Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia—A Microbiome and Gene Profiling Study and Focused Review
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Inclusion Criteria
2.3. Oral Swab Sample Collection
2.4. Tissue Sample Collection
2.5. DNA Extraction
2.6. 16S Amplification and Sequencing
2.7. Sequencing Data Processing
2.8. Operational Taxonomic Unit (OTU) Cluster and Taxonomic Annotation
2.9. Alpha Diversity
2.10. Beta Diversity
2.11. HSC-3 Cell Culture
2.12. Bacterial Culture
2.13. RNAseq
2.14. Statistical Analyses
3. Results
3.1. Demographics of Study Patients
3.2. Oral Dysplasia and OSCC Microbiome Communities Are Distinct from Those in Healthy and Histologically Normal Adjacent Communities
3.3. High-Grade Dysplasia and OSCC Alpha Diversities Are Significantly Different from Those in Histologically Normal Adjacent Specimens
3.4. Significant Increases in Proteobacteria and Decreases in Firmicutes as well as Expansion of Fusobacteria Are Noted When Moving from the Clinically/Histologically Normal Oral Mucosa to Dysplasia and to Cancer
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Variable | Healthy Mucosa (No Dysplasia) | Histologically Normal Adjacent/Low Grade Dysplasia (Matched) |
---|---|---|
Age (Years ± SD) | 50.67 ± 13.58 | 67.50 ± 9.98 |
Sex | ||
Male | 2 | 1 |
Female | 2 | 3 |
Swab Collection Location | ||
Left Tongue | 0 | 1 |
Right Lingual Gingiva | 0 | 0 |
Left Lingual Gingiva | 0 | 1 |
Mandibular Gingiva | 4 | 2 |
Dysplasia diagnosis | - | Mild to moderate |
Smoking Status | ||
Current | 0 | 0 |
Past | 0 | 0 |
Never | 4 | 2 |
N/A | 0 | 2 |
Year | Site | Diagnosis | Age | Sex | HPV Status | Smoking Status | Alcohol Use | Other Relevant History | |
---|---|---|---|---|---|---|---|---|---|
Unmatched Tissue Samples | 2015 | Left posterior floor of mouth | Mild dysplasia | 60 | M | Negative | Never smoker | One standard drink per week | History of dysplasia and squamous cell carcinoma in the left posterolateral tongue |
2010 | Gingiva, between 1st and 2nd premolars | Squamous cell carcinoma in situ | 82 | F | Negative | n/a | n/a | History of squamous cell carcinoma in Gingiva, #7–9 | |
2013 | Left tongue, posterior dorsal mucosal margin | Histologically normal adjacent tumor | 76 | M | Negative | Never smoker | 2 standard drinks per week | Negative margin in a patient with left tongue squamous cell carcinoma | |
2005 | Left lateral tongue | Low-grade dysplasia | 33 | F | Negative | Never smoker | None | ||
2005 | Left lateral tongue | High-grade dysplasia | 33 | ||||||
2012 | Left lateral tongue | Mild dysplasia | 60 | M | Negative | Former smoker (0.25 packs per day, 1.5 pack-year) | 12 standard drinks per week | History of left tongue squamous cell carcinoma in situ | |
2015 | Left soft palate | Moderate dysplasia | 63 | ||||||
2012 | Right base of tongue mucosal margin | Carcinoma in situ | 54 | F | Negative | Former smoker (1 pack-year, 15 pack-years, quit 25 years prior) | <1 standard drink per day | Margin in a patient with oral tongue multifocal squamous cell carcinoma | |
2009 | Right tongue | Moderate dysplasia | 53 | M | Negative | n/a | n/a | History of right lateral tongue squamous cell carcinoma and HIV+ | |
2017 | Right tongue | Severe dysplasia | 60 | M | Negative | Daily tobacco chew user for 22 years | Longtime drinker | Adjacent to right tongue squamous cell carcinoma in resection specimen | |
2013 | Gingiva, lower left 2nd premolar region | Atypical papillary verrucous proliferation | 70 | F | Negative | Former smoker (1 packs per day, 24 pack-years, quit 26 years prior) | None | History of proliferative verrucous leukoplakia | |
2015 | Right ventral lateral tongue | Moderate dysplasia | 95 | F | Negative | Former smoker (0.25 packs per day, 10 pack-years, quit 35 years prior) | None | History of right tongue leukoplakia | |
2014 | Left tongue | Histologically normal adjacent tumor | 66 | F | Negative | Never smoker | None | Adjacent to left tongue squamous cell carcinoma in resection specimen | |
