INTRODUCTION Microorganisms which persists and multiply inside the root canal system is the main causative factor in pulpal and peri radicular lesions .Because endodontic infections are polymicrobial, many microorganisms take part in constitution ofbiolms and become more resistant to endodontic procedures. Candida albicans and Enterococcus faecalis are considered the most resistant species microorganisms which are responsible for root canal treatment failures(Hancock, Sigurdsson, Trope, & Moiseiwitsch, 2001) .Consequently, antimicrobial agents can be combined to be effective against these polymic robialora. Using this idea, different vehicles used to carry the medicaments can have adirect inuence on the release, time of onset of action of the medicament, penetration of the intracanal medicaments into dentinal tubules, and also the dissociation of drugs(Athanassiadis, Abbott, George, & Walsh, 2009). Calcium hydroxide(CH) is the most commonly used medicament in endodontics with signicant antibacterial effects on intracanal microorganisms. The efcacy of this material depends on the penetration ofhydroxyl ions into the dental tubules and accessory canals, where bacteria and their products accumulate. (Siqueira & Lopes, 1999), CH can prevent reinfection of the canal and impair nutritional supply of the residual microorganisms in the root canal system by forming a physical barrier. In order to have optimal efcacy in the root canal system, CH should be spread all over the canal walls to be in close contact with them(Haapasalo, Shen, Wang, & Gao, 2014). So calcium hydroxide powder is mixed with different vehicle for the continuous release of hydroxyl ions. So in this study we have combined CH with chlorhexidine, chitosan, glycerin which have proven antimicrobial action. Different studies have shown controversial results over the antimicrobial efcacy of calcium hydroxide mixed with CHX. Therefore, thepurpose of the present study was to assess antimicrobial activity of CH when mixed with different vehicles against E.faecalis and C. albicans by agar diffusion method and to examine the invitrosusceptibilityof these microorganisms to amixture of CH and CHX. MATERIALS AND METHODS This in vitro study was conducted in the department of Microbiology, Kannur medical college, Anjarakandy, Kannur. Calcium hydroxide(CH) powder was mixed with 2% chlorhexidine digluconate CHX (RC prep), glycerin (100%), 0.5% chitosan and saline to form a slurryat 1.5:1(vol/wt). Freshly prepared pastes of CH and a vehicle were used for each test. Standard E.faecalis (ATCC 29212) and C. albicans (ATCC 60193) strains were used for this study. In the present study CH+CHX(group1),CH+Glycerine (group2),CH+chitosan(group3) andCH+Saline(group 4)as the positive control group. AGAR DIFFUSION TEST Hundred microliters of test organisms E.faecalis and C albicans suspensions were obtained from prepared culture and inoculated in a culture plate with previously set layers of Muller Hinton Agar and Sabouraud dextrose agar IN VITRO STUDY OF ANTIMICROBIAL ACTIVITY OF CALCIUM HYDROXIDE MIXED WITH DIFFERENT VEHICLES AGAINST E.FAECALIS AND CANDIDA ALBICANS Original Research Paper Dr. Noushad M. C, Professor ,Department of conservative Dentistry and endodontics ,Anjarakandy, Kannur dental college, Anjarakandy, Kannur,Kerala X 81GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS Dental Science OBJECTIVE. : The purpose of this in vitro study was to investigate antimicrobial activity of calcium hydroxide (CH) in combination with chlorhexidine gluconate (CHX),glycerine, chitosan and saline against Enterococcus faecalis and Candida albicans. STUDY DESIGN: Antimicrobial activity was determined using agar diffusion test. Standard well in the cultivated agar plates were lled with one of the calcium hydroxide preparations and control agents. The zones of microbial inhibition were measured after incubation period. The combination of Calcium hydroxide with chlorhexidine demonstrated most antibacterial activity of all RESULTS. : other preparations. All the tested groups had signicant antimicrobial action against E faecalis and C albicans. CONCLUSION : Antimicrobial activity of Calcium