After the LC-MS metabolic analysis, the PLS-DA scores plot showed clear separation between S. mutans UA and S. mutans FR, suggesting its potential for identification of biological variations that occur in S. mutans. In our metabolomics study, it is a practical approach to separate S. mutans FR from S. mutans UA by LC-MS. The discriminant metabolites identified with p < 0.05 were validated with VIP (threshold set at 2.0). Successfully, 5 discriminant metabolites were identified in early log phase and 13 discriminant metabolites in stationary phase. Our results indicate that the metabolites from S. mutans FR may be related to the pathways of vitamin B6 metabolism (amino acid, carbohydrate, and fatty acid metabolism) and nucleotide metabolism (purine and pyrimidine metabolism).

Fructose-1,6-bisphosphate (FBP) was identified as distinct markers from S. mutans in the carbohydrate metabolism pathway in this study. Acid production in the carbohydrate metabolism (the glycolysis process) is one of the virulent features of S. mutans. In comparison with the wild-type strains, S. mutans FR can produce more acid in the presence of fluoride [10, 23, 24]. However, in the absence of fluoride, S. mutans FR acid production rates are inconsistent and can be higher [10], lower [25], or the same [23]. In terms of acid tolerance, there are two controversial results. One shows S. mutans FR was more acid-sensitive, which could be more easily killed at a low pH value [6]. The other exhibits S. mutans FR had stronger ability to survive in acidified media than the wild-type strains [22].

FBP can activate phosphofructokinase (PFK) and pyruvate kinase in the glycolysis process and can promote the conversion of glucose to lactate [26]. Thus, the activity of lactate dehydrogenase in Streptococcus depends on the presence of FBP [27]. The fructose phosphotransferase system (PTS) transporters from S. mutans produce fructose-1-phosphate and fructose-6-phosphate, which can be converted into FBP by PFK [28].

In our study, we found that the FBP levels from fluoride-resistant S. mutans increased in the stationary phase compared to the levels from wild-type one. FBP concentration as a sensor of carbon influx was positively correlated with the growth rate [29]. FBP participates in the upstream process of glycolysis, as an important intermediate. It suggests the glycolysis of fluoride-resistant S. mutans might be more active than wild-type one. Further research includes the effect of FBP from fluoride-resistant strains on the glycolysis and why the acid-producing ability of fluoride-resistant strains enhances.

Fatty acids in cell membrane play a critical role in maintaining normal physiological function of cells. When cells are subjected to some environmental stress, such as temperature, ion, salt, drug, and oxidative stress, they can rapidly change their fatty acid composition in the cell membrane, alter their morphology, or increase mobility to resist the damage caused by the external environment [30, 31]. In cariogenic bacteria, the membranous fatty acids keep the acid-base balance inside and outside the cell membrane. At pH 5, the proportion of long chain monounsaturated fatty acids in the cell membrane of S. mutans is increased in response to the acid-resistant stress [32]. In our previous work, we also found that the amount of long chain monounsaturated fatty acids in the cell membrane of S. mutans increased under acidic conditions, and that the amount of monounsaturated fatty acids in S. mutans FR increased to a greater extent than the parental strains with the enhanced ability of acid resistance after S. mutans FR induced [22]. In this study, we found that the FBP level of fluoride-resistant strains was higher than that of wild-type ones under the same conditions.

It is widely accepted that FBP can affect the lipid metabolism [26]. It can activate acetyl coenzyme A carboxylase (acetyl CoA), and thereby can regulate lipid synthesis in yeast [33]. In addition, FBP can activate fatty acid synthetase in E. coli, which is probably because FBP increased the affinity of acetyl CoA in this bacterium, or because FBP promotes the stability of acetyl CoA [34].

Collectively, it is likely that the increased concentration of FBP in the fluoride resistant strains promotes unsaturated fatty acid synthase activity, and thus the amount of unsaturated fatty acids in fluoride-resistant strains increases. The exact interaction between FBP and fatty acid remains unclear. Further studies are warranted.

