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Table of Contents   
ORIGINAL RESEARCH ARTICLE  
Year : 2017  |  Volume : 20  |  Issue : 2  |  Page : 125-128
Effect of conventional irrigation and photoactivated disinfection on Enterococcus faecalis in root canals: An in vitro study


Department of Conservative Dentistry and Endodontics, K.L.E. Society's Institute of Dental Sciences, Bengaluru, Karnataka, India

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Date of Submission30-Dec-2015
Date of Decision14-Apr-2016
Date of Acceptance22-Aug-2016
Date of Web Publication4-Aug-2017
 

   Abstract 

Aims: A study was done to evaluate the antimicrobial efficacy of sodium hypochlorite (NaOCl) and photoactivated disinfection (PAD) on Enterococcus faecalis.
Settings and Design: Random sampling, in-vitro study.
Subjects and Methods: Access opening and biomechanical preparation were performed on fifty freshly extracted mandibular second premolars. The specimens were sterilized; 15 μm of E. faecalis was inoculated into each canal and incubated at 36°C for 24 h. Later, specimens were randomly divided into two groups of fifty each and following procedures was carried out: (i) conventional irrigation with 2.25% NaOCl (ii) PAD using diode laser, and toluidine blue photosensitizer. Samples were collected from each canal using sterile paper points which were deposited in brain heart infusion broth, and microbiological evaluation was carried out.
Statistical Analysis Used: Student's t-test was used to find the significant difference in the reduction of colony forming unit (CFU) between the groups.
Results: The mean CFUs of the two groups showed statistically significant difference (P = 0.001). Improved antibacterial efficacy was seen with PAD group compared to conventional NAOCL irrigation.
Conclusions: NaOCl alone was not effective in eliminating E. faecalis completely from the root canals. PAD compared to conventional irrigation showed the best results in removing E. faecalis from root canals.

Keywords: Enterococcus Faecalis; Photoactivated Disinfection; Sodium hypochlorite

How to cite this article:
Balakrishna N, Moogi P, Kumar G V, Prashanth B R, Shetty NK, Rao KR. Effect of conventional irrigation and photoactivated disinfection on Enterococcus faecalis in root canals: An in vitro study. J Conserv Dent 2017;20:125-8

How to cite this URL:
Balakrishna N, Moogi P, Kumar G V, Prashanth B R, Shetty NK, Rao KR. Effect of conventional irrigation and photoactivated disinfection on Enterococcus faecalis in root canals: An in vitro study. J Conserv Dent [serial online] 2017 [cited 2020 Feb 24];20:125-8. Available from: http://www.jcd.org.in/text.asp?2017/20/2/125/212244

   Introduction Top


The successful outcome of root canal treatment is based on the efficient disinfection of the root canal system and prevention of reinfection. Enterococcus faecalis has long been implicated species from root canals of teeth with posttreatment lesions.[1] The most common reason for reinfection is related to improper instrumentation and inadequacy of conventional irrigation solutions to cleanse the root canal completely. Of the irrigants used, sodium hypochlorite (NaOCl) is preferred by most clinicians as it exhibits a proteolytic effect as well as being a disinfectant.[2],[3] It is a potent antimicrobial agent, killing most bacteria instantly on direct contact. It also effectively dissolves vital and necrotic pulpal remnants and collagen.[4] However, there is evidence that hypochlorite is not effective against all pathogenic bacteria, such as E. faecalis which is associated with recalcitrant canals.[5]

To overcome these limitations, a novel disinfecting system which includes the use of photoactivated disinfection (PAD) has been tested as an adjunct to conventional root canal therapy using NaOCl as a root canal irrigant.

PAD is an antimicrobial strategy in which laser energy is used to activate a nontoxic photosensitizer, the singlet oxygen released from these photosensitive dyes damages the membrane and DNA of microorganisms.[6] The photosensitizers have a high degree of selectivity for killing microorganisms without affecting host cell viability.[7] Hence, this study was undertaken to compare and evaluate the antibacterial efficacy of NaOCl irrigation alone, and PAD as adjuncts to conventional irrigation with NaOCl on E. faecalis in root canals.


