IADR Abstract Archives
Modified SHI-Media Supports Growth Of Difficult To Culture Bacteria
Objectives:
Many oral bacteria in subgingival plaque communities have unknown requirements for growth and are difficult to culture in-vitro. Of the Candidate Phyla Radiation (a group of uncultivated phyla with reduced genomes), only the strain TM7x in the phylum Saccharibacteria has been successfully cultured in-vitro as an ultrasmall epibiont on its bacterial host. In this study, we modified the composition of SHI-media (known to support TM7x growth) to create conditions for the growth of members of the phylum Saccharibacteria along with other difficult-to-culture subgingival bacteria.
Methods:
A subgingival aggressive periodontitis plaque sample was inoculated in SHI-media and grown for 48hrs with varying concentrations of sucrose (0, 0.1, 0.5%), fetal bovine serum (FBS) (0, 10, 30, 50%), and mucin (0.1, 2.5, 8.0g/L). The study was conducted in two replicates with un-inoculated media controls. A 16S rRNA sequencing run was conducted on the resulting polymicrobial biofilms for each condition.
Results: Interestingly, our results revealed growth of a G3 member, an as of yet uncultivated group within the Saccharibacteria phylum. G3 grew in SHI-media containing 10% FBS, and SHI-media without sucrose. Our data supports that serum (a major component of gingival crevicular fluid) supports growth of subgingival anaerobes, as well as G3 members along with their potential hosts. Likewise, an absence of sucrose in the media appears to limit growth of acidogenic bacteria and may better simulate in-vivo subgingival plaque conditions. However, we found that organisms like Treponema spp. and Tannerella forsythia (otherwise hard to grow in-vitro) were found in the polymicrobial communities grown in SHI-media with 10% FBS as well as SHI-media with 0.5% sucrose. The presence of certain acidogenic bacteria in higher sucrose content media may help the growth of these organisms.
Conclusions:
These results suggest altering the FBS and sucrose concentration in SHI-media may support growth of difficult-to-culture organisms from subgingival biofilms.
Many oral bacteria in subgingival plaque communities have unknown requirements for growth and are difficult to culture in-vitro. Of the Candidate Phyla Radiation (a group of uncultivated phyla with reduced genomes), only the strain TM7x in the phylum Saccharibacteria has been successfully cultured in-vitro as an ultrasmall epibiont on its bacterial host. In this study, we modified the composition of SHI-media (known to support TM7x growth) to create conditions for the growth of members of the phylum Saccharibacteria along with other difficult-to-culture subgingival bacteria.
Methods:
A subgingival aggressive periodontitis plaque sample was inoculated in SHI-media and grown for 48hrs with varying concentrations of sucrose (0, 0.1, 0.5%), fetal bovine serum (FBS) (0, 10, 30, 50%), and mucin (0.1, 2.5, 8.0g/L). The study was conducted in two replicates with un-inoculated media controls. A 16S rRNA sequencing run was conducted on the resulting polymicrobial biofilms for each condition.
Results: Interestingly, our results revealed growth of a G3 member, an as of yet uncultivated group within the Saccharibacteria phylum. G3 grew in SHI-media containing 10% FBS, and SHI-media without sucrose. Our data supports that serum (a major component of gingival crevicular fluid) supports growth of subgingival anaerobes, as well as G3 members along with their potential hosts. Likewise, an absence of sucrose in the media appears to limit growth of acidogenic bacteria and may better simulate in-vivo subgingival plaque conditions. However, we found that organisms like Treponema spp. and Tannerella forsythia (otherwise hard to grow in-vitro) were found in the polymicrobial communities grown in SHI-media with 10% FBS as well as SHI-media with 0.5% sucrose. The presence of certain acidogenic bacteria in higher sucrose content media may help the growth of these organisms.
Conclusions:
These results suggest altering the FBS and sucrose concentration in SHI-media may support growth of difficult-to-culture organisms from subgingival biofilms.