A case study of salivary microbiome in smokers and non-smokers in Hungary: analysis by shotgun metagenome sequencing

Abstract

Objective: To investigate the role of cigarette smoking in disease-development through altering the composition of the oral microbial community. Periodontitis and oral cancer are highly prevalent in Hungary; therefore, the salivary microbiome of smoker and non-smoker Hungarian adults was characterized.

Methods: Shotgun metagenome sequencing of salivary DNA samples from 22 individuals (11 non-smokers and 11 current smokers) was performed using the Ion Torrent PGM^TM platform. Quality-filtered reads were analysed by both alignment-based sequence similarity searches and genome-centric binning.

Results: Prevotella, Veillonella and Streptococcus were the predominant genera in the saliva of both groups. Although the overall composition and diversity of the microbiota were similar, Prevotella was significantly more abundant in salivary samples of current smokers compared to non-smokers. Members of the genus Prevotella were implicated in the development of inflammatory diseases and oral cancer. The abundance of the genus Megasphaera also increased in current smokers, whereas the genera Neisseria, Oribacterium, Capnocytophaga and Porphyromonas were significantly reduced. The data generated by read-based taxonomic classification and genome-centric binning mutually validated the two distinct metagenomic approaches.

Conclusion: Smoking-associated dysbiosis of the salivary microbiome in current cigarette smokers, especially increased abundance of Prevotella and Megasphaera genera, may facilitate disease development.

Keywords: Megasphaera; Prevotella; Smoking; genome-centric binning; oral cancer; read-based taxonomy; saliva metagenome.

Figure 1.

Summary of the data analysis workflow and the employed software packages. The main steps of the initial data filtering and bioinformatics steps to extract the read-based and genome-based metagenome data are boxed separately.

Figure 2.

Overall composition of the salivary microbiome of study participants including the metagenomes of both smokers and non-smokers. Due to space limitations, only the most relevant bacterial genera, families, orders and phyla are indicated; the bacterial classes are not shown.

Figure 3.

Relative abundance of the 10 most abundant bacterial genera in non-smokers and current smokers. Circos plot illustrating the most abundant bacterial genera listed in inset from 1 to 10. The widths of the bands are proportional to the abundance of the particular taxon in the two study groups.

Figure 4.

Diversity of the salivary microbiome in non-smokers and current smokers.

Figure 5.

Bacterial genera which differ significantly in relative abundance between the salivary samples of non-smokers and current smokers. The statistical analysis was performed and visualized using the STAMP package. Mean abundance (mean proportion) and difference in mean proportion for genera showing significant difference in abundance are shown. The 95% confidence intervals and statistical significance (corrected q value) are indicated as well.

Figure 6.

Analysis of shotgun sequences by genome-centric binning. Distribution of contigs built from filtered sequences of salivary bacterial communities. The grouping of contigs based on sequence-assignments of automated binning programs METABAT2, MAXBIN2 and CONCOCT as well as manually defined bins were visualised by the Anvi’o platform. SCG: single-copyy genes; GC: guanine-cytosine (GC) content.

Figure 7.

Relative abundance of bacterial genera identified by genome-centric binning. Prevotella (bin 3) showed a significantly higher relative abundance in the salivary microbiome of current smokers, whereas the family Porphyromonadaceae (bin 6) and the genus Neisseria (bin 8) were significantly more abundant in the salivary microbiome of non-smokers.