Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Sep 1.
Published in final edited form as: J Endod. 2024 May 28;50(9):1314–1320.e1. doi: 10.1016/j.joen.2024.05.010

An In-Vitro Evaluation of the Antimicrobial Efficacy of a Novel Irrigant Using Next-Generation Sequencing

Mallory Hackbarth 1, Meghan Montoya 1, Craig W Noblett 1, Bruno Lima 2, Matthew Dietz 3, Christopher Staley 3, Ronald Ordinola Zapata 1
PMCID: PMC11402568  NIHMSID: NIHMS1999710  PMID: 38815857

Abstract

Introduction

To evaluate the antimicrobial activity of Triton irrigation versus 4% NaOCl utilizing a direct contact test and an extracted tooth model.

Methods

In the first experiment, a direct contact test was conducted to compare bacterial DNA removal and microbial diversity changes following irrigation with sodium hypochlorite (4% NaOCl) or Triton. Hydroxyapatite and dentin discs were inoculated with subgingival human-derived dental plaque for 2 weeks utilizing a CDC biofilm reactor and subsequently challenged with the root canal irrigants for 5 min. In the second experiment, teeth contaminated with a multispecies biofilm (n=24) were assigned into two treatment groups, NaOCl or Triton irrigation. Samples were obtained for qPCR and next-generation sequencing (NGS) analysis before and after instrumentation. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray–Curtis dissimilarity and ANOSIM was used to measure beta diversity. Differences in abundances of genera were evaluated using Kruskal–Wallis test with Bonferroni corrections.

Results

The direct contact test revealed no significant differences in the bacterial load based on 16S rRNA gene molecules/μL, reads, or differences in the Shannon index among groups. In the extracted tooth model, a bacterial load reduction of log10 3.08 ± 0.69 and 2.76 ± 0.91 were found for NaOCl and Triton respectively (P = 0.348). NGS showed fewer reads, lower Chao1 and beta diversity values when pre- and post-treatment samples were assessed in both experimental groups (P < 0.0001). The Kruskal Wallis analysis found that 17 genera of bacteria were overrepresented in minimal values in the Triton post-treatment group, 14 of these genera represented less than 1% of the microbial community.

Conclusions

Both irrigants had limited antimicrobial activity in the direct contact test. When used in conjunction with mechanical instrumentation both irrigants were able to reduce the bacterial DNA load and diversity in comparison with pretreatment communities. The NaOCl irrigation, followed by EDTA flush, was more effective in decreasing DNA counts from low-abundance organisms.

Keywords: biofilms, next-generation sequencing, microbiome, sodium hypochlorite

INTRODUCTION

The goal of endodontic treatment is reduction of the microbial population within the root canal system followed by obturation and restoration, which prevent post-treatment infection 1. This has conventionally been achieved through mechanical debridement and chemical dissolution using proteolytic solutions such as sodium hypochlorite 2, 3.

Chelators such as ethylenediaminetetraacetic acid (EDTA) are useful during chemo-mechanical debridement of the root canal space to remove the smear layer and inorganic debris 4. The concept of continuous chelation was introduced by Zehnder et al 5 and Neelakantan et al 6. This involves combining a weak chelator, such as etidronic acid, with NaOCl to achieve continuous smear layer removal during canal preparation. The literature suggests that continuous chelation could provide improved antimicrobial activity, enhanced smear layer and dentinal debris removal, effective tissue dissolution and improved bonding of endodontic sealers to dentin 6–10. Triton is a novel irrigating solution proposed to be an all-in-one, dual-action root canal irrigant 11. It is known that NaOCl is a reactive agent, however, its chlorine content decreases when mixed with other chemical compounds 5. To date, the antimicrobial efficacy of the Triton solution has been explored by only one study 11. The purpose of this in-vitro study is to compare the antimicrobial efficacy of the novel Triton irrigation system to a NaOCl irrigation protocol by using a direct contact test and an extracted tooth model.

