Dentinal tubule invasion and adherence by Enterococcus faecalis

Aim To investigate dentinal tubule invasion and the predilection of Enterococcus faecalis for dentinal tubule walls. Methodology The invasion of dentinal tubules in extracted human teeth by E. faecalis was measured ex vivo after 8 weeks of incubation. The canal walls of 16 root sections were either intact or instrumented with or without smear layer present. Extent and maximum depth of tubule invasion were assessed histologically and compared between groups. In the adherence study, 44 vertically split root samples were prepared to expose longitudinally aligned dentinal tubules and fractured orthodentine (OD). Surfaces were exposed to E. faecalis (erythromycin resistant strain, JH2‐2 carrying plasmid pGh9:ISS1) and incubated aerobically for 2 h. Samples were processed for analysis using scanning electron microscopy. Bacterial adhesion to tubule walls versus fractured OD was calculated as number of cells per 100 μm². Results The strain of E. faecalis used in this study showed moderate to heavy tubule invasion after 8 weeks. In the adhesion studies, significantly more bacteria adhered to fractured OD than to dentinal tubule walls (anova , P < 0.001). With respect to the tubule wall, adherence was greater in inner versus outer dentine (P = 0.02) and greater when bacterial adhesion was tested in chemically defined medium than in phosphate‐buffered saline (anova , P < 0.001). Conclusions Although E. faecalis readily invaded tubules, it did not adhere preferentially to tubule walls. Initial colonization of dentinal tubules by E. faecalis may depend primarily on other factors.     

Effect of the quorum‐sensing luxS gene on biofilm formation by Enterococcus faecalis

Enterococcus faecalis is the species of bacterium most frequently isolated from the root canals of teeth that exhibit chronic apical periodontitis refractory to endodontic treatment. In this study, we evaluated the effect of the S‐ribosylhomocysteine lyase (luxS) quorum‐sensing gene on E. faecalis biofilm formation by constructing a knockout mutant. The biofilms formed by both E. faecalis and its luxS mutant strain were evaluated using the MTT method. Important parameters that influence biofilm formation, including cell‐surface hydrophobicity and the nutrient content of the growth medium, were also studied. Biofilm structures were observed using confocal laser scanning microscopy (CLSM), and expression of biofilm‐related genes was investigated using RT‐PCR. The results showed that the luxS gene can affect biofilm formation, whereas it does not affect the bacterial growth rate. Deletion of the luxS gene also increased cell‐surface hydrophobicity. Biofilm formation was accelerated by the addition of increasing concentrations of glucose. The CLSM images revealed that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures, whereas the wild‐type strain appears confluent and more evenly distributed. All genes examined were up‐regulated in the biofilms formed by the luxS mutant strain. The quorum‐sensing luxS gene can affect E. faecalis biofilm formation.     

Mechanisms Involved In The Resistance Of Enterococcus Faecalis To Calcium Hydroxide

Introduction The species Enterococcus faecalis has been recovered in a high proportion of endodontic failures – approximately one‐third of failed cases with recoverable bacteria contain monoinfections of the microorganism. Eradication of E. faecalis from an infected root canal is difficult, which means that it is a significant clinical problem in endodontic treatment. E. faecalis is known to be resistant to the alkaline bactericidal effect of calcium hydroxide, yet the survival mechanisms of E. faecalis that enable it to tolerate exposure to the high pH of calcium hydroxide are poorly understood. Because endodontic treatment involves repeated or alternate use of antimicrobial agents at various stages, these conditions could adapt or ‘pre‐treat’ cells by eliciting a stress response that enhances their survival. An ability to mount a stress response involving synthesis of general or specific stress‐induced proteins is an important survival mechanism for many bacteria. The production of these stress‐induced proteins protects cells from adverse conditions, facilitating cell recovery when more favourable conditions are re‐established. This study sought to clarify the mechanisms that enable E. faecalis to survive the high pH of calcium hydroxide. Methods E. faecalis strain JH2–2. was exposed to sub‐lethal concentrations of calcium hydroxide, with and without various pre‐treatments. Blocking agents were added to determine the role of stress‐induced protein synthesis and the cell wall‐associated proton pump. Results E. faecalis was resistant to calcium hydroxide at a pH of 11.1, but not pH 11.5. Pre‐treatment with calcium hydroxide pH 10.3 induced no tolerance to further exposure at pH 11.5. No difference in cell survival was observed when protein synthesis was blocked by Chloramphenicol during stress induction. However, addition of the proton pump inhibitor, CCCP, resulted in a dramatic reduction of cell viability of E. faecalis in calcium hydroxide. Conclusions This study confirmed that E. faecalis is resistant to killing by calcium hydroxide. An adaptive response in alkaline pH and stress‐induced protein synthesis do not play a role in cell survival. However, a functioning proton pump with the capacity to drive protons into the cell and acidify the cytoplasm is critical for survival of E. faecalis at high pH. These findings contribute to our understanding of the response by E. faecalis to the antimicrobial action of calcium hydroxide and may open the possibility of novel approaches to address this clinical problem in endodontic treatment.     