hydroxide may change with the type of the vehicle. ABSTRACT KEYWORDS : Calcium Hydroxide, Chitosan, Chlorhexidine, Glycerine, Antimicrobial Activity, Vehicles Dr. Rakhi R* Senior lecturer, Department of conservative Dentistry and endodontics ,Anjarakandy, Kannur dental college, Anjarakandy, Kannur, Kerala *Corresponding Author Dr. Shaheen Abootty Senior Lecturer, Department Of Conservative Dentistry And Endodontics ,anjarakandy, kannur Dental College,anjarakandy,kannur,kerala Dr. Suneetha M. P Post graduate student, Department of conservative dentistry and endodontics, Kannur dental college,Anjarakandy, Kannur, Kerala Dr. Kavya Maheesan Post graduate student, Department of conservative dentistry and endodontics, Kannur dental college,Anjarakandy, Kannur, Kerala Dr Ashraf. K Post graduate student, Department of conservative dentistry and endodontics, Kannur dental college ,Anjarakandy ,Kannur ,Kerala VOLUME-8, ISSUE-11, NOVEMBER-2019 • PRINT ISSN No. 2277 - 8160 • DOI : 10.36106/gjra respectively for each organism. The strains were inoculated in brain heart infusion and incubated at 37°C for 24 hours. Microbial cells were resuspended in saline to give a nal 8concentration of 1.5 10 cells/ml, similar to that of tube #0.5 of the McFarland scale. For agar diffusion test, Petri plates with 20 ml Muller Hinton Agar (Merck) were inoculated with 0.1 ml of one of the microbial suspensions. Holes (4 mm in depth, 6 mm in diameter) were punched in the cultivated agar plates and lled with one of the CH preparations or control agents. Each agar plate contained only one medicament. Nystatin (antifungal; Mycostatin; Bristol-Myers Squibb, NJ) and cefotaxime (antibacterial; Fortum; GlaxoSmith Kline) were the control agents. The plates were re incubated aerobically at 37°C for 24 hours. Then the diameter of microbial inhibition zones around each well was measured and recorded in millimeters. Statistical analysis was performed with one way anova and post hoctukey test. RESULT Analysis of variance (one way anova) was performed as parametric test to compare different groups for both E faecalis and C albicans. Fig 1:Zone of inhibition Fig 2:Zone of inhibition formed against E faecalis formed against C albicans 82 X GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-8, ISSUE-11, NOVEMBER-2019 • PRINT ISSN No. 2277 - 8160 • DOI : 10.36106/gjra TABLE 1: one way anova with post hoc tukey test Graph 1: INHIBITON OF EACH AGENT AGAISNT E FAECALIS TABLE 2:ONE WAY ANOVA WITH POSTHOC TUKEY TEST AGAISNT CANDIDA ALBICANS Graph 2: INHIBITON OF EACH AGENT AGAISNT E FAECALIS Number of values Minimum 25% Percentile Median 75% Percentile Maximum Mean Std. Deviation Std. Error CHLORHEXIDINE-CH 5 16 16.25 17 17.75 18 17 0.7906 0.3536 GLYCERINE-CH 5 12.5 13.25 14.5 15 15 14.2 1.037 0.4637 CHITOSAN-CH 5 11 11.25 12 12.75 13 12 0.7906 0.3536 SALINE-CH 5 9 9.25 10 10.75 11 10 0.7906 0.3536 F 61.2 p value <0.0001 Number of values Minimum 25% Percentile Median 75% Percentile Maximum Mean Std. Deviation Std. Error CHLORHEXIDINE-CH 5 20 20.75 22 23.25 23.5 22 1.369 0.6124 GLYCERINE-CH 5 16.5 16.5 17 18 19 17.2 1.037 0.4637 CHITOSAN-CH 5 17 17.25 18 18.75 19 18 0.7906 0.3536 SALINE-CH 5 14 14.25 15 15.75 16 15 0.7906 0.3536 F 40.68 p value <0.0001 Utilizing abiocompatible intracanal medicament between appointments,help to diminish or eradicate bacteria in the root canal system and it enhance the success of root canal therapy(El Karim, Kennedy, & Hussey, 2007). As E. faecalis is the commonly seen organism in infected root canals ,it was chosen as the test organism. Virulency of E. faecalis in failed endodontically treated teeth may be related to its ability maintain the capability to invade dentinal tubules and adhere to collagen in the presence of human serum(Love, 2001).In addition to this, different fungal speciesare also given special attention in failure of endodontic treatments. Almost all the isolated species of fungi belong to the Candida clade, predominantly Candida albicans(Mozayeni, Hadian, Bakhshaei, & Dianat, 2015); which is the culprit responsible for development of pulpal and periapical infections.C.albicans promotes colonization the root canal by its