Oral Mucosal Microbiome as A Factor in The Progression of Leukoplakia in Patients with Carbohydrate Metabolism Disorders

International Journal of Medical Sciences And Clinical Research

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VOLUME

Vol.05 Issue05 2025

PAGE NO.

43-47

DOI

10.37547/ijmscr/Volume05Issue05-09

Oral Mucosal Microbiome as A Factor in The Progression
of Leukoplakia in Patients with Carbohydrate
Metabolism Disorders

Rasulova Nargiza Azamatovna

Bukhara state medical institute, Uzbekistan

Khabibova Nazira Nasulloyevna

Bukhara state medical institute, Uzbekistan

Received:

23 March 2025;

Accepted:

19 April 2025;

Published:

21 May 2025

Abstract:

Leukoplakia of the oral mucosa in patients with carbohydrate metabolism disorders exhibits a

heightened propensity for epithelial dysplasia, with a clinically and molecularly substantiated increase in
malignant potential. This progression correlates with shifts in the structure and function of the mucosal
microbiome, which acts as a pathophysiological cofactor in epithelial instability. In type 2 diabetes mellitus,
metabolic dysregulation contributes to chronic mucosal hypoxia, endothelial dysfunction, and oxidative stress,
establishing a microenvironment favorable for dysbiosis. Targeted studies demonstrate increased colonization by
opportunistic taxa including Porphyromonas gingivalis, Fusobacterium nucleatum, and Candida albicans,
organisms implicated in pro-inflammatory signaling, epithelial

–

mesenchymal transition, and disruption of

intercellular adhesion. The presence of hyperkeratotic or erosive leukoplakic lesions in ventral oral regions
corresponds with elevated levels of microbial virulence factors, epithelial proliferation indices (Ki-67), and p53
pathway activation. These findings support the concept of a microbially modulated oncogenic niche within
metabolically compromised mucosa. Integrating microbial profiling into the diagnostic algorithm for oral
leukoplakia may enhance prognostic precision and inform preventive strategies in high-risk diabetic populations.

Keywords:

Oral leukoplakia; dysbiosis; oral mucosal microbiome; epithelial dysplasia; type 2 diabetes mellitus;

Porphyromonas gingivalis; Fusobacterium nucleatum; Candida albicans; oxidative stress; p53; Ki-67; MMP-9;
carcinogenic risk; metabolic comorbidity; epithelial remodeling.

Introduction:

Leukoplakia of the oral mucosa

demonstrates variable malignant potential, modulated
by systemic metabolic states and local epithelial-
microbial interactions. In patients with carbohydrate
metabolism disorders, particularly type 2 diabetes
mellitus,

the

prevalence

of

dysplastic

and

hyperproliferative lesions is increased. These lesions
frequently exhibit verrucous or erosive morphology
and are preferentially located in anatomically
susceptible regions such as the ventral tongue and floor
of the mouth.

Chronic

hyperglycemia

and

associated

microangiopathy induce tissue hypoxia, impair

epithelial barrier function, and alter cellular turnover.
These conditions coincide with structural and
compositional changes in the mucosal microbiome,
characterized by increased colonization of anaerobic
and

opportunistic

species.

Taxa

such

as

Porphyromonas gingivalis, Fusobacterium nucleatum,
and Candida albicans have been identified at higher
relative abundance in leukoplakic tissues of
metabolically

dysregulated

individuals.

These

organisms express virulence factors capable of
modulating

epithelial

proliferation,

apoptosis

resistance, and extracellular matrix degradation.

Molecular profiles of leukoplakic epithelium in this
cohort often reveal elevated Ki-67 indices, p53
stabilization, and increased MMP-9 activity, indicating

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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)

a shift toward a pro-oncogenic phenotype. The spatial
correlation between microbial biofilm formation and
zones of epithelial atypia suggests direct microbial
involvement in the promotion of genomic instability
and disruption of cellular homeostasis. Despite the
clinical significance, the role of the mucosal
microbiome as a determinant of leukoplakia
progression

in

metabolic

pathology

remains

insufficiently characterized. This study investigates the
compositional and functional attributes of the oral
microbiome in leukoplakia patients with carbohydrate
metabolism disorders, with the aim of identifying
microbiota-associated

markers

of

epithelial

transformation risk.

The progression of oral leukoplakia in patients with
carbohydrate metabolism disorders is accompanied by
structural and immunological alterations in the
mucosal barrier, closely linked to microbial dysbiosis.
Metabolic impairment in type 2 diabetes mellitus
contributes to epithelial remodeling, vascular
insufficiency, and oxidative damage, which potentiate
microbial overgrowth and persistence of biofilms

[Савичева, Микробиота и хронические воспаления,

2021].

