Patients with OLP exhibit enhanced prevalence of H. pylori infection
The saliva samples of 30 patients with OLP (OLP group) and 21 normal control volunteers (NC group) were collected to analyze the correlation between H. pylori infection status and OLP clinical subtypes. Age and gender were not significantly different between the OLP and NC groups (Table 1).
H. pylori infection in the OLP and NC groups was detected using the urease breath test. The prevalence of H. pylori infection in the OLP group (70%) was markedly higher than that in the NC group (50%)(Table 1).
Erosive OLP was characterized by a red erosive mucosal surface (Figure S1 in Additional file). The lesion severity and the risk of malignant transformation in erosive OLP are higher than those in reticular OLP[9]. In the OLP group, the incidence of erosive OLP in H. pylori-positive patients (61.9%) was significantly (Chi-square test) higher than that in H. pylori-negative patients (11.1%) (p=0.0041). Additionally, H. pylori infection was correlated with the OLP subtypes (Table 2).
Patients with OLP exhibit altered oral microbiome composition
Previous studies have demonstrated that the oral microbiome composition in patients with OLP was different from that in normal control individuals[20–22]. This study demonstrated that the α diversity of oral microbiota in the OLP group was significantly higher than that in the NC group (Fig.1A and B). Principal coordinate analysis (PCoA) revealed that the β diversity of oral microbiota in the OLP group was significantly different from that in the NC group (Fig. 1C).
The oral microbiome composition was significantly different between the OLP and NC groups at the phylum (Fig. 1D) and genus levels (Fig. 1E). The dominant phyla in the oral microbiota were Firmicutes, Proteobacteria, and Bacteroidetes (Fig. 1D). Compared with the NC group, the OLP group exhibited a significantly decreased abundance of Firmicutes and a significantly increased abundance of Bacteroidetes (Fig. 1D). At the genus level, compared with the NC group, the OLP group exhibited a decreased abundance of Streptococcus and increased abundances of Neisseria, Prevotella, and Prevotella7 (Fig. 1E).
The bacterial genera with an average relative abundance higher than 1% in the oral microbiome of the OLP and NC groups are listed in Fig. 2A. The volcano plot was used to represent the differences in the bacterial composition at the genus level between the OLP and NC groups (Fig. 2B). Bacteria with relative abundance greater than 1% and significant differences in relative abundance were screened out (Fig. 2B). Compared with the NC group, the OLP group exhibited decreased relative abundances of Streptococcus and Rothia and increased relative abundances of Alloprevotella, Prevotella, Fusobacterium, and Porphyromonas (Fig. 2B and C).
H. pylori infection alters the salivary microbiome composition in patients with OLP.
To analyze the effect of H. pylori infection on the salivary microbiome composition of the OLP and NC groups, the saliva samples were divided into the following four groups based on the H. pylori infection status: OLP+ (n=21); OLP− (n=9); NC+ (n=10); NC− (n=11).
The α diversity of salivary microbiota in the OLP+ group was significantly higher than that in the OLP− group (Fig. 3A, B, C, D and E). In contrast, α diversity was not significantly different between the NC+ and NC− groups (Fig. 3A, B, C, D, and E). PCoA revealed that the β diversity of salivary microbiota was significantly different between the OLP+ and OLP− groups. However, the β diversity of salivary microbiota was not significantly different between the NC+ and NC− groups (Fig. 3F and G). Additionally, the bacterial composition at the genus and phylum levels was not significantly different between the NC+ and the NC− groups (Figure S2 in Additional file).
The salivary microbiome composition was significantly different between the OLP+ and OLP− groups at the phylum and genus levels (Fig. 4A and B). The predominant bacterial phyla were Proteobacteria, Firmicutes, and Bacteroidetes (Fig. 4A), the relative abundance of Bacteroidetes was significantly high in the OLP+ group. At the genus level, the relative abundance of Alloprevotella in the OLP+ group was significantly higher than that in the OLP− group (Fig. 4B).
The bacteria in the salivary microbiota of the OLP+ and OLP− groups with an average relative abundance higher than 1% are listed in Fig. 4C. The volcano plot was constructed to determine the differences in the bacterial composition between the OLP+ and the OLP− groups at the genus level (Fig. 4D). The bacteria with relative abundance higher than 1% and significant differences in the relative abundance were screened out (Fig. 4D). Compared with the OLP− group, the OLP+ group exhibited significantly increased relative abundances of Alloprevotella and Haemophilus and a significantly decreased relative abundance of Actinomyces (Fig. 4D and E).
Comparative analysis of salivary inflammatory factors
H. pylori infection can induce the gastric mucosa to secrete inflammatory factors, such as IL-6, IL-8, and IL-17[23]. Previous studies have reported the dysregulated expression of various inflammatory factors, such as IL-6, IL-8, IL-17, and TNF-α in patients with OLP[24]. In this study, the salivary levels of IL-6, IL-8, and IL-17 in the OLP and NC groups were analyzed using ELISA. The salivary levels of IL-6, IL-8, and IL-17 in the OLP group were significantly higher than those in the NC group (Fig. 5A).
