The Anaerobic Rose Chamber (ARC): Novel Applications for Probing Clostridioides Difficile Dynamics

Clostridioides difficile infection (CDI) is an urgent public health threat with a high rate of recurrence and limited treatment options. Caused by the anaerobic intestinal pathogen C. difficile, this disease affects up to 500,000 Americans annually. Despite progress in our understanding of CDI pathophysiology, the lack of effective and accessible experimental platforms compatible with obligate anaerobe physiology hinders the advancement of treatment therapy. The application of in vivo models has been indispensable in understanding CDI pathophysiology, probing the complexity of the host response to the microbe, and establishing treatment protocols, and it continues to be essential in preclinical testing. Nonetheless, constraints related to animal availability, cost, ethical considerations, and regulatory control limit accessibility for research. In contrast, anaerobic in vitro CDI models offer accessibility and control over experimental parameters, enabling the dynamic analysis of microbial behavior. However, these platforms lack host representation due to the mutually exclusive oxygen requirements of host and microbe environments. This effectively leaves the challenge of modeling early CDI unanswered and creates a substantial gap in the field. The present work demonstrates novel applications for the anaerobic rose chamber imaging method (ARCIM). The ARCIM provides gas- and liquid-tight insulation of the anaerobic environment, enclosing epithelial monolayers infected with live bacteria inside an optically clear container. The proposed host-pathogen adaptation of the platform enables real-time, single-cell fluorescent microscopy, invaluable for probing C. difficile dynamics. Validation of the approach begins with confirming the compatibility of the host model with ARCIM’s anaerobic conditions, assessed through measurements of electrical impedance. Next, C. difficile toxin-mediated breakdown of the host cytoskeleton is characterized with ARCIM, validating live fluorescent microscopy application in aerobe-anaerobe co-culture. Finally, a novel oxygen-independent, far-red C. difficile reporter is used to visualize the real-time formation of biofilm using the ARCIM. If successful, this work addresses major gaps in C. difficile research, including accessibility and real-time detection of live C. difficile dynamics at single-cell resolution.