The root canal system (RCS) has a complex structure that is characterized by the presence of various anatomical features, including accessory, lateral, apical delta, and collateral canals (Pagonis et al., 2010; Vera et al., 2012; Haapasalo et al., 2014; Wong et al., 2021). This structure is also distinguished by anatomical variations such as canal flattening, isthmuses, and indentations, which collectively prevent the effective removal of residual tissue materials and bacteria (Leoni et al., 2014; Versiani et al., 2016). Therefore, anatomical complexity promotes the formation of bacterial biofilms (Nair et al., 2005; Siqueira, Roças, 2008; Vera et al., 2012; Wong et al., 2021).
Biofilms are microbial communities in which cells adhere to a substrate via an extracellular polymer matrix, altering growth phenotypes (Sadiq et al., 2022; Pandey et al., 2022). Within biofilm communities, microorganisms benefit from several favorable conditions, the most notable of which is protection from antimicrobial agents. The mechanisms underlying antimicrobial tolerance within biofilm-structured cells have been thoroughly investigated, revealing four distinct pathways. The first mechanism involves the matrix, which is primarily composed of exopolysaccharides (EPS) and serves as a natural antibiotic barrier. This matrix concentrates extracellular enzymes, including β-lactamase, which effectively inhibit the activity of β-lactam antibiotics (Anderl et al., 2000; Singh et al., 2021). The second is due to biofilm growth dynamics, whose microorganisms show slower growth rates than planktonic cells (He et al., 2024). Consequently, if nutrient availability is reduced, bacteria enter a latent or stationary growth phase, making them less vulnerable to nutritional cutback (Portenier et al., 2006; Singh et al., 2021). The third mechanism is related to oxygen metabolism, which is influenced by biofilm surface cells that can deplete available oxygen, resulting in anaerobic niches for the bacterial community (De Beer et al., 1994). This phenomenon has a differential effect on biofilm cells’ responses to certain antibiotics, such as aminoglycosides, which are more effective against aerobic bacteria (Tresse et al., 1995; Duggan, Sedgley, 2007). Finally, a persistent subpopulation of microorganisms, despite accounting for a minor fraction of the original population, can evade antimicrobial agents (Spoering, Lewis, 2001).
The human oral cavity contains a large and diverse microbiome, with approximately 700 microbial species present in both forms of biofilm and planktonic cells (Paster et al., 2001; Valera et al., 2009; Zhao et al., 2017; Deo, Deshmukh., 2019). These microorganisms have varying penetration capacities into dentinal tubules, typically around 500 µm (Love, Jenkinson., 2002; Parmar et al., 2011; Brittan et al., 2016; Teoh et al., 2023). Located within the root canal, a conical-shaped structure with distinct diameters in its thirds, cervical (3.20 µm), middle (2.80 µm), and apical (2.11 µm), the dentinal tubules can suffer bacterial infiltration, which allows microorganisms to reach deeper dentin regions (Camargo et al., 2007; Ribeiro et al., 2010).
It is worth highlighting that Enterococcus faecalis has a remarkable penetration ability, reaching depths of 800–1000 µm after three weeks of incubation (Haapasalo, Orstavik., 1987; Ran et al., 2014; Vatkar et al., 2016). This bacterium is significant as a nosocomial pathogen capable of causing various infections in humans (Jett, Huycke., 1994; Richards et al., 2000; Ali et al., 2017). Despite its small population in the oral microbiota, E. faecalis is important in the etiology of persistent periapical lesions (Evans et al., 2002; Sunde et al., 2002; Roças et al., 2004; Johnson et al., 2006; Arias-Moliz et al., 2009) and persistent apical periodontitis (Siqueira, Roças., 2022).
Biofilm formation on the dentin walls of root canals is a common occurrence that can be prevented with biomechanical interventions (Stojicic et al., 2013; Neelakantan et al., 2017). However, E. faecalis is extremely resistant to biomechanical preparation and various intracanal medications, including calcium hydroxide (Vivacqua-Gomes et al., 2005; Dunavant et al., 2006; Abu Hasna et al., 2020). This microorganism has an exceptional ability to survive in alkaline pH conditions within dentinal tubules for extended periods (Kaufman et al., 2005; Berber et al., 2006; Gomes et al., 2006).
Accordingly, to assess the efficacy of antimicrobial medications, the number of colonies forming units (CFUs) must be determined. To accomplish this, biofilm must be removed from the root canal system while microbial cells are still viable. This procedure usually involves the use of sterilized absorbent paper points, which is the same method used in the in vivo collection stage. Nonetheless, this approach has limitations, particularly in terms of achieving efficient biofilm dislodgement during the removal step. Thus, in the current study, a novel methodology was proposed to improve the displacement of mature E. faecalis biofilms from the root canal system, ensuring concomitant bacterial viability while minimizing potential lysis or cell death.