Next, the effect of H. pylori infection on the salivary levels of inflammatory factors in the OLP and NC groups was evaluated. Additionally, the salivary levels of IL-6, IL-8, and IL-17 were comparatively analyzed between the following groups: OLP+ and OLP− groups; NC+ and NC− groups. Compared with those in the OLP− group, the salivary levels of IL-6, IL-8, and IL-17 were significantly upregulated in the OLP+ group (Fig. 5B). However, the salivary levels of IL-6, IL-8, and IL-17 were not significantly different between the NC+ and NC− groups (Fig. 5C).
Next, the correlation between key bacterial genera and inflammatory factors (IL-6, IL-8, and IL-17) was analyzed by constructing the heat map of Spearman’s rank correlation coefficients (Fig. 6A and B). In the OLP and NC groups, the abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated with IL-6 and IL-17, while the abundances of Prevotella and Fusobacterium genera were positively correlated with IL-8. Furthermore, the abundances of Streptococcus and Rothia genera were negatively correlated with IL-7, IL-6, and IL-8 (Fig. 6A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were significantly and positively correlated with IL-17, while those of Actinomyces genus were negatively correlated with IL-7, IL-6, and IL-8 (Fig. 6B).
Correlation of salivary microbiome function with key bacterial genera
PICRUSt was used to predict the metagenome functional content based on 16S rRNA gene sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 7). Compared with those in the NC group, the expression levels of genes involved in various metabolic pathways, such as histidine metabolism, phenylalanine metabolism, novobiocin biosynthesis, lipopolysaccharide (LPS) biosynthesis, LPS biosynthesis proteins, biotin metabolism, ubiquinone, and other terpenoid-quinone biosynthesis were upregulated, while those of genes involved in galactose metabolism, phosphotransferase system, and protein kinase were downregulated in the OLP group (Fig. 7A and B).
Furthermore, the levels of LPS biosynthesis proteins and LPS biosynthesis in the OLP+ group were upregulated when compared with those in the OLP− group (Fig. 7C and D).
LPS can induce inflammatory reaction[25]. PICRUSt analysis predicted that the microbial metabolic pathways involved in the pathogenesis of OLP are LPS biosynthesis proteins and LPS biosynthesis. The correlation between key bacterial genera and these two metabolic pathways was examined by constructing the heat map of Spearman’s rank correlation coefficients (Fig. 8A and B). In the OLP and NC groups, the relative abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated, while those of Rothia were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were positively correlated, while those of Actinomyces were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8B).
Patients with OLP exhibit enhanced prevalence of H. pylori infection
The saliva samples of 30 patients with OLP (OLP group) and 21 normal control volunteers (NC group) were collected to analyze the correlation between H. pylori infection status and OLP clinical subtypes. Age and gender were not significantly different between the OLP and NC groups (Table 1).
H. pylori infection in the OLP and NC groups was detected using the urease breath test. The prevalence of H. pylori infection in the OLP group (70%) was markedly higher than that in the NC group (50%)(Table 1).
Erosive OLP was characterized by a red erosive mucosal surface (Figure S1 in Additional file). The lesion severity and the risk of malignant transformation in erosive OLP are higher than those in reticular OLP[9]. In the OLP group, the incidence of erosive OLP in H. pylori-positive patients (61.9%) was significantly (Chi-square test) higher than that in H. pylori-negative patients (11.1%) (p=0.0041). Additionally, H. pylori infection was correlated with the OLP subtypes (Table 2).
Patients with OLP exhibit altered oral microbiome composition
Previous studies have demonstrated that the oral microbiome composition in patients with OLP was different from that in normal control individuals[20–22]. This study demonstrated that the α diversity of oral microbiota in the OLP group was significantly higher than that in the NC group (Fig.1A and B). Principal coordinate analysis (PCoA) revealed that the β diversity of oral microbiota in the OLP group was significantly different from that in the NC group (Fig. 1C).
The oral microbiome composition was significantly different between the OLP and NC groups at the phylum (Fig. 1D) and genus levels (Fig. 1E). The dominant phyla in the oral microbiota were Firmicutes, Proteobacteria, and Bacteroidetes (Fig. 1D). Compared with the NC group, the OLP group exhibited a significantly decreased abundance of Firmicutes and a significantly increased abundance of Bacteroidetes (Fig. 1D). At the genus level, compared with the NC group, the OLP group exhibited a decreased abundance of Streptococcus and increased abundances of Neisseria, Prevotella, and Prevotella7 (Fig. 1E).
The bacterial genera with an average relative abundance higher than 1% in the oral microbiome of the OLP and NC groups are listed in Fig. 2A. The volcano plot was used to represent the differences in the bacterial composition at the genus level between the OLP and NC groups (Fig. 2B). Bacteria with relative abundance greater than 1% and significant differences in relative abundance were screened out (Fig. 2B). Compared with the NC group, the OLP group exhibited decreased relative abundances of Streptococcus and Rothia and increased relative abundances of Alloprevotella, Prevotella, Fusobacterium, and Porphyromonas (Fig. 2B and C).
