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ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 5  |  Page : 41-44  

Detection of adherence of Enterococcus faecalis in infected dentin of extracted human teeth using confocal laser scanning microscope: An In vitro Study


1 Department of Conservative Dentistry and Endodontics, Sri Sankara Dental College, Trivandrum, Kerala, India
2 Department of Conservative Dentistry and Endodontics, KVG Dental College, Sullia, Karnataka, India
3 Department of Pedodontics and Preventive Dentistry, Asian Institute of Medicine, Science and Technology University, Malaysia
4 Department of Oral Pathology, Faculty of Dentistry, Asian Institute of Medicine, Science and Technology University, Malaysia

Date of Web Publication27-Nov-2017

Correspondence Address:
G Sivadas
Department of Pedodontics and Preventive Dentistry, Faculty of Dentistry, Asian Institute of Medicine, Science and Technology University, Kedah
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.JPBS_92_17

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   Abstract 


Aim: The aim of this study is to detect in vitro adherence of Enterococcus faecalis to the infected dentinal tubules of human extracted teeth using confocal laser scanning microscope. Subjects and Methods: Roots from human premolar teeth (n = 40) were infected with E. faecalis strain the American Type Culture Collection 29212 in brain heart infusion for 21 days. After the experimental period, specimens were divided into two groups, Group A (n = 20), Group B (n = 20), and Group A specimens were stained with fluorescein diacetate dye for the detection of viability and adherence Group B were stained with acridine orange dye for detection of metabolic activity and adherence. Samples were washed, thoroughly sectioned and examined by confocal laser scanning microscopy. Computer-assisted determinants of fluorescence, bacterial viability, metabolic activity, and adherence were compared statistically. Results: E. faecalis was able to invade the dentinal tubules to a depth of 1–400 μm and adhere to 1–200 μm depth. Adherence (90%) was significantly higher in 1–100 μm using fluorescein diacetate and acridine orange dye. Conclusion: Adherence of E. faecalis as evaluated by confocal laser scanning microscope was highest at the depth of 1–100 μm which may have an impact on the shaping and cleaning procedures on the root canal.

Keywords: Adherence, confocal laser scanning microscopy, Enterococcus faecalis


How to cite this article:
Nair V S, Nayak M, Ramya M K, Sivadas G, Ganesh C, Devi S L, Vedam V. Detection of adherence of Enterococcus faecalis in infected dentin of extracted human teeth using confocal laser scanning microscope: An In vitro Study. J Pharm Bioall Sci 2017;9, Suppl S1:41-4

How to cite this URL:
Nair V S, Nayak M, Ramya M K, Sivadas G, Ganesh C, Devi S L, Vedam V. Detection of adherence of Enterococcus faecalis in infected dentin of extracted human teeth using confocal laser scanning microscope: An In vitro Study. J Pharm Bioall Sci [serial online] 2017 [cited 2022 Jul 6];9, Suppl S1:41-4. Available from: https://www.jpbsonline.org/text.asp?2017/9/5/41/219307




   Introduction Top


Enterococcus faecalis, a facultative anaerobe, Gram-positive, nonspore-forming cocci which can survive extreme challenges.[1],[2],[3] Its pathogenicity ranges from life-threatening diseases in compromised individuals to less severe conditions such as infection of obturated root canals with chronic apical speriodontitis.[2]E. faecalis is an extensively evaluated biological Indicator. Its high prevalence in cases with posttreatment disease is associated with virulence factors that can facilitate the adherence of host cells and extracellular matrix, tissue invasions, immunomodulation effect, and cause toxin-mediated damage.[4] Adherence of bacteria to hard tissues or artificial biomaterials is the first step toward biofilm-mediated infections. E. faecalis possess different virulence factors that enable them to adhere to dentin and invade dentinal tubules.[5]

A variety of methods including autoradiography, traditional colony count, and live dead backlight staining for the evaluation of the metabolic activity of bacteria has been used to evaluate bacterial viability.[6] The bacteria's ability to penetrate into the dentin can be determined through microbiological analyses by the number of colony-forming units (CFUs), the number of tubules infected in the histologic sample or the presence of bacteria in the root canal walls. Microbiological culturing has been popularly used to detect bacteria in endodontics. Transmission electron microscope has been used for the visualization of root canal infections. However, this technique is time-consuming and requires multiple steps for specimen preparation.[7],[8]

Laser scanning confocal microscopy has become an invaluable tool for a wide range of investigations in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 μm.[9] Confocal laser scanning microscope provides the capacity for direct, noninvasive serial optical sectioning of intact, thick, living specimens with minimum of sample preparation and is shown to be a useful complement to the established microbiological, histological, standard electron microscopy, and polymerase chain reaction (PCR)-based technique for identification of bacteria in bovine teeth.[10] To date, no literature has been evaluated on the adherence, metabolic activity, and viability of E. faecalis to human teeth using confocal laser scanning microscopy.