Matched Samples | 2011 | Anterior dorsal tongue | Histologically normal adjacent (margin in resection) | 60 | M | Negative | Former smoker (2 packs per day, 68 pack-years) | ≥15 standard drinks per week | History of other recreational drug use |
Mild to moderate dysplasia (margin in resection) | |||||||||
Moderately differentiated SCC | |||||||||
2011 | Left posterior ventral tongue | Histologically normal adjacent (margin in resection) | 45 | M | Negative | Never smoker | One standard drink per week | History of non-Hodgkin lymphoma, esophageal squamous cell carcinoma, and thyroid cancer | |
Mild to moderate dysplasia (margin in resection) | |||||||||
Moderately differentiated SCC | |||||||||
2009 | Anterior dorsal tongue | Hyperkeratosis, no dysplasia (margin in SCC resection) | 41 | M | Negative | n/a | n/a | History of proliferative verrucous leukoplakia | |
Mild dysplasia (adjacent to SCC) | |||||||||
Moderately differentiated SCC | |||||||||
2009 | Ventral tongue, anterior margin | Histologically normal adjacent (margin in resection) | 57 | M | Negative | Former smoker (60 pack-years) | One standard drink per day | ||
Moderate dysplasia (margin in SCC resection) | |||||||||
Moderately differentiated SCC | |||||||||
1999 | Left lateral tongue | Histologically normal adjacent (to SCCIS) | 76 | M | Negative | Former smoker (25 years prior) | <2 standard drink per day | ||
Squamous cell carcinoma in situ with possible superficial microinvasion | |||||||||
2010 | Left lateral tongue | Histologically normal adjacent (to SCCIS) | 77 | M | Negative | None | None | ||
Mild to moderate dysplasia | |||||||||
Squamous cell carcinoma in situ |
Author/Year | Specimen Type | Method | Sample Size | Group Comparison | Microbiome | Other Remarks |
---|---|---|---|---|---|---|
Shen et al. (2023) [26] | - | Systematic Review and Metanalysis | 802 | - | Dysplasia—increase in phylum Bacteroidetes Dysplasia and OSCC—increase in Fusobacterium and decrease of Streptococcus | Analyzed studies presented a high risk of bias due to non-negligible heterogeneity in the type and size of the sample and inconsistent oral microbiome composition, strongly limiting the analysis. However, only 6 out of 11 analyzed studies histopathologically diagnosed their OPMDs as dysplasia, which could account for the discrepancies found. |
Wright et al. (2023) [72] | Oral Swabs | 16S Sequencing | 90 | Progressing vs. non-progressing dysplasia | Increase in Campylobacter in progressing dysplasia compared to non-progressing ones | No significant differences between progressing vs. non-progressing dysplasia |
Herreros-Pomares et al. (2021) [73] | Tissue | 16S Sequencing | 10 | Healthy vs. Leukoplakia + Dysplasia | Leukoplakia + dysplasia—increase in Oribacterium sp. oral taxon 108, Campylobacter jejuni, uncultured Eubacterium sp., Tannerella, and Porphyromonas | Authors have not controlled for dysplasia and mixed no dysplasia samples with mild, moderate, and severe dysplasia samples. |
Sami et al. (2020) [74] | - | Reveiw | - | - | Fusobacterium nucleatum and Candida species has been associated with high-grade dysplasia and its severity | - |
Gopinath et al. (2020) [41] | Whole Mouth Fluid | 16S Sequencing | 74 | Healthy vs. Leukoplakia + Dysplasia vs. OSCC | Leukoplakia + dysplasia—increase in Bacteroidetes and decrease in Firmicutes Leuloplakia + dysplasia and OSCC—increase in Actinobacteria | Shift in bacterial communities of leukoplakia + dysplasia and oral cancer patients; no significant difference in richness and diversity |
Al-Hebshi et al. (2019) [42] | - | Review | - | - | Significantly increased frequency and yeast colony counts (predominantly Candida) in dysplasia and OSCC; significant yeast colony increase correlated with dysplasia severity | Some of the studies did not have healthy controls, rather compared to other OPMD |
Ganly et al. (2019) [27] | Saliva | 16S Sequencing | 38 | Healthy vs. Leukoplakia + Dysplasia vs. OSCC | Leukoplakia + dysplasia—increase in Fusobacterium and Veillonella OSCC—increase in Fusobacterium, Prevotella, Alloprevotella; decrease in Streptococcus | Significantly increase in HSP90 gene and ligands for TLRs 1, 2, and 4 along the healthy → leukoplakia/dysplasia → OSCC progression |
