Multiple assessment methodologies in determining the antibiofilm actions of sodium hypochlorite mixed with clodronate or etidronate in endodontic irrigation

Highlights

• Clodronate/etidronate-NaOCl mixtures have the same antibiofilm action as NaOCl

• Colony counting may be an inaccurate biofilm assessment method

• The use of multiple assessment methodologies in biofilm assessment is recommended

Abstract

This study aimed to use multiple methodologies, including a novel usage of scanning electron microscopy (SEM), to evaluate the antimicrobial actions of sodium hypochlorite (NaOCl) admixed with clodronate or etidronate in root canal irrigation. The study also examined the usefulness of colony counting as a biofilm assessment methodology. Seven day Enterococcus faecalis biofilms were grown on hydroxyapatite discs. The discs were disinfected with 0.26 M clodronate-5% NaOCl, 0.26 M etidronate-5% NaOCl, 5% NaOCl, or treated with phosphate buffered saline (PBS). Assessments were performed using colony counting, SEM and the XTT reduction assay. The XTT assessment used the same groups but with 2.5% NaOCl. For colony counting, bacteria were removed from the discs by vortex mixing, followed by plating. The discs were subsequently fixed for SEM imagining and evaluators scored the SEM micrographs for remaining bacteria. Antibiofilm actions were assessed with the Kruskal-Wallis and Dunn's multiple comparison tests. SEM micrographs and the XTT assay revealed no differences between the NaOCl controls and the clodronate or etidronate mixtures with NaOCl (P > 0.05). It was concluded that the chelator mixtures with NaOCl had antibiofilm actions comparable to NaOCl. Furthermore, vortex mixing incompletely removed biofilm from HA discs in the PBS controls and hence colony counting using E. faecalis biofilms on hydroxyapatite discs could not be used for intergroup comparisons involving PBS. Additionally, colony counting could not be used for comparisons between the NaOCl treatment groups because the removal of bacteria from the substrate by vortex mixing was affected by the irrigant type.

Introduction

Several authors have written on the importance of choosing appropriate methodologies to assess antibiofilm actions. A common theme is the discouragement of the use of a single methodology (Stiefel et al. 2016; Azeredo et al. 2017). Rather, because of the differing strengths, weaknesses and suitability of particular techniques, the use of a variety of methodologies is stressed.

Colony counting is the most commonly encountered tool in endodontic (root canal) antimicrobial research and the majority of studies use the species Enterococcus faecalis (Swimberghe et al. 2019). In this methodology, E. faecalis biofilms are grown on teeth, blocks of dentine or hydroxyapatite (HA) discs, disinfected, and the remaining bacteria are removed by vortex mixing or sonication, subsequent to plating and counting (Deng et al. 2009; Ali et al. 2020). One of the problems associated with colony counting is that the removal of biofilm bacteria from the substrate for both test and control groups is typically not experimentally verified. Thus, calculating percentage reductions and performing intergroup comparisons may be inaccurate.

Scanning electron microscopy (SEM) has been used to qualitatively assess the removal of E. faecalis biofilm from teeth with different irrigation protocols employing NaOCl (NaOCl) (Seet et al. 2012). Because NaOCl effectively dissolves biofilm (Zehnder 2006), SEM could also be applied in a quantitative manner to examine antibiofilm effects by counting bacteria remaining on substrates such as HA discs following treatment. Secondly, after vortex mixing or sonication, SEM could demonstrate the effectiveness of removing E. faecalis biofilm from discs, and thus, it would help ascertain the usefulness of colony counting in this situation.

Sodium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium inner salt (XTT) (Bandara et al. 2016) is a measure of cellular metabolic activity and cell viability (Kuhn et al. 2003). In this assay, XTT undergoes chemical reduction due to the action of bacterial dehydrogenase enzymes with a resulting colour change that is quantified spectrophotometrically (Koban et al. 2012). It has been used as an adjunctive tool in antibiofilm assessments (Philip et al. 2019).

In root canal disinfection and irrigation, continuous chelation is a technique that involves the use of a single irrigant mixture consisting of a chelator combined with NaOCl. This mixture is used throughout the entire preparation of the root canal (Neelakantan et al. 2012). Continuous chelation aims to simultaneously perform the three key functions of an endodontic irrigant, that is, to disinfect, to dissolve organic matter and to remove the instrument created smear layer (Zehnder et al. 2005). In contrast, in standard endodontic irrigation, NaOCl is used first as the main irrigant, to disinfect and dissolve organic matter. Subsequently a rinse of EDTA removes the smear layer (Zehnder 2006).

The majority of research in continuous chelation is centred on the weak chelator etidronate. Several studies have assessed the ability of etidronate-NaOCl mixtures to disinfect biofilms. The in vitro studies have employed a model incorporating 5–7 day E. faecalis biofilms, and based predominantly on the use of live/dead staining in conjunction with confocal laser scanning microscopy (Arias-Moliz et al. 2014; Morago et al. 2016). In vitro studies, in addition to the live/dead technique, have, on occasion, also incorporated colony counting as the assessment methodology (Arias-Moliz et al. 2014). There is a general consensus among studies that etidronate mixtures with NaOCl are at least as antimicrobial as NaOCl.

Recently, another weak chelator, clodronate at alkaline pH, has been identified for use in continuous chelation. Clodronate-NaOCl mixtures can remove smear layer (Wright et al. 2020a) and maintain free available chlorine levels at root canal temperature over clinically relevant time frames (Wright et al. 2020b). Given sustained levels of NaOCl, it is probable that clodronate-NaOCl mixtures will be at least equally as antimicrobial as etidronate-NaOCl mixtures and NaOCl controls. However, to date, there is no published data on the capacity of clodronate mixtures to disinfect biofilms.

The principal aim of this research was to compare the antimicrobial effectiveness of plain NaOCl with clodronate and etidronate mixtures with NaOCl, using three assessment methods. The current study tested two hypotheses using colony counting, SEM and the XTT assay as assessment methods. The first was that the mixtures 0.26 M clodronate-NaOCl and 0.26 M etidronate-NaOCl are equally as effective against E. faecalis biofilms as NaOCl of the same concentration. The second hypothesis was that E. faecalis biofilms can be removed from treatment and control HA discs by vortex mixing, and thus colony counting can provide useful information about the antibiofilm actions of antimicrobial agents used in endodontics.