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Year : 2009  |  Volume : 1  |  Issue : 2  |  Page : 47-55

Effects of alkaline biocides on enterococcus faecalis - An in vitro study

1 Professor, Department of Conservative Dentistry, Endodontics & Aesthetic Dentistry, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune, India
2 Postgraduate Student, Department of Conservative Dentistry, Endodontics & Aesthetic Dentistry, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune, India

Date of Web Publication4-Mar-2011

Correspondence Address:
Lotika Beri
Professor, Department of Conservative Dentistry, Endodontics & Aesthetic Dentistry, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune
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Source of Support: None, Conflict of Interest: None

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The purpose of this study was to do a comparative microbial assessment of disinfecting potential of Calcium hydroxide and Bioactive glass against Enterococcus faecalis when used as an intracanal medicament.
Thirty single rooted premolars undergoing extraction for orthodontic treatment were selected. Root canals of teeth with fully formed apices were instrumented using size 40 K file 1 mm short of canal length and irrigated with 2.5 % sodium hypochloride and EDTA. Teeth were then suspended in tryptic soya broth (TSB) and autoclaved. Positive control and study teeth, were inoculated with Enterococcus faecalis for two weeks in tryptic soya broth. Subsequently teeth were dressed with Calcium hydroxide or Bioactive glass (BAG) suspension for 10 days. Dentin samples were obtained from teeth using ISO size 45 H file to working length, cultured in tryptic soya agar and transferred to TSB for detection of growth by checking the turbidity of broth.
Unpaired t test and ANOVA test were used for statistical analysis. Both the alkaline biocides did not show complete elimination of Enterococcus faecalis. Calcium hydroxide had a stronger antibacterial effect than Bioactive glass.
Calcium hydroxide was an effective disinfectant.

How to cite this article:
Beri L, Kumar S, Aggarwal S. Effects of alkaline biocides on enterococcus faecalis - An in vitro study. J Int Clin Dent Res Organ 2009;1:47-55

How to cite this URL:
Beri L, Kumar S, Aggarwal S. Effects of alkaline biocides on enterococcus faecalis - An in vitro study. J Int Clin Dent Res Organ [serial online] 2009 [cited 2018 Apr 19];1:47-55. Available from:

   Introduction Top

The contribution of microorganisms to the development of pulpal and periapical disease has been well-documented (Kakehashi et al 1965, Sundqvist 1979). The bacteria associated with primary endodontic infections are polymicrobial but are predominantly gram-negative anaerobic rods, while secondary root canal infections are composed of fewer species, which are dominated by facultative anaerobic gram-positive bacteria, principally Enterococcus faecalis (Sundqvist et al 1994, Sjogren 1998). The success of root canal treatment totally depends on the elimination of microbial contamination from the root canal system. Although mechanical instrumentation of root canals can reduce bacterial population, effective elimination of bacteria, especially during retreatment is by the use of antimicrobial root canal medicament.

The main benefit of calcium hydroxide, as an intracanal medicament lies, in the bacterial effect conferred to its high pH related to the release of hydroxyl ions in an aqueous environment [1] . However against some microorganisms, calcium hydroxide may not be the optimal root canal medicament, enterococci and yeasts tolerate an alkaline environment (Siren et al 1997, Waltimo et al 1997, 1998), it was suggested that alternative medicament may sometimes be needed to resolve the infection and to gain healing [2] .

Bioactive glasses are interesting materials in dentistry because of their ability to mineralize dentine (Forsback et al. 2004, Vollenweider et al. 2007) and their antimicrobial effect on oral microorganisms Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Actinomyces naeslundii, Streptococcus mutans, and Streptococcus sanguis[3],[4] . Consequently bioactive glasses could potentially be used as topical endodontic disinfectants without the purported negative side effects of calcium hydroxide on dentine stability [5] . The release of Na + ,Ca + ions, silica and the incorporation of H 3 0 + protons into the corroding glass results in a high pH environment in a closed system, which is not well tolerated by microbiota and thus are used for treatment of demineralized and infected dentine found in deep carious lesions and necrotic root canals (Zehnder et al 2006).

Bioactive glasses are special glass systems composed of SiO 2 , CaO, P 2 O 5 , and Na 2 O. The bioactive behavior of these glasses is defined as the ability to bond to soft and hard tissues by means of a series of reactions, which produces a strong, compliant interface between the glass and the tissue [6] . Due to their high level of tissue integration and regeneration [7] , bioactive glasses have been used clinically in a variety of situations. Bioactive glasses are now available to treat alveolar ridge resorption, bone loss due to periodontal disease and to fill cystic and surgically created defects.

