Quantification of Bacterial DNA from Infected Human Root Canals Using QPCR and DAPI After Disinfection with Established and Novel Irrigation Protocols

Overview

Journal Materials (Basel)

Publisher MDPI

Date 2022 Mar 10

PMID 35269141

Authors

Marie-Theres Weber

Yousef Alkhafaji

Anne Pioch

Evelyn Trips

Sabine Basche

Martin Dannemann

Alan Kilistoff

Christian Hannig

Torsten Sterzenbach

Affiliations

Soon will be listed here.

Abstract

The removal of bacterial infections within the root canal system is still a challenge. Therefore, the cleansing effect of established and new irrigation-protocols (IP) containing silver diamine fluoride (SDF) 3.8% on the whole root canal system was analyzed using quantitative PCR (qPCR) and 4',6-diamidino-phenylindole-(DAPI)-staining. Extracted human premolars were instrumented up to F2 (ProTaper Gold) under NaCl 0.9% irrigation and incubated with for 42 days. Subsequently, different ultrasonically agitated IP were applied to the roots: control (no irrigation), 1. NaOCl 3%, EDTA 20%, CHX 2%, 2. NaOCl 3%, EDTA 20%, 3. NaOCl 3%, EDTA 20%, SDF 3.8%, 4. SDF 3.8%, and 5. NaCl 0.9%. One half of the root was investigated fluorescent-microscopically with DAPI. The other half was grinded in a cryogenic mill and the bacterial DNA was quantified with qPCR. The qPCR results showed a statistically significant reduction of bacteria after the application of IP 1, 2, and 3 compared to the control group. While IP 4 lead to a bacterial reduction which was not significant, IP 5 showed no reduction. These data corresponded with DAPI staining. With qPCR a new molecular-biological method for the investigation of the complete root canal system was implemented. The novel IP 3 had an equally good cleansing effect as the already established IP.

Citing Articles

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References

Tzanetakis G, Azcarate-Peril M, Zachaki S, Panopoulos P, Kontakiotis E, Madianos P . Comparison of Bacterial Community Composition of Primary and Persistent Endodontic Infections Using Pyrosequencing. J Endod. 2015; 41(8):1226-33. PMC: 4578635. DOI: 10.1016/j.joen.2015.03.010. View

Mohmmed S, Vianna M, Penny M, Hilton S, Mordan N, Knowles J . Confocal laser scanning, scanning electron, and transmission electron microscopy investigation of Enterococcus faecalis biofilm degradation using passive and active sodium hypochlorite irrigation within a simulated root canal model. Microbiologyopen. 2017; 6(4). PMC: 5552959. DOI: 10.1002/mbo3.455. View

Nair P, Sjogren U, Krey G, Kahnberg K, Sundqvist G . Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: a long-term light and electron microscopic follow-up study. J Endod. 1990; 16(12):580-8. DOI: 10.1016/S0099-2399(07)80201-9. View

Tan K, Yu V, Quah S, Bergenholtz G . Rapid method for the detection of root canal bacteria in endodontic therapy. J Endod. 2015; 41(4):447-50. DOI: 10.1016/j.joen.2014.11.025. View

Rocas I, Siqueira Jr J . Root canal microbiota of teeth with chronic apical periodontitis. J Clin Microbiol. 2008; 46(11):3599-606. PMC: 2576597. DOI: 10.1128/JCM.00431-08. View

Plotino G, Cortese T, Grande N, Leonardi D, Di Giorgio G, Testarelli L . New Technologies to Improve Root Canal Disinfection. Braz Dent J. 2016; 27(1):3-8. DOI: 10.1590/0103-6440201600726. View

Codony F, Dinh-Thanh M, Agusti G . Key Factors for Removing Bias in Viability PCR-Based Methods: A Review. Curr Microbiol. 2019; 77(4):682-687. DOI: 10.1007/s00284-019-01829-y. View

