The relationship of microbial biodiversity and clinical forms of oral lichen planus: analysis based on 16S rRNA sequencing

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Background

Lichen planus (LP) of the oral mucosa (OLP) is a chronic inflammatory disease of the oral mucosa (OM) of unknown etiology. It is characterized by inflammation, erosion, and damage to the stratified squamous epithelium and the OM connective tissue plate, sometimes accompanied by skin and nail damage [1].

In the Russian Federation, the incidence of LP among the population aged >18 years has reached 12.7 per 100 thousand people. LP most often occurs in people aged 30–60 years. Women account for 60%–75% among patients with OM lesions, and approximately 50% among patients with skin lesions [2].

LP of the OM and vermilion surface manifests in six clinical forms, namely, typical (reticular), hyperkeratotic, exudative-hyperemic, erosive-ulcerative, bullous, and atypical [3, 4].

Contemporary research of the OLP is increasingly recognizing the role of the oral microbiome and its interaction with the environment of the host organism. This is attributed to the importance of the human microbiota in the development of various diseases, making the regulation of microbiocenosis a key aspect of personalized medicine [5].

Various microorganisms have been studied as potential factors associated with OLP development, including Helicobacter pylori, Mycoplasma salivarium, periodontopathogenic bacteria, Candida albicans, human papillomavirus, Epstein–Barr virus, and hepatitis C virus. However, data on such associations are controversial and require further investigations. Existing studies reported conflicting results, and the mechanisms underlying these relationships are not fully understood [4].

Despite studies searching for specific OLP-associated microorganisms, a clear correspondence between their presence and disease development has not been revealed. This suggests the more significant role of the functional characteristics of the oral microbiota in the pathogenesis of OLP than its species composition. Currently, no microorganism can be recognized as the cause of this disease [5].

In this study, the 16S rRNA-sequencing method was used, which enabled the assessment of the biodiversity of microorganisms in patients with OLP. The microbial composition in patients with OLP was compared with those of the control group, followed by pairwise comparisons of the microbiota in different disease forms (typical, erosive-ulcerative, and hyperkeratotic). Thus, the microbial profile was characterized for each clinical form, and unique microbial signatures associated with each type of the disease were identified.

This study aimed to analyze comprehensively the oral microbiota composition in patients with OLP to identify possible pathogenetic microbial associations.

Materials and methods

Study design

The study employed a cross-sectional one-stage design.

Compliance criteria

The inclusion criteria were as follows: OLP diagnosis established earlier or for the first time; voluntary participation, provision of written informed consent for study participation, and consent to the processing of personal data; age ≥18 years; patients of different sexes; non-intake of systemic antibiotics 30 days before and application of topical agents 3 days before sample collection.

The non-inclusion criteria were as follows: failure to meet the inclusion criteria, history of severe concomitant pathology or other autoimmune diseases, and patient’s reluctance to participate in the study.

The exclusion criteria were as follows: the patient’s desire to discontinue participation in the study and non-compliance to the regimen, prescribed examination, and treatment schedule.

Conditions

The study was conducted at the V.A. Rakhmanov Clinic of Skin and Sexually Transmitted Diseases of the Sechenov University (Moscow) and the National Research Center “Kurchatov Institute” (Moscow).

Study duration

The study was conducted from January 2022 to November 2023.

Methods of outcome registration

A comparative study of the OM microbiota was performed by DNA-sequencing in groups. The main group consisted of 45 patients with OLP, and the control group consisted of 40 patients with other diseases of the OM, including 15 patients with pemphigus vulgaris, 10 with recurrent oral ulceration, and 15 with leukoplakia. Depending on the clinical disease form, the main group was divided into four subgroups, namely, typical (n = 9), hyperkeratotic (n = 17), erosive-ulcerative (n = 17), and exudative-hyperemic (n = 2) groups. In the exudative-hyperemic subgroup, owing to the small sample size, a comparative study of the OM microbiota was not conducted.