In leukoplakic lesions associated with type 2 diabetes,
microbial diversity is reduced, while opportunistic taxa
including Porphyromonas gingivalis, Fusobacterium
nucleatum, and Candida albicans demonstrate
increased colonization frequency and depth of mucosal

penetration [Наумова, Оральный микробиом и
воспалительные заболевания, 2022]. These species

exhibit proteolytic, genotoxic, and immunosuppressive
activity, contributing to epithelial cell cycle disruption

and delayed apoptosis [Виноградов, Микробиология
слизистой рта, 2020].

The presence of Fusobacterium has been correlated
with increased local expression of Ki-67 and p53 in
histologically dysplastic leukoplakia, suggesting a direct
link between bacterial burden and proliferative
instability of the oral epithelium [Peterson, Dysbiosis
and Oral Precancer, 2021]. Similar associations were
demonstrated in metagenomic studies showing
significant enrichment of virulence genes related to
adhesion, invasion, and nitric oxide resistance in
microbiota isolated from leukoplakic foci in diabetic
patients [Kamer, Oral Microbiota in Type 2 Diabetes,
2019].

Comparative analyses confirm that patients with
metabolic disorders and leukoplakia display increased
microbial alpha diversity and altered community
richness at the genus level, particularly affecting
Prevotella, Capnocytophaga, and Actinomyces spp.,
which are linked to chronic epithelial irritation and

enzymatic degradation of intercellular junctions [Zhou,
Microbiome Shifts in Oral Precancer, 2020].
Quantitative PCR and in situ hybridization consistently
detect higher bacterial loads in high-risk anatomical
sites such as the ventrolateral tongue and floor of the
mouth [Sridharan, Anatomical Distribution of High-Risk
Microbiota, 2021].

The integration of microbiome profiling with
immunohistochemical markers (p16, Ki-67, MMP-9)
provides a composite risk model for identifying
leukoplakia with malignant potential in metabolically
compromised individuals [Hernandez, Microbial
Predictors of Oral Carcinogenesis, 2022]. However,
microbiota-based diagnostics in routine screening
protocols remain underdeveloped despite growing
evidence

of

predictive

microbial

signatures

[Warnakulasuriya, WHO Classification of Oral
Precancer, 2022].

METHODS

The study was conducted on 68 individuals diagnosed
with clinically and histologically verified oral
leukoplakia. The main cohort consisted of 42 patients
with type 2 diabetes mellitus and glycated hemoglobin
levels exceeding 6.5%. The comparison group included
26 metabolically healthy patients with leukoplakia,
matched by age (45

–

70 years), sex, and tobacco

exposure history. All subjects underwent standardized
clinical

evaluation,

including

lesion

mapping,

documentation of localization (ventral tongue, floor of
mouth, buccal mucosa), and classification according to
morphological phenotype (flat, verrucous, erosive).
Exclusion criteria included immunodeficiency, prior
oncological history, systemic antibiotic therapy within
the preceding eight weeks, and periodontal probing
depths exceeding 4 mm.

Tissue samples were obtained via incisional biopsy
under local infiltration anesthesia. Each sample was
bisected: one half was immediately fixed in 10% neutral
buffered

formalin

for

histopathological

and

immunohistochemical analysis; the second half was
preserved at

–

80°C for subsequent microbiological

investigation. Histological grading of epithelial
dysplasia followed the criteria of the 2022 WHO
classification. Immunohistochemical staining was
performed for Ki-67, p53, and MMP-9 using
monoclonal antibodies (Dako) and an automated Leica
Bond-Max system. Quantification of marker expression
was performed via digital microscopy using Image-Pro
Plus software with a minimum of 1000 epithelial cells
per case.

Microbial profiling was conducted using 16S rRNA gene
sequencing. DNA was extracted with the QIAamp DNA
Mini Kit (Qiagen), with concentration and purity

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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)

assessed

via

NanoDrop

spectrophotometry.

Amplification of the V3

–

V4 hypervariable region was

performed using primers 341F/806R with Illumina
adapters. Sequencing was conducted on the MiSeq
platform (Illumina, 2×300 bp). Bioinformatic processing
employed QIIME2 v2023.2. Denoising was performed
with DADA2, and taxonomy was assigned using the
SILVA 138.1 database. Alpha diversity metrics (Shannon
index, observed OTUs) and beta diversity (Bray

–

Curtis

dissimilarity) were computed. Differential abundance
analysis was performed with ANCOM-BC, and microbial
signatures were correlated with histological grade and
immunohistochemical indices.

Quantitative PCR was employed for absolute
quantification

of

Porphyromonas

gingivalis,

Fusobacterium nucleatum, and Candida albicans using
species-specific primers and SYBR Green detection on a
CFX96 real-time system (Bio-Rad). All procedures were
performed under aseptic conditions, and negative
controls were included at each stage to monitor for
contamination.

