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Journal of Indian Society of Pedodontics and Preventive Dentistry Official publication of Indian Society of Pedodontics and Preventive Dentistry
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Year : 2018  |  Volume : 36  |  Issue : 3  |  Page : 268-272

Efficacy of various intracanal medicaments against aerobic and facultative anaerobic microorganism found in human primary teeth with necrotic pulp: A randomized clinical trial

1 Department of Pedodontics, MES Dental College, Malappuram, Kerala, India
2 Department of Oral Surgery, MES Dental College, Malappuram, Kerala, India
3 Department of Pedodontics, Government Dental College, Thiruvananthapuram, Kerala, India
4 Department of Microbiology, Government Medical College, Kozhikode, Kerala, India
5 Department of Pedodontics, Government Dental College, Kozhikode, Kerala, India

Date of Web Publication24-Sep-2018

Correspondence Address:
Dr. Jophie Varghese Paikkatt
Department of Pedodontics, MES Dental College, Perinthalmanna, Malappuram - 679 338, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JISPPD.JISPPD_152_17

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Aim: The aim of this in vivo study is to evaluate the antimicrobial efficacy of calcium hydroxide (Ca(OH)2), chlorhexidine (CHX), and metronidazole gel as intracanal medicaments against aerobic and facultative anaerobic microorganisms found in root canals of human primary teeth with necrotic pulp. Setting and Design: It is a double-blinded randomized clinical trial. Materials and Methods: Pulp canals of 45 single-rooted primary maxillary anterior teeth with pulp necrosis in 34 children were included in the study. They were divided into three groups of 15 samples each: Group I: Ca(OH)2; Group II: 1% CHX gel; and Group III: 1% metronidazole gel. Microbial count was obtained from each tooth at two different stages – (1) after instrumentation and (2) after placement of the medication. Statistical analysis using the SPSS 10.0 software program (SPSS Inc., Chicago, IL, USA) with Wilcoxon signed-rank test after grouping the samples was performed. Results: Ca(OH)2, 1% CHX gel, and 1% metronidazole gel were ineffective in completely eliminating aerobic and facultative anaerobic microorganism from root canal of human primary teeth with necrotic pulp. Conclusion: None of the commonly used intracanal medicaments, that is Ca(OH)2, 1% CHX gel, and 1% metronidazole gel, was effective in completely eliminating aerobic and facultative anaerobic microorganism from root canal of human primary teeth with necrotic pulp. Ineffectiveness of these medicaments against aerobic and facultative anaerobic microorganism has opened new door of research regarding the manner of bacterial growth in unfavorable environmental and nutritional conditions, the way root canal environment favors biofilm formation and the use of suitable intracanal medicaments against single and multispecies biofilms.

Keywords: Aerobic, calcium hydroxide, chlorhexidine, facultative anaerobic, metronidazole

How to cite this article:
Paikkatt JV, Aslam S, Sreedharan S, Philomina B, Kannan V P, Madhu S. Efficacy of various intracanal medicaments against aerobic and facultative anaerobic microorganism found in human primary teeth with necrotic pulp: A randomized clinical trial. J Indian Soc Pedod Prev Dent 2018;36:268-72

How to cite this URL:
Paikkatt JV, Aslam S, Sreedharan S, Philomina B, Kannan V P, Madhu S. Efficacy of various intracanal medicaments against aerobic and facultative anaerobic microorganism found in human primary teeth with necrotic pulp: A randomized clinical trial. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2023 Feb 1];36:268-72. Available from: http://www.jisppd.com/text.asp?2018/36/3/268/241961

   Introduction Top

The presence of microorganisms and their toxins in the infected root canal results in apical periodontitis.[1] Several studies have shown that endodontic infection is polymicrobial[2],[3] and that in necrotic pulp it is represented by obligatory and facultative anaerobes, microaerophilic bacteria, and fungi. Even though obligatory anaerobes, particularly black pigmented Gram-negatives, produces signs and symptoms, aerobes and facultative anaerobes, such as Enterococcus, Candida, and alpha Streptococcus, though in low proportion are considered one of the most resistant species and possible cause of root canal treatment failures.[4] Hence, the treatment of apical periodontitis should also aim at eradication of these aerobic and facultative anaerobic microorganisms. Because instrumentation and irrigation do not reliably eliminate bacteria,[5] the need of using different intracanal medicaments becomes logical. Among the medicaments available, calcium hydroxide (Ca(OH)2), 1% chlorhexidine (CHX) gel, and 1% metronidazole gel were selected for the study.[6] Medication was selected in gel form as they have both antimicrobial and lubricating action during instrumentation.[7]

