# _microorganisms_
## 4 Discussion
A molecule called \"autoindicers\" was involved in biofilm formation [42]. E. _f faecalis_ biofilm formation is regulated by the fecal streptococci regulator (_fsr_) locus [43]. Enterococcus do not only communicate through Fsr quorum sensing but are also capable of communicating by peptide pheromones, including Cpd, Cob, and Ccf [44,45]. On the other hand, _P. gingivalis_'s capacity to form biofilms is related to the caseionlytic proteases (Clp), which upregulate the expression levels of fimA, mfa1, and luxS [46]. _E. faecalis_' biofilm development was influenced by growth medium and nutrient availability [16]. In this study, TSB broth in Pg broth contained 3 g of glucose which helped to promote _E. faecalis_' biofilm formation. It was reported that _E. faecalis_ showed highly aggregated biofilms with higher bacteria counts and EPS bio-volume when the medium contained sucrose compared to the medium containing glucose or without sugar substrate [16]. _E. faecalis_ possesses many virulence factors, such as enterococcal surface protein (_esp_), gelatinase (_gelE_), aggregation substance (_asa1_), cytolysin B (_cylB_), and endocarditis-specific antigen A (_efaA_) gene, ArgR family transcription factor (_alhr_C), endocarditis and biofilm-associated pili (_elppA_), enterococcal polysaccharide antigen (_epal_), epal and OG1 RF_11715 (_epaOX_), and (p)ppGpp-syntherase/hydrolase (_relA_) genes [47, 48, 49, 50, 51, 52, 53, 54, 55]. Meanwhile, _P. gingivalis_ also possesses various virulence factors, such as lipopolysaccharide-Charg, gingipains, fimbiae/pili, collagenase, (erythrocyte) lectins, capsule, protease, and superoxide dismutase [56,57]. These virulence factors are predominantly proteins of different types, which might have attributed to the higher protein contents observed and thicker biofilm formation in this study when both microorganisms were grown together. A previous dual-species biofilm proteomic study consisting of _F. nucleatum_ and _P. gingivalis_ showed that more proteins were found when they were co-cultured together as compared with _P. gingivalis_ as mono-culture but to a lesser extent than _F. nucleatum_ mono-culture biofilm [58]. The reasons were not fully elucidated. It might be due to the antagonist effect of _P. gingivalis_ upon _F. nucleatum_. To date, no studies have characterized the proteome of dual-species biofilm composed of _E. faecalis_ and _P. gingivalis_. However, some facultative anaerbes such as _C. albicans_ and _Streptococcus gordonii_ (_S. gordonii_) have been shown to co-adhere to _P. gingivalis_ in co-culture [59, 22]. The co-adhesion between _P. gingivalis_ and _S. gordonii_ might have been mediated by the SspB protein of _S. gordonii_, whereas Mp65 adhesin and enolase might have played a role in the interaction between _C. albicans_ and _P. gingivalis_ ([PERSON] et al., 2000, [PERSON] et al., 2019). It was postulated that the cross-talk between these proteins might have rendered the _P. gingivalis_ in the dual-species biofilm the protective mechanism to thrive in an oxic environment. _E. faecalis_, a facultative anaerbe like _S. gordonii_ and _C. albicans_, might have also cross-talked with _P. gingivalis_ when they were co-cultured, resulting in an increased protein production. [PERSON] and co-workers reported that 100-kDa protein of _P. gingivalis_ interacted with _E. faecalis_ to induce the cellular surface's expression of SspB [59]. Furthermore, _E. faecalis_ had been shown to take up heme and synthesize heme proteins, called hemoproteins [60]. The hemin content in the culture medium used in this study could have potentiated the hemoprotein synthesis, contributing to the increased protein contents when _E. faecalis_ cells were present in the co-cultured biofilms. In future studies, scanning electron microscopy or fluorescence in situ hybridization (FISH) combined with CLSM could be used to determine the spatial distribution of the bacteria in the biofilm [61], which may reveal the actual architecture of the dual-species biofilm.