   Subjects and Methods Top


The present study was conducted to detect in vitro the viability, metabolic activity, and adherence of E. faecalis to the infected dentinal tubules of human extracted teeth using confocal laser scanning microscopy. In addition, an attempt was made to detect in vitro the viability of E. faecalis in infected dentin of human teeth using confocal laser scanning microscope with application of fluoroscein diacetate dye, evaluate in vitro the metabolic activity of E. faecalis in infected dentin of human teeth using confocal laser scanning microscope with the application of 0.01% acridine orange dye and determine in vitro the adherence and depth of penetration of E. faecalis in infected dentin of human teeth using confocal laser scanning microscopy.

Forty noncarious premolar teeth undergoing orthodontic extraction 18–30 years age group reporting to K.V.G Dental College and Hospital, Sullia, was selected for the study. E. faecalis strain (American Type Culture Collection [ATCC] 29212) was obtained from Kasturba Medical College, Manipal University, Mangalore. The collection, storage, sterilization, and handling of extracted teeth were followed according to Occupational Safety and Health Administration and the Centre for Disease Control and Prevention recommendations and guidelines.

  1. The teeth were cleaned of visible blood and gross debris
  2. The teeth were stored in a liquid chemical germicide. Then, the teeth were autoclaved
  3. The teeth were kept in a well-constructed container with a secure lid to prevent leaking during transport
  4. The container was labelled with the biohazard symbol.


The confocal laser scanning microscopic study was done at Sri Chithra Institute of Medical Sciences and Technology, Trivandrum.

Specimen preparation

Forty noncarious human extracted teeth were selected and stored in 5% formalin buffer. All 40 samples were divided into two groups of twenty teeth each designated as Group A (20 teeth) – to detect viability and adherence and Group B (20 teeth) – to detect metabolic activity and adherence.

A root segment with a length of about 7–8 mm was prepared by sectioning the root tip and after that crown was removed at 2–3 mm below the cemento enamel junction. Each root canal was enlarged to a size of a Gates Glidden bur #3 under 1% sodium hypochlorite irrigation. The teeth were then washed with sterile water for 10 min and stored in sterile water for 1 week (IS) to remove any residual chemical compound. The specimens were sterilized by autoclave for 20 min at 121°C. Sterility was checked by incubating of each specimen in 5 mL of brain heart infusion (BHI) broth at 37°C for 24 h.

The strain E. faecalis (ATCC 29212) from the ATCC was used in this study. To create the bacterial inoculum, isolated colonies (24 h) of pure cultures of E. faecalis grown aerobically on BHI agar plates was suspended in 3.0 mL BHI. For dentin infection, under laminar flow, twenty specimens were transferred individually into 3 mL BHI inoculated with of the E. faecalis suspension for 21 days. BHI was continuously changed every 48 h, and the purity of the broth was verified to avoid contamination. After the incubation period (21 days), the root canals of dentin blocks were washed with 1 ml phosphate-buffered saline to remove nonadherent bacteria of the root canal walls. Superficial longitudinal grooves were made on the buccal and lingual surface to facilitate the fracture of the specimens.

The determination of bacterial adherence

All the 40 specimens were assessed for adherence using confocal laser scanning microscopy.

The area of increased fluorescence determined the adherence of bacteria.


   Results Top


E. faecalis adhered to a depth of 1–200 μm. Significantly, greater bacterial adherence was observed at a depth of 1–100 μm. Bacterial adherence was less at a depth of 100–200 μm. Bacterial adherence was not observed at a depth of 200–300 μm. Adherence (Group A and B) of E. faecalis as observed by confocal laser scanning microscopy was highest at the depth of 1–100 μm [Table 1].
Table 1: Adherence of bacteria (Group A and B)

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   Discussion Top


Bacteria are commonly found within dentinal tubules of clinically infected canals. E. faecalis is of interest because it is the most frequently detected species in root filled teeth with persistent lesions as shown by culture or molecular methods based studies[7] and prevalence in such infections ranges from 24% to 77%. The strain E. faecalis has shown the ability to invade dentinal tubules, to tolerate the high pH levels to withstand long periods of starvation with subsequent recovery in the presence of serum and to survive in obturated root canals.[7],[11] This strain of E. faecalis is the most prevalent bacterial strain in endodontic cases with persistent endodontic lesions as shown by culture or molecular methods-based studies. In the present study, E. faecalis ATCC 29212 was chosen as the test organism.