collagenolytic activity and also it uses dentin as a nutrient source leading to its high virulence(Yadav, Chaudhary, Saxena, Talwar, & Yadav, 2017). Calcium hydroxide is the most commonly used intracanal medicament and its antibacterial effect is mainly due to thehigh PH by release of hydroxyl ions (Sjogren, Figdor, Spangberg, & Sundqvist, 1991).This hydroxyl ions diffuse through the dentin and reach sufcient levels to be lethal 3against the bacteria located inside the dentinal tubules . Because of the buffering property of dentin, pH value of Ca(OH) may be insufcient to kill some bacterial strains, 2 particularly E. faecalis, which can survive at a pH value of 11.5(Kim & Kim, 2014). Although calcium hydroxide can effectively eliminate most of the root canal pathogens, E. faecalis and C. albicans are resistant to calcium hydroxide according to TurkBT and BallalV et al(Turk, Sen, & Ozturk, 2009).Its resistance is due to the basic pH of calcium hydroxide,which in a basic medium ,proton pump activity occurs inside the microorg anismwhich acidies the environment and forms a biolm(Maekawa et al., 2013). Candida albicans is also resistant to calcium hydroxide. According to Ballal V et al CH showed higher efcacy against Candida albicans at the rst 24 hours and its effect was reduced after 72 hours. Its resistance mechanism has yet to be fully understood but it seems that C. albicans due to biolm formation has a strikingly biphasic killing pattern in response to antibacterial agent(Delattin, Cammue, & Thevissen, 2014). In this study all experimental groups were found to be effective against E faecalis and C albicans. Group1 CHX-CH combination showed the largest inhibition zone against E. faecalis and C.albicans compared to all other group. Chlorhexidine has a broad spectrum antimicrobial activity and substantivity effect, and its optimal antimicrobial activity is achieved at pH range of 5.5-7.0.(Athanassiadis et al) . Therefore it is likely that alkalinizing the pH by adding CH to CHX will lead toprecipitation of CHX molecules and thereby . decreases its effectiveness (Mohammadi & Abbott, 2009).But it has been demonstratedthat ,there is no change in alkalinity of CH when mixed with CHX. Nevertheless, CHX alone does not act as a physical barrier in solution or gel form, but combination of CHX with CH paste act as a barrier in the root canal system long enough to e l iminate exis t ing (microorganisms and to stop recontamination Ballal, Kundabala, Acharya, & Ballal, 2007)Another factor which explain antimicrobial synergism consist of production of more reactive oxygen species (ROS), by combination of CHX and CH.(Turk et al., 2009) Valera et al stated that although 2% CHX gel signicantly decreased the microbial count, intracanal medicaments including CH and CH+CHX completely eliminated the microorganisms from the canals(Pavaskar et al., 2012). Their ndings conrmed the synergistic effects of CH and CHX. However, Delgado et al ,found no signicant difference in terms of antibacterial properties of CHX gel with and without CH. Such controversy in the results may be attributed to the different culture techniques, root canal irrigating solutions, type of microorganisms and root canal anatomy.(Saatchi, Shokraneh, Navaei, Maracy, & Shojaei, 2014)Ina study by Lin et al. CHX alone and in combination withCH showed . antibacterial efcacy greater than that of CH alone (Saatchi et al., 2014) Study conducted by C. Maniglia-Ferreira et al on the evaluation of the antimicrobial effects of different intracanal medications in necrotic immature teeth showed that Ca(OH)2 paste in combination with 2 % CHX gel was devoid of any antimicrobial activity against E faecalis which is in contradiction to the present study(Maniglia-Ferreira et al., 2016).Systemic review and meta analysis by Saatchi et al showed thatCHX does not have synergetic antibacterial effect with CH. This may be due to deprotonation of CHX at high pH, which reduces its solubility and alters its interaction with bacterial surfaces as a result of the altered charge of the .