Application of Anaerobic Fluorescence Activated Cell Sorting to Isolate and Cultivate Inflammatory Bowel Disease Associated Gut Microbes

Abstract

The gut microbiome has been an area of intense study over the last decade and numerous studies have highlighted it as an important environmental factor that influences both health and disease. The microbiome has been implicated in the etiology of chronic inflammatory conditions such as ulcerative colitis and Crohn's disease, collectively referred to as inflammatory bowel disease (IBD). Examination of IgA coated bacteria in IBD patients has led to the identification of specific bacteria that are involved in disease progression. Techniques to isolate and culture viable bacteria anaerobic bacteria from the gut microbiome based on surface phenotype, such as high levels of IgA, are extremely limited. The integration of a fluorescence activate cell sorting (FACS) instrument within an anaerobic culture environment was explored in this study to determine the feasibility of directly isolating IgA+ bacteria from IBD patients. Construction of a custom anaerobic FACS sorting platform enabled the technical development of methods to isolate viable IgA+ and IgA- obligate anaerobes from human fecal material for cultivation. These methods were deployed to generate a IBD patient IgA coated bacteria culture library containing over 2000 isolates, representing 147 unique species. This collection of IBD patient bacteria can now be phenotypically characterized in vitro and in vivo to understand their contributions to IBD pathophysiology.