Antimicrobial effectiveness of grape seed extract against Enterococcus faecalis biofilm: A Confocal Laser Scanning Microscopy analysis

This study evaluated the antimicrobial effectiveness of 6.5% Vitis vinifera grape seed extract (GSE) against Enterococcus faecalis biofilm using confocal laser scanning microscopy (CLSM). Saline solution (SS), 5.25% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) were used for comparison. Dentin discs were inoculated with E. faecalis strain establishing a 3‐week‐old biofilm. Discs (n = 10) were exposed to 5.25% NaOCl, 2% CHX, 6.5% GSE and SS (negative control) for 10 min. Discs were stained with the fluorescent LIVE/DEAD‐BacLight? dye and analysed using CLSM. The proportion of dead cells in biofilm was analysed using one‐way anova and Tukey tests (P < 0.05). A higher proportion of dead cells was found in GSE group compared with CHX and SS (P < 0.05). NaOCl group was associated with the highest proportion of dead cells (P < 0.05). GSE presented antimicrobial activity against E. faecalis; however, NaOCl was the most effective irrigant solution. GSE was more effective than CHX and SS.     

Iron‐ and hemin‐dependent gene expression of Porphyromonas gingivalis

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron‐dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron‐ and hemin‐dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole‐genome microarrays of P. gingivalis genes were used to compare the levels of gene expression under iron‐replete and iron‐depleted conditions as well as under hemin‐replete and hemin‐depleted conditions. Under iron‐depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron‐depleted conditions to those from hemin‐depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron‐dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron‐regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron‐stressed bacterium than the iron‐replete P. gingivalis cells. The major iron‐regulated proteins encoded by PG1019–20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole‐genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P. gingivalis.     

Starvation survival,growth and recovery of Enterococcus faecalis in human serum

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose‐, phosphate‐ or amino acid‐limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient‐limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 10⁶ colony‐forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.     

Antimicrobial efficacy of clindamycin and triple antibiotic paste as root canal medicaments on tubular infection: An in vitro study

This study compared the antibacterial effect of 2% clindamycin and 2% and 100% concentration of triple antibiotic paste (TAP) on an Enterococcus faecalis biofilm. Dentinal tubules of 100 root specimens were infected and randomly assigned to five groups. A total of 1000 mg mL^?1 of TAP, 20 mg mL^?1 of TAP and clindamycin, calcium hydroxide or methylcellulose (control) were placed in the root canal for 1 week. After treatment, dentine shavings were collected from 200 and 400 μm dentine depth and the number of colony‐forming units (CFU) per mg was determined. Reduction in viable bacteria in first three groups was significantly better than calcium hydroxide and control groups. However, the antimicrobial effectiveness among these three groups was not significantly different from each other. There was no significant difference between data at 200 and 400 μm in all groups except the Ca(OH)₂ group. The antibiofilm effect of clindamycin was comparable with TAP, so it may be used instead of TAP.     

Effectiveness of propolis and calcium hydroxide as a short‐term intracanal medicament against Enterococcus faecalis: A laboratory study

The aim of this study was to investigate the antimicrobial activity of propolis‐based intracanal medicament against Enterococcus faecalis using infected dentine models, and to compare its antimicrobial efficacy with that of the non‐setting calcium hydroxide paste when used as a short‐term medication for 1 and 2 days. A total of 50 dentine discs of 7‐mm length was obtained from extracted human teeth. Five dentine discs were kept sterile to serve as a negative control. The remaining 45 were contaminated with E. faecalis and divided into two groups (n = 20) in addition to five discs that served as a positive control. The discs were treated as follow: 20 discs were filled with propolis, while the other 20 discs were filled with non‐setting calcium hydroxide. Microbiological sampling was performed utilising sterile paper point, headstrom file and disc immersion. Results showed that propolis was significantly more effective than non‐setting calcium hydroxide against E. faecalis after short‐term application, which made comparison from this prospect unlevelled. The most effective microbiological sampling technique was abrading the lumen with headstrom file. Propolis is very effective as intracanal medicament in rapidly eliminating E. faecalis ex vivo.     

