Fecal microbiota transplantation is a compelling treatment for recurrent Clostridium difficile infections, with promising applications in other diseases. But fecal processing methods that are effective for C. difficile infections may not translate to other conditions. Investigating potential uses of fecal transplants beyond C. difficile infections requires a tool that quantifies the effects of processing methods on different microbial species.
Standard molecular tools that profile microbial communities do not discriminate between living and dead microbes. To address this problem, we used a DNA-sequencing method (PMA-seq) that uses propodium monoazide (PMA) to differentiate between living and dead microbes in fecal samples, providing a species-specific view of processing effects on the gut microbial community.
The chemical PMA binds to DNA, preventing amplification by polymerase chain reaction. Because PMA cannot pass through intact cell membranes, however, it does not bind DNA within living cells. Thus, amplification of DNA from a PMA-treated sample amplifies DNA only from living cells. We treated fecal microbiota samples with PMA and profiled the microbial communities using 16S rRNA sequencing to evaluate the effects of oxygen exposure and freeze-thaw cycles during processing.
PMA-seq revealed that oxygen exposure degrades the microbial community (Figure 1), particularly, obligate anaerobic bacteria. Notably, abundance of Faecalibacterium prausnitzii---an anti-inflammatory commensal bacterium that moderates inflammatory bowel disease---decreased greatly with oxygen exposure. Freeze-thaw cycles, in contrast, only moderately change the microbial composition of fecal samples (Figure 2).
Our results suggest that PMA-seq is an effective tool for identifying living fecal microbes and that current practices adversely affect the microbial content of fecal samples. Reduction in anti-inflammatory bacteria may be a factor in what has been modest efficacy of fecal transplants in treating inflammatory diseases. PMA-seq could serve as a valuable tool to inform best practices and, eventually, as a screen to evaluate the suitability of clinical fecal material.
A. Perrotta, None
Z. Kassam, OpenBiome: Employee , Salary
E. Alm, OpenBiome: Board Member , None