Multiple assessment methodologies in determining the antibiofilm actions of sodium hypochlorite mixed with clodronate or etidronate in endodontic irrigation
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.
Section snippets
Materials
The following solutions were used.
1. 0.26 M (7.6%) clodronate-5% NaOCl, (pH = 12.5), derived from 0.52 M Na2 clodronate (Enke Pharma-tech, Cangzhou, China) (99% pure), adjusted to pH 10.7 with sodium hydroxide (NaOH) (SA178-500G, Chem-supply, Gillman, SA, Australia) and mixed with equal volumes of 10% NaOCl.
2. 0.26 M clodronate-2.5% NaOCl (pH = 12.4) derived from 0.52 M Na2 clodronate (pH = 10.7) mixed with equal volumes of 5% NaOCl.
3. 0.26 M clodronate (pH = 10.7).
4. 0.26 M (7.7%) etidronate (9%
Microbial counts
The SEM results reported in the next section (3.2), showed that the microbial counts could not be used for intergroup comparisons in a valid manner. Therefore, statistical analysis was not performed for the CFU experiments. However, the counts are nonetheless presented because they form a basis for a discussion of this methodology. Fig. 1 shows the logarithmic transformation of data for colony forming units (CFU)/mL for the median and 95% confidence interval from the average of plating in
Discussion
The results of this study add to the body of knowledge supporting the view that etidronate mixtures with NaOCl are at least equally as effective against E. faecalis biofilms as NaOCl of the same concentration. The findings further indicate that these effects can be extended to the chelator clodronate, that is that clodronate mixtures with NaOCl are equally effective as NaOCl. The first hypothesis was thus accepted. A second important outcome was the SEM result which demonstrated that, in the
Conclusions
Clodronate or etidronate mixtures with sodium hypochlorite were equally as effective against Enterococcus faecalis biofilms as sodium hypochlorite of the same concentration. However, the mixtures showed no additional benefits. Colony counting should be avoided as a biofilm assessment methodology in conjunction with hydroxyapatite discs and E. faecalis biofilms. A variety of methodologies is needed to better assess biofilm disinfection.
Author declaration
We declare that all authors have contributed significantly to this study and that all authors are in agreement with the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
While this article did not receive any specific grant for funding from the public, commercial or not-for-profit sector, PPW was supported by a University of Queensland Graduate School, St Lucia, Queensland Australia, Research Training Program Scholarship.
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