Abstract
Three-dimensional (3D) scaffold systems have proven instrumental in advancing our understanding of polymicrobial biofilm dynamics and probiotic interactions within the oral environment. Among oral probiotics, Streptococcus salivarius K12 (Ssk12) has shown considerable promise in modulating microbial homeostasis; however, its long-term therapeutic benefits are contingent upon successful and sustained colonization of the oral mucosa. Despite its clinical relevance, the molecular mechanisms underlying the adhesion, persistence, and integration of Ssk12 into the native oral microbiome/biofilm remain inadequately characterized. In this pilot study, we explored the temporal colonization dynamics of Ssk12 and its impact on the structure and functional profiles of salivary-derived biofilms cultivated on melt-electrowritten poly(ε-caprolactone) (MEW-mPCL) scaffolds, which emulate the native oral niche. Colonization was monitored via fluorescence in situ hybridization (smFISH), confocal microscopy, and RT-qPCR, while shifts in community composition and function were assessed using 16S rRNA sequencing and meta-transcriptomics. A single administration of Ssk12 exhibited transient colonization lasting up to 7 days, with detectable presence diminishing by day 10. This was accompanied by short-term increases in Lactobacillus and Bifidobacterium populations. Functional analyses revealed increased transcriptional signatures linked to oxidative stress resistance and metabolic adaptation. These findings suggest that even short-term probiotic colonization induces significant functional changes, underscoring the need for strategies to enhance probiotic persistence.