In vitro study of the inactivation by dentine of some endodontic medicaments and their bases

Background: The aim of this study was to investigate the antimicrobial effect of endodontic medicaments and their bases in the presence of dentine powder. Methods: The medicaments tested were Ledermix paste, Pulpdent paste, a 50:50 combination of the Pulpdent:Ledermix and their bases. The test organism was Enterococcus faecalis ATCC 29212. The presence or absence of dentine was examined as well as the effect of autoclaving dentine. Serial dilutions of samples at 1 hour, 1 day and 3 days were used for colony counting. The effects of dentine powder on pH for saturated Ca(OH)₂ solution and Pulpdent paste at 1 hour and 24 hours were also measured. Results: Pulpdent and the 50:50 combination of Pulpdent:Ledermix completely inhibited the growth of E. faecalis from 1 hour onwards, and these results were not affected by the presence/absence of dentine powder, pre‐incubation period, timing of autoclaving, or exposure time. Saturated solutions of Ca(OH)₂ are prone to inactivation by dentine powder unlike Pulpdent paste. Ledermix paste took 3 days to exert a significant effect on the growth of E. faecalis. Conclusions: In this laboratory study, both Pulpdent and the 50:50 mixture of Pulpdent with Ledermix were effective medicaments against E. faecalis in the presence of dentine powder.     

Effectiveness of calcium and sodium hypochlorite in association with reciprocating instrumentation on decontamination of root canals infected with Enterococcus faecalis

The aim of this study was to evaluate the antimicrobial activity of sodium and calcium hypochlorite utilising reciprocating instrumentation. Sixty root canals were inoculated with E. faecalis for 14 days. Samples were divided into six groups according to decontamination protocol: G1: no treatment, G2: distilled water, G3: 2.5% sodium hypochlorite, G4: 2.5% calcium hypochlorite, G5: 5.25% sodium hypochlorite and G6: 5.25% calcium hypochlorite. Instrumentation was performed with Wave One reciprocating system (Dentsply Sirona Endodontics, York, PA, USA) in groups G2 to G6. Colony‐forming units (CFUs) counting was performed and the data were subjected to Anova and Tukey (α  =  0.05). Group 1 and 2 showed the highest mean contamination, with a significant difference between them (P < 0.05). Groups 3, 4, 5 and 6 showed the lowest contamination means with no significant difference between them (P < 0.05). Sodium and calcium hypochlorite, in association with reciprocating instrumentation, can be an effective decontamination protocol in root canals infected with E. faecalis.     

PCR detection of Scardovia wiggsiae in combination with Streptococcus mutans for early childhood caries‐risk prediction

The microbial factor is an important determinant in caries risk assessment. This study aimed to use detection, by PCR, of Scardovia wiggsiae, in combination with Streptococcus mutans, for the accurate prediction of caries risk in children. Detection of Lactobacillus, as a caries‐specific species, was also performed. Dental plaque, as well as infected dentine when available, was collected from children who were caries‐free (n = 30) or diagnosed with early childhood caries (n = 30), and the prevalence and abundance of S. wiggsiae and S. mutans were estimated using quantitative PCR. Lactobacillus was amplified by Lactobacillus genus‐specific primers and then sequenced. Both S. wiggsiae and S. mutans were concurrently detected in 19 children diagnosed with early childhood caries, but in none of the caries‐free children. The positive predictive value was 1 in children with S. wiggsiae‐ and S. mutans‐positive test results, compared with 0.58 when only S. mutans was detected and 0.9 when only S. wiggsiae was detected. The abundance of S. wiggsiae and S. mutans in infected dentine was higher than that in dental plaque from children. Diverse Lactobacillus species were observed in dental plaque but none appeared to be caries‐specific. In conclusion, the detection of S. wiggsiae in combination with S. mutans improves the positive predictive value and the specificity of the test.     