Sequencing resulted in 7956–121,460 reads per sample. After filtering and removing chimeric sequences, the analysis included 4,874–68,898 reads per sample. Data were processed in the R programming language (v 4.2.0) using the dada2 package (v 1.24.0). A rarefaction curve of amplicon sequence variants (ASV) was plotted, and most samples became saturated at 10,000 reads.

For the ecological analysis of the buccal epithelium microbiome in the oral cavity, the following methods were used:

• Alpha diversity using the estimate_richness function of the phyloseq package (v 1.40.0): the significance of the between-group difference was determined by the Wilcoxon T-test, and the null hypothesis was rejected at p-value < 0.05.
• Beta diversity using the cal_betadiv function of the microeco package (v 0.19.5): compositional dissimilarities between groups were considered Bray–Curtis dissimilarity, and the significance of group differences was determined using PERMANOVA.
• Representation analysis, differential analysis of representation, and Venn analysis were calculated in the microeco package (v 0.19.5).

Ethical considerations

The study was approved by the local ethics committee of Sechenov University (Protocol No. 01-22 of 01/20/2022). All patients provided signed voluntary informed consent to participate in the study. The patients were fully informed about the study, therapy courses, possible outcomes, and side effects of the therapy.

Statistical analysis

IBM SPSS Statistics version 27.0 (IBM Corp., Armonk, NY, USA) was used to perform statistical analysis. Descriptive statistics included the calculation of means and standard deviations for quantitative data and frequencies and percentages for categorical data. The R programming language (v 4.2.0) was used to analyze DNA-sequencing data by employing the dada2 (v 1.24.0) and phyloseq (v 1.40.0) packages. A p-value of <0.05 was considered significant.

Results

Objects (participants) of the study

The main group consisted of 45 patients with OLP [10 (22.22%) men, 35 (77.78%) women; average age, 55.3 ± 13.4 years]. The control group consisted of 40 patients with other OM diseases, including 15 patients with pemphigus vulgaris, 10 with recurrent oral ulceration, and 15 with leukoplakia. The study revealed significant differences between the groups. The main group had significantly higher proportion of women (77.7% versus 55% in the control group, p < 0.05) and higher prevalence of smoking (33.3% vs. 10%, p < 0.05); gastritis associated with H. pylori (22.2% vs. 10%, p < 0.05), type 2 diabetes mellitus (33.3% vs. 2.5%, p < 0.05), and obesity (22.2% vs. 2.5%, p < 0.05) (Table 1).

 

Table 1. Main characteristics of patients with red squamous lichen planus of the oral mucosa

Parameter	Group		p
	Main, n=45	Control, n=40
Sex
• Male	10 (22.2)	8 (20)	-
• Female	35 (77.7)	22 (55)
Age, years, M ± m	55.3±13.4	53.7±11.9	>0.05
Type:
• Typical	17 (37.7)	-	-
• Erosive-ulcerative	17 (37.7)	-	-
• Hyperkeratotic	9 (20)	-	-
• Exudative-hyperemic	2 (4.4)	-	-
Smoking	15 (33.3)	4 (10)	<0.05
Gastritis associated with H. pylori	10 (22.2)	4 (10)	<0.05
Chronic esophagitis	2 (4.4)	1 (2.5)	>0.05
Hypertonic disease	15 (33.3)	10 (25)	>0.05
Type 2 diabetes mellitus	15 (33.3)	1 (2.5)	<0.05
Obesity	10 (22.2)	1 (2.5)	<0.05

Note. Significant differences between the main and control groups with p <0,05 are highlighted in bold.

 

Main research results

Alpha diversity analysis. The bacterial composition was more diverse in the samples of the main group (Fig. 1); however, when comparing different OLP forms, significant differences were revealed for the typical and erosive-ulcerative forms when using the Chao1 and Shannon indices in the control group samples (Fig. 2).

 

Fig. 1. Analysis of alpha diversity of the bacterial composition of the oral mucosa depending on the presence of the disease using the Chao1 and Shannon indices.

