Biofilms are microbial communities enmeshed in a three-dimensional (3D) extracellular polysaccharide matrix (Costerton et al., 1995) that provide several advantages to microbial growth, which may be physical, physiological, or molecular (Yin et al., 2019). As soon as oral microorganisms adhere to biotic and abiotic surfaces on the oral cavity, such as oral mucosal tissues, teeth, or to the surface of prosthetic materials (such as dental implants), they secrete extracellular polymeric material forming these highly organized and adhered communities (Marsh et al., 2011). While biofilms forming on dental surfaces have been reported to be responsible for dental caries and periodontal disease for centuries (Berg and Fosdick, 1946, Hopkins, 1899), biofilms growing on dental implant surfaces, usually made of titanium (Ti), are a much newer issue, described in the late 1990 s (Alcoforado et al., 1991) as a condition that led to the development of an inflammatory response in the peri-implant tissues (Lang et al., 1993).

Biofilm formation on Ti surfaces has been considered the main etiological factor triggering inflammatory disease processes known as peri-implant mucositis and peri-implantitis (Heitz-Mayfield and Salvi, 2018, Salvi et al., 2017). It has been proposed that biofilm accumulation around teeth in periodontal disease can produce an increased inflammatory host response that changes the local environmental conditions, favoring the growth of proteolytic and anaerobic gram-negative bacteria (Marsh et al., 2011). The same process has been hypothesized for biofilms growing on implant surfaces (Costa et al., 2021, Souza et al., 2021). Importantly, although there is a well-recognized gold standard treatment for periodontal disease based on mechanical debridement often combined with antibiotics (Sanz et al., 2020), there is still no consensus regarding the effective treatment strategies for implant-related infections (Heitz-Mayfield & Mombelli, 2014). Thus, it is imperative to understand better factors that promote biofilm growth, hamper bacterial killing or biofilm removal, and identify specific pathogenic processes related to biofilms growing on Ti surfaces. Such goals will shed light on the development of future therapeutic strategies for peri-implantitis.

In this context, the extracellular matrix (ECM) plays an essential functional role in the biofilm accretion and structure (Flemming & Wingender, 2010). Although biofilm matrix is composed of a complex array of components, such as proteins and nucleic acids (eDNA and eRNA), the bacterial-derived exopolysaccharides (EPS) have been responsible for promoting bacterial cell adhesion and growth, cross-kingdom interaction, and microbiological shift with an increased load of pathogenic species (Bowen et al., 2018, Karygianni et al., 2020). Within the oral bacterial colonizers, Streptococcus mutans is known to be the main contributor to the formation of EPS matrix in dental biofilms due to their extracellular or cell-associated exoenzymes, known as glucosyltransferases (GTFs). Such GTFs hydrolyze sucrose from the host diet to synthesize extracellular polymers, known as glucans, formed in various proportions of α-1,6 and α-1,3-linkages (Bowen and Koo, 2011, Klein et al., 2015, Kopec and Vacca-Smith, 1997). The role of S. mutans to synthesize EPS has been widely explored on dental surfaces, mostly linked to enamel demineralization and dental caries, showing significant influence on biofilm growth, increased virulence and decreased susceptibility to most antimicrobial treatments in vivo, in situ and in vitro (Bowen et al., 2018). Importantly, this bacterium has been also found on early and late stages of peri-implant disease (Kumar et al., 2012; Souza et al., 2017) but still, little to no efforts have been made to characterize its role on biofilm virulence when growing on Ti surfaces presents in the oral cavity. S. mutans is an important EPS producer, which promotes peri-implant pathogens growth (Costa et al., 2021), thus its role on biofilm architecture cannot be neglected when biofilms are formed on Ti surfaces, as it could play an accessory role on peri-implant infections, by reducing biofilm susceptibility to antimicrobials, such as CHX, protecting the biofilm, which could allow peri-implant pathogens to grow and accumulate.

Interestingly, a previous study has shown that the surface where the biofilm is growing modulates the gtf expression, and a higher expression was found for the Ti substrate (Souza et al., 2020b). This data corroborated the existing literature suggesting that the chemical and physical properties of different surfaces can directly affect biofilm formation and its molecular pathways (Song et al., 2015, Yang et al., 2022). Previous studies described that salivary pellicle composition can significantly vary on different surfaces present in the oral cavity, such as dental surfaces (biotic surfaces) and Ti material (abiotic surfaces) (Cavalcanti et al., 2016, Mukai et al., 2020), which can then affect biofilm maturation (Laosuwan et al., 2018). Interestingly, evidence has shown that bacterial-derived EPS can reduce the antifungal susceptibility of Streptococcus-Candida mixed-biofilms (Kim et al., 2018). The same effect has been described for polymicrobial biofilms formed on Ti surface and exposed to antibiotics (Costa et al., 2020a). This protective effect has been attributed to the ability of EPS to create microenvironments that restrict the access of antimicrobial agents; the negative charge of EPS, reducing the diffusion of positively charged antimicrobials (Xiao et al., 2012); and the binding ability of EPS to create a protective layer surrounding microbial cells (Falsetta et al., 2014). As more information emerges on the characterization of peri-implant microbiome, more we understand that this disease is not caused by a single or even a few selected oral bacteria, but rather by polymicrobial communities with different species playing different roles on biofilm pathogenicity. Importantly, the comparison between dental surface and Ti in modulating the ability of ECM to enhance the antimicrobial resistance of biofilms has not been experimentally tested.

Moreover, when we consider the complexity of biofilms, the use of bactericidal and/or bacteriostatic agents alone do not lead to their complete eradication since dead cells can remain attached to the bacterial community and serve as coaggregation sites for new viable cells (Koo et al., 2017), further allowing the re-colonization by new pathogens. Since the biofilm ECM has been recognized as the main factor promoting microbial adhesion and co-aggregation (Schilling & Bowen, 1992), it is expected that after the use of antimicrobial treatment, the remaining and not removed ECM from biofilms will show enhanced recolonization by new microbial cells. However, this has not been experimentally tested yet. Therefore, this study compared the role of EPS to reduce antimicrobial susceptibility on biotic (dental surface) and abiotic (Ti biomaterial) surfaces and the effect of remaining ECM-enriched biofilms to promote bacterial recolonization.