Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Schinagl, B.
Molecular analysis of oral biofilm
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Graz Medical University; 2019. pp.
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Santigli Elisabeth
- Wimmer Gernot
- Altmetrics:
- Abstract:
- Aim: The aim of this study was the analysis of the native oral microbiota using different molecular methods in two setups. Materials und Methods: The first part of this thesis concentrated on the establishment of a method for the FISH staining and CLSM analysis of oral microbiota grown on orthodontic appliances. In the second part a certain dental splint for research was developed. It contained standardized human enamel-dentin slabs as a surface to grow biofilm intraorally on. These slabs could then be clipped out and transferred into an in vitro test-system. Microbiota survival and compositional changes over time were then tested in a biofilm reactor. In parallel two temperature loggers were embedded in these research splints to measure oral temperatures over 48 hours. The microbiome composition was subsequently analyzed with 454-pyrosequencing and data explored with QIIME and R. Live/dead staining and CLSM were used to monitor bacterial survival in vitro and stack data evaluated with a self-written Matlab®-script and Python. FISH was used for the visualization of bacteria down to genus level. Results: As a first result a method for the FISH staining and CLSM analysis of biofilm on orthodontic appliances was developed. Secondly, a research splint was created that allows intraoral growth of native microbiota on human enamel-dentin slabs. They can subsequently be transferred to in vitro test systems. Microbiota on top of the slabs is kept alive in a stable composition on phylum level over 48 hours. On genus level significant growth was found in Rothia, Prevotella, Granulicatella und Haemophilus. The live/dead ratio approximated initial values after 48 hours in vitro. Discussion: Both test systems allowed the analysis of native oral microbiota. The developed research splint extends conventional splints through the incorporation of sensors. They allow for intraoral growth of biofilm on human enamel-dentin slabs and their transfer to in vitro systems in a pristine condition. Strong temperature oscillations found during the day should be respected in further microbiome models.