Ca(OH)2 is the “Gold Standard” endodontic medicament used widely to eliminate microbes that survive instrumentation.[8],[9] CHX is an alternate medicament used in endodontic practice as an effective antimicrobial means for disinfecting the root canal.[7],[9] Despite its promising results from in vitro studies, there are not enough in vivo studies published so far on the effectiveness of the above medicaments against aerobic microorganism.

The objective of this clinical study was to evaluate the antimicrobial efficacy of Ca(OH)2 paste, 1% CHX gel, and 1% metronidazole gel against aerobic and facultative anaerobic microorganism found in root canal of primary teeth with necrotic pulp. As the percentage of aerobic microorganisms in tooth with necrotic pulp is in low proportion, a total count in colony forming unit (CFU) of the following pathological microorganisms, that is, Enterococcus, alpha Streptococcus, and Candida, was taken to make the study more reliable.

   Materials and Methods Top

Eligible participants for this double-blinded randomized clinical trial were selected from patients of both gender aged 4–6 years that had been referred for dental treatment at Pediatric Dentistry clinic. The 34 patients who met all of these inclusion criteria were enrolled providing a total sample of 45 teeth.

Inclusion criteria

The inclusion criteria were as follows: Healthy cooperative children in the age group of 4–6 years who had at least one primary maxillary anterior teeth with confirmed pulpal necrosis owing to caries (with or without periapical lesion), but with sufficient coronal structure to permit isolation of operative field with rubber dam, less than two-third of root resorption, presence or not of fistula, mobility degree 0 or 1, and no periodontal pocket. If present, the periapical lesion should not be invading the follicles of germ of permanent successor.

Exclusion criteria

Patients with underlying systemic conditions and special health care needs and patients giving positive history of antibiotics use within the last 1 month were excluded from the study.

The study purposes were fully explained to parents/guardians, who signed a written informed consent form after getting the verbal consent from the children. The research protocol was received and approved by Institutional Research Ethics.

Medicaments tested

  1. Ca(OH)2 paste (RC Cal, Prime Dental Product, India)
  2. 1% CHX gel (Hexigel, ICPA Ltd, India)
  3. 1% metronidazole gel (Metrogyl DG gel, Unique Pharmaceuticals, India).

Method of the study

For every appointment, three teeth were selected who met the inclusion criteria. The three maxillary incisors were divided into three experimental groups as described below:

  • Group I: Ca(OH)2 paste
  • Group 2: 1% metronidazole gel
  • Group 3: 1% CHX gel.

After isolation with a rubber dam, the teeth were disinfected with 30% hydrogen peroxide and 10% tincture iodine.[10] The pulp chambers were opened using aseptic conditions. Airotor with sterile cooled water and high-speed diamond round burs was used. After confirming the working length, instrumentation was performed 1 mm above the apices with Hedstroem (H) files, up to size 50. After the mechanical preparation and irrigation, a sterile, size 20 paper point was introduced into the length of the root canals for the initial microbiological sampling. The paper point was placed for 60 s in the canal and then immediately transferred to a sterile test tube. The test tubes containing paper point sample were taken to the Department of Microbiology for processing within 1 h.[11]

After collecting the sample, the root canals were dried and medicaments placed depending on the group. All medicaments were applied with a syringe and a 26-gauge needle. Subsequently, a sterile cotton pellet was placed at the entrance, and the cavities were temporarily sealed with zinc oxide eugenol cement.

For microbiological procedures, the paper point samples were rolled in blood agar plates, and the plate was incubated in an incubator for 48 h at 37°C. Enterococcus was seen as small translucent colonies with alpha hemolysis, as pair of oval cocci, the cells in pair arranged at an angle to each other. Candida was seen as a nonhemolytic dry white colony oval budding yeast 2–6 μm in diameter; alpha Streptococcus was seen as long chain, the colony morphology was studied, and a total count of Enterococcus, alpha Streptococcus, and Candida was determined in CFU depending on growth on number of streaks. A majority opinion from the microbiology panel of Government Medical College Kozhikode was taken as result. The growth was further confirmed by Gram staining. To evaluate the effect of the medicaments against aerobic microorganism, the patients were recalled after 2 weeks.[7] After irrigation with saline, a second microbiological sampling was carried out with sterile paper points in the root canals as described earlier.

