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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 25  |  Issue : 5  |  Page : 504-509
Antibacterial efficacy of nisin, calcium hydroxide, and triple antibiotic paste in combination with chitosan as an intracanal medicament against Enterococcus faecalis – An in vitro study


Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India

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Date of Submission25-Feb-2022
Date of Decision15-May-2022
Date of Acceptance20-May-2022
Date of Web Publication12-Sep-2022
 

   Abstract 

Aims: Evaluate and compare antibacterial efficacy of nisin, calcium hydroxide (Ca (OH)2), and Triple antibiotic paste (TAP) mixed with distilled water and chitosan as an intracanal medicament on Enterococcus faecalis after 1st and 7th day.
Settings and Design: Experimental in vitro study.
Materials and Methods: One hundred and twenty Decoronated single-rooted premolars were prepared and autoclaved. Specimens were placed in brain heart infusion (BHI) broth of a 24 h old E. faecalis (ATCC 29212) suspension. The canal was infected for 21 days, followed by grouping as-Group 1: Nisin + distilled water; Group 2: Nisin + 2% chitosan; Group 3: Ca (OH)2 + distilled water; Group 4: Ca (OH)2 + 2% chitosan; Group 5: TAP + distilled water; Group 6: TAP + 2% chitosan and were incubated for 1 and 7 days. Dentin scrapings from 10 specimens at each time interval were transferred into BHI broth and incubated, Serially diluted and colony-forming unit (CFU) count was assessed after 1st and 7th day.
Statistical Analysis Used: Kruskal–Wallis test to compare mean CFUs after day 1 and day 7. Mann–Whitney post hoc Analysis to estimate significance. Wilcoxon Signed-Rank Test to compare mean CFUs between day 1 and day 7.
Results: After 1 day, Group 6 showed least CFUs than other groups and highest CFUs was with Group 3. There was no difference statistically when Group 5 and Group 2 were compared. After 7 days, Group 6 showed least CFUs than other groups with no significant difference between Group 1 and Group 5 as well as no significant difference between Group 2, Group 5 and Group 6.
Conclusions: TAP with chitosan can be the medicament of choice for 1-day placement and was equivalent to nisin with chitosan combination after 7 days.

Keywords: Calcium hydroxide; Enterococcus faecalis; intracanal medicament; nisin, triple antibiotic paste

How to cite this article:
Harshitha V S, Ranjini M A, Nadig RR. Antibacterial efficacy of nisin, calcium hydroxide, and triple antibiotic paste in combination with chitosan as an intracanal medicament against Enterococcus faecalis – An in vitro study. J Conserv Dent 2022;25:504-9

How to cite this URL:
Harshitha V S, Ranjini M A, Nadig RR. Antibacterial efficacy of nisin, calcium hydroxide, and triple antibiotic paste in combination with chitosan as an intracanal medicament against Enterococcus faecalis – An in vitro study. J Conserv Dent [serial online] 2022 [cited 2022 Sep 25];25:504-9. Available from: https://www.jcd.org.in/text.asp?2022/25/5/504/355898

   Introduction Top


The main cause of apical periodontitis with endodontic origin is the presence of microbes in root canal system. Treatment outcome of endodontic infections depends on effective eradication of causative micro-organisms. Certain microbes cannot be completely eliminated by cleaning and shaping procedures and survives within anatomical complexities posing clinical challenge.[1] Enterococcus faecalis is seen in persistent periapical lesions due to its ability to invade and live within dentinal tubules to variable depth.[2] To ensure complete elimination of root canal bacteria, effective antimicrobial agents are required for a predetermined time period.[3]

Commonly used medicament is calcium hydroxide (Ca (OH)2). It acts by providing initial bactericidal effect then bacteriostatic, neutralizes low pH of acids, inexpensive and easy to use.[4] However, many studies report it to be ineffective against E. faecalis, organism that can tolerate a variety of growth conditions. Further, low solubility and diffusibility of (Ca (OH)2 might make it difficult to penetrate dentinal tubules to exert any action.[5]

Triple antibiotic paste (TAP) (metronidazole, ciprofloxacin, and minocycline), successfully used intracanal medicament (ICM) has proven its effectiveness against E. faecalis. Although TAP has been most successful in achieving root canal disinfection, discoloration of crown was disadvantage reported.[6] Therefore, determining if experimental medicament alone or combination can have a beneficial outcome with less disadvantage seems to be clinically valuable.