MATERIAL AND METHODS

Direct Contact Test

This in-vitro study protocol was approved by IRB #00014237. The antimicrobial activity of irrigants 4.4% NaOCl (Brasseler, Savannah, GA, USA), Triton (Brasseler, Savannah, GA, USA), and distilled water (negative control), was tested by direct contact test against a two-week-old multispecies biofilm on two substrates, hydroxyapatite and dentin. The hydroxyapatite (Clarkson Chromatography, South Williamsport, PA, USA) and bovine dentin discs were stored in sterile saline and autoclaved prior to their use.

The Center for Disease Control (CDC) biofilm reactor (BioSurface Technologies, Bozeman, MT, USA) was used to generate biofilms on the samples. There was a lidded vessel through which growth media (350 mL Columbia medium; Difco) flowed at a constant, defined rate while a stir bar generated shear force 12. The medium was inoculated with human-derived subgingival dental plaque (100 μl) provided by one donor. This sample was diluted in anaerobic transport medium (Anaerobe System, Morgan Hill, CA, USA). Environmental conditions of the reactor were maintained at an internal temperature of 37°C and 5% humidity and stirring rate was 90 rpm. The reactor was incubated under conditions of shear, without media flow, for the first 24 h. Columbia medium then circulated through the reactor at a rate of 2.5 L/24 h flow rate for 14 days. After two weeks, the hydroxyapatite and dentin discs were removed with sterile instruments and a direct contact test was conducted with a minimum of five replicates in each group (between 11 and 12 per irrigant solution). The blocks were randomly assigned to 1mL of distilled water, 1mL of 4.4% sodium hypochlorite, or 1mL of Triton irrigant for 5 minutes. Triton was mixed according to the manufacturer instructions (Brasseler, Savannah, GA, USA) (See Supplementary Table 1). After the contact test, discs were placed in a one-minute soak in a solution of 10% sodium thiosulfate to inactivate the sodium hypochlorite. Discs were processed for DNA extraction and immediately stored at −80 Celsius until 16S rRNA amplicon analysis.

Extracted Tooth Model

Twenty-four extracted mandibular single rooted teeth (premolars and canines) were collected. Teeth were accessed and instrumented to a size #15 K File. Teeth were then autoclaved and stored in distilled water. Prepared root canal specimens were inoculated with subgingival human-derived dental plaque (100 μl), provided by one donor previously diluted in anaerobic transport medium. Samples were mounted onto a custom-built stand that held the samples within a glass reactor vessel. The biofilm development protocol matched that of the direct contact test.

Before instrumentation, the foramen was blocked with sterile red wax to create a closed system. Sterile Hedstrom files (Dentsply Sirona, Ballaigues, Switzerland) size 20 were used to create dentine shavings before instrumentation, and sterile paper points were used to collect preoperative samples in Eppendorf tubes containing 1.5 mL of Tris HCl buffer. The teeth had previously been assigned randomly into two treatment groups, NaOCl irrigation or Triton irrigation group.

Root Canal Preparation and Sampling

All specimens had working lengths recorded and were prepared with Vortex Blue rotary instruments (Dentsply Sirona, Ballaigues, Switzerland) within 0.5 mm of the apex. The final preparation size was 35/04. In the NaOCl group, irrigation was performed by delivering a total of 10 mL of 4% NaOCl during instrumentation using a 30G side vented needle placed 2–3 mm from the working length. Passive ultrasonic irrigation (PUI) was used for 30s three times with 1mL of NaOCl each time (3mL/90 sec total). The canals were irrigated with a final rinse of 1 mL of 17% EDTA. In the Triton group, the two-part root canal cleanser was prepared according to the manufacturer’s instructions. The Triton group irrigation was performed similar to NaOCl technique with 10 mL of Triton cleanser, using a 30G side vented needle placed 2–3 mm from the working length. PUI was also used for 30 seconds separately 3 times (3mL/90sec total). After disinfection procedures, 2 mL of 10% sodium thiosulfate were used for both the NaOCl and Triton groups.

Post-cleaning microbiologic specimens were collected using sterile Hedstrom files size 20 in a filling motion for 30 seconds. Three sterile paper points were used to collect the post-treatment samples. All samples (pre-and post-treatment) were placed in 1.5mL Tris-HCl buffer and immediately stored at −80°C until analysis.

