canal system and radicular dentin. However, the most common cause of root canal treatment failure is the inadequate elimination of these bacteria, which can be attributed to the complex morphology of the root canal space and agglomeration of intracanal bacteria into biofilms.
E. faecalis is the most prevalent species isolated from the canals of teeth displaying posttreatment disease. This microorganism has demonstrated the capacity to survive in an environment in which there are scant available nutrients and the commensality with other bacteria is minimal. E. faecalis can form biofilms on the root canal walls and invade dentinal tubules up to a depth of 653 μm, and it is therefore probable that cells within dentinal tubules survive chemo-mechanical instrumentation and intracanal medication could colonize the tubules and reinfect the obturated root canal.
Proper cleaning and shaping of the root canal space are considered to be essential for success in endodontic therapy. Technological innovations in rotary NiTi files have led to new concepts of root canal instrumentation including an increased taper of preparation, cutting efficiency, and flexibility. However, Shuping et al. demonstrated that the ability of these instruments to fully eliminate bacteria with the addition of NaOCl is no better than previous means using conventional instruments.
It is generally approved that the fracture resistance of an endodontically treated tooth is directly related to the amount of remaining sound tooth structure. Variations in root dimensions may affect the residual dentin thickness after instrumentation with different tapers. Khandale et al. compared the fracture resistance of teeth instrumented with differently tapered NiTi files and hand files and concluded that the force required to fracture the treated teeth was maximum in 2% taper whereas least in the 6% taper group. Zandbiglari et al. evaluated the influence of instrument taper on the resistance to fracture of endodontically treated roots and concluded that the roots were significantly weakened by the preparation with greater taper instruments. Khandale et al. remarked that instrumented teeth have a higher risk of fracture than uninstrumented counterparts and roots were significantly weakened by the preparation with instruments having greater tapers. In the present study, cleaning and shaping were done using NiTi files and the tapers used were 0.04 and 0.06. Hence, 0.04 taper is preferred over 0.06 when NaOCl is used as an irrigant alone or in combination with CHX and saline.
Intracanal cleaning and disinfecting procedures are highly dependent on the mechanical and chemical effects of the irrigants. Irrigating solutions in different concentrations with antimicrobial activity have been used during biomechanical instrumentation. Root canal irrigation protocols commonly employ the proteolytic agent NaOCl (tissue dissolution and bacterial reduction) and a chelating agent such as EDTA (removes the smear layer).
NaOCl is the most commonly employed root canal irrigant, which is effective in a concentration range of 2.6% to 5.25%. The bactericidal ability of NaOCl results from the formation of hypochlorous acid (HOCl) when in contact with organic debris. HOCl exerts its effects by oxidizing sulphydryl groups within bacterial enzyme systems, thereby disrupting the metabolism of the microorganism and resulting in the killing of the bacterial cells. Its antimicrobial property is proportional to the drug concentration as well as its toxicity. Concentrations over 0.5% are considered to be cytotoxic; therefore, they not only kill microorganisms but also disrupt the cells from the root canals. Acute inflammation followed by necrosis results when NaOCl comes into contact with vital tissue especially if extruded beyond the apex. Hence, in the present study, the concentration of NaOCl used is 3%, which is less cytotoxic compared to the 5.25% concentration.
CHX is widely used as a mouth rinse for periodontal diseases and has been used as irrigating solution and intra-canal dressing. CHX is a broad-spectrum antimicrobial agent that has an antibacterial effect against Gram-positive and Gram-negative bacteria and acceptable biocompatibility. CHX can be useful as an alternate endodontic irrigant for patients allergic to NaOCl or in teeth with patent apices. It is effective against certain NaOCl resistant bacterial strains, and hence in the present study chlorhexidine gluconate 2% was used once for a period of 6 minutes. However, the inability of CHX to dissolve organic matter is a perceived drawback.
Hence, a combination of NaOCl followed by saline and CHX has been advocated to enhance their antimicrobial properties. Kuruvilla suggested that the antimicrobial effect of 2.5% NaOCl and 0.2% CHX used in combination was better than that of either component. Zehnder proposed an irrigation regimen in which NaOCl would be used throughout instrumentation followed by EDTA, and CHX would be used as a final irrigant.– NaOCl mixed with CHX formed a thick dark precipitate para-chloroaniline, which is hazardous to the periapical tissues. The formation of this precipitate must be avoided by flushing the NaOCl with alcohol or EDTA before using CHX. Therefore, in the present study when NaOCl and CHX are used as an irrigant, saline is used to flush the NaOCl before using CHX.