Enterococcus faecalis is resistant to calcium hydroxide and since the mechanism of action of bioactive glass is similar to calcium hydroxide, this study was carried out to check the efficacy of bioactive glass to eliminate Enterococcus faecalis and to compare the antimicrobial properties of calcium hydroxide with bioactive glass.

   Materials and Methods Top

Preparation of teeth and instrumentation procedures

Thirty human single rooted premolar teeth free of caries and restoration were selected for the study. The teeth were placed in 3% solution of sodium hypochlorite for 30 minutes for cleaning. Access cavity preparation was done with diamond-coated bur and canals were instrumented using K-File (Dentsply Maillefer). An ISO size 10 no. K-File was inserted into the root canal until the tip was just visible beyond the foramen and the working length determined by subtracting 1 mm from this length and apical stop was created at this level. Canal was instrumented till ISO size 40 no. K-File. During instrumentation root canals were irrigated with 2.5% NaOCl solution and 15% EDTA followed by copious amount of distilled water.

After the instrumentation of the samples, the teeth were divided into two test groups with ten teeth each and two control groups with five teeth each [Table 1]. Test and control teeth were rinsed with 2ml tryptic soya broth and teeth were placed in the test tubes and autoclaved for 15 mins at 121 o C.
Table 1

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Enterococcus faecalis strain was used as test organism (procured from Armed Forces Medical College, Pune). The strain was identified by gram stain, catalase reaction and by patterns of carbohydrate fermentation. An overnight culture on tryptic soya agar plates was checked for purity and adjusted to give cell density of 10 7 cells/ml for stock solution. (Done at Department of Microbiology at Dr.D.Y.Patil Medical College, Pune)

All the tubes except those containing negative control were seeded with 0.1ml of overnight culture of Enterococcus faecalis [Figure 1]. Tubes were incubated for two week at 37 0 C. The broth was aseptically changed after 2 to 3 days interval. Turbidity was measured at day 7 and 14 [Table 2],[Figure 2]; gram staining and culture were done to check purity of growth [Figure 3],[Figure 4]. All teeth were rinsed with sterile saline solution. Sterile suspension of bioactive glass (Perioglas, NovaBone Products, LLC, Florida; USA) and Calcium hydroxide in distilled water was prepared according to the manufacturer's instructions. Teeth in test group A were medicated with calcium hydroxide and group B were medicated with bioactive glass. The medicaments were placed into root canals using lentulo spiral in a contra- angle hand piece. Positive control teeth (Group C) were infected but not medicated. Negative control teeth (Group D) were neither infected nor medicated. All teeth were incubated for 10 days at 37 0 C.
Table 2: Mean values of Optical density/Turbidity (P < 0.001)

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Figure 1: Teeth in TSB and infected with E.faecalis

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Figure 2: Samples showing turbidity

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Figure 3: Culture

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Figure 4: Gram Staining

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After 10 days, before sampling of dentin chips, teeth were rinsed with sterile saline solution. Using ISO 45 no. H-file till working length dentine chips were obtained. With help of a sterile wire cutter, 5mm of file from tip was separated and placed in sterile test tubes containing TSB. Samples were incubated for 10 days to detect any growth. Turbidity of samples was assessed at day 3 and 7 [Table 3]. Gram staining and culture were done to assess the efficacy of medicaments [Figure 5],[Figure 6],[Figure 7].
Table 3: Mean values of Optical density/Turbidity *(P < 0.001) After placement of medicament in group A and B

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Figure 5: Gram Staining of Group A after placement of medicament

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Figure 6: Gram Staining of Group B after placement of medicament

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Figure 7: Gram Staining of Group C

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   Results Top

Data was analyzed statistically between groups A, B, C and D using ANOVA test. Groups A and B were compared using unpaired t test.

   Discussion Top

The current in vitro study revealed a superior disinfecting potential of a calcium hydroxide compared with bioactive glass. In the present study, it was difficult to standardize dentine yield as H-file was used to harvest dentin from the canal which produced disproportionate dentin chips, so instead of quantification of bacteria, change in turbidity of the broth was evaluated as growth or no growth. One visible characteristic of growing bacterial culture is the increase in cloudiness of the medium (turbidity). Turbidity measurements employ a variety of instruments like colorimeter to determine the amount of light scattered by a suspension of cells. Particulate objects such as bacteria scatter light in proportion to their numbers. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number. Optical density is a measure of the extent to which a substance transmits light. Darker the material, higher the density.

All specimens showed broth turbidity when infected with Enterococcus faecalis at day 7 and 14 respectively. Medicated samples (Group A & B) displayed reduction in turbidity as compared to GroupC. (positive control) indicating a good antimicrobial activity. Negative control (Group D) showed relatively less turbidity when compared with rest of specimens. Significant difference in antimicrobial efficacy of two medicaments was observed at day 3 (p<0.005). The results obtained after day 7, p > 0.005 between groups A & B showed no statistical difference. Incomplete eradication of Enterococcus faecalis by both the medicaments was found by day 7 confirming the hypotheses that alkaline biocides are less effective againstEnterococcus faecalis[5].