( )molecule Saatchi et al., 2014 Chitosan is a natural unbranched homopolymer obtained from chitin, an abundant by-product of seafood processing, via a de acetylation reaction with alkali.Positivelycharged - +NH groupof glucosamine present in chitosan, interact with 3 negatively charged surface components of bacteria, resulting in extensive cell surface alterations, leakage of intracellular substances and ultimately causing damage of vital bacterial activities.(Raafat& Sahl, 2009) Chitosan binds to DNA and inhibits mRNAs ynthesis by penetrating toward the nuclei of microorganisms and interfering with the synthesisof mRNA andproteins .Consequently, it is possible that Ca(OH) 2 combined with chitosan inhibits the growth of E. faecalis and subsequently it may inhibit bacterial re-entry and recolonization(Elsaka& Elnaghy, 2012). A synergistic antibacterial effect was found in Ca(OH) combined with 2 chitosan against E. faecalis. Another tested vehicle was 100% glycerin which is a sweet, syrupy liquid obtained from animal fats and oils or by the fermentation of glucose. Glycerol consists of a propane molecule attached to three hydroxyl (OH) groups. Recently glycerine has been recommended for use as vehicle as they possess better therapeutic and handling properties. According to this study, glycerin-CH combinations had potential antifungal effects and antibacterial effect. This activity can be due to continued dissociation of CH by imbibitions of water into paste, by hygroscopic nature of glycerine thus ensuring a continued therapeutic effect. Sylvia et al have reported this combination have extensive gradual and sustained release of Ca and hydroxyl X 83GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-8, ISSUE-11, NOVEMBER-2019 • PRINT ISSN No. 2277 - 8160 • DOI : 10.36106/gjra Tukey's Multiple Comparison Test Mean Diff. q Signicant? P < 0.05? Summary 95% CI of diff CHLORHEXIDINE-CH vs GLYCERINE-CH 4.800 10.47 Yes *** 2.946 to 6.654 CHLORHEXIDINE-CH vs CHITOSAN-CH 4.000 8.729 Yes *** 2.146 to 5.854 CHLORHEXIDINE-CH vs SALINE-CH 7.000 15.28 Yes *** 5.146 to 8.854 GLYCERINE-CH vs CHITOSAN-CH -0.8000 1.746 No ns -2.654 to 1.054 GLYCERINE-CH vs SALINE-CH 2.200 4.801 Yes * 0.3459 to 4.054 CHITOSAN-CH vs SALINE-CH 3.000 6.547 Yes ** 1.146 to 4.854 DISCUSSION lion.Rivera and Williams showed that glycerin help in easy placement of CH in the root canals. According to Sudeep et al CH- glycerine combination showed a gradual increase in the zone of inhibition upto 7 days of incubation. The negative results which is obtained in other studies with glycerine may be due to their inability to diffuse through agar due to their viscous nature more than absence of any antimicrobial activity.(Liu, Wei, Ling, Wang, & Huang, 2010) Safavi and Nakayama concluded that higher concentrations of glycerin reduce the conductivity of CH that would decrease the antibacterial activity of CH which is in contrast to our 30study . Because of the pH of glycerin-CH combination would not be high enough to eliminate E. faecalis, because it could tolerate very high pH values with the durability of cell membrane and by using specic proton pumps and enzymatic systems. (Portenier, Waltimo, Orstavik, & Haapasalo, 2005)Unlike other invitro tests, Agar diffusion method, mainly depend upon the molecular size, solubility and diffusion of the materials through the aqueous agar medium, the sensitivity of the drug, bacterial source (wild strains or collection species), the number of bacteria inoculated, pH of the substrates in plates, agar viscosity, storage conditions of the agar plates, incubation time and the metabolic activity of the microorganisms. Therefore, the inhibition zones may be more related to the materials' solubility and diffusabilityin agar than to their actual efcacy against the microorganisms which might be a limitation in our study. CONCLUSION In summary the antimicrobial effect of Ca(OH) is related to 2 the hydroxyl ions released in an aqueous environment, which affects cytoplasmic membranes, proteins, and the DNA of microorganisms. 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J Clin Exp Dent, 9(3), e361-e367. doi:10.4317/jced.53210 84 X GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-8, ISSUE-11, NOVEMBER-2019 • PRINT ISSN No. 2277 - 8160 • DOI : 10.36106/gjra