Nisin is antimicrobial agent, highly active against Gram-positive bacteria and their spores even against multidrug-resistant E. faecalis isolates. The pH value in the environment influences antibacterial activity and stability of nisin and it has high antibacterial activity and stability at low pH values.[7] However, Guiga et al. showed solubility and stability of nisin reduced its antibacterial efficacy at pH above 4.[8] This necessitates the addition of another chemical to have prolonged action.

Distilled water, the most common vehicle lack antimicrobial property.[9] In recent years, chitosan is evaluated for its possible use as vehicle in endodontics. It is aminopolysaccharide, produced by partial deacetalylation of chitin. The most important properties of chitosan are high bioactivity, biocompatibility, hydrophilicity, chelating, and antibacterial. It has shown to be effective against several oral microorganisms, such as Candida albicans, Streptococcus mutans, and E. faecalis. Several evidences have indicated the role of chitosan as an ICM.[10],[11]

To increase the stability and insolubility of nisin as ICM, chitosan can be tried as vehicle that can have added advantage of slow and controlled release of ICM in short time. Moreover, this combination of nisin with chitosan, its dental application has not been fully exploited.

Hence, this study was performed to evaluate and compare antibacterial efficacy of experimental combination against E. faecalis.


   Materials and Methods Top


Tooth preparation

One hundred and twenty single-rooted premolar teeth were decoronated at cementoenamel junction to length of 15 mm. 15k file was used to scout, establish patency till tip of file is seen at apical foramen, from which 1 mm was subtracted to establish a working length. Samples were instrumented with ProTaper rotary system up to size F3. 3% NaOCL was used as irrigant during instrumentation followed by 17% Ethylenediaminetetraacetic acid as final rinse. Roots were sterilized in autoclave at 121°C for 15 min and subjected to root canal infection.[5]

Root canal infection with Enterococcus faecalis

The test organism used for this study is E. faecalis (ATCC 29212), which was grown in brain heart infusion (BHI) agar for 24 h. Each tooth specimen was placed in presterilized microcentrifuge tubes containing 2 mL BHI broth with 1 × 107 colony-forming units per mL (CFU/mL) of a 24-h old E. faecalis suspension. Root canal infection was carried out for 21 days.[5]

Preparation and formulation of medicaments

  1. NISIN: Commercial nisin powder (Freda Biotechnology Co., Ltd. India) was dissolved in each vehicle to concentration of 100 mg/mL to paste
  2. Ca (OH)2: Commercial Ca (OH)2 powder (Deepti dental, India) was dissolved in each vehicle to concentration of 100 mg/mL to paste
  3. TAP: Coatings of antibiotic ciprofloxacin (Ciplox 500 mg, Cipla, India), metronidazole (Metrogyl 400 mg, J. B. Chemicals Ltd., India), and minocycline (Minoz 100 mg, Cipla, India) tablets were removed and crushed using mortar and pestle. Crushed powder was passed through fine sieve to obtain fine powder, weighed separately, and mixed in a 1:1:1 proportions respectively, to obtain mixture. Prepared TAP powder was dissolved in each vehicle to concentration of 100 mg/mL to paste
  4. Two percent CHITOSAN GEL: 2 g of chitosan (85% degree of deacetylation) was diluted with 100 ml of 1% acetic acid and mixture was stirred for 2 h using magnetic stirrer.


    • Group 1: Nisin + Distilled Water
    • Group 2: Nisin + Chitosan
    • Group 3: Ca (OH)2 + Distilled Water
    • Group 4: Ca (OH)2 + Chitosan
    • Group 5: TAP + Distilled Water
    • Group 6: TAP + Chitosan.


Medicaments mixed to paste consistency were placed into canals using lentulo spirals and sealed coronally and apically with cavit. Specimens were placed into petri dishes and covered with humid sterile gauzes and incubated at 37°C for 1 and 7 days. Root canals were irrigated with saline and dried with sterile paper points. Harvesting of dentin was performed at two depths (200 μm and 400 μm) with gates Glidden drills numbers 4 and 5, respectively, with 10 specimens at each time interval. Collected dentin scrapings were transferred into 1 ml sterile BHI broth and incubated at 37°C for 24 h. After the incubation period, content of each tube was serially diluted, five times with 100 μl broth in 100 μl sterile saline. Fifty microliter of dilution was plated on BHI agar plates and incubated for 24 h. Colonies were counted and total number of CFUs per milliliter was calculated. The number of CFUs of E. faecalis served as measure of antibacterial activity. Data were statistically analyzed.[5]


   Results Top


The level of significance (P Value) was set at P < 0.05.

