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      Dentinal tubule disinfection with 2% chlorhexidine, garlic extract, and calcium hydroxide against Enterococcus faecalis by using real-time polymerase chain reaction: In vitro study

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          Abstract

          Aim:

          To compare the efficacy of garlic extract with 2% chlorhexidine (CHX) and calcium hydroxide Ca(OH) 2 in disinfection of dentinal tubules contaminated with Enterococcus faecalis by using real-time polymerase chain reaction (PCR).

          Materials and Methods:

          Agar diffusion test was done to evaluate the minimum inhibitory concentration of garlic extract against E. faecalis. Forty human extracted mandibular premolar teeth were selected for this study, access cavity was prepared and cleaning and shaping was done. Middle third of the root was cut using a rotary diamond disc. The teeth specimens were inoculated with E. faecalis for 21 days. Specimens were divided into four groups---Group 1: 2% CHX, Group 2: Garlic extract, Group 3: Ca(OH) 2, and Group 4: Saline (negative control). The intracanal medicaments were packed inside the tooth specimens and incubated for 5 days. The dentinal chips were collected at 400 μm depth using a Gates-Glidden drill, following which DNA isolation was done. The specimens were analyzed using real-time PCR. The results were then statistically analyzed using one-way analysis of variance, followed by post hoc Tukey's honestly significant difference (HSD) multiple comparison of means.

          Results:

          Threshold cycle (Ct) values of 2% CHX was found to be 32.4, garlic extract to be 27.5, and Ca(OH) 2 to be 25.6.

          Conclusion:

          A total of 2% CHX showed the maximum efficacy against E. faecalis, followed by garlic extract and Ca(OH) 2.

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          Most cited references34

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          In vitro infection and disinfection of dentinal tubules.

          Dag Ørstavik, M Haapasalo (1987)
          An in vitro model for dentinal tubule infection of root canals was developed. Cylindrical dentin specimens, 4 mm high with a diameter of 6 mm and a canal 2.3 mm wide, were prepared from freshly extracted bovine incisors. The cementum was removed from all dentin blocks. The tubules were opened by four-minute treatments with 17% EDTA and 5.25% NaOCl before being infected with Enterococcus faecalis ATCC 29212 in yeast extract-glucose broth. Bacteria rapidly invaded the tubules. After three weeks of incubation, a heavy infection was found 400 micron from the canal lumen, and the front of the infection reached 1000 micron in some blocks. Camphorated paramonochlorophenol (CMCP) and a calcium hydroxide compound, Calasept, were tested for their disinfecting efficacy toward E. faecalis-infected dentin. Liquid CMCP rapidly and completely disinfected the dentinal tubules, whereas CMCP in gaseous form disinfected tubules less rapidly. Calasept failed to eliminate, even superficially, E. faecalis in the tubules. The method used in bacteriological sampling allowed for sequential removal of 100-micron-thick zones of dentin from the central canal toward the periphery. Control specimens were uniformly infected and yielded growth in bur samples up to some 500 microns from the surface. The model proved quite sensitive and seems suitable for in vitro testing of root canal medicaments.
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            Biofilm formation in medicated root canals.

            Timothy Distel, F Hatton, Steven Gillespie (2002)
            The hypothesis that Enterococcus faecalis resists common intracanal medications by forming biofilms was tested. E. faecalis colonization of 46 extracted, medicated roots was observed with scanning electron microscopy (SEM) and scanning confocal laser microscopy. SEM detected colonization of root canals medicated with calcium hydroxide points and the positive control within 2 days. SEM detected biofilms in canals medicated with calcium hydroxide paste in an average of 77 days. Scanning confocal laser microscopy analysis of two calcium hydroxide paste medicated roots showed viable colonies forming in a root canal infected for 86 days, whereas in a canal infected for 160 days, a mushroom-shape typical of a biofilm was observed. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed no differences between the protein profiles of bacteria in free-floating (planktonic) and inoculum cultures. Analysis of biofilm bacteria was inconclusive. These observations support potential E. faecalis biofilm formation in vivo in medicated root canals.
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              In vitro mechanism of inhibition of bacterial cell growth by allicin.

              R S Feldberg, S. Chang, A Kotik … (1988)
              Diallyl thiosulfinate (allicin) is the agent found in garlic which is responsible for the antibacterial and antifungal activity of extracts of this plant. The effect of bacteriostatic concentrations of allicin (0.2 to 0.5 mM) on the growth of Salmonella typhimurium revealed a pattern of inhibition characterized by: (i) a lag of approximately 15 min between addition of allicin and onset of inhibition, (ii) a transitory inhibition phase whose duration was proportional to allicin concentration and inversely proportional to culture density, (iii) a resumed growth phase which showed a lower rate of growth than in uninhibited controls, and (iv) an entry into stationary phase at a lower culture density. Whereas DNA and protein syntheses showed a delayed and partial inhibition by allicin, inhibition of RNA synthesis was immediate and total, suggesting that this is the primary target of allicin action.
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                Author and article information

                Journal
                Journal ID (nlm-ta): J Conserv Dent
                Journal ID (iso-abbrev): J Conserv Dent
                Journal ID (publisher-id): JCD
                Title: Journal of Conservative Dentistry : JCD
                Publisher: Medknow Publications & Media Pvt Ltd (India )
                ISSN (Print): 0972-0707
                ISSN (Electronic): 0974-5203
                Publication date (Print): May-Jun 2013
                Volume: 16
                Issue: 3
                Pages: 194-198
                Affiliations
                [1]Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, Sri Ramachandra University, Chennai, Tamil Nadu, India
                Author notes
                Address for correspondence: Dr. Kandaswamy Eswar, Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, Sri Ramachandra University, Chennai - 600 116, Tamil Nadu, India. E-mail: frozenfire.eswar@ 123456gmail.com
                Article
                Publisher ID: JCD-16-194
                DOI: 10.4103/0972-0707.111312
                PMC ID: 3698579
                PubMed ID: 23833449
                SO-VID: 176f2e21-c4b1-4f57-8c54-11e848c6c937
                Copyright © Copyright: © Journal of Conservative Dentistry
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 10 December 2012
                Date revision received : 02 January 2013
                Date accepted : 04 March 2013
                Categories
                Subject: Original Article

                ScienceOpen disciplines: Dentistry
                Keywords: calcium hydroxide,dentinal tubules,garlic extract,intracanal medicament,real- time polymerase chain reaction 2% chx
                Data availability:
                ScienceOpen disciplines: Dentistry
                Keywords: calcium hydroxide, dentinal tubules, garlic extract, intracanal medicament, real- time polymerase chain reaction 2% chx

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