After the microbiological sampling, all the root canals were filled with zinc oxide eugenol paste and all treated teeth were given fiber-reinforced post and crown.

Statistical analysis was carried out using the Statistical Package for the Social Sciences (SPSS) 10.0 software program (SPSS Inc., Chicago, IL, USA) with Wilcoxon signed-rank test after grouping the samples.

   Results Top

A total of 45 teeth were studied. They included three groups of 15 teeth each. Group I was medicated with Ca(OH)2, Group II with 1% CHX gel, and Group III with 1% metronidazole gel.

[Table 1] outlines that Ca(OH)2, 1% CHX gel, and 1% metronidazole gel has no statistically significant score on aerobic and facultative anaerobic microorganisms.
Table 1: Effect of various medicaments on aerobic and facultative anaerobic microorganisms

Click here to view

After taking the average count of aerobic and facultative anaerobic microorganisms in CFUs just before and after various medicaments, [Graph 1] shows that none of the commonly used medicaments, that is, Ca(OH)2, 1% CHX gel, or 1% metronidazole gel, was able to completely eradicate aerobic and facultative anaerobic microorganisms from the root canal system.

   Discussion Top

Aerobic and facultative anaerobic microorganism plays a major role in change of environment conditions, particularly in regard to oxygen tension and nutrient availability, and also the role of Enterococcus and Candida in endodontic treatment failures is well established.[12],[13] The antimicrobial effectiveness of 1% CHX gel, Ca(OH)2, and 1% metronidazole gel is well investigated in vitro.[7],[8],[9] It appeared that they have wide antimicrobial activity against microorganisms isolated from root canal system. However, the results found from in vitro study cannot be directly extrapolated to in vivo study because of the optimum contact between medication and dentin under in vitro condition and because of variable imposed by clinical situation and not reproduced under experimental model. Hence, the present in vivo study was undertaken after taking into consideration the above objective.

Aerobic and facultative anaerobic microorganisms in nature rarely exist in planktonic state but are organized in biofilm structures, which form a complex community, composed of great variety of organisms with different ecological requirements and pathogenic potentials. Biofilm growth is a continuous process which goes through various stages from young to mature, structurally complex biofilm.[14] In necrotic pulp with harsh ecological milieu, it is likely that the physiological state of biofilm is closest to mature, hence making it resistant to various antimicrobial agents. The protective mechanism underlying biofilm antimicrobial resistance is not fully understood, although several mechanisms have been proposed.[15] These mechanisms include physical or chemical diffusion barrier to antimicrobial penetration into the biofilm, slow growth of biofilm owing to nutrient limitation, activation of general stress response, and emergence of biofilm-specific phenotype.[16]

CHX is a cationic bisguanide with broad antimicrobial activity, low mammalian toxicity, and strong affinity to binding to dentin and mucous membrane. The CHX molecules react with negatively charged groups on the cell surface, causing an irreversible loss of cytoplasmic constituents, membrane damage, and enzyme inhibition. It is likely that ionic interaction occurs between the positively charged CHX molecules and negatively charged extracellular matrix. This ionic interaction is understood to reduce the diffusion of fluorescent probes within biofilms about 50 folds, thus explaining the possible mechanism of aerobic and facultative anaerobic microorganism resistant to 1% CHX gel.[17]

As seen in previous studies, Enterococcus and Candida were resistant to Ca(OH)2, Its inability to completely eradicate Enterococcus and Candida from the root canal system is due to the buffering nature of root dentin which prevents attaining a high alkalinity to kill. The proton pump provides a means of maintaining pH homeostasis for Enterococcus.[18],[19]

Metronidazole is bactericidal against most anaerobes that contain electron transport components, such as ferrodoxin, which donates electron to metronidazole, forming highly reactive nitro radical anions that kill susceptible organisms by a radical mediated mechanism. About 1% metronidazole gel in this study has not been effective as it is active against strict anaerobes but is ineffective against facultative anaerobes.[20],[21][22]

   Conclusion Top

On the basis of observations made during the present study, the following conclusions were drawn.