CFUs for different medicaments were obtained after day 1 and day 7.

Comparison of mean CFUs after day 1 and day 7 was made by Kruskal–Wallis test followed by Mann–Whitney post hoc Analysis [Table 1] and [Table 2] to estimate significance of use of various medicaments.
Table 1: Mean colony-forming units and multiple comparison of different study groups on day 1 using Tukey's post hoc analysis

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Table 2: Mean colony-forming units and multiple comparison of different study groups on day 7 using Tukey's post hoc analysis

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A comparison of mean CFUs between day 1 and day 7 in each study group was made using Wilcoxon Signed-Rank Test [Table 3] and [Graph 1].
Table 3: Comparison of mean colony-forming units between Day 1 and Day 7 in each study group using Wilcoxon signed rank test

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[Table 1] shows mean CFUs and multiple comparison of different study groups on day 1.

TAP + 2% Chitosan group showed lesser mean CFUs as compared to others P < 0.001. This was statistically significant, except for Nisin + 2% Chitosan (P = 0.003) and TAP + Distilled Water (P = 0.08) implying there was not much difference between these groups against E. faecalis.

Followed by TAP + Distilled Water group showing lesser mean CFUs compared to other groups at P < 0.001 which was statistically significant, except for Nisin + 2% Chitosan (P = 0.31), having no significant difference. Therefore, TAP + distilled water and nisin + 2% chitosan showed a similar effect.

Nisin + 2% Chitosan group showed lesser mean CFUs as compared to other remaining study groups at P < 0.001 which was statistically significant. Nisin + Distilled Water group showed lesser Mean CFUs as compared to Ca (OH) 2 + Distilled Water group and Ca (OH) 2 + 2% Chitosan groups at P < 0.001 and P = 0.001 respectively. Finally, Ca (OH) 2 + 2% Chitosan group also showed lesser mean CFUs as compared to Ca (OH) 2 + Distilled Water group at P = 0.01 [Table 1].

[Table 2] shows mean CFUs and multiple comparison of different study groups on day 7.

TAP + Chitosan group showed least mean CFUs as compared to other groups at P < 0.001 which was statistically significant, except TAP + Distilled Water (P = 0.03) and with no significance with Nisin + Chitosan (P = 0.36), implying there was not much difference between these groups against E. faecalis. Followed by Nisin + Chitosan group that showed lesser mean CFUs as compared to Ca (OH)2 + Distilled Water and Ca (OH)2 + Chitosan groups at P < 0.001 which was statistically significant. But no significant difference was found with TAP groups implying nisin with chitosan group was comparable with TAP group with both vehicles (distilled water and 2% chitosan) after 7 days of testing.

TAP + Distilled Water group showed lesser CFUs as compared Ca (OH)2 + Distilled Water group and Ca (OH)2 + Chitosan groups at P < 0.001 which was statistically significant. Followed with Nisin + Distilled Water showing significantly lesser CFUs as compared Ca (OH)2 + Distilled Water group and Ca (OH)2 + Chitosan groups at P < 0.001 and P = 0.003 respectively. And finally, Ca (OH)2 + Chitosan group showed lesser mean CFU as compared to Ca (OH)2 + Distilled Water group at P = 0.004. However, no significant difference was observed between Nisin + Distilled Water and TAP + Distilled Water (P = 0.14) and also between Nisin + Chitosan and TAP + Distilled Water (P = 0.15) [Table 2].

[Table 3] shows comparison of mean CFUs between day 1 and day 7 in each study group.

All medicament groups showed lesser mean CFUs at day 7 as compared to day 1 at P < 0.001. This was statistically significant. The above value of CFU shows effect of these medicament improved with time. TAP + Distilled Water showed a relative reduction in mean CFUs at day 7 as compared to day 1 and similarly TAP + Chitosan group showed relative reduction of mean CFUs at day 7 as compared to day 1. However, no significant reduction was observed in both TAP groups [Table 3] and [Graph 1].