DNA Extraction and Sequence Analysis

The DNeasy® PowerSoil® Pro Kit (Qiagen, Hilden, Germany) was used to extract the DNA on the automated QIAcube platform. Samples were obtained for quantitative real-time polymerase chain reaction (qPCR) and 16S rRNA gene sequencing. Amplification and sequencing were done by the institution’s Genomic Center. For DNA sequencing, the V3-V4 hypervariable region of the 16S rRNA gene was amplified and sequenced using paired-end sequencing at 301 nucleotides (nt) read length on the Illumina MiSeq platform by the dual-index method 13. Raw sequence data are deposited in the Sequence Read Archive at NCBI under accession number SRP366904.

Amplicon Processing and Analysis

Sequence data were processed and analyzed using mothur ver. 1.41.1 14. Sequences were first trimmed to the first 250 nt and paired-end joined using fastq-join software 15. Quality trimming was performed at a threshold of 35 over a sliding window of 50 nt. In addition, sequences with homopolymers >6 nt, ambiguous bases, or >2 mismatches from primer sequences were removed. High-quality sequences were aligned against SILVA database ver. 138.1 16 for downstream processing. Chimeras were identified and removed using UCHIME ver. 4.2.40 17. Sequencing errors were further removed using a 2% pre-clustering step. Operational taxonomic units (OTUs) were binned at a similarity of 99% using the furthest-neighbor algorithm and were classified against the version 18 release from the Ribosomal Database Project 18. Different databases were used for alignment and classification due to processing considerations described previously 19.

Statistical Analysis

For qPCR (16S rRNA gene molecules/μL) from the direct contact test, data were transformed to Log10 values and assessed using ANOVA. In the second experiment, Log10 reduction in NaOCl and Triton samples was analyzed using the T-student test with Welch’s correction (GraphPad Software, La Jolla, CA, USA). To analyze microbiome data, ANOVA and Kruskal Wallis were used to compare alpha diversity and genus abundances, using Tukey's post-hoc test and Bonferroni adjustment for multiple comparisons (XLSTAT ver. 2020.2.3, Addinsoft, Belmont, MA, USA). The Shannon and Chao1 indices were used to measure alpha (within-sample) diversity and were calculated using mothur14. The Bray-Curtis dissimilarity was used to measure beta (between-groups) diversity and was visualized by ordination using principal coordinate analysis (PCoA) 20. Differences in community composition were evaluated using analysis of similarity 21 with Bonferroni correction for multiple comparisons.

RESULTS

Direct Contact Test

No significant differences were detected between the hydroxyapatite and dentin substrates according to the number of reads, OTUs, Shannon and Chao1 indices (P = 0.14, 0.76, 0.83 and 0.53 respectively). Therefore, subsequent statistical analysis combined dentin and hydroxyapatite samples (n=11 per irrigant solution). The analysis with qPCR showed that Triton, NaOCl and distilled water promoted log10 values of 2.37 ± 2.30, 2.82 ± 2.52, 4.56 ± 0.98, respectively. No significant differences between the experimental groups and control were observed ANOVA (P = 0.0539).

Overall, there was no significant difference in number of OTUs, and alpha diversity (Chao1) between water, sodium hypochlorite and Triton (Table 1). The number of reads and Shannon alpha diversity were significantly lower in Triton and NaOCl in relation to distilled water (P < 0.05). There was a significant difference in beta diversity between Triton and water (R= 0.17, P=0.015) as well as between NaOCl and water (R=0.13, P=0.03). No significant difference in beta diversity was found between Triton and NaOCl (R= −0.007, P > 0.99). Biofilm communities were similar in all groups (See Figure 1), except the control group exhibited significant differences in Alloprevotella, Prevotella, Megasphera, Candidatus saccharibacteria (further unclassified), Solobacterium, Clostridiales (unclassified), Peptoanaerobacter, Staphylococcus, Prevotellaceae (unclassified), Escherichia and Neisseriaceae (unclassified).

Table 1.

Mean and standard deviation of sequencing data from all samples including OTUs, Shannon and Chao1 alpha diversity indices. Different uppercase letters in each column indicate significant differences by Tukey’s post-hoc test (P < .05).

