Alteration of oral flora in betel nut chewers

doi:10.21203/rs.3.rs-4971375/v1

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Alteration of oral flora in betel nut chewers

https://doi.org/10.21203/rs.3.rs-4971375/v1

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Objectives: To analyzed microbiota changes in gingival crevicular fluid of betel nut chewers and non-chewers.

Materials and methods: We collected gingival crevicular fluid samples from betel nut chewers and non-chewers and analyzed using 16srRNA high-throughput sequencing technology. Various statistical methods were used for the test. P values < 0.05 were considered to be statistically significant.

Results: Data analysis of all the abundant species revealed a lower abundance of Tannerella and Treponema in the chewer without periodontitis group than in the other groups(P < 0.05), and Haemophilus were more abundant in that group(P < 0.05). A statistically significant difference in PD Whole tree was observed (P < 0.05).

Conclusions: The results suggested that betel nut chewing altered the microbial flora in gingival crevicular fluid. However, the link between microbial changes and betel nut-induced periodontitis is speculative.

Scientific rationale for study: Betel nut chewing is known as a crucial risk factor of periodontitis. Abnormal microbiota is associated with the progression of periodontitis, but little information is currently available on microbiota changes in betel nut chewers.

Principal Findings： A lower abundance of Tannerella and Treponema was detected in the betel nut chewer without periodontitis group than in the other groups, conversely, parainfluenzae were more abundant.

Practical Implications：Betel nut chewing altered the microbial flora in gingival crevicular fluid.It can be speculated that the pathogenic mechanism of periodontitis caused by chewing betel nut is different from that of traditional periodontitis.

Periodontitis is an infectious disease caused by bacteria colonizing the tooth surfaces and Areca, Meta-analysis, microbiota, Periodontitis, high-throughput sequencing technology

forming a biofilm at the gingival margin and in the gingival/periodontal pockets.^{[1, 2]} Decades of investigations have demonstrated that the microbial communities associated with periodontitis differ from those in health.^[3] The results of DNA-DNA hybridization demonstrated that the microbial abundance of the 14 species was generally higher in the periodontitis environment than in the healthy environment, and the microbial proportions varied between conditions.^[4] Studies using pyrosequencing of 16S rRNA gene amplicons showed that community diversity in disease was high, with 123 species identified to be significantly enriched in disease and 53 species abundant in health.^[5] The transition of community structure from healthy to periodontitis resembles ecological succession, with the emergence of new dominant taxa in periodontitis without replacing the main health-related species. That is, the periodontitis community had a higher proportion of groups such as Spirochetes, Synergists, Firmicutes, and Aspergillus aeruginosa, while Actinobacteria had a higher proportion in the healthy community.^[5]

Chewing betel nut is widespread in in Taiwan, India, and numerous Southeast Asian countries.^{[6, 7]} Arecoline, a natural alkanoid in betel nut, has genotoxic, carcinogenic, embryotoxic, and immunotoxic potential.^[8] Several epidemiological investigations have established that betle nut chewing may cause oropharyngeal cancer, interferes with the microbial mechanism of neutrophils, and inhibits the growth of various cells including oral mucosal keratinocytes, fibroblasts, and gingival fibroblasts.^[9-12] . This in turn promotes bacterial colonization and periodontal infection. It will also increase the chewing load and directly lead to soft and hard tissue damage, causing excessive tooth wear, sensitivity, loosening, etc.^[13] Betel nut extract induces prostaglandin E2 production, activation of intracellular calcium concentration, P-38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase inhibitor. These findings suggest that betel nut chewing may induce an inflammatory response and affect the periodontal health of consumers. In addition, studies have shown that betel nut chewing can damage periodontal tissue because the cholinergic action of betel nut and calcium salts in saliva can produce excessive saliva-induced calculus deposition. Large deposits of calculus attract bacteria to attach and form plaque on the tooth surface, leading to the destruction of gingival tissue and periodontal ligament. It was found that there was a higher incidence of periodontal disease in betel nut chewers than in nonchewers, however the etiological role is less clear.^[14-17] Studies have shown that chewing betel nut has a certain specific effect on subgingival periodontal pathogens. For example, Pseudomonas intermedius, Pseudomonas forsythia, Pseudomonas endodonticum, and Pseudomonas gingivalis were significantly less abundant in betel nut chewers than in non-betel nut chewers.^[2] In vitro evidence suggested that areca nut extract may suppress the growth of several oral microorganisms, including Streptococcus mutans and Fusobacterium nucleatum.^[18] This conclusion is contrary to that of Ling et al.^[8] Therefore, the effect of chewing betel nut on periodontal pathogens remains unclear.

