Oral microbiome dysbiosis among cigarette smokers and smokeless tobacco users compared to non-users

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Subjects

Location of subjects: United States of America

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: Smoking behaviour measurement Smoking behaviour measurement,smoking behaviour measurement

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

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

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group smoked 6–10 cigarettes per day and smoked filtered, menthol, and full-flavour cigarettes.

Group 0 sample size Number of subjects in the control (unexposed) group: 43

Group 1 sample size Number of subjects in the case (exposed) group: 24

Antibiotics exclusion Number of days without antibiotics usage (if applicable) and other antibiotics-related criteria used to exclude participants (if any): 6 months

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).: relative abundances

Statistical test: Kruskall-Wallis

Significance threshold p-value or FDR threshold used for differential abundance testing (if any): 0.05

MHT correction Have statistical tests be corrected for multiple hypothesis testing (MHT)?: No

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: increased abundance in Cigarettes users (CG )

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Leptotrichia

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: decreased abundance in Cigarettes users (CG )

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Actinomyces
Granulicatella
Neisseria
Oribacterium
Prevotella
Veillonella

Revision editor(s): Shulamite, Victoria

Experiment 2

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Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

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

Group 0 sample size Number of subjects in the control (unexposed) group: 18

Lab analysis

Statistical Analysis

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: increased abundance in Cigarettes users (CG )

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Leptotrichia

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/29

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: decreased abundance in Cigarettes users (CG )

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Pseudomonas

Revision editor(s): Victoria

Experiment 3

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Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

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

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

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group had used tobacco at least once per week for the previous year.

Group 0 sample size Number of subjects in the control (unexposed) group: 43

Group 1 sample size Number of subjects in the case (exposed) group: 18

Lab analysis

Statistical Analysis

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

Alpha Diversity

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

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: decreased abundance in Smokeless tobacco users (ST)

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Prevotella
Porphyromonas
Oribacterium
Leptotrichia
Granulicatella
Actinomyces

Revision editor(s): Shulamite, Victoria

Experiment 4

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Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology: Saliva Sailva normalis,Saliva atomaris,Saliva molecularis,Salivary gland secretion,Saliva,saliva

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

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

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group smoked 6–10 cigarettes per day and smoked filtered, menthol, and full-flavour cigarettes.

Group 1 sample size Number of subjects in the case (exposed) group: 24

Lab analysis

Statistical Analysis

Matched on Factors on which subjects have been matched on in a case-control study: Not specified

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: increased abundance in Cigarettes users (CG )

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Actinomyces
Leptotrichia
Porphyromonas

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: decreased abundance in Cigarettes users (CG )

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Fusobacterium
Haemophilus
Pseudomonas
Rothia

Revision editor(s): Shulamite, Victoria

Experiment 5

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Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

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

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

Group 0 sample size Number of subjects in the control (unexposed) group: 18

Lab analysis

Statistical Analysis

MHT correction Have statistical tests be corrected for multiple hypothesis testing (MHT)?: Not specified

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: increased abundance in Cigarette users (CG)

NCBI	Quality Control	Links
Prevotella

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: decreased abundance in Cigarette users (CG)

NCBI	Quality Control	Links
Fusobacterium
Haemophilus
Leptotrichia
Oribacterium
Porphyromonas

Revision editor(s): Shulamite, Victoria

Experiment 6

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

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

Group 1 name Corresponds to the case (exposed) group for case-control studies: Smokeless tobacco user (ST)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group had used tobacco at least once per week for the previous year.

Group 0 sample size Number of subjects in the control (unexposed) group: 43

Group 1 sample size Number of subjects in the case (exposed) group: 18

Lab analysis

Statistical Analysis

MHT correction Have statistical tests be corrected for multiple hypothesis testing (MHT)?: No

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

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: increased abundance in Smokeless tobacco user (ST)

NCBI	Quality Control	Links
Prevotella

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 4

Description: Box plots of relative abundance of the top 14 bacterial genera by user group: cigarette user (CG, orange), smokeless tobacco user (ST, green), and non-user (NU). (*) represents p-value < 0.05.

Abundance in Group 1: decreased abundance in Smokeless tobacco user (ST)

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Fusobacterium

Revision editor(s): Shulamite, Victoria

Experiment 7

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

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

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Non-tobacco users at Time 1 (NU)

Group 1 name Corresponds to the case (exposed) group for case-control studies: Non-tobacco users at Time 4 (NU)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants who had either never used tobacco products or had smoked less than 20 cigarettes or used smokeless tobacco less than 20 times in their lifetime at time point 4.