Reads OTUs Shannon Chao1 Coverage
NaOCl 15858±16216A 134.64±173.55A 2.19±0.67A 378.60±580.18A 0.971±0.048A
Triton 15717±15718A 127.27±170.26A 2.28±0.49A 309.11±504.93A 0.988±0.026A
Water 29408±3220B 269.09±168.27A 2.85±0.23B 796.58±793.85A 0.995±0.005A
p-value 0.031 0.107 0.008 0.170 0.214
Open in a new tab

Figure 1.

Figure 1.

Open in a new tab

(A) qPCR log10 molecules found in the direct contact test (P = 0.0539). (B) Microbial diversity of the analyzed samples (relative abundance). Genera reflecting a mean < 2.0% of sequence reads among all samples were consolidated. (C) Principal coordinate analysis of Bray-Curtis dissimilarity matrices (beta-diversity). Differences between groups (ANOSIM R-value =0.17, p-value =0.015)

Extracted Tooth Model

The analysis with qPCR showed that the NaOCl and Triton groups promoted an average log10 reduction after instrumentation of 3.08 ± 0.69 and 2.76 ± 0.91, respectively (P = 0.348) (See Figure 2).

Figure 2.

Figure 2.

Open in a new tab

(A) qPCR log10 molecules reduction after instrumentation and irrigation using NaOCl and triton (P = 0.348). (B) Microbial diversity of the analyzed samples (relative abundance) before and after root canal instrumentation and irrigation. Genera reflecting a mean < 1.0% of sequence reads among all samples were consolidated. (C) Principal coordinate analysis of Bray-Curtis dissimilarity matrices (beta-diversity). A significant difference in beta diversity between pre-treatment and post-treatment communities for both irrigation test groups was observed, NaOCl (ANOSIM R = 0.62, P < 0.001) and Triton, (R = 0.64, P < 0.001).

The number of average reads, OTUs, Shannon Index, and Chao1 Index in pre and post-treatment groups can be found in Table 2. Communities in pre-treatment samples were similar in both groups (ANOSIM R = 0.048, P = 0.18). The results showed a significant difference in Chao1 and beta diversity between pre-treatment and post-treatment communities for both irrigation test groups, NaOCl (ANOSIM R = 0.62, P < 0.001) and Triton, (R = 0.64, P < 0.001) (See Figure 2). Beta diversity analysis also showed significant differences between both post-treatment communities (R = 0.22, P = 0.008, Bonferroni-corrected α = 0.008).

Table 2.

Mean and standard deviation of sequencing data from all samples including OTUs, Shannon and Chao1 alpha diversity indices. Different uppercase letters in each column indicate significant differences by Tukey’s post-hoc test (P < .05).

Reads OTUs Shannon Chao1 Coverage
NaOCl Pre 35992±11631A 100.8±32.6A 2.16±0.04 A 345±115A 0.99±0.002A
Post 10005±11967B 28.0±33.6B 2.2±0.64 A 77±66B 0.98±0.030A
Triton Pre 37722±14435A 105.6±40.4A 2.11±0.07 A 335±171A 0.99±0.002A
Post 22977±20897AB 64.4±58.5AB 2.56±0.62 A 109±60B 0.99±0.002A
p-value 0.0001 0.0001 0.059 0.0001 0.062
Open in a new tab

Overall, the Kruskal Wallis test with Bonferroni corrections found that 17 genera of bacteria were over abundant in the Triton post-treatment groups. These genera mostly represented taxa with less than 1% relative abundance. In comparison there were only 4 genera of bacteria in the NaOCl post-treatment groups in quantities significantly greater than that of the Triton post-treatment groups. (See Supplementary Table 2).

DISCUSSION

An advantage of the methodology used in this study was the mature biofilm established in the CDC biofilm reactor which could be more representative of real-life scenarios compared to the use of monospecies biofilms22–24. Rudney et al 12 found that a biofilm incubated for 72h in this reactor was equivalent to a biofilm that would take thirty days to develop without the reactor and media flow. Further quantitative and qualitative diversity analysis of pre-treatment samples revealed a homogeneous and reproducible microbial community between the experimental groups. The model has proven to be reproducible and consists of microbial genera previously associated with endodontic infections 23, 24.

