The tongue biofilm metatranscriptome identifies metabolic pathways associated with halitosis and its prevention

Abstract

Halitosis is an oral condition caused by an increase in the concentration of volatile sulfur compounds (VSCs), such as methyl mercaptan and hydrogen sulfide, generated as a consequence of bacterial metabolism on the tongue biofilm. Microbial communities on the tongue of halitosis patients have been studied by bacterial culture, 16S rRNA taxonomic studies and metagenomics. However, there are currently no reports on the microbial gene-expression profiles. In this study, we performed RNAseq of tongue coating samples from control individuals and halitosis patients with different levels and composition of VSCs, as determined by gas chromatography. In this metatranscriptomic study, the activity of Streptococcus, Veillonella and Rothia species was associated with halitosis-free individuals while Prevotella, Fusobacterium and Leptotrichia species were associated with halitosis. Although methyl mercaptan is considered an indicator of halitosis, the metatranscriptome of patients in which only this VSC was present in elevated levels was similar to that of halitosis-free individuals. Veillonella dispar, Streptococcus parasanguinis and Rothia mucilaginosa were over-represented in halitosis-free communities in comparison to the rest of the groups, suggesting that these species could be used as a halitosis-free biomarkers. In contrast, the abundance of Prevotella shahi and Fusobacterium nucleatum were significantly higher when hydrogen sulfide concentration was over the established halitosis-threshold, making these species putative halitosis biomarkers. Finally, gene expression profiles showed a significant over-expression of genes involved in L-cysteine and L-homocysteine synthesis in halitosis-free individuals and an over-expression of genes responsible for cysteine degradation into hydrogen sulfide in halitosis patients. In addition, nitrate reduction into nitrite was also over-expressed in halitosis-free patients. In conclusion, halitosis was associated with communities that degrade amino acids and reduce sulfide, whereas tongue communities that produce L-cysteine from hydrogen sulfide and that reduce nitrate were associated with the absence of halitosis. The latter could provide new strategies to treat this condition.

The Importance of Nitrate Reduction for Oral Health

“…A recent metatranscriptomic study detected expression of a similar set of genes (Fig. 3, yellow boxes), as well as a transcript ( nrfA ) involved in dissimilatory nitrate reduction to ammonium and a nitrification transcript ( nifX ), which indicates that nitrogen could be fixed by oral bacteria, and this requires further investigation (Carda-Diéguez et al 2021).…”

“…As long as more energy-efficient denitrification takes place, sulfate reduction is inhibited, and this could occur in the oral cavity, where sulfate reduction is associated with halitosis (Ren et al 2016). In addition, hydrogen sulfide (H 2 S) can be used as an electron donor when nitrite is reduced (Koch et al 2017), which might reduce halitosis, while the nitrate-reducing species R. mucilaginosa and V. dispar were higher in halitosis-free participants, whose nitrate reduction genes were overexpressed (Carda-Diéguez et al 2021). Collectively, the data suggest that nitrate could be beneficial in patients with halitosis by eliminating halitosis-related pathogens, by suppressing metabolic pathways involved in production of VSCs, and by stimulating the utilization of H 2 S, and this should be explored in future in vitro and clinical studies.…”

“…The graph represents genes identified in 3 studies. Brown boxes are genes identified by Schreiber et al (2010) with quantitative polymerase chain reaction in dental plaque; blue boxes, by Rosier, Moya-Gonzalvez, et al (2020) in the full genomes of Rothia isolates; and yellow boxes, by Carda-Diéguez et al (2021) in the metatranscriptomes of tongue communities. The metabolites in this figure are nitrate (NO 3 − ), nitrite (NO 2 − ), nitric oxide (NO), nitrous oxide (N 2 O), nitrogen (N 2 ), ammonium (NH 4 + ), and ammonia (NH 3 ).…”

“…Interestingly, Rothia and Neisseria are genera associated with oral disease–free individuals (i.e., oral health) when compared with patients with caries (Agnello et al 2017; Rosier et al 2018) and periodontal diseases (Meuric et al 2017; Feres et al 2020; Chen et al 2021). Additionally, R. mucilaginosa —the most common Rothia species on the tongue (Rosier et al 2021)—is associated with halitosis-free individuals (Carda-Diéguez et al 2021).…”

“…On the tongue, genera associated with halitosis include Prevotella, Porphyromonas , and Fusobacterium (Seerangaiyan et al 2017; Carda-Diéguez et al 2021), which have representatives sensitive to nitric oxide and/or can decrease in the presence of nitrate (Rosier, Buetas, et al 2020). Interestingly, nitrate has been used as a biological agent to treat malodor from sewer networks by limiting microbial VSC production resulting from sulfate reduction (Mathioudakis et al 2006).…”