Smokeless tobacco impacts oral microbiota in a Syrian Golden hamster cheek pouch carcinogenesis model

Abstract

The use of smokeless tobacco products (STPs) can cause many serious health problems. The oral microbiota plays important roles in oral and systemic health, and the disruption in the oral microbial population is linked to periodontal disease and other health problems. To assess the impact of smokeless tobacco on oral microbiota in vivo, high-throughput sequencing was used to examine the oral microbiota present in Syrian Golden hamster cheek pouches. Sixteen hamsters were divided into four groups and treated with the STP Grizzly snuff (0, 2.5, 25, or 250 mg) twice daily for 4 weeks. After 0, 1, 2, 3, and 4 weeks of treatment, bacterial genomic DNA was extracted from oral swabs sampled from the cheek pouches of the hamsters. The oral bacterial communities present in different hamster groups were characterized by sequencing the hypervariable regions V1-V2 and V4 of 16S rRNA using the Illumina MiSeq platform. Fifteen phyla, 27 classes, 59 orders, 123 families, and 250 genera were identified from 4,962,673 sequence reads from the cheek pouch samples. The bacterial diversity and taxonomic abundances for the different treatment groups were compared to the non-treated hamsters. Bacterial diversity was significantly decreased after 4 weeks of exposure to 2.5 mg, and significantly increased by exposure to 250 mg STP. Treatment with 250 mg STP significantly increased Firmicutes, transiently increased Cyanobacteria and TM7, and decreased Bacteroidetes and Fusobacteria compared to the control group. At the genus level, 4 weeks of administration of 250 mg STP significantly increased Granulicatella, Streptococcus, Oribacterium, Anaerococcus, Acidaminococcus, Actinomyces, Eubacterium, Negativicoccus, and Staphylococcus, and decreased Bacteroides, Buleidia, Dialister, and Leptotrichia, and transiently decreased Arcanobacterium compared to the control group. For the first time, an animal model was used for evaluating the effects of STP on oral microbiota by metagenomic sequencing. Our results provide a view of the shift of the oral microbiota in response to STP exposure in Syrian Golden hamster. Our findings indicate that the use of smokeless tobacco significantly disrupts the oral microbiota.

Keywords: Bacterial diversity; Oral microbiota; Smokeless tobacco; Toxicity.

Fig. 1. Comparison of data derived from the V1-V2 and V4 variable regions

A) Alpha diversity was compared for V1-V2 and V4 sequence data. Error bars indicate standard errors of the means. B) The total number of named taxa with found in each of the data sets at the levels of genus and phylum.

Fig. 2. Abundance of commensal microbiota present in the hamster cheek pouch

A) Relative abundance of phyla. B) Relative Abundance of genera.

Fig. 3. The impact of STP on the alpha diversity of microbiota present in the hamster cheek pouch

A) Diversity of the control group (0 mg STP). B) Diversity of the 2.5 mg STP exposure group. C) Diversity of the 25 mg STP exposure group. D) Diversity of the 250 mg STP exposure group. The combined data from V1-V2 region and V4 region were applied (compared to the combined data, the separated data from V1-V2 region or V4 region did not change the trends of diversity caused by STP).

Fig. 4. The impact of STP on the relative abundance of phyla

The mean relative abundance (%) of OTUs found in oral swab of hamster is presented. Samples were grouped based on the treatment dose and treatment time of STP.

Fig. 5. The impact of STP (250 mg) on the relative abundance of phyla

5A: Heatmap analysis of the most abundant 7 phyla. The heatmap was clustered by phyloseq [34] and the heatmap colors represent the relative abundance of the bacterial phylum assignments within each sample. 5B–5F: Significant impact of STP on the relative abundance of bacterial classes. The blue lines represent control group (no exposure to STP) and the red lines represent the exposure group (exposure to 250 mg STP). Error bars indicate standard errors of the means. The ρ value between the 250 mg STP exposure group and the control group was less than 0.05 for all taxa. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. The impact of STP (250 mg) on the relative abundance of bacterial classes

6A: Heatmap analysis of the most abundant 13 bacterial classes. The heatmap was clustered by phyloseq [34] and the heatmap colors represent the relative abundance of the bacterial class assignments within each sample. 6B–6F: Significant impact of STP on the relative abundance of bacterial classes. The blue lines represent the control group (no exposure to STP), and the red lines represent the exposure group (exposure to 250 mg STP). Error bars indicate standard errors of the means. The ρ value between 250 mg STP exposure group and control group was less than 0.05 for all taxa. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7. The impact of STP on the relative abundance of bacterial orders

7A: Heatmap analysis of the most abundant 17 bacterial orders. The heatmap was clustered by phyloseq [34] and the heatmap colors represent the relative abundance of the bacterial order assignments within each sample. 7B–7I: Significant impact of STP on the relative abundance of bacterial orders. The blue lines represent the control group (no exposure to STP), and the red lines represent the exposure group (exposure to 250 mg STP). Error bars indicate standard errors of the means. The ρ value between 250 mg STP exposure group and control group was less than 0.05 for all taxa. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8. The impact of STP (250 mg) on the relative abundance of bacterial families

8A: Heatmap analysis of the most abundant 33 bacterial families. The heatmap was clustered by phyloseq [34]. And the heatmap colors represent the relative abundance of the microbial family assignments within each sample. 8B–8M: Significant impact of STP on the relative abundance of bacterial families. The blue lines represent the control group (no exposure to STP), and the red lines represent the exposure group (exposure to 250 mg STP). Error bars indicate standard errors of the means. ^# highlighted abundance from V1-V2 data only and * highlighted abundance from V4 data only. The ρ value between 250 mg STP exposure group and control group was less than 0.05 for all families. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9. The impact of STP (250 mg) on the relative abundance of bacterial genera

9A: Heatmap analysis of the most abundant 67 bacterial genera. The heatmap was clustered by phyloseq [34] and the heatmap colors represent the relative abundance of the microbial genus assignments within each sample. 9B–9Y: Significant impact of STP on the relative abundance of bacterial genera. The blue lines represent the control group (no exposure to STP), and the red lines represent the exposure group (exposure to 250 mg STP). Error bars indicate standard errors of the means. ^# highlighted abundance from V1-V2 data only and *highlighted abundance from V4 data only. The ρ value between 250 mg STP exposure group and control group was less than 0.05 for all genera. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9. The impact of STP (250 mg) on the relative abundance of bacterial genera

9A: Heatmap analysis of the most abundant 67 bacterial genera. The heatmap was clustered by phyloseq [34] and the heatmap colors represent the relative abundance of the microbial genus assignments within each sample. 9B–9Y: Significant impact of STP on the relative abundance of bacterial genera. The blue lines represent the control group (no exposure to STP), and the red lines represent the exposure group (exposure to 250 mg STP). Error bars indicate standard errors of the means. ^# highlighted abundance from V1-V2 data only and *highlighted abundance from V4 data only. The ρ value between 250 mg STP exposure group and control group was less than 0.05 for all genera. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10. The overall impact of 250 mg STP on the oral microbiome in hamster’s cheek pouch

The taxonomic name in red indicates that its abundance was increased by exposure to 250 mg STP. The taxonomic name in blue indicates that its abundance was decreased by exposure to 250 mg STP. * means the abundance change caused by STP had statistical significance. ‘g_’ and ‘Other’ were unclassified taxa. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)