Group 1 sample size Number of subjects in the case (exposed) group: 43

Lab analysis

Statistical Analysis

Matched on Factors on which subjects have been matched on in a case-control study: Not specified

Alpha Diversity

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

Richness Number of species: unchanged

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 2a

Description: Differential abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: increased abundance in Non-tobacco users at Time 4 (NU)

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unclassified Granulicatella
unclassified Leptotrichia
unclassified Prevotella
Neisseria subflava
unclassified Rhodococcus (in: high G+C Gram-positive bacteria)
unclassified Ochrobactrum

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/29

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 2a

Description: Differential abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: decreased abundance in Non-tobacco users at Time 4 (NU)

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unclassified Lactobacillus
unclassified Blautia
unclassified Staphylococcus
Enterobacter cloacae
Streptococcus lutetiensis

Revision editor(s): Victoria

Experiment 8

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Cigarette users at time 1 (CG)

Group 1 name Corresponds to the case (exposed) group for case-control studies: Cigarette users at time 4 (CG)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants who smoked 6–10 cigarettes per day and smoked filtered, menthol, and full-flavour cigarettes at time point 4.

Group 0 sample size Number of subjects in the control (unexposed) group: 24

Group 1 sample size Number of subjects in the case (exposed) group: 24

Lab analysis

Statistical Analysis

Alpha Diversity

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

Richness Number of species: unchanged

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 2a

Description: Differential abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: increased abundance in Cigarette users at time 4 (CG)

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unclassified Peptoniphilus
unclassified Finegoldia
unclassified Anaerococcus
unclassified Porphyromonas
unclassified Prevotella
Campylobacter ureolyticus
unclassified Corynebacterium
unclassified Varibaculum
unclassified Wal_1855Dunclassified Wal_1855D
unclassified 1-68unclassified 1-68

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 2a

Description: Differential abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: decreased abundance in Cigarette users at time 4 (CG)

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unclassified Pseudomonas

Revision editor(s): Shulamite, Victoria

Experiment 9

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Smokeless tobacco users at time 1 (ST)

Group 1 name Corresponds to the case (exposed) group for case-control studies: Smokeless tobacco users at time 4 (ST)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants who had used tobacco at least once per week for the previous year at time point 4.

Group 0 sample size Number of subjects in the control (unexposed) group: 18

Group 1 sample size Number of subjects in the case (exposed) group: 18

Lab analysis

Statistical Analysis

Alpha Diversity

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

Richness Number of species: unchanged

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 2a

Description: Differential abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: increased abundance in Smokeless tobacco users at time 4 (ST)

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unclassified Prevotella

Revision editor(s): Shulamite, Victoria

Experiment 10

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology: Saliva Sailva normalis,Saliva atomaris,Saliva molecularis,Salivary gland secretion,Saliva,saliva

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Cigarette users at time 1 (CG)

Group 1 name Corresponds to the case (exposed) group for case-control studies: Cigarette users at time 4 (CG)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants who smoked 6–10 cigarettes per day and smoked filtered, menthol, and full-flavour cigarettes at time point 4.

Group 0 sample size Number of subjects in the control (unexposed) group: 24

Group 1 sample size Number of subjects in the case (exposed) group: 24

Lab analysis

Statistical Analysis

Alpha Diversity

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

Richness Number of species: unchanged

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 2b

Description: Differential abundance of bacterial OTUs in saliva samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: increased abundance in Cigarette users at time 4 (CG)

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unclassified Achromobacter

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 2b

Description: Differential abundance of bacterial OTUs in saliva samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: decreased abundance in Cigarette users at time 4 (CG)

NCBI	Quality Control	Links
unclassified Pseudomonas
unclassified Rhodococcus (in: high G+C Gram-positive bacteria)

Revision editor(s): Shulamite, Victoria

Experiment 11

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Smokeless tobacco users at time 1 (ST)

Group 1 name Corresponds to the case (exposed) group for case-control studies: Smokeless tobacco users at time 4 (ST)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants who had used tobacco at least once per week for the previous year at time point 4.