H. pylori infection alters the salivary microbiome composition in patients with OLP.
To analyze the effect of H. pylori infection on the salivary microbiome composition of the OLP and NC groups, the saliva samples were divided into the following four groups based on the H. pylori infection status: OLP+ (n=21); OLP− (n=9); NC+ (n=10); NC− (n=11).
The α diversity of salivary microbiota in the OLP+ group was significantly higher than that in the OLP− group (Fig. 3A, B, C, D and E). In contrast, α diversity was not significantly different between the NC+ and NC− groups (Fig. 3A, B, C, D, and E). PCoA revealed that the β diversity of salivary microbiota was significantly different between the OLP+ and OLP− groups. However, the β diversity of salivary microbiota was not significantly different between the NC+ and NC− groups (Fig. 3F and G). Additionally, the bacterial composition at the genus and phylum levels was not significantly different between the NC+ and the NC− groups (Figure S2 in Additional file).
The salivary microbiome composition was significantly different between the OLP+ and OLP− groups at the phylum and genus levels (Fig. 4A and B). The predominant bacterial phyla were Proteobacteria, Firmicutes, and Bacteroidetes (Fig. 4A), the relative abundance of Bacteroidetes was significantly high in the OLP+ group. At the genus level, the relative abundance of Alloprevotella in the OLP+ group was significantly higher than that in the OLP− group (Fig. 4B).
The bacteria in the salivary microbiota of the OLP+ and OLP− groups with an average relative abundance higher than 1% are listed in Fig. 4C. The volcano plot was constructed to determine the differences in the bacterial composition between the OLP+ and the OLP− groups at the genus level (Fig. 4D). The bacteria with relative abundance higher than 1% and significant differences in the relative abundance were screened out (Fig. 4D). Compared with the OLP− group, the OLP+ group exhibited significantly increased relative abundances of Alloprevotella and Haemophilus and a significantly decreased relative abundance of Actinomyces (Fig. 4D and E).
Comparative analysis of salivary inflammatory factors
H. pylori infection can induce the gastric mucosa to secrete inflammatory factors, such as IL-6, IL-8, and IL-17[23]. Previous studies have reported the dysregulated expression of various inflammatory factors, such as IL-6, IL-8, IL-17, and TNF-α in patients with OLP[24]. In this study, the salivary levels of IL-6, IL-8, and IL-17 in the OLP and NC groups were analyzed using ELISA. The salivary levels of IL-6, IL-8, and IL-17 in the OLP group were significantly higher than those in the NC group (Fig. 5A).
Next, the effect of H. pylori infection on the salivary levels of inflammatory factors in the OLP and NC groups was evaluated. Additionally, the salivary levels of IL-6, IL-8, and IL-17 were comparatively analyzed between the following groups: OLP+ and OLP− groups; NC+ and NC− groups. Compared with those in the OLP− group, the salivary levels of IL-6, IL-8, and IL-17 were significantly upregulated in the OLP+ group (Fig. 5B). However, the salivary levels of IL-6, IL-8, and IL-17 were not significantly different between the NC+ and NC− groups (Fig. 5C).
Next, the correlation between key bacterial genera and inflammatory factors (IL-6, IL-8, and IL-17) was analyzed by constructing the heat map of Spearman’s rank correlation coefficients (Fig. 6A and B). In the OLP and NC groups, the abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated with IL-6 and IL-17, while the abundances of Prevotella and Fusobacterium genera were positively correlated with IL-8. Furthermore, the abundances of Streptococcus and Rothia genera were negatively correlated with IL-7, IL-6, and IL-8 (Fig. 6A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were significantly and positively correlated with IL-17, while those of Actinomyces genus were negatively correlated with IL-7, IL-6, and IL-8 (Fig. 6B).
Correlation of salivary microbiome function with key bacterial genera
PICRUSt was used to predict the metagenome functional content based on 16S rRNA gene sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 7). Compared with those in the NC group, the expression levels of genes involved in various metabolic pathways, such as histidine metabolism, phenylalanine metabolism, novobiocin biosynthesis, lipopolysaccharide (LPS) biosynthesis, LPS biosynthesis proteins, biotin metabolism, ubiquinone, and other terpenoid-quinone biosynthesis were upregulated, while those of genes involved in galactose metabolism, phosphotransferase system, and protein kinase were downregulated in the OLP group (Fig. 7A and B).
Furthermore, the levels of LPS biosynthesis proteins and LPS biosynthesis in the OLP+ group were upregulated when compared with those in the OLP− group (Fig. 7C and D).
LPS can induce inflammatory reaction[25]. PICRUSt analysis predicted that the microbial metabolic pathways involved in the pathogenesis of OLP are LPS biosynthesis proteins and LPS biosynthesis. The correlation between key bacterial genera and these two metabolic pathways was examined by constructing the heat map of Spearman’s rank correlation coefficients (Fig. 8A and B). In the OLP and NC groups, the relative abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated, while those of Rothia were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were positively correlated, while those of Actinomyces were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8B).