   Subjects and Methods Top


Fifty freshly extracted mandibular second premolars from patients for orthodontic or periodontal purposes were collected for the study. Ethical clearance was obtained by the Institution Ethical Committee.

Conventional access to the root canal system was performed. Patency of each canal was established by placing a size 10 K-file (Mani Inc., Tochigi, Japan) until it was visible in the apical foramen. Working length was established 1 mm short of the apex, and the canals were enlarged sequentially up to a size F2 protaper (Dentsply, Maillefer, Switzerland) as per the manufacturer's recommendation. EDTA (RC Help) was used as a lubricant, and canals were irrigated with 2.25% NaOCl (VIP Vensons, India) during the preparation.

After root canal preparation, the enlarged apical foramina were sealed with epoxy resin to prevent bacterial leakage, and the specimens were then sterilized in an autoclave at 121°C for 20 min at 20 psi pressure.

Pure culture of E. faecalis (ATCC 29212) grown in brain heart infusion (BHI), broth was used to contaminate the root canals. The root canals were inoculated with 15 μm of the turbid suspension of E. faecalis ATCC 29212 using a micropipette (Kasablanka, Digital Variable Micropipette, Mumbai, India). The turbidity was verified using the McFarland turbidity scale, and adjusted to 0.5, corresponding to 10[8] organisms per milliliter. The specimens were incubated at 36.5°C for 24 h. Colony-forming units (CFUs) of E. faecalis was counted for one sample in each group to ensure growth in root canals. Autoclavable foam with punch holes was used to hold the prepared specimens. Asepsis was maintained throughout the procedures using standard precautions with two flames in a biosafety cabinet.

The teeth were randomly divided into two groups of 25 each:

  • Group I - Conventional irrigation with 2.25% NaOCl solution
  • Group II - PAD as an adjunct to conventional irrigation with 2.25% NaOCl.


Group I: (n = 25) Conventional irrigation

After incubation, samples were retrieved from the incubator. The canals were subjected to copious irrigation with 20 ml of 2.25% NaOCl solution (VIP Vensons, India) and 2 ml of 17% EDTA (Dent Wash; Prime Dental, Chicago) alternately for 2 min. Finally, canal was washed with 2 ml of saline to remove any residual irrigant. A sterile paper point was used to obtain the sample from the canal which was deposited in a sterile Eppendorf tube containing 200 μl of BHI broth.

Group II: (n = 25) Photoactivated disinfection

After conventional irrigation of root canals was done as discussed in Group I, the canals were dried using sterile paper points. 1 ml of toluidine blue (concentration of 100 μg/ml) solution was injected into the canals and agitated for 15 s using a sterile size 10 K-file and then left undisturbed for 1 min. The fiber optic tip (diameter = 200 μm) of the Diode laser having a wavelength of 980 nm was inserted into each canal and irradiated at 100 mW for a period of 120 s [Figure 1]. Following this sterile, paper point was used to take sample from the canal and deposited in a sterile Eppendorf tube containing 200 μl of BHI broth.
Figure 1: Canal irradiation using fiber optic tip

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Microbiological evaluation

Later, the specimens were incubated at 36.5°C for 24 h. Using sterile micropipettes, decimal series of dilutions were made up to 10−4 for each sample. The BHI agar plates were incubated at 36.5°C for 24 h. Colony count of E. faecalis was done using semi-quantitative method and expressed as CFU/mL [Figure 2] and [Figure 3].
Figure 2: Colonies formed in Group I

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Figure 3: Colonies formed in Group II

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   Results Top


The microbiological evaluation of E. faecalis CFU revealed a significant reduction in CFUs in the two study groups. The results were analyzed by counting the number of CFU of Enterococcus fecalis after disinfecting the canals and calculating the mean values for the two groups [Graph 1] and [Table 1].

