Research

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Gut microbiota plays an integral role in the maintenance of gut health and bolstering resistance against pathogens. Antibiotic use often damages gut microbiota and makes the host susceptible to infections, such as Clostridioides difficile infection (CDI). Paradoxically, CDI is treated with antibiotics and consequently, CDI recurrence is a major clinical problem. Up to 35% of patients develop recurrent CDI (rCDI) which could be lethal. Fecal microbiota transplant (FMT) is a highly effective treatment (>90% efficacy) for recurrent CDI (rCDI). However, specific species and their functions contributing to the efficacy of FMT are poorly understood. Moreover, there are reports of transmission of undetected pathogens from donor to receipt during FMT. Thus, there is an urgent and critical need to identify key microbes involved in CDI resistance, leading to the creation of defined and well characterized treatments.

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Gut microbiota structure and function is highly complex and is difficult to recapitulate in-vitro, thus requiring the use of live animals such as mice. Ex-germ-free (ex-GF) mice are frequently used as a model to study C. difficile pathogenesis and to identify key microbes involved in CDI resistance. These studies involve enumeration of C. difficile in fecal samples over the course of CDI in ex-GF mice. The suspension of mouse fecal material has suspended undissolved feces particles that interfere with subsequent procedures. Therefore, mouse fecal suspensions are subjected to very low speed (500g) centrifugation to remove particulate matter. In our preliminary studies, we discovered that low speed centrifugation results in pelleting of C. difficile, leading to 10-100-fold underestimation of C. difficile counts. Based on this observation we hypothesized that in mouse fecal suspensions, C. difficile cells aggregate or adhere to undissolved fecal particles and consequently pellet during low-speed centrifugation. Our preliminary data also revealed the presence of bacterial clumps in the fecal suspensions. In order to develop a better method for preparation of mouse fecal suspensions, we propose the following aims:

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Aim 1: To optimize a method to disrupt bacterial clumping. We will study the impact of various vortexing times and bead homogenization on C. difficile counts. We are aiming to disrupt the clumps without affecting the viability of the bacterial cells.

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Aim 2: To determine the effect of low speed centrifugation on commensal gut microbiota. We aim to determine the impact of low speed centrifugation on sedimentation of commensal gut bacteria and to develop a method to obtain single cell suspension of viable gut bacteria.

Significance: The long-term objective of our lab is to identify and culture gut microbes key to C. difficile resistance. This study will lead to development of an optimized method to accurately enumerate C. difficile in fecal samples of ex-GF mice colonized with defined microbes and identify gut bacteria that provide C. difficile resistance. A single cell suspension of viable gut bacteria is necessary to culture commensal bacteria. An optimized method to prepare fecal suspensions not only helps ongoing lab projects, but also the broader scientific community studying gut microbiota.

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Investigator: Sukhman Sidhu has been volunteering in Dr. Wang’s lab since July 2022.​