Cultivating Previously Uncultured Bacteria from the Human Oral Cavity

Our knowledge of the bacteria that make up the human microbiome and the roles they play in health and disease is severely limited, and one of the greatest causes of that limitation is the inability to culture many of these organisms. It is estimated that 50% of the human oral flora is uncultured (Aas et al., 2005) and the essential challenge is to develop methods for cultivating these elusive organisms, in order to understand the role of the oral microbiome in human health. It was previously discovered that many natural bacterial isolates from environments outside of the human body were uncultured due to their dependence on growth factors that are normally provided by other organisms in the environment (D'Onofrio et al., 2010). The hypothesis is that similar interactions are responsible for the failure to culture many of the organisms that make up the human microbiome. The goal of this study was to cultivate previously uncultured organisms from the oral cavity using co-culture techniques, identify their limiting growth factors and to determine the ubiquity of these growth factor-requiring organisms in the oral cavity. Several dependent bacteria from the oral cavity were isolated using co-culture techniques. One previously uncultured bacterium, KLE1280, was chosen as the model organism to identify its growth factor requirement. KLE1280 is related to Porphyromonas catoniae by 16S rRNA sequencing, and KLE1280 was isolated in co-culture with an oral isolate closely related to Staphylococcus hominis which acts as a helper. We found that this isolate (KLE1280) would also grow in the presence of E. coli, which allowed for the screening of a library of deletion mutants in search of a growth factor. E. coli mutants lacking menaquinone biosynthesis genes were unable to induce growth of Porphyromonas sp KLE1280. Exogenously added menaquinone 4 (MK4) induces growth of KLE1280. Along with MK4, heme (naturally occurring or synthetic) is also required for its growth. Based on these results, whole genome sequencing was done by our collaborator George Weinstock's group at The Genome Institute at Washington University. It was confirmed that this isolate is indeed missing the menaquinone biosynthesis genes. It appears to be very specific in its requirement for MK4, as KLE1280 was not induced by any quinone except MK4 or an intermediate of the menaquinone pathway, 1,4--dihydroxy-2-naphthoquinoic acid (DHNA). Two other species of Porphyromonas were also dependent on MK4. We hypothesize that other uncultured bacteria might be deficient in the same or similar growth factors, and similar to the model organism studied here, could be very specific in their growth factor requirement. It is therefore necessary to identify more growth factors in order to cultivate more organisms from the human microbiome. Using this approach may allow us to isolate many more uncultured organisms.