Lee et al. (2017) [75] | Saliva | 16S sequencing | 376 | Healthy vs. Dysplasia vs. OSCC | Significantly different levels of Bacillus, Enterococcus, Parvimonas, Peptostreptococcus, and Slackia in dysplasia compared to cancer | - |
Mok et al. (2017) [76] | Oral Swabs | 16S Sequencing | 27 | Healthy vs. Dysplasia vs. OSCC | Dysplasia—increase in Neisseria and Granulicatella; decrease in Streptococcus | Analysis of Molecular Variance (AMOVA) showed no significant difference between dysplasia and other groups |
Amer et al. (2017) [39] | Oral Swabs | 16S Sequencing | 6 | Healthy vs. Leukoplakia + Dysplasia | Severe dysplasia was associated with elevated levels of Leptotrichia spp. and Campylobacter concisus | - |
Hebbar et al. (2013) [77] | Tissue and Oral Rinse | Periodic Acid–Schiff Staining | 50 | Healthy vs. Dysplasia vs. OSCC | Significant increase in yeast infection and colony number with higher dysplasia grades and OSCC | - |
Spolidorio et al. (2003) [78] | Tissue | Periodic Acid–Schiff Staining | 832 | Healthy vs. Dysplasia | 27.2% of dysplasia samples were PAS-positive; significant association of yeast infection and dysplasia | Tongue was the significant most affected site by yeast infection |
McCullough et al. (2002) [79] | Tissue and Oral Rinse | 223 | Healthy vs. Dysplasia vs. OSCC | Dysplasia and OSCC—significantly higher frequency of oral yeast carriage and higher number of yeast (>1000 cfu/mL) than control. Correlation between dysplasia degree and yeast amount in oral cavity | ||
Barrett et al. (1998) [80] | Tissue | Periodic Acid–Schiff Staining | 4724 | Healthy vs. Dysplasia | 4.7% of the biopsies contained PAS-positive fungi; significant positive association of fungal infection with moderate and severe dysplasia | Significantly higher number of males infected compared to females; 21.9% of fungi-infected dysplasia worsened in histological severity, compared to 7.6% of non-infected dysplasia |
Rindum et al. (1994) [81] | Tissue | Periodic Acid–Schiff Staining and Smear Culture | 153 | Healthy vs. Leukoplakia/erythroleukoplakia + Dysplasia | 4 Candida albicans strains were found in moderate and severe dysplasia, but none on mild dysplasia | - |
Krogh et al. (1987) [70] | Swab over biopsy | Yeast culture | 12 | Healthy vs. leukoplakia/erythroleukoplakia +Dysplasia | 4 different strains of Candida albicans, one strain of Candida parapsilosis found in dysplasia samples | Samples positive for dysplasia showed yeast strains with lower nitrosation potential compared to the ones on samples negative for dysplasia (no statistical analysis) |
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Radaic, A.; Shamir, E.R.; Jones, K.; Villa, A.; Garud, N.R.; Tward, A.D.; Kamarajan, P.; Kapila, Y.L. Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia—A Microbiome and Gene Profiling Study and Focused Review. Microorganisms 2023, 11, 2250. https://doi.org/10.3390/microorganisms11092250
Radaic A, Shamir ER, Jones K, Villa A, Garud NR, Tward AD, Kamarajan P, Kapila YL. Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia—A Microbiome and Gene Profiling Study and Focused Review. Microorganisms. 2023; 11(9):2250. https://doi.org/10.3390/microorganisms11092250
Chicago/Turabian StyleRadaic, Allan, Eliah R. Shamir, Kyle Jones, Alessandro Villa, Nandita R. Garud, Aaron D. Tward, Pachiyappan Kamarajan, and Yvonne L. Kapila. 2023. "Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia—A Microbiome and Gene Profiling Study and Focused Review" Microorganisms 11, no. 9: 2250. https://doi.org/10.3390/microorganisms11092250
APA StyleRadaic, A., Shamir, E. R., Jones, K., Villa, A., Garud, N. R., Tward, A. D., Kamarajan, P., & Kapila, Y. L. (2023). Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia—A Microbiome and Gene Profiling Study and Focused Review. Microorganisms, 11(9), 2250. https://doi.org/10.3390/microorganisms11092250