Methods: Ten adults who had not taken antibiotics within the past 3 months were sampled at 9 oral sites each: supragingival, subgingival (4 deepest sites), cheek, palate, tongue, tonsils (throat sampled for two subjects without tonsils). Soft tissue sites were sampled using nylon brushes, hard tissue sites with scalers. DNA was purified from the clinical samples, PCR amplified using 16S rRNA V3V4 primers, purified, and sequenced using an Illumina Miseq instrument. The 16S rRNA reads were parsed, and BLASTN analyzed using HOMD RefSeq Version 13.2.
Results: A total of 5,586,237 sequences (V3-V4 assembled overlapped paired reads) matched HOMD reference sequences at >98.5% BLASTN identity. The reads were identified to 481 HOMD taxa: 317 cultivated and 164 uncultivated phylotypes. The number of uncultivated taxa per sample ranged from 15-68 with a mean of 40.6. The total number of oral taxa/subject-site ranged from 91-251 with a mean of 182. The mean number of uncultivated taxa/individual sample at oral sites was as follows: cheek 45.9, palate 38.4, tongue 29.0, tonsils 46.3, throat 45.0, supragingival 39.8 and subgingival 39.6. Uncultivated taxa identified included 22 from the rare phyla: Chloroflexi, 1; Synergistetes genus Fretibacterium, 4; GNO2, 3; SR1, 3; and TM7, 11 taxa.
Conclusion: In screening just 10 subjects, we identified subject-sites with 164/244 (67%) of the uncultivated taxa currently in HOMD. The majority of these uncultivated taxa are thought to be uncultivable using standard cultivation methods; however, this study shows they are readily available for study and attempted cultivation.