Enterococcus faecalis
was chosen as the test organism because it is associated with persistent apical inflammation and is cultivated from periapical lesions refractory to endodontic treatment (Engstrom 1964, Sundquist et al 1998, Sunde et al 2002). Enterococcus faecalis is resistant to alkaline biocides probably due to an effective proton pump mechanism which maintains optimal cytoplasmic pH levels (Evans et al 2002). Invitro studies show that Enterococcus faecalis invades dentinal tubules, colonizes root canal and survives without the support of other bacteria (Ψrstavik2004). Enterococcus faecalis is able to form a biofilm that helps it resist destruction, by enabling the bacteria to become 1000 times more resistant to phagocytosis, antibodies, and antimicrobials as compared to nonbiofilm producing organisms (Hatton et al 2002) [6] .

Calcium hydroxide is a potent antimicrobial agent but the action may not only be beneficial as the proteolytic action affects dentin stability (Andreasen et al 2002). Unlike calcium hydroxide, bioactive glass promotes Ca/P precipitation in its environment (Brunner et al 2006) and has substantial potential to remineralize dentine; this study found bioactive glass to be less effective than calcium hydroxide in eliminating the microorganism tested.

The antimicrobial activity of calcium hydroxide in suspension was related to quick deposit of hydroxyl ions (Proell et al 1949, Sjogren 1991), high pH : 12.5 12.8 (Estrela 1995) [8] and it also acted as a physical barrier for the bacteria to invade(Lopes et al 1999). The antimicrobial BAG properties have also been related to its ability to raise pH high and osmotic pressure (M.Zehnder et al 2008, Jukka et al 1998) [9],[10] and due to diffusion of Ca 2+ , Na + , Po4 3- and Si 4+ ions (Patricia 1998, Gubler et al 1998).

Just as Calcium hydroxide, Bioactive glass slowly but steadily releases alkaline species which interact with its environment hence there is a cumulative effect over time. Hence we need to leave alkaline biocides for a longer period in the canal for effective antimicrobial action.

   Conclusion Top

In this study it was shown that though both the medicaments reduced the growth of Enterococcus faecalis but could not completely eliminate it and bioactive glass was found to be less effective as compared to calcium hydroxide. So further search for other intracanal medicaments is required.[Table 4]
Table 4: Comparison between A and B

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   References Top

1.B Athanassiadis et al. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics .Aust. Dent. J 2007; 52(1Suppl):S64.  Back to cited text no. 1
2.H.K.Haapasalo & E.K.Siren et al. Inactivation of local root canal medicaments by dentine: an in vitro study.International Endodotic Journal 2000; 33,126-131.  Back to cited text no. 2
3.Patricia Stoor, Eva S. derling, Jukka Salonen. Antibacterial effects of a bioactive glass paste on oral microorganisms. Acta Odontologica Scandinavica, 1998;Vol.56(3),161-165.  Back to cited text no. 3
4.T.Waltimo et al. Antimicrobial Effect of Nanometric Bioactive Glass. J Dent Res 2007; 86(8):754.  Back to cited text no. 4
5.M.Gubler et al. Do bioactive glasses convey a disinfecting mechanism beyond a mere increase in pH? International Endodontic Journal 2008; 41, 670678.  Back to cited text no. 5
6.Hench, L. L., R. J. Splinter, W. C. Allen, and T. K. Greenlee. 1972. Bonding mechanism at the interface of ceramic prosthetic materials. J. Biomed. Mater. Res. 2:117141.  Back to cited text no. 6
7.Hench, L. L. 1998. Bioactive materials: the potential for tissue regeneration. J. Biomed. Mater. Res. 41:511518.  Back to cited text no. 7
8.P.C.Foreman & I.E.Barnes. A review of calcium hydroxide. International Endodontic Journal;1990,23:283-297.  Back to cited text no. 8
9.Charles H. Stuart, Scott A. Schwartz, Thomas J. Beeso, and Christopher B. Owatzx Enterococcus faecalis: It's Role in Root Canal Treatment Failure and Current Concepts in Retreatment. JOE 2006; 32(2): 93.  Back to cited text no. 9
10.Guven kayogule, Dag Ørstavik. Virulence factors of Enterococcus faecalis: relationship to endodontic disease Oral. Biol. Med. 2004; 15(5):30.  Back to cited text no. 10


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

  [Table 1], [Table 2], [Table 3], [Table 4]


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