The present study aimed to identify alterations in oral flora of betel nut chewers and investigate the effect of the microbiota on periodontal disease. We took the gingival crevicular fluid of betel nut chewers and non-chewers as samples and used 16srRNA sequencing technology.

2.1 Sample collection

We enrolled 51 participants in this study. 13 of these participants were assigned to the betel nut chewer with periodontitis group, 22 were assigned to the chewer without periodontitis group, while 7 were assigned to the non-chewer with periodontitis group and 9 were assigned to the non-chewer without periodontitis group. These participants had no systemic disease and no oral disease other than periodontitis. The filter paper strip method was used to collect gingival crevicular fluid.^[19]

2.2 DNA extraction

DNA was extracted from gingival crevicular fluid using BIOG DNA Saliva Kit, following the manufacturer’s instructions. The extracted DNA was stored at 80℃ until PCR.

2.3 Sequencing

The amplicon PCR targeted the V3-V4 regions of the bacterial 16S ribosomal RNA (rRNA) gene and used illumina high-throughput sequencing. PCR amplification was performed using a two-step method to generate DNA clusters. The DNA amplicons are linearized into single strands. The modified DNA polymerase and dNTPs with 4 fluorescent labels were added, and only one base was synthesized per cycle. Scan the surface of the reaction plate with a laser to read the nucleotide species polymerized in the first round of reaction of each template sequence. The "fluorophore" and "termination group" are chemically cleaved to restore the 3' end stickiness and continue to polymerize the second nucleotide. The fluorescent signal results collected in each round were counted, and the sequence of the template DNA fragment was obtained. The PEreads obtained by sequencing first distinguish each sample according to the barcode, and then perform quality control and filtering on the sequence quality, and then splicing according to the overlap relationship.

Analysis of sequencing data. The above-processed CleanTags were subjected to OTU clustering, and then the species classification of OTUs was completed by annotating the OTUs. The assembled sequences were clustered into OTUs using the software USEARCH. Based on OTU, we performed taxonomic analysis, Alpha diversity analysis, Beta diversity analysis, and compositional difference analysis.

2.4 Statistical analysis

Difference analysis was performed on the Alpha diversity index between groups, including parametric and nonparametric tests. The wilcox method was used between two groups of samples; the krustal method was used between three groups of samples or multiple groups of samples. The species difference analysis between multiple samples generally uses Chi-Squaretest to compare the abundance differences of species between two or more samples. The significance of differences in species across multiple contrasting samples can be obtained from this analysis. MRPP group difference analysis was used in conjunction with PCoA to analyze whether the differences in microbial community structure between groups were significant. We considered P-values <0.05 to indicate statistical significance.

Fifty-one samples were sequenced and a total of 3,537,171 sequences were amplified, ranging from a minimum of 58,853 to a maximum of 74,594 sequences per sample, with a mean of 69,356 sequences per sample.

A total of 148 different bacterial species were detected. The most abundant species among all the samples was Porphyromonas_gingivalis (mean 17.82%), followed by Leptotrichia_buccalis (3.78%) and Neisseria_elongata (2.09%) (Fig. 1A). Data analysis of all the abundant species revealed a lower abundance of Tannerella_forsythia and Treponema_medium in the chewer without periodontitis group than in the other groups (P < 0.05)( Fig. 1B). Conversely, Haemophilus_parainfluenzae were more abundant in the chewer without periodontitis group (P < 0.05).

3.1 Species richness and diversity (alpha diversity)

Diversity analysis (Alpha diversity) can reflect the abundance and diversity of microbial communities. A statistically significant difference in PD Whole tree was observed (PD Whole tree, P=0.049) (Fig. 2). No significant differences were observed in Chao1, Observed Species,Simpsion and Shannon index among groups (Chao1, P=0.597; Simpsion, P=0.367, Observed Species, P=0.444; Shannon index, P=0.204) (Fig. 2).

3.2 PCoA and MRPP analysis (beta diversity)

To evaluate diversity difference, PCoA and MRPP analysis were analyzed. PCoA plots demonstrated clustering among groups (Fig. 3A). The unweighted UniFrac distance metric significantly differed (P=0.002) (Fig. 3B, Tab.1). Based on species and genus-level analysis, there were significant differences in distance between samples (species.bray, P=0.023; genus.bray, P= 0.04)(Fig.3CD, Tab.1).

