IADR Abstract Archives
Bacterial Diversity Within Oral Squamous Cell Carcinoma by Pyrosequencing
Objectives: Diversity of bacteria within oral squamous cell carcinoma (OSCC) tissues has been assessed using culture-based methods, DGGE, 16S rRNA clonal analysis, and next generation sequencing (NGS). While the latter technology enables diversity analysis at greater breadth and depth, it has been so far used for assessment of bacterial diversity in OSSC at the genus level only. Therefore, the purpose of this preliminary study was to employ NGS for assessment of bacterial diversity within OSCC at the species-level.
Methods: DNA was extracted from fresh biopsies of three OSSC cases. The 27F / 534R degenerate primers were used to prepare barcoded libraries of the 16S rRNA gene’s V1-V3 region. The libraries were then sequenced using the 454’s GS FLX chemistry at a depth of 10,000 reads per sample. Reads were bioinformatically filtered for low quality and chimeric sequences, aligned, and then assigned to species-level OTUs by blasting to HOMD (≥ 98.5% sequence similarity).
Results: Over 150 species were identified representing 10 phyla; the most abundant were, in order: Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Actinobacteria. Seventy-five genera were identified, of which 20 were detected in all samples. Prevotella, Neisseria, Fusobacteria, Porphyromonas, Streptococcus and Aggregatibacter were the most abundant genera in order. Forty five species were detected in two of the samples and 27 were detected in all samples. Abundant species (≥ 1%) were Prevotella oris, Neisseria flava, Fusobacterium nucleatum_ss_polymorphum, unclassified Aggregatibacter sp. , Alloprevotella tannerae, Fusobacterium periodonticum, Fusobacterium nucleatum_ss_vincentii, Gemella morbillorum, unclassified Pasteurellaceae sp., Catonella morbi, Parvimonas micra, Porphyromonas catoniae and Leptotrichia oral taxon 218.
Conclusions: This is the highest diversity ever reported for bacteria within OSSC. Proportions of the identified genera/species suggest a microbial composition characteristic of OSSC. Further investigation involving larger sample size and control tissue to identify species associated with OSSC is warranted.
Methods: DNA was extracted from fresh biopsies of three OSSC cases. The 27F / 534R degenerate primers were used to prepare barcoded libraries of the 16S rRNA gene’s V1-V3 region. The libraries were then sequenced using the 454’s GS FLX chemistry at a depth of 10,000 reads per sample. Reads were bioinformatically filtered for low quality and chimeric sequences, aligned, and then assigned to species-level OTUs by blasting to HOMD (≥ 98.5% sequence similarity).
Results: Over 150 species were identified representing 10 phyla; the most abundant were, in order: Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Actinobacteria. Seventy-five genera were identified, of which 20 were detected in all samples. Prevotella, Neisseria, Fusobacteria, Porphyromonas, Streptococcus and Aggregatibacter were the most abundant genera in order. Forty five species were detected in two of the samples and 27 were detected in all samples. Abundant species (≥ 1%) were Prevotella oris, Neisseria flava, Fusobacterium nucleatum_ss_polymorphum, unclassified Aggregatibacter sp. , Alloprevotella tannerae, Fusobacterium periodonticum, Fusobacterium nucleatum_ss_vincentii, Gemella morbillorum, unclassified Pasteurellaceae sp., Catonella morbi, Parvimonas micra, Porphyromonas catoniae and Leptotrichia oral taxon 218.
Conclusions: This is the highest diversity ever reported for bacteria within OSSC. Proportions of the identified genera/species suggest a microbial composition characteristic of OSSC. Further investigation involving larger sample size and control tissue to identify species associated with OSSC is warranted.