Table 1: T-test to analyze the difference between the two groups

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   Discussion Top


Enterococci is usually isolated in root canals undergoing standard endodontic treatment because of low sensitivity to antimicrobial agents or their ability to inactivate antimicrobial agents.[8],[9] Authors have reported that E. faecalis has the capacity to survive under various environmental stresses.[10],[11],[12] It has also been speculated that E. faecalis can enter the canal, survive the antibacterial treatment, and then persist after obturation.[13],[14] Hence, in the present study, the root canals were contaminated with E. faecalis (ATCC 29212) that was obtained by growing the cells in BHI broth.

The antibacterial efficacy of various irrigating solutions has been tested against E. faecalis. NaOCl is considered a gold standard for irrigants and is the most popular irrigating solution.[15] NaOCl is commonly used in concentrations between 0.5% and 6%.[4] However, there is considerable variation in the literature regarding the antibacterial effect of NaOCl. It is reported to kill the target microorganisms in seconds, even at low concentrations, although some reports have shown that considerable longer time for the elimination of the same microorganisms.[5],[16],[17],[18] Furthermore, there is evidence that hypochlorite is not effective against all pathogenic bacteria specifically E. faecalis which is associated with recalcitrant canals.[5]

PAD is a new treatment modality that has been developing rapidly within various medical specialties since the 1960s and has been defined as “the light-induced inactivation of cells, microorganisms, or molecules.” On laser irradiation of an appropriate wavelength, the photosensitizer undergoes a transition from low-energy level “ground state” to a higher-energy “triplet state.” This triplet-state sensitizer can react with biomolecules to produce free radicals and radical ions or with molecular oxygen to produce singlet oxygen. These cytotoxic species can cause oxidation of cellular constituents such as plasma membranes and DNA, resulting in cell death.[19],[20] Another type of damage caused by PAD is the damage caused to the cytoplasmic membrane of the bacteria by cytotoxic species generated by antimicrobial PDT, resulting in inactivation of the membrane transport system, inhibition of plasma membrane enzyme activities, and lipid peroxidation.[21]

In Group I, the mean value of E. faecalis CFUs was 163.72. Conventional irrigation with NaOCl showed the least efficacy in removing E. faecalis colonies compared to the other groups.

In Group II, the mean value of E. faecalis CFU was 14.68, which was highly significant (P = 0.001) compared to Group I, implying that PAD, used as an adjunct to conventional irrigation with NaOCl, was significantly more effective in removing E. faecalis from the root canals.

The result of this study was in accordance with a study wherein PAD with Diode laser was found to be more effective at reducing or eliminating bacterial load from the canals compared to NaOCl irrigation alone.[22] Another study demonstrated greater reduction in number of CFUs of E. faecalis in PAD group than conventional 2.5% NaOCl syringe irrigation.[6]


   Conclusions Top


Within the limitation of this study, it was found that PAD was more effective than NaOCl in reducing E. faecalis counts. NaOCl alone was not effective in eliminating E. faecalis completely from the root canals. However, further in-vivo studies are required to corroborate the present in-vitro study to intra-oral conditions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Engstrom B. The significance of enterococci in root canal treatment. Odontol Revy 1964;15:87-106.  Back to cited text no. 1
    
2.
Siqueira JF Jr., Batista MM, Fraga RC, de Uzeda M. Antibacterial effects of endodontic irrigants on black-pigmented Gram-negative anaerobes and facultative bacteria. J Endod 1998;24:414-6.  Back to cited text no. 2
    
3.
Siqueira JF Jr., Rôças IN, Favieri A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1%, 2.5%, and 5.25% sodium hypochlorite. J Endod 2000;26:331-4.  Back to cited text no. 3
    
4.
Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin North Am 2010;54:291-312.  Back to cited text no. 4
    
