Dual-Stable-Isotope-Probed Raman microspectroscopy reveals the metabolic dynamic of Streptococcus mutans

Streptococcus mutans (S. mutans) is regarded as a cariogenic pathogen with the ability to metabolize sugars and form organic acids. However, its actual timely level of glucose consumption and cellular vitality in a polymicrobial culture system remains largely unknown. To tackle this challenge, we employed the S. mutans UA159 as a model and developed a dual-stable-isotope-probed Raman microspectroscopy method (Dual SIP-Raman) to simultaneously profile the general metabolic activity and glucose assimilative activity in situ. (i) Mono-SIP substrate feeding revealed that 0.5% ¹³C-glucose and 30% D₂O were proper doses in the medium to obtain prominent and quantitative band shifts along with the ¹³C or D₂O incorporation. In addition, the intensity of the ¹³C peak of phenylalanine (Phe) is proposed as a Raman-based biomarker for glucose utilization in a cell. (ii) The state of dual SIP substrate incorporation of ¹³C-glucose and D₂O could be visualized by the corresponding spectral "red shifts" of Raman-scattered emissions; moreover, we also demonstrated that ¹³C/¹²C analysis was closely correlated with the C-D ratio. (iii) The application of the dual ¹³C-glucose and D₂O feeding approach on a mock microbiota of S. mutans UA159 and C. albicans ATCC14053 revealed a stimulatory effect of fungus on both the glucose intake rate and general metabolic vitality of S. mutans UA159 (p < 0.05). Therefore, the ¹³C-glucose and D₂O dual-feeding Raman Microspectroscopy approach is a valuable new tool for evaluating the glucose intake rate and general metabolic levels in situ, tracing the changing trend of the above metabolic activities, which is helpful to clarify the changes in the cariogenicity of oral microorganisms caused by the external environment at the single-cell level.