The infection and proliferation of microorganisms within the root canal results in inflammation of pulpal tissue, developing apical periodontitis (Kakehashi et al., 1965). Therefore, root canal treatment was purposed to medicate and to prevent the inflammation of the tissues around the root apex. Persistent infection is a major cause of failure of endodontics treatment (Lin et al., 1992; Siqueira Jr, 2001). In this type of secondary infection, the diversity of microbial strains has been found to be lower than in the primary infection due to the limitations on the limiting conditions in the root canal environment (Sundqvist, 1992; Sundqvist and Figdor, 2003), with facultative gram-positive bacteria, including Enterococcus and Streptococcus, being among the most prevalent species (Siqueira, 2002, Siren et al., 1997). In addition, Candida albicans is the most common fungal species detected in persistent root canal infection (Siqueira and Rôças, 2008).

Living in the biofilm communities renders the residential to promote each other’s resistance to the harmful external environment (Yin et al., 2019) and antimicrobial agents (Gilbert et al., 1997, Johnson et al., 2002). Inter-kingdom bacterial and fungal biofilms have been found to be more complex due to the interaction between species, enhancing their fitness in many areas (Krüger et al., 2019). In mixed-species biofilmss, resident microbes interact with one to each other in different modes, including synergism, antagonism, and mutualism. C. albicans and streptococci have well-demonstrated mutualistic relations, and when they coexist in mixed-species biofilms they enhance one another’s virulence (Xu et al., 2014). The dual-species biofilm of C. albicans and S. gordonii indicates that the bacteria enhance fungal filamentation by physical and chemical influences, due to the quorum-sensing molecule auto-inducer-2 pathway. Therefore, the biomass of the mixed-species biofilms is often higher than single species ones (Bamford et al., 2009, Dutton et al., 2014). However, the relationship of E. faecalis and C. albicans is antagonistic due to E. faecalis inhibition of hyphal formation of C. albicans via the Fsr quorum-sensing system, which is the virulence factor of E. faecalis (Cruz et al., 2013). Meanwhile, the positive interaction between S. gordonii and E. faecalis in the production of a thick biofilm enable them to resist nutrient deprivation (Gao et al., 2016). Furthermore, these two species can transfer viulence factors to increase antibiotic resistance (Chávez de Paz et al., 2005, Sedgley et al., 2008).

Bouea macrophylla (commonly known as Maprang) is a tropical fruit tree which is native to and widely cultivated in Southeast Asia, including in Indonesia, Burma, Malaysia, and Thailand. B. macrophylla is an economically significant fruit crop in Thailand, popular both for local consumption and for export. The trees grow well in the central and lower parts of the country’s northern region. Therapeutic potential has been attributed to every part B. macrophylla, such as the root, bark, leaves, flower, and fruit. The kernel of the fruit possesses high phytochemical potential, due to phenolic and flavonoid compounds (Dechsupa et al., 2019) which have anti-inflammatory, antioxidant, and antimicrobial properties, the latter affecting both gram-positive and gram-negative bacteria as well as fungi (Dechsupa et al., 2019, Leelapornpisid and Poomanee, 2021, Nguyen et al., 2020, Zainah et al., 2018). Moreover, B. macrophylla has been successful in eliminating oral microbes such as E. faecalis, S. gordonii and C. albicans (Leelapornpisid & Poomanee, 2021). However, the efficacy of this novel extract against root canal-associated strains in the form of a mixed-species root canal biofilm are yet to be determined.

This study aimed to determine the antimicrobial activity of B. macrophylla kernel extract in eradicating mixed-species biofilms of E. faecalis, S. gordonii and C. albicans. The hypotheses were that B. macrophylla kernel extract is effective in eradicating mixed-species biofilms of E. faecalis, S. gordonii, and C. albicans both in vitro and in tooth model, and that its efficacy is comparable to that of conventional medicaments, including calcium hydroxide (Ca(OH)2) and chlorhexidine.