Title: 0558 - Extended Incubation of Periodontal Biofilms Promotes Growth of Uncultured "Pathogens"


Flavia Teles (Presenter)
University of Pennsylvania

Sandra Henz, University of North Carolina
Shawn Dua, University of North Carolina
Di Wu, University of North Carolina
MARIA ANDREA AZCARATE-PERIL, University of North Carolina
Jorge Frias-Lopez, University of Florida
Lynn Martin, University of North Carolina


Objectives: Out of the 688 bacterial species present in the oral cavity, 208 remain uncultured. Virtually nothing is known about them, precluding inferences regarding their pathogenic role. This study aimed at growing uncultured taxa using extended incubation of ex vivo biofilms and multiplex cultivation.

Methods: Subgingival biofilm samples were collected from 6 periodontitis patients (from 4 sites with PD≥5 mm/patient), with minimal dispersal, into PRAS and transferred to Calgary Biofilm Devices (CBD) containing 12 different growth media. CBDs were incubated anaerobically and capnophilically for up to 112 days. Biofilms formed and their spent media were collected at days 4,10,14, 25, 35, 54, 84 and 112. Inocula aliquots were plated in HNK agar (viability plate) and incubated anaerobically for 7 days. The microbial composition of samples was determined using 16S rRNA sequencing (MiSeq, Illumina). Relative abundance (% of total reads) of taxa and species-level taxonomy were analyzed with QIIME and HOMINGS, respectively.

Results: 182 uncultured taxa were sought in 2,118 samples. Inocula aliquots harbored several phylotypes, but not the growth in viability plates, which were dominated by 1-4 species, typically Parvimonas micra, Streptococcus anginosus and Veillonella dispar, comprising up to 43% of a sample. Twenty-five phylotypes were consistently detected in the CBD samples. The most abundant were Fretibacterium sp HOT 360 (mean %±SD; 2.1%±7.0%; range: 0.01%-57.0%) and Stomatobaculum sp HOT 373 (0.3±1.7%; 0.01%-20.3%), which grew preferably in anaerobiosis and reached levels comparable to that of periodontal pathogens: Porphyromonas gingivalis (0.61%±7.5%; 0.1%-31.4%), Tannerella forsythia (0.3%±3.83%; 0.01%-12.2%) and Treponema denticola (0.2%±1.8%; 0.01%-7.3%). Aggregatibacter sp HOT 513 (1.2%±7.5%; 0.01%-84.1%) and Megasphaera sp HOT 123 (0.1%±2.18%; 0.01%-44.8%) were abundant in capnophilic incubation. Anaerobic incubation for 10-54 days in six of the media tested provided the best enrichment of phylotypes.

Conclusions: Extended incubation of ex vivo biofilms in different types of media fosters the growth of uncultured oral bacteria.

This abstract is based on research that was funded entirely or partially by an outside source:
NIH;NIDCR: R01-DE024767(FRFT), NIH: P30DK 34987 (UNC Microbiome Core)

Disclosure Statement:
The submitter must disclose the names of the organizations with which any author have a relationship, the nature of the relationship, and the clinical or research area involved. The following is submitted: None

Sponsoring Group/Network