First, Ca(OH)2, 1% CHX gel, and 1% metronidazole gel, which are commonly used intracanal medicaments, were found to be ineffective in completely eliminating aerobic and facultative anaerobic microorganisms from root canals of primary teeth with necrotic pulp. Second, ineffectiveness of these medicaments has opened new door of research regarding the use of intracanal medicaments on single and multispecies biofilms.


The authors deny any conflicts of interest related to this study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Mohammadi Z. Antibiotics as intracanal medicaments: A review. J Calif Dent Assoc 2009;37:98-108.  Back to cited text no. 1
Ferrari PH, Cai S, Bombana AC. Effect of endodontic procedures on enterococci, enteric bacteria and yeasts in primary endodontic infections. Int Endod J 2005;38:372-80.  Back to cited text no. 2
Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340-9.  Back to cited text no. 3
Pinheiro ET, Gomes BP, Ferraz CC, Teixeira FB, Zaia AA, Souza Filho FJ, et al. Evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility. Oral Microbiol Immunol 2003;18:100-3.  Back to cited text no. 4
Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8.  Back to cited text no. 5
Walton RE. Intracanal medicaments. Dent Clin North Am 1984;28:783-96.  Back to cited text no. 6
Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007;52:S64-82.  Back to cited text no. 7
de Rezende GP, da Costa LR, Pimenta FC, Baroni DA. In vitro antimicrobial activity of endodontic pastes with propolis extracts and calcium hydroxide: A preliminary study. Braz Dent J 2008;19:301-5.  Back to cited text no. 8
Basrani B, Ghanem A, Tjäderhane L. Physical and chemical properties of chlorhexidine and calcium hydroxide-containing medications. J Endod 2004;30:413-7.  Back to cited text no. 9
Möller AJ. Microbiological examination of root canals and periapical tissues of human teeth. Methodological studies. Odontol Tidskr 1966;74:Suppl:1-380.  Back to cited text no. 10
Forbes AB, Daniel FS, Weissfeld AS. Upper respiratory infection and other infections of oral cavity and neck. In: Bailey RW, Scott EG, editor. Textbook of Diagnostic Microbiology. 11th ed. Missouri: Mosby Publishers; 2002. p. 903.  Back to cited text no. 11
Brook I. Microbiology and management of endodontic infections in children. J Clin Pediatr Dent 2003;28:13-7.  Back to cited text no. 12
Pazelli LC, Freitas AC, Ito IY, Souza-Gugelmin MC, Medeiros AS, Nelson-Filho P, et al. Prevalence of microorganisms in root canals of human deciduous teeth with necrotic pulp and chronic periapical lesions. Pesqui Odontol Bras 2003;17:367-71.  Back to cited text no. 13
Stewart PS, Rayner J, Roe F, Rees WM. Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates. J Appl Microbiol 2001;91:525-32.  Back to cited text no. 14
Mah TF, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001;9:34-9.  Back to cited text no. 15
Stewart PS. Diffusion in biofilms. J Bacteriol 2003;185:1485-91.  Back to cited text no. 16
Gulot E, Georges P, Brun A, Fontaine-Aupart MP, Bellon-Fontaine MN, Briandet R, et al. Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation. Photochem Photobiol 2002;75:570-8.  Back to cited text no. 17
Evans MD, Baumgartner JC, Khemaleelakul SU, Xia T. Efficacy of calcium hydroxide: Chlorhexidine paste as an intracanal medication in bovine dentin. J Endod 2003;29:338-9.  Back to cited text no. 18
Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267-75.  Back to cited text no. 19
Siqueira JF Jr., de Uzeda M. Intracanal medicaments: Evaluation of the antibacterial effects of chlorhexidine, metronidazole, and calcium hydroxide associated with three vehicles. J Endod 1997;23:167-9.  Back to cited text no. 20
Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive glass when compared with calcium hydroxide as intracanal medicaments. J Endod 2007;33:1473-6.  Back to cited text no. 21
McDonald JH, editor. Handbook of Biological Statistics, 3rd ed. Maryland: Sparky House Publishers;2 014. p.186-9.  Back to cited text no. 22


  [Table 1]

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