# _microorganisms_
## 4 Discussion  
A large body of evidence has suggested that bacterial behaviors in multi-species biofilm differ from the monospecies biofilm. Hence, research into the interactions between microbial species in biofilms can reveal the crucial factors that drive stability and changes leading to dysbiosis and the development of preventive and therapeutic approaches [23; 24]. In vitro studies on dual- or multi-species biofilms have been carried out under controlled conditions to address the microbial community lifestyle in the root canal and periodontal pocket, assess their interactions and effects of stresses, as well as investigate and modify current therapeutic and preventive protocols [25; 26; 27]. This in vitro laboratory study employed dual-species bacteria, namely _E. faecalis_ and _P. gingivalis_, to develop a simple and reproducible biofilm model. This dual-species biofilm model produced laboratory biofilm samples with minimal variation, which are readily assessable by microscopic and molecular analyses. A combination of techniques, including RT-PCR using viable cell staining and confocal laser scanning microscopy, were applied to study the interactions of this in vitro biofilm community. Three different models of dual-species infections were established in this study to identify biofilm-favoring conditions. In the first two sequential models, a single species of either _E. faecalis_ or _P. gingivalis_ was seeded prior to its colonization by the second microorganism. The co-culture model, as postulated to be more likely to occur in combined endodontic-periodontal lesions, involved the common contact of both species that shared and cross-infected two habitat niches, i.e., the root canal system and periodontal pockets. All models considered both anaerobic (anoxic) and aerobic (normoxic) conditions. It was found by next-generation sequencing methods that the key genera in primary and persistent infected root canals included _Prevotella_, _Fusobacterium_, _Porphyromonas_, _Parximonas_, and _Streptococcus_ and the most abundant phyla were _Firmicutes_, _Bacteroidetes_, _Proteobacteria_, _Actinobacteria_, and _Fusobacteria_[28; 29]. Using the checkerboard DNA-DNA  
Figure 7: Representative CLSM images of protein content in the biofilm stained with the SyPRO biofilm matrix stain: **(a)**_E. faecalis_ as a monospecies culture under aerobic conditions; **(b)**_E. faecalis_ as co-culture under aerobic conditions; **(c)**_E. faecalis_ as a monospecies culture under anaerobic conditions; **(d)**_E. faecalis_ as co-culture under anaerobic conditions; **(e)**_P. gingivalis_ as a monospecies culture under aerobic conditions; **(f)**_P. gingivalis_ as co-culture under aerobic conditions; **(g)**_P. gingivalis_ as a monospecies culture under anaerobic conditions; and **(h)**_P. gingivalis_ as co-culture under anaerobic conditions. Note: Protein contents are stained red. Images with a greater degree of redness denote a greater amount of protein contents, as seen in **(b,f)**. hybridization method, root canal and periodontal niches were found to share similar microbial profiles [18], and Gram-positive _E. faecalis_ and Gram-negative _P. gingivalis_ were detected in both root canals with secondary endodontic involvements and periodontal pockets in primary periodontal lesions [18]. A recent scientific report highlighted that the obligate anaerobic _P. gingivalis_ could survive in the presence of oxygen when grown along with _C. albicans_, a facultative yeast-like anaerbe [22]. It was shown that _C. albicans_ grew in their filamentous form, i.e., hyphae, and this could create a microenvironment with reduced oxygen tension and support the growth of _P. gingivalis_[22]. Obligate anaerbes usually do not possess any anti-oxidation mechanisms like superoxide dismutase to detoxify the oxygen challenge [19]. _C. albicans_ is able to create a reduced redox potential environment to support the growth of some anaerobic bacteria, and a gradient of oxygen concentration down to ca. 4% has been observed in C. albicans-formed biofilms [30; 31].