There are over 700 bacterial species in the oral cavity but over half of these cannot be cultivated in vitro and remain inaccessible for study. The principal goal of this research is to gain access to the """"""""uncultivable"""""""" missing species.
Aim 1 utilizes a fundamentally new approach to grow """"""""uncultivables"""""""" in their natural environment (Kaeberlein, T., Lewis, K., and Epstein, S.S. 2002. Isolating """"""""uncultivable"""""""" microorganisms in pure culture using a simulated natural environment (Science 296:1127-1129) combined with miniaturizing the growth devices to nanoscale. The nanochambers will be inoculated with single cells of target microorganisms and incubated in situ. The nanochambers are separated from the environment by membranes restricting movement of cells, but allowing for diffusion. This will provide the cells with the complete suite of growth components of their natural environment. This bypasses the limitations of standard cultivation techniques, and will allow growth and isolation in pure culture of presently uncultivated strains.
In Aim 2, we will focus on species implicated in oral diseases, and domesticate these species for growth in a standard Petri dish. We have previously successfully domesticated uncultured bacteria from environmental samples. The main outcomes of this project will be (1) a new method of cultivating """"""""uncultivable"""""""" representatives of human oral microflora, and (2) a collection of novel species potentially important to human health. The newly cultivated species can be readily characterized, i.e. biochemically and genetically (including whole genome sequencing). We will make this collection available to other researchers and will create a free web-based database characterizing this collection.
These aims will be achieved in cooperation between three academic labs, one clinical practitioner, and one industrial partner. This multidisciplinary project across five different fields will allow us to achieve the principal goal of the application: gaining access to clinically important members of human oral microflora. Uncultivable bacteria make up more than half of the human oral microflora among which new pathogens or symbionts are likely to be found. We will develop a novel method of isolation and characterization in pure cultures previously uncultivable oral bacteria. This will lead to identifying previously unknown microorganisms that impact human health, and will lay the basis for development of improved therapeutics against oral pathogens.