Bovine teeth were used in the majority of in vitro investigations related to infected root dentin. In the present study, extracted human teeth were selected as the test specimens as bovine root dentine differs from human dentine; dentinal tubules of bovine teeth have wider diameters and in some cases giant tubules are present. Therefore, infected human root specimens seem to be more suitable for investigating the microbial vitality status than infected bovine root dentine.[12]

Procedures such as histologic sections, scanning electron microscope used to evaluate the presence of bacteria in root canals does not give clear information about the spatial distribution of bacteria inside the dentin. The bacteria's ability to penetrate into the dentin can be determined through microbiological analysis. Confocal laser scanning is shown to be a useful complement to the established microbiological, histological, standard electron microscopy, and PCR-based technique for the identification of bacteria is advantageous for several reasons such as the painstaking and difficult preparation of thin sections is not required; consequently, the procedure becomes very easy and fast, specimens examined do not require any special preparation and therefore are not subjected to any distortions caused by dehydration as required for other procedures such as scanning electron microscopy, the operator is not exposed to the risks associated with the use of X-ray radiation used for microradiography, the methodology allows subsurface examination of intact tooth samples, disregarding irregularities, or contaminants present on the surface since the scattered, reflected, and fluorescent light from planes out of focus is eliminated providing a subsurface image only from a thin layer on which it is focused.[13],[14]

To the best of our knowledge, we described for the first time the visualization of adherence in human-infected single dentinal tubules using confocal laser scanning microscopy. Although bacterial infection can be substantially reduced by standard intracanal procedures, it is difficult to render the root canal free from bacteria as they are located in inaccessible areas such as complicated root canal anatomy and dentinal tubules and is difficult to deliver antimicrobial agents to those locations. Understanding these mechanisms may lead to the development of novel clinical strategies to inhibit bacterial colonization and to manage these infections in the future by thorough biomechanical preparation and disinfection procedures.


   Conclusion Top


E. faecalis is a normal inhabitant of the oral cavity. It is associated with different forms of peri-radicular diseases including primary endodontic infections and persistent infections.

The visualization and quantification of adherent bacteria are still one of the challenges in dentistry. Laser scanning confocal microscopy has become an invaluable tool in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 μm.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Gomes BP, Pinheiro ET, Sousa EL, Jacinto RC, Zaia AA, Ferraz CC, et al. Enterococcus faecalis in dental root canals detected by culture and by polymerase chain reaction analysis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:247-53.  Back to cited text no. 1
    
2.
Kayaoglu G, Østavik D. Virulence factors of Enterococcus faecalis: Relationship to endodontic disease. Crit Rev Oral Biol Med 2004;15:308-20.  Back to cited text no. 2
    
3.
Kishen A, Chen NN, Tan L, Asundi A. Chairside sensor for rapid monitoring of Enterococcus faecalis activity. J Endod 2004;30:872-5.  Back to cited text no. 3
    
4.
Estrela C, Silva JA, de Alencar AH, Leles CR, Decurcio DA. Efficacy of sodium hypochlorite and chlorhexidine against Enterococcus faecalis – A systematic review. J Appl Oral Sci 2008;16:364-8.  Back to cited text no. 4
    
5.
Kishen A, Sum CP, Mathew S, Lim CT. Influence of irrigation regimens on the adherence of Enterococcus faecalis to root canal dentin. J Endod 2008;34:850-4.  Back to cited text no. 5
    
6.
Shen Y, Stojicic S, Haapasalo M. Bacterial viability in starved and revitalized biofilms: Comparison of viability staining and direct culture. J Endod 2010;36:1820-3.  Back to cited text no. 6
    
7.
Zapata RO, Bramante CM, de Moraes IG, Bernardineli N, Gasparoto TH, Graeff MS, et al. Confocal laser scanning microscopy is appropriate to detect viability of Enterococcus faecalis in infected dentin. J Endod 2008;34:1198-201.  Back to cited text no. 7
    
8.
Love RM. Enterococcus faecalis – A mechanism for its role in endodontic failure. Int Endod J 2001;34:399-405.  Back to cited text no. 8
    
9.
Claxton NS, Fellers TJ, Davidson MJ. Laser Scanning Confocal Microscopy; 2006.  Back to cited text no. 9
    
10.
Watson TF. Fact and artefact in confocal microscopy. Adv Dent Res 1997;11:433-41.  Back to cited text no. 10
    
11.
Chivatxaranukul P, Dashper SG, Messer HH. Dentinal tubule invasion and adherence by Enterococcus faecalis. Int Endod J 2008;41:873-82.  Back to cited text no. 11
    
12.
Weiger R, de Lucena J, Decker HE, Löst C. Vitality status of microorganisms in infected human root dentine. Int Endod J 2002;35:166-71.  Back to cited text no. 12
    
13.
McLean JS, Ona ON, Majors PD. Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy. ISME J 2008;2:121-31.  Back to cited text no. 13
    
14.
Coder DM. Assessment of cell viability. Curr Protoc Cytometry 1997;9:1.14.  Back to cited text no. 14
    



 
 
    Tables

  [Table 1]


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