3.3 Biomarker analysis ( LDA Effect Size)

To evaluate microbial contents among groups, linear discriminant analysis (LDA) was performed using LEfSe. From 946 biomarkers or higher taxa, 79 taxa were statistically significantly different among groups (P< 0.05) and had a linear discriminant analysis (LDA) score above 2 (Fig. 4). Taxa are ranked by the effect size in LEfSe(Fig. 4).

The most discriminative taxa in the samples of chewer with periodontitis group were Spirochaetae; in the samples of chewer without periodontitis group, the taxa were Treponema_denticola and Lentimicrobiaceae; in the samples of non-chewer with periodontitis group, the taxa were Bacilli and Lactobacillales; in the samples of non-chewer without periodontitis group, the taxa were Fretibacterium and Synergistetes.

Table 1 MRPP analysis

The present study is the first to use high-throughput 16S rRNA gene sequencing to demonstrate the effect of chewing betel nut on the gingival sulcus microbiota. In the study, we demonstrated differences in bacterial community composition among samples from each group. A lower abundance of Tannerella and Treponema was detected in the betel nut chewer without periodontitis group than in the other groups, conversely, parainfluenzae were more abundant. ( Fig. 1B). Tannerella and Treponema are classical carious pathogens, this result may prove the anti-carious effect of betel nut.^[20-22] The possible reason is tannin content of betel may have antimicrobial properties which may contribute to the cariostatic role of betel. ^[23] Long-term chewing of betel nut causes the occlusal sockets and fissures to disappear, creating an open environment that is not conducive to the survival of anoxic bacteria and thus reduces the risk of caries. Haemophilus grows well in blood‑enriched culture media^[24], its presence may be explained by the bleeding gums typically featured in periodontal disease.^[25] This may be related to the physical stimulation of the periodontal tissue caused by betel nut. Mechanical friction leads to bleeding gums, favoring the colonization of Haemophilus. The reason for the low abundance of Haemophilus bacillus in the periodontitis group after chewing betel nut may be that long-term chewing of betel nut leads to submucosal fibrosis, decreased blood supply, and declined bleeding.

The PD whole tree revealed a large difference in the degree of homogeneity for bacterial species among groups, which means the degree of homogeneity species may show a large difference. Observed Species, Simpsion and Shannon index were not significantly different, which is inconsistent with the decreased bacterial diversity in patients with periodontitis^[14]. This may be related to the habit of chewing betel nut changing the periodontal environment. It can be speculated that the pathogenic mechanism of periodontitis caused by chewing betel nut is different from that of traditional periodontitis.

Biomarker analysis confirmed that chewing betel nut resulted in the accumulation of Treponema in the gingival sulcus. Treponema is closely related to the progression of periodontitis. The deeper the periodontal pocket, the more severe the loss of attachment and the easier the detection of Treponema.^[26] Treponema can convert proMMP-8 to aMMP-8，aMMP-8 activity in oral fluids has been widely studied as biomarker of periodontitis.^[27] The enrichment of Treponema may explain the microbiological etiology of betel nut aggravation of periodontitis.

In conclusions, we demonstrated differences in bacterial community composition among groups. Haemophilus and Treponema changes are particularly prominent. This study showed that betel nut altered the microbial flora in gingival crevicular fluid. However, the link between microbial changes and betel nut-induced periodontitis is speculative. Therefore, further studies are needed to elucidate the clinical significance of oral microbiota changes, including the potential role of chewing betel nut in the development of periodontitis.

Acknowledgements

None.

Authors’ contributions

Hongbo Zhou: study conception and design. Yu Han、Yanxu Lu: drafting of the article、data collection and analysis、data analysis and interpretation. Ran Wang、Chengxin Chen: data acquisition.

Funding

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this article and are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The study received ethical approval from the Institutional Research Ethics Review Committee of Xiangya School of Stomatology, Central South University. All procedures and methods adhered to the relevant guidelines and regulations.

Author details

¹Xiangya Stomatological Hospital&Xiangya School of Stomatology, Central South University, Changsha, Hunan, 410008, China. ²Workstation for Oral-maxilofacial and Regenerative Medicine.³Beijing Nuclear Industry Hospital, Beijing, 100000, China.⁴Department of Stomatology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361101, China.

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No competing interests reported.

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Alteration of oral flora in betel nut chewers

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Abstract

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1. INTRODUCTION

2. MATERIALS AND METHODS

3. RESULTS

4. DISCUSSION

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