Compositional profile of mucosal bacteriome of smokers and smokeless tobacco users

Study information

Reviewed Marked as Reviewed by Lwaldron on 2023-6-16

study design

case-control

Citation

PMID PubMed identifier for scientific articles.

34436669

DOI Digital object identifier for electronic documents.

10.1007/s00784-021-04137-7

URI

Authors

Gopinath D, Wie CC, Banerjee M, Thangavelu L, Kumar R P, Nallaswamy D, Botelho MG, Johnson NW

Journal

Clinical oral investigations

Year

2022

Keywords:

Microbiome, Microbiota, Oral microbiome, Smokeless tobacco, Smoking, Tobacco

INTRODUCTION: Smoked, and especially smokeless, tobacco are major causes of oral cancer globally. Here, we examine the oral bacteriome of smokers and of smokeless tobacco users, in comparison to healthy controls, using 16S rRNA gene sequencing. METHODS: Oral swab samples were collected from smokers, smokeless tobacco users, and healthy controls (n = 44). Microbial DNA was extracted and the 16S rRNA gene profiled using the Illumina MiSeq platform. Sequencing reads were processed using DADA2, and taxonomical classification was performed using the phylogenetic placement method. Differentially abundant taxa were identified using DESeq2, while functional metagenomes based on KEGG orthology abundance were inferred using LIMMA. RESULTS: A significantly higher microbial diversity was observed in smokeless tobacco users and smokers relative to controls (P < 0.05). Compositional differences in microbial communities were observed in all comparisons with healthy controls (PERMANOVA P < 0.05) but not between smokers and smokeless tobacco users. Levels of Fusobacterium spp., Saccharibacterium spp., and members of Shuttleworthia were elevated in smokers when compared to controls (BH adj P < 0.01). In addition, the relative abundance of three bacterial taxa belonging to genera Fusobacterium spp., Catonella, and Fretibacterium spp. was significantly increased in smokeless tobacco users relative to controls (BH adj P < 0.01). Major functional pathways significantly increased in smokeless tobacco users relative to both controls, and smokers were similar and involved amino acid metabolism including glutamate and aspartate biosynthesis and degradation (log FC > 1.5; BH adj P < 0.01). CONCLUSIONS: A distinct taxonomic and functional profile of oral microbiome in smokers and smokeless tobacco users as compared to healthy controls implicates a significant role of microbes and their metabolites in diseases associated with tobacco use including oral cancer. CLINICAL RELEVANCE: Future efforts in preventive, diagnostic, curative, and prognostic strategies for diseases associated with tobacco use in smokers and smokeless tobacco users could incorporate the oral microbiome.

Experiment 1

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Reviewed Marked as Reviewed by Lwaldron on 2023-6-16

Curated date: 2023/06/13

Curator: Atrayees

Revision editor(s): Atrayees, Lwaldron, Victoria

Subjects

Location of subjects: India

Host species Species from which microbiome was sampled. Contact us to have more species added.: Homo sapiens

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology: Buccal mucosa Buccal mucosa,buccal mucosa

Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology: Chewing tobacco behavior Chewing tobacco behavior,chewing tobacco behavior

Group 0 name Corresponds to the control (unexposed) group for case-control studies: age-matched controls

Group 1 name Corresponds to the case (exposed) group for case-control studies: smokeless tobacco users

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Smokeless tobacco users with or without areca nut.

Group 0 sample size Number of subjects in the control (unexposed) group: 13

Group 1 sample size Number of subjects in the case (exposed) group: 14

Lab analysis

Sequencing type: 16S

16S variable region One or more hypervariable region(s) of the bacterial 16S gene: V3-V4

Sequencing platform Manufacturer and experimental platform used for quantifying microbial abundance: Illumina

Statistical Analysis

Data transformation Data transformation applied to microbial abundance measurements prior to differential abundance testing (if any).: raw counts

Statistical test: DESeq2

Significance threshold p-value or FDR threshold used for differential abundance testing (if any): 0.01

Matched on Factors on which subjects have been matched on in a case-control study: age, sex

Alpha Diversity

Pielou Quantifies how equal the community is numerically: increased

Shannon Estimator of species richness and species evenness: more weight on species richness: increased

Simpson Estimator of species richness and species evenness: more weight on species evenness: increased

Signature 1

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Reviewed Marked as Reviewed by Lwaldron on 2023-6-16

Curated date: 2023/06/13

Curator: Atrayees

Revision editor(s): Atrayees, Lwaldron

Source: Figure 5

Description: Differentially abundant taxa identified in smokeless tobacco users and controls using DESeq2.

Abundance in Group 1: increased abundance in smokeless tobacco users

NCBI	Quality Control	Links
Catonella
Fretibacterium
Streptomyces plicatus

Revision editor(s): Atrayees, Lwaldron

Experiment 2

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Reviewed Marked as Reviewed by Lwaldron on 2023-6-16

Curated date: 2023/06/13

Curator: Atrayees

Revision editor(s): Lwaldron, Atrayees, Victoria

Differences from previous experiment shown

Subjects

Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology: Smoking behavior smoking,Smoking behavior,smoking behavior

Group 1 name Corresponds to the case (exposed) group for case-control studies: Smokers who were using either bidis or cigarettes

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Smokers who were using either bidis (thin, hand-rolled cigarettes composed of tobacco wrapped in a “tendu” or “temburni” leaf) or cigarettes

Group 1 sample size Number of subjects in the case (exposed) group: 17

Lab analysis

Statistical Analysis

Alpha Diversity

Pielou Quantifies how equal the community is numerically: increased

Shannon Estimator of species richness and species evenness: more weight on species richness: increased

Simpson Estimator of species richness and species evenness: more weight on species evenness: increased

Signature 1

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Reviewed Marked as Reviewed by Lwaldron on 2023-6-16

Curated date: 2023/06/13

Curator: Atrayees

Revision editor(s): Atrayees, Lwaldron

Source: Figure 5

Description: Differentially abundant taxa identified in smokers and controls using DESeq2.

Abundance in Group 1: increased abundance in Smokers who were using either bidis or cigarettes

NCBI	Quality Control	Links
Fusobacterium
Shuttleworthella
unclassified Candidatus Saccharibacteria

Revision editor(s): Atrayees, Lwaldron

Experiment 3

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Reviewed Marked as Reviewed by Lwaldron on 2023-6-16

Curated date: 2023/06/13

Curator: Atrayees

Revision editor(s): Lwaldron, Atrayees, Victoria

Differences from previous experiment shown

Subjects

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Smokeless tobacco users

Group 1 name Corresponds to the case (exposed) group for case-control studies: Smokers

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Smokers who were using either bidis or cigarettes