Investigation of the microbiological profile of oral leukoplakia

Abstract

Oral leukoplakia (OLK) presents as a white patch on the oral mucosa and is recognised as a potentially malignant lesion. Although colonisation of these patches with yeast such as Candida albicans has been reported, little is known about the bacterial microbiome of these lesions. Recent studies have indicated the microbiome of oral squamous cell carcinoma (OSCC) is different from healthy mucosa and we wished to determine if similar changes occurred in OLK prior to malignant progression. In the current study, analysed of the OLK microbiome in 36 patients compared to contralateral healthy mucosal tissue from the same patients and 36 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 firstly 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 ( p < 0.01). Candida colonisation was also more prevalent in leukoplakia patients relative to healthy controls. Bacterial colonisation patterns on oral leukoplakia were highly variable and 5 distinct bacterial clusters were discerned. These clusters exhibited co-occurrence of Fusobacterium, Leptotrichia, and Campylobacter species (Pearson p < 0.01), which is very similar to the microbial co-occurrence patterns observed on colorectal cancers. Severe dysplasia was associated with elevated levels of Leptotrichia spp., and Campylobacter concisus (p < 0.05). Further sequence analysis using primers specific for the V3-V4 region of the 16S rRNA confirmed these data. In general, it was found that V1-V2 sequencing was more effective and could identify more OTUs than V3-V4 sequence analysis. Smoking, Candida spp. carriage and alcohol consumption were all shown to have specific effects on the oral microbiome. Candida carriage was associated with increased carriage of F. nucleatum whereas smoking was negatively associated with carriage of F. nucleatum. Alcohol consumption was associated with increased levels of Campylobacter spp. An increasing of abundance some species including Rothia mucilaginosa and other Gram-positive were observed on OLK samples. R. mucilaginosa spp., were isolated from several OLK sites and assessed its capacity to generate acetaldehyde (ACH) in vitro using aldehyde detection kits and Gas-Chromatography Mass-Spectrometry (GC-MS). R. mucilaginosa was shown to produce ACH from ethanol at similar levels to C. albicans and Neisseria mucosa, with only one isolate (41C2.2) producing >150 μM ACH from 10 mM ethanol. These levels of ACH were shown to induce oxidative stress in cultured oral keratinocytes. In summary, OLK exhibits an altered microbiota that has similarities to the microbiome of colorectal cancer. R. mucilaginosa has the potential to drive malignant change through the production of mutagenic levels of ACH.