Group 0 sample size Number of subjects in the control (unexposed) group: 43

Group 1 sample size Number of subjects in the case (exposed) group: 18

Lab analysis

Statistical Analysis

Alpha Diversity

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

Richness Number of species: unchanged

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Figure 2b

Description: Differential abundance of bacterial OTUs in saliva samples that were statistically significantly different (α = 0.001) between time-points 1 and 4. The OTUs are colored by user groups (cigarette user (CG), orange; smokeless tobacco user (ST), green; non-user (NU), grey). A positive log2-fold change value denotes an OTU that is significantly higher in time-point 1 samples, while a negative log2-fold change indicates an OTU that is significantly higher in time-point 4 samples.

Abundance in Group 1: increased abundance in Smokeless tobacco users at time 4 (ST)

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unclassified Mycoplasmataceae

Revision editor(s): Shulamite, Victoria

Experiment 12

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology: Buccal mucosa , Saliva Buccal mucosa,buccal mucosa,Sailva normalis,Saliva atomaris,Saliva molecularis,Salivary gland secretion,Saliva,saliva

Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology: Specimen Specimen,specimen

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Cigarettes users (CG - Buccal)

Group 1 name Corresponds to the case (exposed) group for case-control studies: Cigarettes users (CG - Saliva)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Saliva samples of participants who smoked 6–10 cigarettes per day and smoked filtered, menthol, and full-flavour cigarettes.

Group 0 sample size Number of subjects in the control (unexposed) group: 24

Group 1 sample size Number of subjects in the case (exposed) group: 24

Lab analysis

Statistical Analysis

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Supplementary Figure 9

Description: Box plots of relative abundance of the top 14 bacterial genera by sample type (buccal swab (red), saliva (blue) across each user group: cigarette users (CG), smokeless tobacco users (ST), and non-users (NU).

Abundance in Group 1: increased abundance in Cigarettes users (CG - Saliva)

NCBI	Quality Control	Links
Fusobacterium
Actinomyces
Leptotrichia
Neisseria
Prevotella
Streptococcus
Veillonella

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Supplementary Figure 9

Description: Box plots of relative abundance of the top 14 bacterial genera by sample type (buccal swab (red), saliva (blue) across each user group: cigarette users (CG), smokeless tobacco users (ST), and non-users (NU).

Abundance in Group 1: decreased abundance in Cigarettes users (CG - Saliva)

NCBI	Quality Control	Links
Pseudomonas
Lactobacillus
Granulicatella

Revision editor(s): Victoria

Experiment 13

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria, Tosin

Differences from previous experiment shown

Subjects

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology: Saliva , Buccal mucosa Sailva normalis,Saliva atomaris,Saliva molecularis,Salivary gland secretion,Saliva,saliva,Buccal mucosa,buccal mucosa

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Non-users (NU - Buccal)

Group 1 name Corresponds to the case (exposed) group for case-control studies: Non-users (NU - Saliva)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Saliva samples of participants who had either never used tobacco products or had smoked less than 20 cigarettes or used smokeless tobacco less than 20 times in their lifetime.

Group 0 sample size Number of subjects in the control (unexposed) group: 43

Group 1 sample size Number of subjects in the case (exposed) group: 43

Lab analysis

Statistical Analysis

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Supplementary Figure 9

Description: Box plots of relative abundance of the top 14 bacterial genera by sample type (buccal swab (red), saliva (blue) across each user group: cigarette users (CG), smokeless tobacco users (ST), and non-users (NU).

Abundance in Group 1: increased abundance in Non-users (NU - Saliva)

NCBI	Quality Control	Links
Leptotrichia
Neisseria
Porphyromonas
Actinomyces
Prevotella

Revision editor(s): Shulamite, Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/04/30

Curator: Shulamite

Revision editor(s): Shulamite, Victoria

Source: Supplementary Figure 9

Description: Box plots of relative abundance of the top 14 bacterial genera by sample type (buccal swab (red), saliva (blue) across each user group: cigarette users (CG), smokeless tobacco users (ST), and non-users (NU).

Abundance in Group 1: decreased abundance in Non-users (NU - Saliva)

NCBI	Quality Control	Links
Lactobacillus
Veillonella
Streptococcus
Rothia
Pseudomonas
Granulicatella

Revision editor(s): Shulamite, Victoria

Experiment 14

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/28

Curator: Victoria

Revision editor(s): Victoria, Tosin

Differences from previous experiment shown

Subjects

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

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

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Saliva samples of participants who had used tobacco at least once per week for the previous year.