   Discussion Top


E. faecalis is one of the species widely studied for its capacity to survive in the most unfavorable conditions. Popular ICM in endodontic treatment is Ca (OH)2 which has low solubility in water making environment alkaline. pH of Ca (OH)2 pastes used in endodontics is 12.3, this value drops to 7.8 in 15 days because of buffering effect of dentin, making it not high enough to kill E. faecalis.[12] To sustain high ph-value for prolonged time, combination of Ca (OH)2 pastes with different vehicles has been suggested.

An in vitro study, with chitosan as vehicle for Ca (OH)2, presented alkaline pH of 10.32 maintained for 1 month and was significantly higher than distilled water.[13] Hence, this combination was included in the present study and compared with distilled water as vehicle. Nisin was chosen for its antimicrobial property.

TAP (metronidazole, ciprofloxacin, and minocycline) with normal saline can remove bacteria from deep areas such as 200 μm from dentinal tubules.[14] However coronal discoloration is commonly reported following treatments using TAP.[14] Therefore, traditionally used TAP in combination with chitosan is tested and compared in the present study.

This study aimed at evaluating experimental medicaments after 1 day to check the possibility of having better antibacterial efficacy than medicament suggested to be used for more than week period (>7 days). Also, prolonged duration of medicament in canal can weaken their effectiveness.

The present study emphasized the importance of time duration on the effectiveness of antimicrobial activity. Mean CFUs of E. faecalis reduced with medicament retained in canals for 7 days than after 1 day.

TAP-TAP was superior medicament compared to others at both intervals. Among vehicles used, TAP with 2% chitosan showed the highest effectiveness than its distilled water group and other combinations used. This can be substantiated by Kotadia et al.'s study who reported that TAP + 2% chitosan produced significantly better and faster wetting of root canal walls compared to Ca (OH)2 + 2% chitosan which could be attributed to its decreased particle size in comparison to Ca (OH)2 which enables it to take up more amount of solvent and thereby show superior flow providing synergistic effect.[3],[15]

Nisin-nisin with distilled water had less CFUs compared to Ca (OH)2 with distilled water on both intervals because the mode of action of nisin differs to that of Ca (OH)2 and is not reliant on highly alkaline environment for effective killing. This is in agreement with previous study which examined nisin and Ca (OH)2 against E. faecalis where nisin group showed significantly lower CFU than Ca (OH)2 group.[16],[17] Moreover, a study which explained as the presence of proton pump as a primary resistance mechanism of E. faecalis for higher CFU in Ca (OH)2 group while nisin was tested showed none of CFU.[18] Another study showed use of nisin as ICM for 1 week resulted in lesser reduction in microhardness of root dentin compared to Ca (OH)2.[19] Thus, nisin may be a better material for use as an ICM to eradicate E. faecalis from root canals.

Two percent Chitosan vehicle medicament achieved higher antimicrobial efficacy which is in agreement with findings of Kristl et al.'s study which determined the efficacy of chitosan gels as drug carriers and drug release was slow and sustained.[20] In our study similarly, the additive effect between tested medicament TAP, nisin, and Ca (OH)2 with 2% chitosan vehicle could be reason for enhanced antibacterial activity against E. faecalis on both tested times. Distilled water vehicle provided an alkaline pH but only for a short duration of time.

Ca (OH)2-Chitosan vehicle exhibited controlled release of calcium ions from Ca (OH)2 for prolonged duration and with time reduced CFU (by 7 days).[13],[14] It was shown in an in vitro study, the ability of chitosan to retain high amounts of water as a property, which could be of particular value in relation to slow-release formulations.[13]

Limitations of this study are TAP containing minocycline and 2% chitosan vehicle used have chelating property. This effect of combined ICM on root dentin microstructure is not known. As Chitosan has mucoadhesive property, whether it has a similar adhesive property to root canal dentin needs to be investigated because this property might help in prolonged action of medicament within root canal system.


   Conclusion Top


Antibacterial effect of medicaments tested in the study was enhanced when combined with chitosan indicating chitosan when used as a vehicle provides an additive action.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Adl A, Hamedi S, Sedigh Shams M, Motamedifar M, Sobhnamayan F. The ability of triple antibiotic paste and calcium hydroxide in disinfection of dentinal tubules. Iran Endod J 2014;9:123-6.  Back to cited text no. 14
    
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Correspondence Address:
Dr. V S Harshitha
Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcd.jcd_125_22

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