Does the vitamin B₂ analogue roseoflavin have the potential to influence the composition of the oral microbiome?

Streptococcus mutans is auxotrophic for riboflavin (RF, vitamin B₂) and thus depends on its uptake from the environment [1]. In its natural soil habitat, Streptomyces davaonensis produces the antivitamin roseoflavin (RoF), an antimicrobial RF analogue, to reduce growth of competing microorganisms [2]. We hypothesized that RoF might be useful as a microbiome modulator against potential oral pathogens, e.g. involved in caries formation. However, little is known about the antimicrobial efficacy of RoF against members of the human oral microbiota.

In the cultivation-based arm of our study, more than 300 microbial isolates were obtained from saliva samples of 41 healthy human volunteers and characterized by MALDI-TOF-MS. Securely identified species were analysed with regard to the presence of RF biosynthesis and uptake genes and tested for susceptibility to RoF in RF-free media supplemented with RF and/or RoF, respectively. For cultivation-independent studies, we obtained saliva samples from 15 healthy volunteers, which were pooled and incubated with or without RoF for 24 hours, respectively. After DNA and RNA extraction, 16S rRNA (cDNA) and 16S rRNA gene sequencing was used to investigate shifts in the community composition of the present (DNA-based) or active (cDNA-based) saliva microbiota.

None of the isolated gram-negative species were found to be RF auxotrophic, while 7 out of 19 gram-positive species grew significantly better when supplemented with RF (100 µM). Similarly, growth of 11 out of 12 tested gram-negative species was not negatively affected by RoF (100 µM), whereas 11 out of 19 gram-positives showed growth reduction, including S. mutans. Our data tentatively suggested that RoF-treatment shifts the human oral microbiota towards the gram-negative fraction [3]. However, our 16S-sequencing-based incubation experiment did not confirm a relative increase of the gram-negative fraction, neither on DNA- nor on RNA-basis. We believe that under the growth conditions used, too few cell divisions took place to reduce the cell number of the riboflavin-auxotrophic species. Consequently, future experiments will address monitoring of RF-concentrations and different bacterial growth conditions.

[1] Turner et al. (2020), J Bacteriol 203(2): e00293-20; [2] Pedrolli et al. (2014), Methods Mol Biol 1146: 41-63; [3] Schwendenmann et al. (2024). Folia Microbiol, submitted.