Group 0 sample size Number of subjects in the control (unexposed) group: 18

Group 1 sample size Number of subjects in the case (exposed) group: 18

Lab analysis

Statistical Analysis

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Supplementary Figure 9

Description: Box plots of relative abundance of the top 14 bacterial genera by sample type (buccal swab (red), saliva (blue) across each user group: cigarette users (CG), smokeless tobacco users (ST), and non-users (NU).

Abundance in Group 1: increased abundance in Smokeless tobacco users (ST - Saliva)

NCBI	Quality Control	Links
Actinomyces
Fusobacterium
Leptotrichia
Neisseria
Oribacterium
Streptococcus

Revision editor(s): Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Supplementary Figure 9

Description: Box plots of relative abundance of the top 14 bacterial genera by sample type (buccal swab (red), saliva (blue) across each user group: cigarette users (CG), smokeless tobacco users (ST), and non-users (NU).

Abundance in Group 1: decreased abundance in Smokeless tobacco users (ST - Saliva)

NCBI	Quality Control	Links
Granulicatella
Lactobacillus
Pseudomonas

Revision editor(s): Victoria

Experiment 15

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/29

Curator: Victoria

Revision editor(s): Victoria, Tosin

Differences from previous experiment shown

Subjects

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: Smoking behaviour measurement Smoking behaviour measurement,smoking behaviour measurement

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

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

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group smoked 6–10 cigarettes per day and smoked filtered, menthol, and full-flavour cigarettes.

Group 0 sample size Number of subjects in the control (unexposed) group: 43

Group 1 sample size Number of subjects in the case (exposed) group: 24

Lab analysis

Statistical Analysis

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

Statistical test: DESeq2

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 5a

Description: Relative abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between non-users (NU) and cigarette users (CG).

Abundance in Group 1: increased abundance in Cigarettes users (CG )

NCBI	Quality Control	Links
unclassified Peptostreptococcaceae
unclassified Ochrobactrum
Actinobacillus parahaemolyticusActinobacillus parahaemolyticus
unclassified Leptotrichia
unclassified Neisseria
unclassified Anaerovorax
unclassified Aggregatibacter
unclassified Haemophilus
Neisseria subflava
unclassified Rhodococcus (in: high G+C Gram-positive bacteria)
unclassified Pseudomonas
Solobacterium moorei
unclassified Prevotella
Streptococcus infantis

Revision editor(s): Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 5a

Description: Relative abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between non-users (NU) and cigarette users (CG).

Abundance in Group 1: decreased abundance in Cigarettes users (CG )

NCBI	Quality Control	Links
unclassified Actinomyces
Streptococcus anginosus
unclassified Abiotrophia
unclassified Aggregatibacter
unclassified Leptotrichiaceae

Revision editor(s): Victoria

Experiment 16

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/29

Curator: Victoria

Revision editor(s): Victoria, Tosin

Differences from previous experiment shown

Subjects

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

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

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group had used tobacco at least once per week for the previous year.

Group 1 sample size Number of subjects in the case (exposed) group: 18

Lab analysis

Statistical Analysis

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

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 5b

Description: Relative abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between non-users (NU) and smokeless tobacco users (ST).

Abundance in Group 1: increased abundance in Smokeless tobacco users (ST)

NCBI	Quality Control	Links
unclassified Coprococcus

Revision editor(s): Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 5b

Description: Relative abundance of bacterial OTUs in buccal swab samples that were statistically significantly different (α = 0.001) between non-users (NU) and smokeless tobacco users (ST).

Abundance in Group 1: decreased abundance in Smokeless tobacco users (ST)

NCBI	Quality Control	Links
Haemophilus parainfluenzae
Neisseria subflava
unclassified Achromobacter
unclassified Actinomyces
unclassified Neisseria
unclassified Oribacterium
unclassified Peptostreptococcaceae
unclassified Streptococcus
unclassified Veillonellaceae
unclassified Mogibacteriaceaeunclassified Mogibacteriaceae

Revision editor(s): Victoria

Experiment 17

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/29

Curator: Victoria

Revision editor(s): Victoria, Tosin

Differences from previous experiment shown

Subjects

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology: Saliva Sailva normalis,Saliva atomaris,Saliva molecularis,Salivary gland secretion,Saliva,saliva

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

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

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group smoked 6–10 cigarettes per day and smoked filtered, menthol, and full-flavour cigarettes.

