# _microorganisms_
## 1 Introduction  
The oral cavity is a well-oxygenated environment, where the elevated oxygen tension modulates oxygen-tolerating bacterial cells to increase their enzymatic and non-enzymatic reduction of molecular oxygen to superoxide anions. This results in the formation of hydrogen peroxide and oxygen by dismutation, and the former reacts with superoxide anions to form hydroxyl ions in the presence of iron complexes [1]. These free oxygen radicals are highly reactive and able to cleave nucleic acids and oxidize essential proteins and lipids [2; 3]. In contrast, obligate anaerobic microorganisms, such as _P. gingivalis_, do not possess the mechanism of anti-oxidation. Thus, the reactive oxygen species must be detoxified to minimize the undesirable effects on the obligate anaerobic microorganisms.  
_P. gingivalis_, a group of obligate anaerobic bacteria, is highly associated with periodontal diseases, ranging from reversible gingivitis to irreversible periodontitis [4]. Along with _Tamerella forsythia_ (_T. forsythia_) and _Treponema denticola_ (_T. denticola_), they form the \"red complex\" and have been implicated as the primary causative pathogens in periodontal diseases [5; 6]. They progressively degrade the periodontal collagen and non-collagenous tissues, forming periodontal pockets [7]. _Enterococcus faecalis_ (_E. faecalis_) is the most frequently isolated bacterial species from symptomatic root canal-treated teeth, with reported prevalence in up to 90% of cases [8; 9; 10]. _E. faecalis_ is often found in secondary or persistentcases due to its ability to survive in harsh environments with nutrient deprivation and high alkalinity despite the presence of intracanal medicaments [11; 12; 13; 14]. The pathogenicity and difficulty of their eradication have been attributed to the ability of _E. faecalis_ to form biofilms, which can be 1000-fold more resistant to antimicrobials than their planktonic counterparts [15; 16]. Both _E. faecalis_ and _P. gingivalis_ have been frequently isolated in infected root canals and periodontal pockets in failed cases by culturing and molecular identification techniques [17; 18].  
_P. gingivalis_ can tolerate low levels of oxygen (6-10%) and reach a steady state of growth depending on the availability of hemin [19; 20]. _Fusobacterium nucleatum_ (_F. nucleatum_), another obligate anaerobic bacteria, supports the growth of _P. gingivalis_ in an unfavorable oxygenated and carbon dioxide-depleted environment [19; 21]. _F. nucleation_ creates a microenvironment with reduced oxygen tension that protects _P. gingivalis_ within the niche [19]. A recent study demonstrated the potential of _Candida albicans_ (_C. albicans_), a yeast-like fungus, in protecting anaerobic bacteria in an aerobic environment [22]. As a facultative anaerobe, _C. albicans_ creates a hypoxic microenvironment within the fungal biofilms, which is conducive to the growth of _P. gingivalis_[22]. To date, there is no research conducted to study the interaction between _E. faecalis_ and _P. gingivalis_ when they co-exist. Therefore, this study aimed (i) to develop a simple and reproducible biofilm model consisting of _E. faecalis_ and _P. gingivalis_ and (ii) to study the interaction between these two bacterial species under both aerobic and anaerobic conditions separately, either using single- or dual-species culture models.