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.
Keywords: (13)C-glucose; Heavy water; Metabolism; Single-cell Raman spectrum; Streptococcus mutans.
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