Stojicic et al 25 demonstrated antimicrobial activity of endodontic irrigants on hydroxyapatite discs with a mature biofilm derived from dental plaque, like the methodology utilized in this study. The same authors found that less mature biofilms, defined as <1 or 2 weeks of passive inoculation, were more susceptible to antimicrobials than mature biofilms 3+ weeks old. Wang et al 26 found 6% sodium hypochlorite had the greatest antimicrobial activity of the irrigants studied and could eliminate 99% of a three-day-old biofilm and 95% of a three-week-old biofilm; Maezono et al 27 revealed that 1mL of 2% NaOCl killed 75% of microbial cells in 3 minutes. Similar data was found by Petridis et al 28 after 1 min exposure time, as revealed by this study more mature biofilms were more difficult to eradicate.

Several authors have revealed that active chlorine in sodium hypochlorite is rapidly consumed in the presence of organic matter and dentinal debris 8, 9, 29, 30. Other studies also corroborated that a thicker and denser biofilm requires a greater volume, concentration, or contact time with sodium hypochlorite 26–28, 31–33. The inactivation of irrigants by organic compounds present in the biofilm, the limited contact time (5 minutes), and the lack of mechanical debridement may explain the difference in results between the direct contact test and the extracted tooth model.

Dentinal debris, which is created throughout root canal treatment, could negatively influence the action of sodium hypochlorite for this reason a second experiment was performed. In the extracted tooth experiment, both irrigant solutions were able to significantly impact the pre-treatment microbial population in terms of quantity (qPCR) and quality, NaOCl (R = 0.62, P < 0.001) and Triton, (R = 0.64, P < 0.001). The difference in the post-treatment microbial community beta diversity in the extracted tooth experiment could probably come from the difference in relative abundances in the Triton group. As reported by the manufacturer, 2% citric acid is present in the Triton formulation. This compound has been shown to strongly reduce available chlorine in NaOCl solutions and therefore may cause it to become less reactive 5. While the Kruskal-Wallis test showed greater abundances of bacterial genera in the Triton group, it is important to consider that this demonstrates relative and not absolute abundance.

This study used paper points to collect bacterial samples from the root canal space a technique that has been used to determine the microbial load and associated outcomes in clinical studies34. One limitation is that dentin shavings cannot be collected for posterior weighed since the dentinal debris in the fluid are absorbed by paper points. Limitations related to paper point sampling have been previously addressed including its ability to sample only the main canal, and the inability to determine the location of bacteria (cervical vs apical)35.

The current challenge for developing endodontic biofilms for in-vitro research is the complex isolation and identification of fastidious anaerobic bacteria from multiple patients27. These isolated microorganisms will need to establish first beneficial interspecies relationships to form biofilms that are resilient to root canal procedures ex-vivo. Another limitation of this study is that open and closed-end molecular studies do not provide information on bacterial viability 36. Currently, there is a shortage of research that specify the clinical levels of remaining bacterial load found in root canals that would support the persistence of apical periodontitis.

Although the presence of smear layer is not necessarily associated with endodontic failures, its presence can reduce the penetration of irrigants or inactivate antimicrobials creating a suitable environment for biofilm persistence 9, 26, 30, 37. Smear layer and dentin debris accumulation may also increase the chance for procedural errors such as iatrogenic blockage38. While clinical studies have found that smear layer removal could improve radiographic outcomes in retreatment cases39, current research has yet to demonstrate a direct causative relationship between smear layer removal and healing of apical periodontitis. Future studies should also thoroughly evaluate the organic tissue dissolution and smear layer removing abilities of Triton.

CONCLUSIONS

Both irrigants had limited antimicrobial activity in the direct contact test. When used in conjunction with mechanical instrumentation both irrigants were able to reduce the DNA bacterial load and diversity in comparison with pretreatment communities. The NaOCl irrigation, followed by EDTA flush, was more effective in decreasing bacterial DNA from low-abundance organisms.