 

Fig. 2. Alpha diversity measurement of bacterial composition of oral cavity according to the form of the oral lichen planus disease using Chao1 and Shannon Index.

 

The rarefaction curves indicated that the results represented virtually the entire bacterial population in samples taken from the main group, as indicated by the 97% Good’s coverage (Fig. 3).

 

Fig. 3. Rarefaction curve plot.

 

Analysis of representation. An analysis of the 10 most common taxa showed that in the main group, the relative amounts of bacteria at the level of phyla Actinobacteria, Bacteroidota, and Fusobacteria (Fig. 4a), families Pasteurellaceae and Pseudomonadaceae (Fig. 5a), and genera Pseudomonas and Porphyromonas increased. A wide range of other genera was also recorded (Fig. 6a). In addition, the bacterial populations of the phyla Firmicutes and Proteobacteria (Fig. 4a), families Streptococcaceae, Gemellaceae and Carnobacteriaceae (Fig. 5a), and genera Streptococcus, Granulicatella, and Gemella decreased (Fig. 6a). Note that in the erosive-ulcerative and hyperkeratotic OLP, the Streptococcus population decreased compared with other clinical variants of the disease (Fig. 6b). The significance of the data obtained was confirmed using the Wilcoxon test (p < 0.05).

 

Fig. 4. Representation of the relative abundance of oral microbiota at the phylum level with comparisons between the main group (О-Г) and control group (К-Г) (a), as well as among the typical, erosive-ulcerative, and hyperkeratotic forms (b) (The 10 more frequent taxa).

 

Fig. 5. Representation of the relative abundance of oral microbiota at a family level with comparisons between the main group (О-Г) and control group (К-Г) (a), as well as among the typical, erosive-ulcerative, and hyperkeratotic forms (b) (The 10 more frequent taxa).

 

Fig. 6. Representation of the relative abundance of oral microbiota at a genus level with comparisons between the main group (О-Г) and control group (К-Г) (a), as well as among the typical, erosive-ulcerative, and hyperkeratotic forms (b) (The 10 more frequent taxa).

 

Beta diversity analysis. The calculation of beta diversity did not reveal any differences between the main group and the control group (Fig. 7); however, when compared by disease form, significant differences were recorded (Fig. 8). In particular, the typical and hyperkeratotic forms were clearly grouped (Fig. 8b), as well as the erosive-ulcerative and hyperkeratotic forms (Fig. 8d) with p values of 0.628 and 0.612, respectively. This finding suggests that the typical form demonstrates more significant differentiation in the structure of the microbiome compared with the hyperkeratotic and erosive-ulcerative forms.

 

Fig. 7. Beta-diversity analysis of microbial community compositional differences between the main group (О-Г) and the control group (К-Г) using PCoA (Principal coordinate Analysis).

 

Fig. 8. Beta-Diversity Analysis of microbial community compositional differences according to the form of the disease using PCoA (Principal coordinate Analysis).

 

Venn analysis. This analysis provided information about overlapping and unique ASVs in the groups analyzed. Only half of the ASV overlapped between the main and control groups (Fig. 9a). When comparing disease types, the hyperkeratotic form differed most significantly from the microbiome of the control group, and it also differed from other forms (Fig. 9b).

 

Fig. 9. Venn diagram representation of the main (О-Г, oral lichen planus patients) and control (К-Г) group (a) and according to clinical form (b).

 

Analysis of differential representation. Compared with the control group, the main group exhibited significant differences in the number of nine unique species of microorganisms of various taxonomic levels (Fig. 10). In addition, a comparative analysis of the microbiota by clinical forms revealed diversity in species composition between the hyperkeratotic and typical forms, highlighting the specific microbial profile of each form (Fig. 11).

 

Fig. 10. Differential abundance of amplicon sequence variants (ASV) between the main group (О-Г) and the control group (К-Г) as determined by linear discriminant analysis (LDA).

