Bacterial and microeukaryotic succession in the gut microbiome during infection by Clostridium difficile

Characterizing the bacterial and eukaryotic members of the microbiome and their dynamic response to stimuli is critical to understanding their role in health and disease. We designed and validated novel primers for the selective amplification of microeukaryotic 18S rDNA for further usage in parallel with 16S bacterial community characterization by 454 pyrosequencing. The primer pair 18S-0067a-deg-5\u27/Euk_NSR399 is capable of amplifying microeukaryote 18S rDNA while excluding mammal and plant DNA and therefore is suitable for deep sequencing such as by 454 pyrosequencing. We went on study succession in the hamster gut after treatment with antibiotics and exposure to Clostridium difficile. C. difficile infection is typically lethal in hamsters, but protection can be conferred with neutralizing antibodies against the A and B toxins. We compare treatment with neutralizing monoclonal antibodies (mAb) to treatment with vancomycin, which prolongs the lives of animals but ultimately fails to protect them from death. We carried out longitudinal deep sequencing analysis and found distinctive waves of succession associated with each form of treatment. Clindamycin sensitization prior to infection was associated with the temporary suppression of the previously dominant Bacteroidales and the Oxymonad Saccinobaculus in favor of Proteobacteria. In mAb-treated animals, C. difficile proliferated before joining Proteobacteria in giving way to re-expanding Bacteroidales and the fungus Wickerhamomyces. However, the Bacteroidales lineages returning by day seven were different from those that were present initially, and they persisted for the duration of the experiment. Animals treated with vancomycin showed a different set of late-stage lineages that were dominated by Proteobacteria as well as increased disparity between the tissue-associated and luminal cecal communities. The untreated control animals showed no change in their gut microbiota. These data thus demonstrate different patterns of ecological succession following antibiotic treatment and C. difficile infection