Tzanetakis G, Giannakoulas D, Papanakou S, Gizani S, Lygidakis N . Regenerative endodontic therapy of immature permanent molars with pulp necrosis: a cases series and a literature review. Eur Arch Paediatr Dent. 2020; 22(3):515-525. DOI: 10.1007/s40368-020-00550-w. View

Buetti N, Wassilew N, Rion V, Senn L, Gardiol C, Widmer A . Emergence of vancomycin-resistant enterococci in Switzerland: a nation-wide survey. Antimicrob Resist Infect Control. 2019; 8:16. PMC: 6337856. DOI: 10.1186/s13756-019-0466-x. View

10.

Bhuva B, Patel S, Wilson R, Niazi S, Beighton D, Mannocci F . The effectiveness of passive ultrasonic irrigation on intraradicular Enterococcus faecalis biofilms in extracted single-rooted human teeth. Int Endod J. 2010; 43(3):241-50. DOI: 10.1111/j.1365-2591.2009.01672.x. View

11.

Ruiz-Linares M, Aguado-Perez B, Baca P, Arias-Moliz M, Ferrer-Luque C . Efficacy of antimicrobial solutions against polymicrobial root canal biofilm. Int Endod J. 2015; 50(1):77-83. DOI: 10.1111/iej.12598. View

12.

Urquhart O, Tampi M, Pilcher L, Slayton R, Araujo M, Fontana M . Nonrestorative Treatments for Caries: Systematic Review and Network Meta-analysis. J Dent Res. 2018; 98(1):14-26. PMC: 6304695. DOI: 10.1177/0022034518800014. View

13.

Kayaoglu G, Erten H, Orstavik D . Possible role of the adhesin ace and collagen adherence in conveying resistance to disinfectants on Enterococcus faecalis. Oral Microbiol Immunol. 2008; 23(6):449-54. DOI: 10.1111/j.1399-302X.2008.00446.x. View

14.

Love R . Enterococcus faecalis--a mechanism for its role in endodontic failure. Int Endod J. 2001; 34(5):399-405. DOI: 10.1046/j.1365-2591.2001.00437.x. View

15.

Al-Madi E, Al-Jamie M, Al-Owaid N, Almohaimede A, Al-Owid A . Antibacterial efficacy of silver diamine fluoride as a root canal irrigant. Clin Exp Dent Res. 2019; 5(5):551-556. PMC: 6820571. DOI: 10.1002/cre2.222. View

16.

Kirsch J, Basche S, Neunzehn J, Dede M, Dannemann M, Hannig C . Is it really penetration? Part 2. Locomotion of Enterococcus faecalis cells within dentinal tubules of bovine teeth. Clin Oral Investig. 2019; 23(12):4325-4334. DOI: 10.1007/s00784-019-02865-5. View

17.

Hiraishi N, Yiu C, King N, Tagami J, Tay F . Antimicrobial efficacy of 3.8% silver diamine fluoride and its effect on root dentin. J Endod. 2010; 36(6):1026-9. DOI: 10.1016/j.joen.2010.02.029. View

18.

Herzog D, Hosny N, Niazi S, Koller G, Cook R, Foschi F . Rapid Bacterial Detection during Endodontic Treatment. J Dent Res. 2017; 96(6):626-632. DOI: 10.1177/0022034517691723. View

19.

Mathew V, Madhusudhana K, Sivakumar N, Venugopal T, Reddy R . Anti-microbial efficiency of silver diamine fluoride as an endodontic medicament - An ex vivo study. Contemp Clin Dent. 2013; 3(3):262-4. PMC: 3532785. DOI: 10.4103/0976-237X.103615. View

20.

Sedgley C, Nagel A, Shelburne C, Clewell D, Appelbe O, Molander A . Quantitative real-time PCR detection of oral Enterococcus faecalis in humans. Arch Oral Biol. 2005; 50(6):575-83. DOI: 10.1016/j.archoralbio.2004.10.017. View