Effects of direct-printed aligner eluents on the growth, biofilm, and transcriptome of Streptococcus mutans GS-5

Abstract

Objectives: This study aimed to evaluate the influence of eluents from direct-printed aligners (DPAs) on Streptococcus mutans (S. mutans), a major cariogenic bacterium, by examining bacterial growth, biofilm formation, and transcriptomic changes. Methods: Eluents were collected by immersing 3D-printed aligners in artificial saliva under simulated oral conditions. These eluents were then used to treat S. mutans, and bacterial proliferation was measured by optical density at 600 nm (OD600 ). Biofilm formation was quantified using crystal violet staining, and ultrastructural alterations were assessed via scanning electron microscopy (SEM). Transcriptomic shifts were identified by RNA sequencing (RNA-seq). Results: DPA eluents reduced S. mutans growth and biofilm formation by 29-39% compared to controls (p<0.05). SEM revealed decreased biofilm without morphological alterations. RNAseq analysis detected 10 differentially expressed genes (DEGs), showing downregulated expression of quorum sensing (QS) and biofilm-associated genes, including lantibiotic-related and acyl carrier protein genes. Conversely, genes related to oxidative stress responses-such as aldo-keto reductases, short-chain dehydrogenases, and ATP-binding cassette transporters-were upregulated, suggesting activation of a stress-adaptive mechanism. Conclusions: While DPA eluents effectively suppress biofilm formation, the accompanying activation of oxidative stress responses may enhance bacterial resilience, raising concerns about potential long-term microbiome dysbiosis. Comprehensive microbial evaluations of orthodontic materials remain essential to ensure clinical safety and efficacy.