Microbiological analysis of root canal infections using high throughput sequencing on the Illumina MiSeq platform

Aim: To investigate the microbial diversity of primary and secondary root canal infections using high throughput sequencing on the illumina MiSeq and culture methods. Methods: 19 subjects were recruited for the study; ten primary infections and nine secondary infections. Samples were collected before chemo-mechanical preparation (S1) and prior to obturation (S2), respectively. Microbiological culture aliquots were serially diluted and inoculated onto various non selective and selective media for total anaerobic and total aerobic counts. For high throughput sequencing, DNA was extracted and the V3/V4 region of the 16SrRNA gene was amplified using the 347F/803R primers, sequenced using the Illumina MiSeq instrument. Raw data were analysed using an open-source bioinformatics pipeline called quantitative insights into microbial ecology (QIIME). Results: Culture: Total anaerobic counts from primary infections ranged from 1.7 X10^1- 7.9 X10^6 colony forming units (cfu)/ml (mean log10 cfu/ml ± SD: 3.08 ± 1.51), whilst total aerobic counts ranged from 3 X10^3- 4.17 X10^5 cfu/ml ( mean log10 cfu/ml ± SD:3.09 ± 1.72). The quantity of microorganisms recovered from secondary infections ranged from 3 X10^2- 4.9 X10^3 cfu/ml (mean log10 cfu/ml ± SD: 2.81 ± 0.78) and from 2.7 X10^2- 8 X10^5 (mean log10 cfu/ml ± SD: 2.60 ± 1.48) with regard to total anaerobic and total aerobic viable counts, respectively. Sequencing analysis yielded partial 16S rRNA gene sequences that were taxonomically classified into 10 phyla and 143 genera. The most represented phyla in the total sample were Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Synergistetes and Fusobacteria. The most dominant genera in primary S1 samples were Streptococcus, Bacillaceae and Eubacterium while Alkalibacterium, Bacillaceae and TG5 dominated the secondary infections. The majority of genera occurred at low levels. The mean number (± SD) of species-level phylotypes per canal was 63 (±14.9; range 34– 80), and 69.9 (± 12.0; range 50 – 87) in primary and secondary infections (S1) samples, respectively. A great inter-individual variation in the composition of the root canal microbiota was observed. Conclusions: The study demonstrated the extensive diversity of the bacterial communities present in root canal infections although the majority of the taxa detected were in low abundance. The study indicates that secondary infections seem more diverse than previously anticipated.