# _microorganisms_
## 4 Discussion
On the other hand, _P. gingivalis_ cells were shown to survive and tolerate low oxygen levels (6-10%), depending on the haemin availability [32]. Haemin dimers binding to the surface of _P. gingivalis_ would serve as a catalase-like buffer system to overcome the oxygen stress and reach a steady-state growth [19; 20; 33]. Whether _E. faecalis_ possesses the same ability to form a protective \"biofilm\" to shield anaerobic _P. gingivalis_ in the aerobic environment has never been investigated. PCR is usually used to qualitatively detect the presence or absence of a particular bacterial DNA, while it does not provide quantification of the bacterial species of interest and is unable to differentiate live from dead cells [34]. To counteract these issues, quantitative PCR was used in this study to quantify the relative amount of individual living bacterial cells and propidium monoazide (PMA) stain (PMAX(tm), Biotinum, CA, USA) was used to detect the viable DNA. PMAxx(tm) dye is a photoreactive dye that preferentially binds to the dsDNA [35]. Visible blue light by PMA-Lite LED Photolysis Device will induce a photoreaction of the chemical, leading to a covalent bond with PMA and the dsDNA. PMAxx(tm) dye is designed to be a cell membrane-impermeant [36]; thus, only dead cells are susceptible to DNA modification due to compromised cell membranes. Thus, this unique feature of PMAxx(tm) is highly useful in selectively detecting live bacteria by subsequent qPCR. CLSM, a non-destructive method often used to investigate the biofilm ecosystem and the hydrated spatial arrangement at the cellular scale, was employed in this study to assess biofilm thickness and relative protein contents [15]. This technique allows for three-dimensional reconstruction of the biomass to study the biofilm architecture [15]. SyPRO biofilm matrix stain was used in this study to allow the visualization of various protein molecules produced by the bacterial species, including glycoprotein, lipoprotein, phosphoprotein, calcium-binding protein, and fibrillar protein [37]. It was reported that _C. albicans_ formed filamentous hyphae that facilitate the growth of _P. gingivalis_ under the oxic challenge [22]. Similarly, another Gram-negative obligate anaerbe, _F. nucleatum_, could support the growth of _P. gingivalis_ in the oxygenated environment by its ability to metabolize oxygen [19]. _F. nucleatum_ expresses nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to break down the oxygen molecule [38]. In this study, _P. gingivalis_ was found to maintain its viability in the oxic condition in the presence of _E. faecalis_. Hence, the authors proposed that _E faecalis_ could probably provide some barrier to protect the obligate anaerbe _P. gingivalis_ from the oxygen challenges so that _P. gingivalis_ was able to grow and proliferate in the presence of high oxygen tension. The possible mechanism that _E. faecalis_ could act as an \"oxygen reducing bacteria\" is that it is a facultative anaerbe that can grow and utilizes the oxygen present in the biofilm micro-environment [39; 40]. Additionally, _E. faecalis_ can form a biofilm, and the extracellular polysaccharide (EPS) formed in the biofilm can protect the resident _P. gingivalis_. The EPS also offers protection against various environmental stresses such as pH shifts, osmotic shock, UV radiation, and desiccation [15]. However, a 15-species polymicrobial biofilm model showed that when _E. faecalis_ was added to the basic biofilm that consisted of _P. gingivalis_ and others, the total cell number was reduced [41]. This could be due to the antagonistic effect of the remaining bacteria present in the biofilm models. Biofilm thickness was significantly greater when both species were co-cultured simultaneously, regardless of the oxygen challenge. This was further supported by the findings of increased protein contents in the co-cultured biofilms. Biofilm formation is associated with quorum sensing, which regulates bacterial gene expression in response to large cell population densities.