(1) Objectives: This study is the first one to investigate the molecular composition of the dental biofilm during the exogenous and endogenous prophylaxis stages (use of dentifrice/drug) of individuals with different cariogenic conditions using molecular spectroscopy methods. We suggest that TiO2NPs pave the way for further applications in oral mouthwash formulations and antibiofilm dental coatings. Overall, these results have important implications for the use of tetragonal anatase phase TiO2NPs (size range 5–25 nm, crystalline size 13.7 nm, and spherical shape) as an oral antibiofilm agent against Gram positive cocci infections. Exposure of oral isolates to TiO2NPs (14.7 nm) significantly (p < 0.05) reduced planktonic cell viability and biofilm formation in a concentration-dependent manner, which was confirmed by observing biofilm architecture by scanning electron microscopy (SEM) and optical microscopy. Furthermore, these Gram positive MDR oral cocci showed significant (p < 0.05) variations in their biofilm forming ability under different physicochemical conditions, that is, at temperatures of 28, 30, and 42 ☌, pH of 6.4, 7.4, and 8.4, and NaCl concentrations from 200 to 1000 µg/mL. These three oral isolates exhibited antibiotic-resistance against nalidixic acid, tetracycline, cefuroxime, methicillin, and ceftazidime. Here, we isolated and identified three Gram-positive MDR oral cocci, (1) SJM-04, (2) SJM-38, and (3) SJM-65, and characterized them morphologically, biochemically, and by 16S rRNA gene-based phylogenetic analysis as Georgenia sp., Staphylococcus saprophyticus, and Rothia mucilaginosa, respectively. Multi-drug resistant (MDR) bacterial cells embedded in biofilm matrices can lead to the development of chronic cariogenesis.
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