Group 1 sample size Number of subjects in the case (exposed) group: 24

Lab analysis

Statistical Analysis

Matched on Factors on which subjects have been matched on in a case-control study: Not specified

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 6a

Description: Relative abundance of bacterial OTUs in saliva samples that was statistically significantly different (α = 0.001) between non-users (NU) and cigarette users (CG).

Abundance in Group 1: increased abundance in Cigarettes users (CG )

NCBI	Quality Control	Links
unclassified Corynebacterium
unclassified Leptotrichia
unclassified Propionibacteriaceae
unclassified Anaerovorax
unclassified Hyphomicrobiales
unclassified Haemophilus
unclassified Neisseria
unclassified Peptostreptococcaceae
unclassified Ochrobactrum
unclassified Weeksellaceae
Actinobacillus parahaemolyticusActinobacillus parahaemolyticus
unclassified Aggregatibacter
unclassified Pseudomonas
unclassified Streptococcus
unclassified Fusobacterium
Haemophilus parainfluenzae
unclassified Actinomyces
Neisseria subflava
unclassified Porphyromonas
unclassified Vibrionaceae
unclassified Prevotella
Prevotella melaninogenica
unclassified Neisseriaceae
Veillonella parvula
Rothia aeria
unclassified Lautropia
unclassified Granulicatella
unclassified Pasteurellaceae
unclassified Lachnospiraceae
unclassified Capnocytophaga
Streptococcus infantis
Rothia mucilaginosa
Veillonella dispar
unclassified Cardiobacterium
unclassified Oribacterium

Revision editor(s): Victoria

Signature 2

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 6a

Description: Relative abundance of bacterial OTUs in saliva samples that was statistically significantly different (α = 0.001) between non-users (NU) and cigarette users (CG).

Abundance in Group 1: decreased abundance in Cigarettes users (CG )

NCBI	Quality Control	Links
unclassified Streptococcus
Streptococcus anginosus
unclassified Actinomyces
unclassified Slackia
Prevotella nigrescens
Prevotella melaninogenica
unclassified Dialister
Porphyromonas endodontalis
unclassified Megasphaera
Limosilactobacillus reuteri
unclassified Leptotrichia
unclassified Filifactor
Treponema amylovorum
unclassified Acidaminobacteraceae
unclassified Lactobacillus
unclassified Sharpea
unclassified Enhydrobacter
unclassified Bulleidia
unclassified Eubacteriales
unclassified Scardovia
unclassified Mogibacteriaceaeunclassified Mogibacteriaceae
unclassified Porphyromonas
Treponema socranskii
unclassified Catonella
Ligilactobacillus salivarius
unclassified Bifidobacterium
unclassified Veillonella
unclassified Pseudoramibacter-Eubacteriumunclassified Pseudoramibacter-Eubacterium
unclassified Leptotrichiaceae

Revision editor(s): Victoria

Experiment 18

Your review change was submittedDuplicate this Experiment Add a new Signature

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/29

Curator: Victoria

Revision editor(s): Victoria, Tosin

Differences from previous experiment shown

Subjects

Group 1 name Corresponds to the case (exposed) group for case-control studies: Smokeless tobacco user (ST)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Participants in this group had used tobacco at least once per week for the previous year.

Group 1 sample size Number of subjects in the case (exposed) group: 18

Lab analysis

Statistical Analysis

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

Alpha Diversity

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

Signature 1

Reviewed Marked as Reviewed by KateRasheed on 2025-7-31

Curated date: 2025/07/30

Curator: Victoria

Revision editor(s): Victoria

Source: Figure 6b

Description: Relative abundance of bacterial OTUs in saliva samples that was statistically significantly different (α = 0.001) between non-users (NU) and smokeless tobacco users (ST).

Abundance in Group 1: increased abundance in Smokeless tobacco user (ST)

NCBI	Quality Control	Links
unclassified Leptotrichia
Treponema amylovorum
Porphyromonas endodontalis
unclassified Streptococcus
unclassified Streptococcaceae

Revision editor(s): Victoria

Signature 2