The objective of this proposal is to develop a novel combinatorial labeling and detection technology for imaging and distinguishing between most if not all of the members of the human oral microbiological community. Fluorescence In Situ Hybridization (FISH) for 16S rRNA sequences will visually identify individual members of the microbial community either as collected or in situ. The capabilities of FISH will be extended by employing a combination of fluorescent reporter groups to create """"""""spectral signatures"""""""" that allow simultaneous encoding and imaging of tens to hundreds of different microbial phylotypes. Enhanced, state-of-the-art spectral imaging techniques will record the spectral signatures. Novel algorithms will be developed to deconstruct spectral signatures of labeled microbes. This new imaging capability will allow a comprehensive census of phylotypes in the human oral microbial community as well as determination of the spatial organization of this community. In principle, the combinatorial labeling strategy could be extended by simultaneously labeling microbes with probes against 16S rRNA for phylogenetic identification and probes against mRNA or proteins for metabolic or structural information. The capability to assess the relative abundance, organization and function of particular microbes will open new avenues for comprehensive and detailed study of the ecology of the oral microbial community. Oral microbes are implicated in diseases such as caries, gingivitis, and periodontitis, which are among the most common bacterial infections in humans, as well as systemic diseases including cardiovascular disease, pulmonary infections, and low birth-weight and pre-term pregnancy outcome. No single microbial species is implicated as the etiological agent of these diseases, rather, it is hypothesized that the complex association of multiple microbial phylotypes in communities are responsible for maintaining health and causing disease. Development of the technology proposed here will allow a comprehensive understanding of the normal human oral flora and foster the development of new and effective strategies to study, monitor, and control infection. ? ? ?
|Valm, Alex M; Mark Welch, Jessica L; Borisy, Gary G (2012) CLASI-FISH: principles of combinatorial labeling and spectral imaging. Syst Appl Microbiol 35:496-502|
|Valm, Alex M; Mark Welch, Jessica L; Rieken, Christopher W et al. (2011) Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging. Proc Natl Acad Sci U S A 108:4152-7|