Background/Purpose: The role of the microbiome in the pathogenesis of autoimmune disorders such as axial spondyloarthritis (SpA) has been well-established through means of describing gross microbial community dysbiosis between healthy controls. However specific mechanisms of how pathogenic bacteria cause disease progression in axial SpA have escaped current research. In this novel project we use flow cytometry to interrogate microbial composition using Quantitative Microbial Profiling (QMP), where we couple flow cytometric microbe counts with 16S rRNA sequencing. We compare QMP to the standard microbial analysis technique, Relative Microbial Profiling (RMP), that does not account for potential inter-sample differences in total microbial load. We also investigate the host-immune response to these microbial communities using the IgA-SEQ technique in which we flow sort and sequence IgA-coated bacteria.

Methods: Fecal samples of healthy controls (n = 23) and axial SpA patients (n = 24) were subjected to QMP through the combination of microbial flow cytometry and 16s rRNA sequencing. Antibiotic use was an exclusion criteria and 12/24 (50%) of the axial SpA patients had exposure to biologics for treatment. Saliva and feces of axial SpA and healthy control patients were subsequently subjected to IgA-SEQ (n = 12-13/group) in which IgA-coated bacteria are sorted and 16s rRNA sequenced to produce an IgA-coating index score for comparison of host immune response. This method identifies targets of the intestinal and oral IgA response in patients with axial SpA. The data was analyzed using the DADA2 pipeline implemented in R.

Results: For the first time, QMP methods revealed striking quantitative fecal microbial load differences between axial SpA patients and healthy controls, underpinned by a dramatic near log-fold decrease in total microbial concentration as compared to healthy controls (p < 0.01). Additionally, IgA-SEQ analysis demonstrated a significantly altered microbiota-specific IgA repertoire between axial SpA patients and healthy controls in both saliva and feces. Of note, some of the most significantly altered bacterial populations were from the Lachnospiraceae family, specifically from the genus Roseburia.

Conclusion: Through the use of QMP we identify a number of novel quantitative differences to the composition of the intestinal microbiota in axial SpA patients as compared to healthy controls. We also propose that patients with axial SpA have an altered host immune response to the oral and fecal microbiome that could play a critical role in disease pathogenesis and drive progression. Further research focused on immunogenic microbes, including L. roseburia could yield potential treatments to delay or stop disease progression.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-use-of-microbial-flow-cytometry-to-analyze-the-intestinal-and-oral-microbiota/