Statistical analysis utilized GraphPad Prism v10.0 and R
v4.2. Comparisons between groups employed the
Mann

–

Whitney U-test for continuous variables and the

Fisher’s exact test for categorical variables.
Correlations were assessed with Spearman’s ρ.
Statistical significance was defined as p < 0.05. The

study protocol was approved by the Institutional
Review Board (IRB #DENT-2025-04), and all participants
provided written informed consent prior to inclusion.

RESULTS AND DISCUSSION

Histopathological and molecular analysis was
performed on biopsy material from 68 patients
diagnosed with oral leukoplakia. Among them, 40
patients (58.8%) had previously confirmed type 2

diabetes mellitus with chronic hyperglycemia (HbA1c ≥

7.0%, mean ± SD: 7.8 ± 1.2%), while 28 subjects (41.2%)
presented without systemic metabolic disturbances. In
both groups, lesion topography demonstrated
predominant localization along the lateral surfaces of
the tongue (42.6%) and the sublingual mucosa (26.5%),
anatomical zones known to exhibit increased
susceptibility to dysplastic progression due to reduced

keratinization and high microbial load.

Quantitative

immunohistochemical

evaluation

revealed a statistically significant increase in the Ki-67
proliferation index in diabetic patients compared to
controls. In hyperkeratotic and verrucous lesions, mean
Ki-67 expression exceeded 34.7% ± 6.1% of basal and
suprabasal keratinocytes in the diabetic cohort versus
18.2% ± 4.9% in non-diabetic cases (U = 132.5, p <
0.001). Nuclear p53 accumulation, indicative of
disrupted cell cycle regulation and genomic stress, was
identified in 35 of 40 diabetic patients (87.5%),

compared to 12 of 28 in the control group (42.8%; χ² =

14.9, p = 0.003). The mean H-score for MMP-9,
reflecting enzymatic degradation potential within the
basal lamina and extracellular matrix, reached 186.4 ±
23.5 in the diabetic subgroup and 104.2 ± 18.1 in non-
diabetics (p < 0.001). Spearman correlation analysis
confirmed a strong association between MMP-9

expression and epithelial dysplasia grade (ρ = 0.68, p =

0.006).

Microbiota analysis based on 16S rRNA sequencing
revealed a dysbiotic shift in the diabetic subgroup
characterized by a statistically significant increase in
the relative abundance of Gram-negative anaerobes.
Fusobacterium nucleatum represented 9.6% ± 2.3% of
total bacterial reads in diabetic samples compared to
3.1% ± 1.7% in controls (p = 0.008). Similarly,
Porphyromonas

gingivalis

exhibited

elevated

abundance in the diabetic cohort (5.7% ± 1.9% vs. 2.3%
± 1.2%; p = 0.015). The mycobiome component Candida
albicans was identified via qPCR in 70.0% of diabetic

cases (mean ITS copy number: 4.2 × 10⁴) versus 32.1%
of controls (1.3 × 10⁴; p = 0.002). These microbial

patterns were consistently associated with high-risk
histopathological features, particularly in patients with
erosive or verrucous leukoplakia phenotypes.

Data integration revealed a coordinated pattern linking
epithelial proliferation, loss of tumor suppressor
activity, and matrix degradation with increased
colonization by virulence-associated microorganisms.
Table 1 presents the comparative quantitative
parameters across the two study groups.

Table 1.

Quantitative associations between immunohistochemical markers and microbial taxa in patients with

oral leukoplakia (n = 68)

Parameter

Diabetic patients

(n = 40)

Non-diabetic

patients (n = 28)

p-

value

Ki-67

proliferation

index (%)

34.7 ± 6.1

18.2 ± 4.9

<

0.001

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International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)

p53-positive nuclei (%)

87.5

42.8

0.003

MMP-9 H-score

186.4 ± 23.5

104.2 ± 18.1

<

0.001

F. nucleatum

(% relative

reads)

9.6 ± 2.3

3.1 ± 1.7

0.008

P. gingivalis

(% relative

reads)

5.7 ± 1.9

2.3 ± 1.2

0.015

C. albicans

detection

rate (%)

70.0

32.1

0.002

C.

albicans

copy

number (×10

⁴

)

4.2

1.3

0.002

The convergence of molecular dysregulation and
microbiota expansion suggests the formation of a pro-
oncogenic mucosal microenvironment in patients with
impaired carbohydrate metabolism. The combined
elevation of proliferative markers, p53 dysregulation,
and increased matrix metalloproteinase activity in the
context of microbial virulence underscores the
pathogenic role of dysbiosis in accelerating leukoplakia
progression. The predominance of F. nucleatum and P.
gingivalis correlates with epithelial barrier disruption
and nuclear instability, consistent with patterns
observed in other inflammation-driven carcinogenic
models. Fungal overgrowth, particularly by C. albicans,
further amplifies inflammatory signaling and may
contribute to epithelial

–

mesenchymal transition in

predisposed tissues.

