Mucositis and peri-implantitis are infections caused by fungi and bacteria biofilms [23,24,25]. Understanding this biofilm is essential to guide future therapeutic approaches, such as the use of probiotics. Traditionally, in oral candidiasis models, C. albicans and S. salivarius establish antagonistic relationships, in which the fungal cells are surrounded by S. salivarius, resulting in a decrease adhesive of ability and pathogenic potential [11, 13]. Thus, S. salivarius may be used as an alternative to the treatment of oral candidiasis. However, our findings suggest the antagonistic relationship C. albicans and S. salivarius was not established within the peri-implant microenvironment, in which biofilms were developed on titanium surfaces under low oxygen levels. Therefore, the use of S. salivarius as a probiotic would not be effective in treating peri-implant diseases.
Previous studies demonstrated an antagonistic interaction between C. albicans and S. salivarius using the following bacteria strains SK56, DSM14685, and K12 [11, 13, 26,27,28]. In our study, S. salivarius ATCC 7073 was used in all experiments. Evidence shows that the S. salivarius NCTC 8618 strain, which is a homologous strain of ATCC 7073 (available from https://www.atcc.org/Products/All/7073.aspx#generalinformation) also develops an antagonism relationship with C. albicans tested in vitro and in vivo . These findings reinforce that an antagonistic relationship would be expected between S. salivarius and C. albicans. Of importance, S. salivarius is recognized as a microorganism that does not cause harmful effects on humans and plays an important role in the biofilm composition, by inactivating and establishing antagonistic relationships with oral pathogens, such as Candida albicans [11, 13]. Thus, according to our findings, we suggest the antagonistic relationships did not happen due to the conditions in which biofilms were developed.
In the oral cavity, the oxygen levels, the substratum (e.g. mucosa, teeth, prosthesis, and implants), and variations in nutrient content could hind microorganisms to establish interactions . Our study used RPMI 1640 medium, which is known to mimic the composition of human fluids, due to the presence of amino acids such as L-Glutamine, L-Arginine, and L-Asparagine, as well as vitamins and inorganic salts . Previous investigations showed that RPMI 1640 medium can be used to initiate and develop in vitro biofilms of C. albicans, similarly to yeast nitrogen base and sabouraud dextrose broth medium . Regarding the bacteria growth, RPMI 1640 has the nutritional requirements of S. salivarius . Therefore, RPMI 1640 medium does not hind interactions between the microorganisms tested in this study.
Regarding the cell viability, co-culture biofilms of C. albicans showed differences between 24 and 72 h of biofilm development, in which the higher viability was at 72 h. Moreover, C. albicans did not have its viability changed in single-species and co-culture biofilms. This data suggested that, under the conditions tested in this study, S. salivarius was unable to decrease the number of fungal cells. Observations of C. albicans growth indicate that the mature and dispersion stage is mostly composed of cells in a hyphae-form, which are related to virulence and pathogenicity of Candida biofilms . At these stages, the dispute for nutrients among the cells may be so high that the bacteria can not interfere with the fungus growth. Although this occurs, the viability of the bacteria has not been disabled. Co-cultures biofilms of S. salivarius presented significantly higher number of viable cells.
Interactions between fungi and bacteria occur through physical contact or metabolic products [33,34,35]. S. salivarius can interact with other microorganisms through its bacteriocin-like inhibitory substances (BLIS), which is responsible for maintaining orderly population dynamics within oral microbiota . Although there is a lack of evidence concerning bacteriocin production of the strain S. salivarius ATCC 7073, the bacteriocin production has been reported in several S. salivarius strains [36,37,38]. Thus, it might be possible that S. salivarius ATCC 7073 also produced bacteriocins because it is a behavior characteristic of the streptococcal species. Notwithstanding, future studies should evaluate the S. salivarius ATCC 7073 bacteriocin production.
Despite BLIS contribution to interactions among the microorganisms in an oral candidiasis model, C. albicans was not directly inhibited by bacteriocin action. Indeed, physical cell contact is required to inhibit fungi’s growth . These findings suggest that the ability of S. salivarius inactivating some microorganisms is apart from the bacteriocin's action. Another explanation for S. salivarius did not decreasing C. albicans development is the time in which BLIS operates. Usually, microorganisms’ metabolite products act during the early stages of biofilm development, especially during exponential growth [13, 39]. It is possible that at the times evaluated in this study, the metabolite product was inactive, being insufficient to decrease the C. albicans growth. Thereby, future studies should evaluate the biofilm at the early stages of development.
In addition, the exponential growth of the C. albicans biofilm continues to advance in its maturation stage, resulting in several dense layers of polymorphic cells round in an extracellular matrix. This matrix gives the biofilm a robust and dense structure, which could protect it from chemical and physical injury [40, 41]. One possibility to estimate the contribution of the biofilm’s matrix is by measuring the biomass of the biofilm. Expectedly, single-species biofilms of C. albicans have significantly higher biomass at 72 h compared to 24 h. Besides that, our results indicate that co-culture biofilms at 72 h presented higher biomass. These findings suggest that the matrix of biofilm contributes to its architecture and could act as a protective barrier for C. albicans.
The extracellular matrix of biofilm has around 500 proteins in its structure, most of which are hydrolyzing enzymes that can disrupt biopolymers as both a protective response and a nutrient source . Preliminarily, we also investigate the proteins in the biofilm cultures throughout the biuret assay. Higher protein production was observed at 72 h of biofilm development, and this is possibly due to the longer period these biofilms remained in cultivation. Moreover, there is possible that a fungi-bacteria relationship and a robust extracellular matrix contributed to increasing the total protein production. However, our results are limited to the dosage of total proteins, which could not estimate which protein would make the greatest contribution to this biofilm. The process of identifying this protein could be important for creating therapeutic targets.
In general, our findings suggest that C. albicans growth did not change due to the presence of S. salivarius. To better understand the interactions between both microorganisms and the virulence of C. albicans through the filamentous formation, fluorescence microscopy was performed. In the early stage (24 h), the yeast cells were prevalent, with few pseudo-hyphal cells and hyphae surrounded by S. salivarius. However, at 72 h numerous pseudo-hyphal cells and some hyphae were presented. The filamentous forms of C. albicans (hyphae and pseudo-hyphae) are considered pathogenic . Thus, these results suggest that under the conditions tested in this study and during the dispersion stage, C. albicans inactivation is a challenge to S. salivarius.
Although microscopy evaluations are widely used, future investigations should consider using gene expression analysis or other genetic approaches to evaluate transcriptional regulators of C. albicans (Efg1, Tec1, Bcr1, Ndt80, Brg1, and Rob1) , virulence factors such as Hwp1 and Als3 , as well as invasiveness (Sap family) . In addition, other study designs should consider including relevant bacteria involved with peri-implant infections (i.e. Porphyromonas gingivalis)  and host–pathogen interactions of C. albicans .