 

Fig. 11. Differential abundance of amplicon sequence variants ( ASVs) between the typical and hyperkeratotic forms of oral lichen planus using linear discriminant analysis (LDA).

 

Discussion

In this study, the microbial composition in patients with OLP was analyzed, and the results were compared with the indicators of the control group. Differences in microbial composition depending on the clinical forms (typical, erosive-ulcerative, and hyperkeratotic) were also examined, which enabled detailed comparative analysis of the subgroups.

The results of alpha diversity analysis (Fig. 1) showed a more diverse microbial composition in the main group. Moreover, a comparison of various clinical forms of OLP in the main group with the indicators of the control group revealed significant differences in the biodiversity of microorganisms, particularly the typical and erosive-ulcerative forms, which is confirmed by Chao1 and Shannon indices (Figs. 4–6). The present results differ from those of F.Y. Yu et al. [6]. The differences may be caused by the specific composition of the control group, diversity of sequenced regions, and other confounders that can modify the relationship between the risk factor in question and the study outcome.

The relative abundance of the oral microbiota at all taxonomic levels (phylum, family, and genus) also showed that the dominant bacteria in OLP were significantly different from those detected in the control group. The subgroup analysis of the relative abundance revealed that the Streptococcus population was lower in erosive-ulcerative and hyperkeratotic forms than in the typical form. On the contrary, M.M. Bornstein et al. [7] reported that in patients with LP having nonerosive/asymptomatic lesions, the bacterial loads for Capnocytophaga sputigena, Eikenella corrodens, Lactobacillus crispatus, Mobiluncus curtisii, Neisseria mucosa, Prevotella bivia, Prevotella intermedia, and Streptococcus agalactiae in the LP lesion were significantly higher than those in similar sites of the control group. However, this discrepancy may be due to the analysis of asymptomatic patients, which implies the exclusion of the influence of oral hygiene, which can introduce differences in the species composition of microorganisms [8].

The results also confirm that the microbiota of both groups, despite the presence of numerous ulcers of the OM, differs distinctly. This finding suggests that changes in the oral microbiota may be directly related to the underlying pathological process and not only to the presence of oral ulcers or the inflammatory environment in the oral cavity.

The microbial composition of the main group was significantly different from that of the control group. Increased amounts of periodontitis-associated pathogens such as Pseudomonas and Porphyromonas in the main group compared with the control group were notable, which indicated the significant difference in the microbial ecosystem (Figs. 4–6).

This study confirms the results of previous studies that assessed the prevalence of periodontal pathogens using culture methods. However, in contrast to a previous study, where the control group consisted of patients without OLP but had periodontitis or gingivitis, the control group of the present study did not have any oral diseases. Owing to this selection of a control group, microorganisms with periodontal destructive effects were detected, namely, Aggregatibacter actinomycetemcomitans, Veillonella parvula, Porphyromonas gingivalis, and Treponema denticola, which can be associated with OLP [8].

In contrast to alpha diversity measures, which demonstrated differences in the oral microbiota of the main group compared with the control group, the analysis of beta diversity did not reveal significant differences between the groups. Thus, while the microbiota composition (beta diversity) remains essentially similar between the study groups, the diversity and abundance of individual species (alpha diversity) differ, indicating specific changes in the microbial community, associated with oral health.

This phenomenon can be interpreted by the presence of certain conditions that may promote the growth of specific bacteria existing in the oral cavity. These bacterial species then increase in abundance, whereas others may decrease, resulting in a change in alpha diversity without a marked change in the overall spectrum of bacteria present.

A pairwise comparison of beta diversity indices between the subgroups showed that samples from the typical subgroup were characterized by a more pronounced difference in the microbiota composition compared with the samples from the hyperkeratotic and erosive-ulcerative subgroups. These differences in microbial composition among clinical forms suggest that changes in the oral microbiota not only precede the development of more severe forms but may also influence actively such forms and severity.

Venn diagrams revealed significant similarity in the microflora between the control and main groups, confirming the results of previous beta diversity analysis. However, the presence of unique microorganisms in patients with the disease emphasizes their specific role and indicates a distinct microbiome signature associated with this condition.