These findings reinforce the necessity of incorporating
microbial surveillance and molecular stratification into
the clinical management algorithm of oral leukoplakia,
particularly in patients with metabolic comorbidities.
Microbiota-informed risk profiling may enable earlier
identification of transformation-prone lesions and
support precision-targeted interventions.

CONCLUSION

The present study establishes a clinically and
statistically

substantiated

association

between

metabolic dysregulation and microbial shifts in the oral
mucosa of patients with leukoplakia. The data
demonstrate that carbohydrate metabolism disorders,
particularly type 2 diabetes mellitus, significantly
exacerbate epithelial proliferation, tumor suppressor
pathway

disruption,

and

extracellular

matrix

degradation. These molecular alterations co-occur with
increased colonization by high-risk microbial taxa,
including Fusobacterium nucleatum, Porphyromonas
gingivalis, and Candida albicans. The spatial and

quantitative correlation between dysbiosis and
dysplastic transformation supports the hypothesis of
microbiome-driven

modulation

of

leukoplakia

progression.

Microbiota

profiling,

in

conjunction

with

immunohistochemical risk markers (Ki-67, p53, MMP-
9), offers a promising framework for stratifying
malignant potential in precancerous lesions. Future
diagnostic algorithms should integrate microbial and
molecular data to improve risk prediction and to guide
targeted surveillance in patients with systemic
metabolic impairments. The findings further justify the
inclusion of microbiome modulation strategies as
adjunctive measures in the clinical management of
high-risk leukoplakia.

REFERENCES

Warnakulasuriya, S. (2020). Oral potentially malignant
disorders: A comprehensive review on clinical aspects
and management. Oral Oncology, 102, 104551.

Zhou, Y., & Zheng, W. (2020). Microbiota shifts and
epithelial dysplasia: Mechanistic insights into oral
leukoplakia. Frontiers in Cellular and Infection
Microbiology, 10, 428.

Kamer, A. R., et al. (2019). The oral microbiome in
systemic metabolic disease. Journal of Dental
Research, 98(6), 599

–

605.

Peterson, D. E., & Sonis, S. T. (2021). Dysbiosis and oral
precancer: Emerging paradigms. Cancer Prevention
Research, 14(3), 189

–

197.

Hernandez, M., et al. (2022). Microbial predictors of
oral carcinogenesis in precancerous lesions. Scientific
Reports, 12(1), 4579.

Sridharan, G., et al. (2021). Anatomical distribution and
microbial signatures of high-risk leukoplakia. Journal of
Oral Pathology & Medicine, 50(1), 84

–

91.

International Journal of Medical Sciences And Clinical Research

47

https://theusajournals.com/index.php/ijmscr

International Journal of Medical Sciences And Clinical Research (ISSN: 2771-2265)

Naik,

S.,

&

Deshmukh,

R.

(2017).

Matrix

metalloproteinase-9 as a marker of malignant
transformation. Indian Journal of Pathology and
Microbiology, 60(4), 533

–

537.

Takahashi, N., & Nyvad, B. (2016). The role of bacteria
in the caries process: Ecological perspectives. Journal of
Dental Research, 95(3), 243

–

249.

Al-hebshi, N. N., et al. (2019). Inflammatory microbial
signatures in oral precancer and cancer. Scientific
Reports, 9(1), 1354.

Sharma, S., & Rathore, M. (2021). Role of Candida
albicans in the pathogenesis of oral epithelial dysplasia.
Journal of Clinical and Experimental Dentistry, 13(3),
e244

–

e250.

Al-Hebshi, N. N., et al. (2017). Microbiome analysis of
oral squamous cell carcinoma: Identification of a
microbial signature. NPJ Biofilms and Microbiomes, 3,
23.

Perera, M., et al. (2018). Inflammatory bacteriome in
oral leukoplakia and erythroplakia. NPJ Biofilms and
Microbiomes, 4, 29.

Siqueira, J. F., & Rôças, I. N. (2017). Microbial diversity
in persistent oral infections. Archives of Oral Biology,
83, 151

–

159.

Mager, D. L., et al. (2016). The salivary microbiome in
health and disease. Journal of Clinical Periodontology,
43(6), 492

–

500.

Reichart, P. A., & Philipsen, H. P. (2022). Clinical and
histopathologic classification of leukoplakia and
implications for malignant transformation. Oral
Diseases, 28(1), 36

–

43.