5.
Radcliffe CE, Potouridou L, Qureshi R, Habahbeh N, Qualtrough A, Worthington H, et al. Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis. Int Endod J 2004;37:438-46.  Back to cited text no. 5
    
6.
Bago I, Plecko V, Gabric Panduric D, Schauperl Z, Baraba A, Anic I. Antimicrobial efficacy of a high-power diode laser, photo-activated disinfection, conventional and sonic activated irrigation during root canal treatment. Int Endod J 2013;46:339-47.  Back to cited text no. 6
    
7.
Lee MT, Bird PS, Walsh LJ. Photo-activated disinfection of the root canal: A new role for lasers in endodontics. Aust Endod J 2004;30:93-8.  Back to cited text no. 7
    
8.
Dahlén G, Samuelsson W, Molander A, Reit C. Identification and antimicrobial susceptibility of enterococci isolated from the root canal. Oral Microbiol Immunol 2000;15:309-12.  Back to cited text no. 8
    
9.
Portenier I, Haapasalo H, Orstavik D, Yamauchi M, Haapasalo M. Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heat-killed microbial whole cells. J Endod 2002;28:634-7.  Back to cited text no. 9
    
10.
Flahaut S, Hartke A, Giard JC, Benachour A, Boutibonnes P, Auffray Y. Relationship between stress response toward bile salts, acid and heat treatment in Enterococcus faecalis. FEMS Microbiol Lett 1996;138:49-54.  Back to cited text no. 10
    
11.
Laplace JM, Thuault M, Hartke A, Boutibonnes P, Auffray Y. Sodium hypochlorite stress in Enterococcus faecalis: Influence of antecedent growth conditions and induced proteins. Curr Microbiol 1997;34:284-9.  Back to cited text no. 11
    
12.
Capiaux H, Giard JC, Lemarinier S, Auffray Y. Characterization and analysis of a new gene involved in glucose starvation response in Enterococcus faecalis. Int J Food Microbiol 2000;55:99-102.  Back to cited text no. 12
    
13.
Hartke A, Giard JC, Laplace JM, Auffray Y. Survival of Enterococcus faecalis in an oligotrophic microcosm: Changes in morphology, development of general stress resistance, and analysis of protein synthesis. Appl Environ Microbiol 1998;64:4238-45.  Back to cited text no. 13
    
14.
Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.  Back to cited text no. 14
    
15.
McDonnell G, Russell AD. Antiseptics and disinfectants: Activity, action, and resistance. Clin Microbiol Rev 1999;12:147-79.  Back to cited text no. 15
    
16.
Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ.In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424-8.  Back to cited text no. 16
    
17.
Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ.In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79-84.  Back to cited text no. 17
    
18.
Waltimo TM, Orstavik D, Sirén EK, Haapasalo MP.In vitro susceptibility of Candida albicans to four disinfectants and their combinations. Int Endod J 1999;32:421-9.  Back to cited text no. 18
    
19.
Maisch T, Szeimies RM, Jori G, Abels C. Antibacterial photodynamic therapy in dermatology. Photochem Photobiol Sci 2004;3:907-17.  Back to cited text no. 19
    
20.
Babilas P, Schreml S, Landthaler M, Szeimies RM. Photodynamic therapy in dermatology: State-of-the-art. Photodermatol Photoimmunol Photomed 2010;26:118-32.  Back to cited text no. 20
    
21.
Takasaki AA, Aoki A, Mizutani K, Schwarz F, Sculean A, Wang CY, et al. Application of antimicrobial photodynamic therapy in periodontal and peri-implant diseases. Periodontol 2000 2009;51:109-40.  Back to cited text no. 21
    
22.
Bonsor SJ, Nichol R, Reid TM, Pearson GJ. An alternative regimen for root canal disinfection. Br Dent J 2006;201:101-5.  Back to cited text no. 22
    

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Correspondence Address:
Prashant Moogi
Department of Conservative Dentistry and Endodontics, K.L.E. Society's Institute of Dental Sciences, Bengaluru - 560 022, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.212244

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