Invasion of dentinal tubules by root canal bacteria

Bacterial invasion of dentinal tubules and the clinical consequences have been recognized for over a century. However, while many components of the infected dentinal tubule microflora have been identified, it is likely that there are etiological agents involved in endodontic infections that have not yet been recognized. Bacterial invasion of coronal dentinal tubules occurs when the dentine is exposed to the oral environment and of radicular dentinal tubules subsequent to infection of the root canal system or as a consequence of periodontal disease. The content and architecture of a dentinal tubule can influence bacterial invasion, with tubule patency being important. This can account for regional variations in bacterial invasion and is particularly seen with dentinal sclerosis, where more advanced sclerotic changes in apical radicular tubules, especially in elderly individuals, limit bacterial invasion in this area. While several hundred bacterial species are known to inhabit the oral cavity, a relatively small and select group of bacteria are involved in invasion of dentinal tubules. Gram‐positive organisms dominate the tubule microflora in both carious and non‐carious dentine. The relatively high numbers of obligate anaerobes present, such as Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., Peptostreptococcus micros, and Veillonella spp., suggests that the environment favors the growth of these bacteria. Gram‐negative obligate anaerobic rods, e.g. Porphyromonas spp., are less frequently recovered; however, with time, fastidious obligately anaerobic bacteria become established as principal components of the microflora and can be found within the deep dentine layers. In the early stages of infection, Gram‐positive bacteria dominate the microflora. The identification of adhesins that mediate these initial interactions of bacteria with dentine is important for understanding the development of tubule infection and in designing adhesion‐blocking compounds. Recent evidence suggests that streptococci and enterococci may recognize components present within dentinal tubules, such as collagen type I, which stimulate bacterial adhesion and intra‐tubular growth. Specific interactions of other oral bacteria with invading streptococci may then facilitate invasion of dentine by select bacteria. It is important therefore that the mechanisms of invasion and inter‐bacterial adhesion are understood to assist development of novel control strategies.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

Sodium chloride and potassium sorbate: a synergistic combination against Enterococcus faecalis biofilms: an in vitro study

Incomplete disinfection of the root canal system is a major cause of post‐treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double‐hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high‐throughput resazurin metabolism assay (RMA) was used to test a range of organic acid salts. Then, to gain more insight into the efficacy of sorbate salt solutions, 48‐h E. faecalis biofilms were evaluated in colony‐forming unit (CFU) assays. Chlorhexidine (CHX) and calcium hydroxide [Ca(OH)₂] were tested in parallel as controls. Sorbate salt produced the largest and most significant reduction of fluorescence intensity in the RMA assay. Neither NaCl nor potassium sorbate (KS) alone induced a clinically relevant reduction of CFU counts after 1 h. Surprisingly, the combination of the two in a single solution had a synergistic effect on the inactivation of E. faecalis. Potassium sorbate amplified the efficacy of NaCl. Of the salts tested, NaCl with KS eradicated E. faecalis biofilms within 1 h. This study showed that the double‐hurdle strategy indeed leads to synergistic efficacy and is a possible next step in the complete disinfection of endodontic infections.     

The effect of sodium hypochlorite on Enterococcus faecalis when grown on dentine as a single‐ and multi‐species biofilm

Enterococcus faecalis is often involved in the aetiology of apical periodontitis after endodontic treatment. This project aimed to establish, on dentine in vitro, a multi‐species biofilm containing E. faecalis, and to determine if the organism had an increased resistance to sodium hypochlorite compared with an axenic biofilm. Biofilms were established on dentine discs in flow cells with either E. faecalis alone (axenic) or together with Fusobacterium nucleatum and Streptococcus sanguinis. Following treatment with either 0.9% sodium hypochlorite or saline, the viability of E. faecalis was determined by serial plating and qualitative analysis was performed by scanning electron microscopy and confocal laser scanning microscopy. Viable counts indicated that 0.9% NaOCl is highly effective against E. faecalis grown alone and as part of a multi‐species biofilm (P = 0.0005 and P = 0.001, respectively). No significant difference in its survival in the two biofilm types was found (P = 0.8276).     

Antimicrobial Activity of a Sodium Hypochlorite/Etidronic Acid Irrigant Solution

Introduction

The aim of this study was to evaluate the antimicrobial activity of a 2.5% sodium hypochlorite (NaOCl)/9% etidronic acid (HEBP) irrigant solution on Enterococcus faecalis growing in biofilms and a dentinal tubule infection model.

Methods

The antimicrobial activity of the solutions 2.5% NaOCl and 9% HEBP alone and associated was evaluated on E. faecalis biofilms grown in the Calgary biofilm model (minimum biofilm eradication concentration high-throughput device). For the dentinal tubule infection test, the percentage of dead cells in E. faecalis–infected dentinal tubules treated with the solutions for 10 minutes was measured using confocal laser scanning microscopy and the live/dead technique. Available chlorine and pH of the solutions were also measured. Distilled water was used as the control. Nonparametric tests were used to determine statistical differences.

