Recovery of Previously Uncultured Bacteria from Attached Gingiva

Objectives: Extensive microbiological efforts have allowed cultivation of 65% of the 688 bacterial species inhabiting the human oral cavity, yet 35% (244 taxa) remain uncultivated. We screened 9 oral sites (supragingival, subgingival, attached gingivae, tongue dorsum, cheek, palate, tonsils, throat and saliva) from 20 subjects to determine the prevalence of previously uncultivated oral taxa at each site in each subject. The results identified which oral sites and in which subjects previously uncultivated bacteria were present as the highest proportion of the community. We hypothesized that certain sites, such as the attached gingiva, are underrepresented in cultivation studies and that sampling a lesser studied site could lead to cultivation of previously uncultivated oral taxa.
Methods: Four nylon swab samples of attached gingiva from a healthy individual were dispersed into 5 ml Maximum Recovery Diluent (MRD), diluted and plated in triplicate on Fastidious Anaerobe agar (FAA) supplemented with 5 g/L yeast extract, 5% sheep blood, 0.001 mg/ml 1,4-dihydroxy-2-naphthoate (DHNA), 0.01 mg/ml hemin and nicotinamide adenine dinucleotide (NAD), 1 mg/L (FAAYBHDN). Plates were incubated aerobically, anaerobically and in microaerophilic conditions. Resultant colonies were screened by 16S rDNA sequencing to identify previously uncultivated human bacterial taxa.
Results: A total of 2,304 colonies were screened by 16S rDNA sequencing. We were able to recover three previously uncultivated human oral taxa, namely Haemophilus sp. HOT-036, Actinomyces sp. HOT-897 and Prevotella sp. HOT-313.
Conclusions: We recovered three previously uncultivated bacterial taxa from a single subject from the attached gingivae niche. This study demonstrates that there still exist easily cultured “uncultivated oral taxa” in lesser studied oral niches.