Detailed (linear discriminant) analysis revealed unique bacterial taxa characteristic of the main group; thus, Pseudomonas, Pseudomonadaceae, and Pseudomonadales deserve special attention because of their high ecological resistance and potential for opportunistic pathogenicity. The increase in their abundance in patients with OLP suggests possible pathogenic changes in the oral microbiota composition [9]. Stenotrophomonas, Xanthomonadaceae, and Xanthomonadales are also of concern because of antibiotic resistance and infections, which, given their dominance in the main group, indicates dysbiosis associated with the disease [10]. Finally, Massilia, Oxalobacteraceae, and Microbacteriaceae, which are less dominant in the oral cavity, may represent commensal bacteria characteristic of a healthy oral microbiome.

The subgroup analysis using linear discriminant analysis identified certain bacterial taxa with significantly different abundances between hyperkeratotic and typical forms, consistent with beta diversity.

Conclusion

Changes in the oral microbiota composition may be of key importance in the pathogenesis of OLP, indicating the relationship between microbial imbalance and disease development. Moreover, the discovery of high pathogen load associated with periodontal diseases emphasizes the possible overlap of pathogenetic mechanisms between OLP and periodontitis, which opens up prospects for further study of their relationship. In addition, the unique microbial signatures that are associated with various clinical forms of OLP offer a basis for the development of targeted therapeutic approaches. This approach can contribute to the creation of differentiated treatment strategies adjusted to the specifics of each clinical form.

The importance of additional research to clarify the cause-and-effect relationships between microbiota changes and OLP cannot be overemphasized. A thorough understanding of these relationships may be the key to the development of personalized approaches to OLP treatment and prevention, which will ultimately increase the treatment efficiency and the quality of life of patients.

Additional information

Funding source. The work was carried out as part of the fulfillment of the state task of the Scientific Research Center “Kurchatov Institute”.

Competing interests. The authors declare that they have no competing interests.

Authors' contribution. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work. N.P. Teplyuk, M.A. Stepanov ― research concept, making significant edits to the manuscript in order to increase the scientific value of the article; B.Sh. Damdinova ― analysis of the data obtained, interpretation of the results, a significant contribution to the writing of the article; S.V. Toshchakov. S.A. Noskov, N.A. Tutubalina ― obtaining data, significant edits in order to increase the scientific value of the article.

About the authors

Natalya P. Teplyuk

Sechenov First Moscow State Medical University (Sechenov University)

Email: teplyukn@gmail.com
ORCID iD: 0000-0002-5800-4800
SPIN-code: 8013-3256

MD, Dr. Sci. (Med.), Professor

Russian Federation, Moscow

Mikhail A. Stepanov

Sechenov First Moscow State Medical University (Sechenov University)

Email: Doctor.stepanov@gmail.com
ORCID iD: 0000-0002-1872-9487
SPIN-code: 6524-5665

MD, Cand. Sci. (Med.), Associate Professor

Russian Federation, Moscow

Baira Sh. Damdinova

Sechenov First Moscow State Medical University (Sechenov University)

Author for correspondence.
Email: baira_d@mail.ru
ORCID iD: 0000-0002-4162-2928
Russian Federation, Moscow

Stepan V. Toshchakov

National Research Centre "Kurchatov Institute"

Email: stepan.toshchakov@gmail.com
ORCID iD: 0000-0001-7549-3450
SPIN-code: 8994-5224

Cand. Sci. (Biol.)

Russian Federation, Moscow

Sergey A. Noskov

National Research Centre "Kurchatov Institute"

Email: sergey.noskov.2001@icloud.com
ORCID iD: 0009-0006-1578-1382
Russian Federation, Moscow

Nina A. Tutubalina

National Research Centre "Kurchatov Institute"

Email: nina.tutubalina@gmail.com
ORCID iD: 0009-0004-7016-3670
Russian Federation, Moscow

References

Supplementary files