Results

The highest viability was found in the distilled water group and the lowest in the NaOCl-treated dentin (P < .05). Both NaOCl solutions killed 100% of the E. faecalis biofilms and showed the highest antimicrobial activity inside dentinal tubules, without statistical differences between the 2 (P < .05). The HEBP isolated solution killed bacteria inside dentinal tubules but did not present any significant effect against E. faecalis biofilms. The incorporation of HEBP to NaOCl did not cause any loss of available chlorine within 60 minutes.

Conclusions

HEBP did not interfere with the ability of NaOCl to kill E. faecalis grown in biofilms and inside dentinal tubules.     

Properties of a modified quaternary ammonium silane formulation as a potential root canal irrigant in endodontics

ObjectivesEvaluate a new modified quaternary ammonium silane irrigant solution for its antimicrobial, cytotoxic and mechanical properties of dentine substrate.MethodsRoot canal preparation was performed using stainless steel K-files™ and F4 size protaper with irrigation protocols of 6% NaOCl + 2% CHX; 3.5% QIS; 2% QIS and sterile saline. Biofilms were prepared using E. faecalis adjusted and allowed to grow for 3 days, treated with irrigants, and allowed to grow for 7 days. AFM was performed and surface free energy calculated. MC3T3 cells were infected with endo irrigant treated E. faecalis biofilms. Raman spectroscopy of biofilms were performed after bacterial re-growth on root dentine and exposed to different irrigation protocols and collagen fibers analysed collagen fibers using TEM. Antimicrobial potency against E. faecalis biofilms and cytoxicity against 3T3 NIH cells were also. Resin penetration and MitoTracker green were also evaluated for sealer penetration and mitochondrial viability. Data were analysed using One-way ANOVA, principal component analysis and post-hoc Fisher’s least-significant difference.ResultsElastic moduli were maintained amongst control (5.5 ± 0.9) and 3.5% QIS (4.4 ± 1.1) specimens with surface free energy higher in QIS specimens. MC3T3 cells showed reduced viability in 6%NaOCl+2%CHX specimens compared to QIS specimens. DNA/purine were expressed in increased intensities in control and 6% NaOCl + 2% CHX specimens with bands around 480−490 cm⁻¹ reduced in QIS specimens. 3.5% QIS specimens showed intact collagen fibrillar network and predominantly dead bacterial cells in confocal microscopy. 3.5% QIS irrigant formed a thin crust-type surface layer with cytoplasmic extensions of 3T3NIH spread over root dentine. Experiments confirmed MitoTracker accumulation in 3.5% treated cells.SignificanceNovel QIS root canal irrigant achieved optimum antimicrobial protection inside the root canals facilitating a toxic effect against the Enterococcus faecalis biofilm. Root dentine substrates exhibited optimum mechanical properties and there was viability of fibroblastic mitochondria.     

The effect of various concentrations of iodine potassium iodide on the antimicrobial properties of mineral trioxide aggregate – a pilot study

Abstract – Background: Mineral trioxide aggregate (MTA) is a multi‐purpose dental material with various uses in dentistry. Iodine potassium iodide (IKI) is the most commonly used iodine compound in endodontics. We aimed to assess the antimicrobial activity of tooth‐colored ProRoot MTA combined with IKI. Materials and methods: The antimicrobial activity of IKI was assessed at three concentrations (1%, 2%, and 4%) as the mixing agents combined with MTA against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. For each microorganism, three plates were inoculated with 100 μl of a microbial suspension (McFarland 0.5). Four wells were prepared in each plate. MTA (70 mg) was mixed with any of the three concentrations of IKI (25 μl) or sterile distilled water (25 μl) and placed in each well. The plates were incubated for 24 h at 37°C. Zones of inhibition (ZOI) were measured in millimeters by a blinded observer. Data were analyzed using analysis of variance and the Dunnett t‐test. Results: All MTA mixtures with water or IKI solutions showed inhibitory zones. The mean ZOI of each MTA/IKI mixture was not significantly different from MTA/water mixture (P > 0.05). MTA/1% IKI had smaller ZOI than MTA/water against E. coli, E. faecalis, and C. albicans. MTA/2% IKI showed larger ZOI only against P. aeruginosa. MTA/4% IKI showed larger ZOI against P. aeruginosa and E. coli (P < 0.05). Conclusions: Substitution of IKI solutions (1%, 2%, and 4%) for water did not significantly increase the antimicrobial activity of MTA.