Supplementary Material

1

Funding source

Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1-TR002494.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Siqueira JF. Aetiology of root canal treatment failure: why well-treated teeth can bail. Int Endod J 2001;34:1–10. [DOI] [PubMed] [Google Scholar]
  • 2.Schilder H. Cleaning and shaping the root canal. Dent Clin North Am 1974;18:269–96. [PubMed] [Google Scholar]
  • 3.Zehnder M Root canal irrigants. Journal of Endodontics 2006;32:389–98. [DOI] [PubMed] [Google Scholar]
  • 4.Baumgartner JC, Mader CL. A scanning electron-microscopic evaluation of 4 root-canal irrigation regimens. J Endod 1987;13:147–57. [DOI] [PubMed] [Google Scholar]
  • 5.Zehnder M, Schmidlin P, Sener B, Waltimo T. Chelation in root canal therapy reconsidered. J Endod 2005;31:817–20. [DOI] [PubMed] [Google Scholar]
  • 6.Neelakantan P, Varughese AA, Sharma S, Subbarao CV, Zehnder M, De-Deus G. Continuous chelation irrigation improves the adhesion of epoxy resin-based root canal sealer to root dentine. Int Endod J 2012;45:1097–1102. [DOI] [PubMed] [Google Scholar]
  • 7.Arias-Moliz MT, Ordinola-Zapata R, Baca P, Ruiz-Linares M, Ferrer-Luque CM. Antimicrobial activity of a sodium hypochlorite/etidronic acid irrigant solution. J Endod 2014;40:1999–2002. [DOI] [PubMed] [Google Scholar]
  • 8.Arias-Moliz MT, Morago A, Ordinola-Zapata R, Ferrer-Luque CM, Ruiz-Linares M, Baca P. Effects of Dentin Debris on the Antimicrobial Properties of Sodium Hypochlorite and Etidronic Acid. J Endod 2016;42:771–5. [DOI] [PubMed] [Google Scholar]
  • 9.Morago A, Ordinola-Zapata R, Ferrer-Luque CM, Baca P, Ruiz-Linares M, Arias-Moliz MT. Influence of Smear Layer on the Antimicrobial Activity of a Sodium Hypochlorite/Etidronic Acid Irrigating Solution in Infected Dentin. J Endod 2016;42:1647–50. [DOI] [PubMed] [Google Scholar]
  • 10.Paqué F, Rechenberg DK, Zehnder M. Reduction of hard-tissue debris accumulation during rotary root canal instrumentation by etidronic acid in a sodium hypochlorite irrigant. J Endod 2012;38:692–5. [DOI] [PubMed] [Google Scholar]
  • 11.Sheng X, Yu J, Liu H, Wang Z, Deng S, Shen Y. Dual effectiveness of a novel all-in-one endodontic irrigating solution in antibiofilm activity and smear layer removal. Front Bioeng Biotechnol 2023;11:1254927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Rudney JD, Chen R, Lenton P, Li J, Li Y, Jones RS, et al. A reproducible oral microcosm biofilm model for testing dental materials. J Appl Microbiol 2012;113:1540–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Gohl DM, Vangay P, Garbe J, MacLean A, Hauge A, Becker A, et al. Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies. Nat Biotechnol 2016;34:942–9. [DOI] [PubMed] [Google Scholar]
  • 14.Schloss PD. Reintroducing mothur: 10 Years Later. Appl Environ Microbiol 2020;86: e02343–19 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Aronesty E. Comparison of sequencing utility program. Open Bioinform J 2013;7:1–8. [Google Scholar]
  • 16.Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 2013;41:D590–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011;27:2194–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009;37:D141–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Schloss PD, Westcott SL. Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl Environ Microbiol 2011;77:3219–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Anderson MJ Willis TJ. Canonical analysis of principal coordinates: A useful method of constrained ordination for ecology. Ecology 2003;84:511–25. [Google Scholar]
  • 21.Clarke KR. Non-parametric multivariate analyses of changes in community structure. Austral Ecology 1993;18:117–43. [Google Scholar]