Currently, the database related to S. mutans metabolites has not yet been established. Since the study of S. mutans metabolomics is still at the exploratory stage, the potential biomarkers found in our study can only be speculated according to the database related to the metabolites of Streptococcus pyogenes M1 476 (serotype M1) (KEGG).

In our study, we found that there were many other different metabolites, for instance, pyridoxamine 5′-phosphate (PMP) and UDP, between fluoride-resistant strains and their parental strains. These presumably identified metabolites are generally related to bacterial vitamin B6 metabolism (amino acid, carbohydrate, and fatty acid metabolism) and nucleotide metabolism (purine and pyrimidine metabolism). Our results are consistent with previous studies. In amino acid metabolism pathway, S. mutans was inhibited by PMP acting on the glucosyltransferase [35]. UDP-associated metabolites [17] were reduced in arginine-treated (dental caries preventive agent treated) S. mutans from the nucleotide metabolism pathway view. Although the understanding of the detailed function of these metabolites is still limited, Eva-Maria Decker and colleagues reported that the exposure of S. mutans to xylitol resulted in distinct gene expression patterns. Specifically, GtfC exhibited upregulation exclusively in the presence of xylitol. Furthermore, under xylitol exposure, the upregulation of gtfB was sixfold, whereas under sucrose exposure, it was threefold [36]. Thus, the involved metabolic pathways of S. mutans FR in our study may be due to altered gene functions after the mutation of fluoride resistance.

Meanwhile, it is reported that Lysine lactylation, a posttranslational modification, affects bacterial survival in altered environments and pathogenicity by regulating energy metabolism and amino acid metabolism [37]. A study on the acetylome patterns of S. mutans reveals that the acetyltransferase ActA acetylated lactate dehydrogenase (LDH) and hindered LDH’s enzymatic capacity to facilitate the transformation of pyruvate into lactic acid, consequently diminishing its cariogenicity in a rat caries model [38]. The functional role of lactylation at Lys173 of RNA polymerase subunit α (RpoA) in S. mutans involves the regulation of exopolysaccharides synthesis in glycol-metabolic pathways [39]. In dental caries, therefore, the excess fluoride use may be responsible for the metabolic reprogramming to be more favorable for S. mutans FR associated with its acidogenicity and acid tolerance.

Systematic mapping of identified metabolites to metabolic pathways involves associating metabolites with the biochemical pathways in which they participate. By doing so, we can gain insights into the underlying biological processes that are influenced by the observed changes in metabolite concentrations. Recently, a genome-scale metabolic model for the UA159 strain, named iSMU, encompassing 675 reactions and incorporating 429 metabolites and the outputs of 493 genes, will pave the way to a comprehensive understanding of the metabolism of S. mutans [40]. Further research will probably reveal a largely unexplored facet of metabolomes toward the comprehensive coverage.

This study’s exclusive use of anaerobic cultivation for S. mutans does indeed prompt important considerations regarding the applicability of the findings to aerobic conditions in the oral environment. The metabolism of S. mutans can exhibit significant variations between anaerobic and aerobic environments due to differences in metabolic pathways, including available energy sources, oxygen sensitivity, and redox status. Specifically, S. mutans switches fermentation pathways towards oxidative phosphorylation to generate energy. This shift can lead to variations in metabolic intermediates, potentially impacting the levels of fructose-1,6-bisphosphate and other metabolites [26]. Understanding these differences in metabolic pathways between anaerobic and aerobic conditions is crucial for comprehending the physiological adaptations of S. mutans. This knowledge is valuable in the context of dental caries, where variations in the oral environment can impact the metabolic strategies employed by this bacterium.

Also, the single use of metabolomics technology may limit the identification of the distinguished markers between S. mutans UA and S. mutans FR. Due to a lack of a complete functional database for metabolomics, the unknown markers found in our study were not fully identified. Further research by multiple complementary analytical platforms needs to validate our results, such as transcriptomic analyses, DNA sequencing, and/or quantitative PCR.