Rossetti Blair J.

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Rossetti
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Blair J.
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  • Article
    Dynamics of tongue microbial communities with single-nucleotide resolution using oligotyping
    (Frontiers Media, 2014-11-07) Mark Welch, Jessica L. ; Utter, Daniel R. ; Rossetti, Blair J. ; Mark Welch, David B. ; Eren, A. Murat ; Borisy, Gary G.
    The human mouth is an excellent system to study the dynamics of microbial communities and their interactions with their host. We employed oligotyping to analyze, with single-nucleotide resolution, oral microbial 16S ribosomal RNA (rRNA) gene sequence data from a time course sampled from the tongue of two individuals, and we interpret our results in the context of oligotypes that we previously identified in the oral data from the Human Microbiome Project. Our previous work established that many of these oligotypes had dramatically different distributions between individuals and across oral habitats, suggesting that they represented functionally different organisms. Here we demonstrate the presence of a consistent tongue microbiome but with rapidly fluctuating proportions of the characteristic taxa. In some cases closely related oligotypes representing strains or variants within a single species displayed fluctuating relative abundances over time, while in other cases an initially dominant oligotype was replaced by another oligotype of the same species. We use this high temporal and taxonomic level of resolution to detect correlated changes in oligotype abundance that could indicate which taxa likely interact synergistically or occupy similar habitats, and which likely interact antagonistically or prefer distinct habitats. For example, we found a strong correlation in abundance over time between two oligotypes from different families of Gamma Proteobacteria, suggesting a close functional or ecological relationship between them. In summary, the tongue is colonized by a microbial community of moderate complexity whose proportional abundance fluctuates widely on time scales of days. The drivers and functional consequences of these community dynamics are not known, but we expect they will prove tractable to future, targeted studies employing taxonomically resolved analysis of high-throughput sequencing data sampled at appropriate temporal intervals and spatial scales.
  • Article
    Preservation of three-dimensional spatial structure in the gut microbiome
    (Public Library of Science, 2017-11-27) Hasegawa, Yuko ; Mark Welch, Jessica L. ; Rossetti, Blair J. ; Borisy, Gary G.
    Preservation of three-dimensional structure in the gut is necessary in order to analyze the spatial organization of the gut microbiota and gut luminal contents. In this study, we evaluated preparation methods for mouse gut with the goal of preserving micron-scale spatial structure while performing fluorescence imaging assays. Our evaluation of embedding methods showed that commonly used media such as Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin, and polyester waxes resulted in redistribution of luminal contents. By contrast, a hydrophilic methacrylate resin, Technovit H8100, preserved three-dimensional organization. Our mouse intestinal preparation protocol optimized using the Technovit H8100 embedding method was compatible with microbial fluorescence in situ hybridization (FISH) and other labeling techniques, including immunostaining and staining with both wheat germ agglutinin (WGA) and 4', 6-diamidino-2-phenylindole (DAPI). Mucus could be visualized whether the sample was fixed with paraformaldehyde (PFA) or with Carnoy’s fixative. The protocol optimized in this study enabled simultaneous visualization of micron-scale spatial patterns formed by microbial cells in the mouse intestines along with biogeographical landmarks such as host-derived mucus and food particles.