  • 22.Burmølle M, Webb JS, Rao D, Hansen LH, Sørensen SJ & Kjelleberg S (2006) Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms. Appl Environ Microbiol; 72: 3916–3923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ordinola-Zapata R, Mansour D, Saavedra F, Staley C, Chen R, Fok AS. In vitro efficacy of a non-instrumentation technique to remove intracanal multispecies biofilm. Int Endod J 2022; 55: 495–504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Coaguila-Llerena H, Ordinola-Zapata R, Staley C, Dietz M, Chen R, Faria G. Multispecies biofilm removal by a multisonic irrigation system in mandibular molars. Int Endod J 2022;55:1252–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Stojicic S, Shen Y, Haapasalo M. Effect of the source of biofilm bacteria, level of biofilm maturation, and type of disinfecting agent on the susceptibility of biofilm bacteria to antibacterial agents. J Endod 2013;39:473–7. [DOI] [PubMed] [Google Scholar]
  • 26.Wang Z, Shen Y, Haapasalo M. Dynamics of Dissolution, Killing, and Inhibition of Dental Plaque Biofilm. Front Microbiol 2020;11:964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Maezono et al. , Effects of Sodium Hypochlorite Concentration and Application Time on Bacteria in an Ex Vivo Polymicrobial Biofilm Model. J Endod 2024. in press [DOI] [PubMed] [Google Scholar]
  • 28.Petridis X, Busanello F, So MVR, Dijkstra RJB, Sharma PK, Van der Sluis LWM., Chemical efficacy of several NaOCl concentrations on biofilms of different architecture: new insights on NaOCl working mechanisms. Int Endod J 2019; 52:1773–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Estrela C, Estrela CR, Barbin EL, Spanó JC, Marchesan MA, Pécora JD. Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13:113–7. [DOI] [PubMed] [Google Scholar]
  • 30.Tejada S, Baca P, Ferrer-Luque CM, Ruiz-Linares M, Valderrama MJ, Arias-Moliz MT. Influence of dentine debris and organic tissue on the properties of sodium hypochlorite solutions. Int Endod J 2019;52:114–22. [DOI] [PubMed] [Google Scholar]
  • 31.Del Carpio-Perochena AE, Bramante CM, Duarte MA, Cavenago BC, Villas-Boas MH, Graeff MS, et al. Biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. J Endod 2011;37:1134–8. [DOI] [PubMed] [Google Scholar]
  • 32.Ordinola-Zapata R, Bramante CM, Garcia RB, de Andrade FB, Bernardineli N, de Moraes IG, et al. The antimicrobial effect of new and conventional endodontic irrigants on intra-orally infected dentin. Acta Odontol Scand 2013;71:424–31. [DOI] [PubMed] [Google Scholar]
  • 33.Ruiz-Linares M, Aguado-Pérez B, Baca P, Arias-Moliz MT, Ferrer-Luque CM. Efficacy of antimicrobial solutions against polymicrobial root canal biofilm. Int Endod J 2017;50:77–83. [DOI] [PubMed] [Google Scholar]
  • 34.Molander et al. Clinical and Radiographic Evaluation of One- and Two-visit Endodontic Treatment of Asymptomatic Necrotic Teeth with Apical Periodontitis: A Randomized Clinical Trial. J Endod 2007; 33:1145–48. [DOI] [PubMed] [Google Scholar]
  • 35.Siqueira JF, Roças IN. A critical analysis of research methods and experimental models to study the root canal microbiome. Int Endod J 2022; 55 S1: 46–71. [DOI] [PubMed] [Google Scholar]
  • 36.Young G, Turner S, Davies JK, Sundqvist G, Figdor D. Bacterial DNA persists for extended periods after cell death. J Endod 2007;33:1417–20. [DOI] [PubMed] [Google Scholar]
  • 37.Wang Z, Shen Y, Haapasalo M. Effect of smear layer against disinfection protocols on Enterococcus faecalis-infected dentin. J Endod 2013; 39:1395–1400. [DOI] [PubMed] [Google Scholar]
  • 38.Lambrianidis T. Ledging and blockage of root canals during root canal preparation: causes, recognition, prevention, management, and outcomes. Endod Topics 2009; 15: 56–74. [Google Scholar]
  • 39.Ng YL, Mann V, Gulabivala K. A prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. Int Endod J 2011;44: 583–609. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1999710-supplement-1.pdf (91.7KB, pdf)

RESOURCES