These irrigants are commonly delivered using a syringe and needle. A 26G closed side vented needle with an outer diameter of 0.4636 mm and an inner diameter of 0.26 mm was used in this study. The factors affecting the efficacy of needle irrigation include the diameter of the irrigating needle, needle design, depth of the irrigating needle engaged in the root canal, and the final size of the root canal preparation. The use of a close-ended single side vented needle is considered to be the safest needle design for irrigation. Sedgley et al. recommend positioning the needle as close to the working length as possible to improve debridement and irrigant replacement. However, the eradication of the microbes in the apical canal should be of key importance to the success of endodontic treatment.
The different irrigants and the tapers employed in this study did not eradicate the E. faecalis biofilm completely. Therefore, the use of intracanal medicaments and newer endodontic irrigants and irrigating devices that enable the penetration of irrigants into complex root anatomy and up to working length is important for the success of root canal therapy. However, this study proved the use of NaOCl saline and CHX in combination at both 0.04 taper and 0.06 taper gave better results with no significant change. Use of 0.04 taper is preferred as we know the drawbacks of larger apical preparations, which include undesirable deviation from the original shape of the canal, weakening of the root, and procedural complications such as ledge formation, transportations, and perforations. The conservation of tooth structure and the preventing extrusion of obturating materials have been cited as primary advantages of minimal apical enlargements.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Alim-Uysal BA, Dincer AN, Yurtgezen B, Guneser MB. Does the endodontic education level affect decision-making for endodontically treated teeth with apical periodontitis? A web-based survey. Int Dent J 2021;71:1–7.
Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J. Endod 2006;32:93–8.
Haapasalo M, Shen Y, Wang Z, Gao Y. Irrigation in endodontics. Br Dent J 2014;216:299–303.
Alfadhli HA, Fawzy MI, Hassan M. A comparative in vitro study of rotary versus manual instruments for canal preparation of primary molars. Al-Azhar Dent J 2021;8:501–6.
Bhullar KK, Kaur S, Malhotra S, Singh H, Handa A, Kaur R. Comparative evaluation of antimicrobial efficacy of three endodontic irrigating solutions against Enterococcus faecalis. Eur J Mol Clin Med 2021;7:4800–4.
Arias-Moliz MT, Ferrer-Luque CM, Espigares-García M, Baca P. Enterococcus faecalis biofilms eradication by root canal irrigants. J Endod 2009;35:711–4.
Kuzekanani M, Moaddab S. Isolation of Enterococcus faecalis in previously root-filled canals in Kerman population. Dent News 2006;13:33–6.
Torabinejad M, Walton TE. Periradicular lesions. In: Endodontics. (J.I. Ingle, L.K. Bakland, Eds.), Baltimore: Williams & Wilkins, 4th ed. Biotechnol Biotechnol Equip 1994:439–464.
Siqueira JF, Rocas IN. Clinical implications and microbiology of bacterial persistence after treatment procedures. J Endod 2008;34:1291–301.
Mathew J. Viability and antibacterial efficacy of four root canal disinfection techniques evaluated using confocal laser scanning microscopy. J Conserv Dent 2014;17:444–8.
] [Full text]
Horiba N, Maekawa Y, Matsumoto T, Nakamura H. A study of the distribution of endotoxin in the dentinal wall of infected root canals. J Endod 1990;16:331–4.
Berber VB, Gomes B, Sena NT et al.
, Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules. Int Endod J 2006;39:10–7.
Shuping GB, Ørstavik D, Sigurdsson A, Trope M. Reduction of intracanal bacteria using nickel-titanium rotary instrumentation and various medications. J Endod 2000;26:751–5.
Khandale SH, Rane P, Rudagi K, Reddy K, Nanda Z, Bhojane R. Comparative evaluation of influence of instrument taper on the fracture resistance of endodontically treated teeth, an in-vitro study. J App Dent Med Sci 2020;6:88–94.
Zandbiglari T, Davids H, Schafer E. Influence of instrument taper on the resistance to fracture of endodontically treated roots. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:126–31.
Zehnder M. Root canal irrigants. J Endod 2006;32:389–98.
Sabry HA, Nashaat YM, Omar N, Negm A, Shaheen NA. Comparative study of the antibacterial effect of nano-silver irrigant, sodium hypochlorite and chlorhexidine against Enterococcus faecalis biofilm. Egypt Dent J 2019;65:1503–9.
Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endod 1998;24:472–476.
Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9.
Gopikrishna V. An in vivo assessment of the influence of needle gauges on endodontic irrigation flow rate. J Conserv Dent 2016;19:189–193.
Sedgley CM, Nagel AC, Hall D, Applegate B. Influence of irrigant needle depth in removing bioluminescent bacteria inoculated into instrumented root canals using real-time imaging in vitro. Int. Endod J 2005;38:97–104.
[Table 1], [Table 2], [Table 3]