The Microbiome of Potentially Malignant Oral Leukoplakia Exhibits Enrichment for Fusobacterium, Leptotrichia, Campylobacter, and Rothia Species

Abstract

Oral leukoplakia presents as a white patch on the oral mucosa and is recognized as having significant malignant potential. Although colonization of these patches with Candida albicans is common, little is known about the bacterial microbiota of these patches. In the current study we analyzed the microbiome of oral leukoplakia in 36 patients compared to healthy mucosal tissue from the same patients and healthy control subjects to determine if specific microbial enrichments could be identified early in the malignant process that could play a role in the progression of the disease. This was carried out by sequence analysis of the V1-V2 region of the bacterial 16S rRNA gene using the Illumina MiSeq. Oral leukoplakia exhibited increased abundance of Fusobacteria and reduced levels of Firmicutes (Metastats P < 0.01). Candida colonization was also more prevalent in leukoplakia patients relative to healthy controls (P = 0.025). Bacterial colonization patterns on oral leukoplakia were highly variable and five distinct bacterial clusters were discerned. These clusters exhibited co-occurrence of Fusobacterium, Leptotrichia, and Campylobacter species (Pearson P < 0.01), which is strikingly similar to the microbial co-occurrence patterns observed on colorectal cancers (Warren et al., 2013). Increased abundance of the acetaldehydogenic microorganism Rothia mucilaginosa was also apparent on oral leukoplakias from lingual sites (P 0.0012). Severe dysplasia was associated with elevated levels of Leptotrichia spp. and Campylobacter concisus (P < 0.05). Oral leukoplakia exhibits an altered microbiota that has similarities to the microbiome of colorectal cancer.

Keywords: Campylobacter; Fusobacteria; Rothia mucilaginosa; microbiome; oral cancer; oral leukoplakia.

FIGURE 1

Overview of bacterial phyla distribution and Candida carriage patterns in patients with OLK and healthy controls. (A) Graph showing the abundance of the six most important phyla in healthy control subjects and sites of OLK and contralateral normal sites in patients. ^∗ Indicates Metastats P < 0.05 and ^∗∗P < 0.01. (B) Percentage of Candida positive samples recovered from patients (OLK and contralateral sites) and healthy controls, determined by qPCR.

FIGURE 2

Analysis community structure in samples from OLK and healthy mucosa. (A) Rarefaction curve showing analysis of OTUs identified in samples from healthy controls (green) and patients (red = OLK tissue, orange = contralateral normal tissue). (B) Non-metric multidimensional scaling (NMDS) plots generated from a matrix of Bray–Curtis dissimilarity values using Mothur (Schloss et al., 2009). Separation of OLK communities (red) from contralateral (orange; P = 0.007) and healthy control communities (green; P = 0.001) was determined using analysis of molecular variance (AMOVA).

FIGURE 3

Results of LEfSe analysis to identify significantly enriched bacterial taxa in (A) communities from patients and healthy controls and (B) in communities from sites of OLK and contralateral tissue from patients.

FIGURE 4

Relative abundance of selected taxonomic groups identified by LEfSe analysis. The abundance of each organism (A–F) in OLK and contralateral healthy communities from each patient is plotted side by side. No healthy tissues were available from four patients (Supplementary Table S1) and these are plotted versus the average values for control tissue. P-values refer to Wilcoxon matched pairs test results.

FIGURE 5

(A) Heatmap showing the abundance of taxa identified by LEfSe analysis. Heatmap and dendogram were generated in Vegan using a matrix of Bray–Curtis dissimilarity values. Separation of patient clusters marked 1 to 5 in the dendogram was highly significant (Unweighted UNIFRAC P < 0.001). The color-coded legend indicates whether each sample is from a buccal or lingual site and the degree of dysplasia following biopsy (mild, moderate, or severe). CON (light blue) correspond to the average values in healthy buccal (left) and healthy lingual (right) samples. (B) Co-occurrence map generated from Pearson correlation coefficients (r) generated in Prism (P < 0.01) and graphically presented using Cytoscape 3.2.1. Thickness of edges (connecting lines) are proportional to r-values (0.4–0.99). Clusters within the dotted line were identified in patients who were also colonized with Candida spp.