Date Published: January 26, 2017
Publisher: Public Library of Science
Author(s): Nathaniel D. Chu, Mark B. Smith, Allison R. Perrotta, Zain Kassam, Eric J. Alm, Erwin G Zoetendal.
Fecal microbiota transplantation is a compelling treatment for recurrent Clostridium difficile infections, with potential applications against other diseases associated with changes in gut microbiota. But variability in fecal bacterial communities—believed to be the therapeutic agent—can complicate or undermine treatment efficacy. To understand the effects of transplant preparation methods on living fecal microbial communities, we applied a DNA-sequencing method (PMA-seq) that uses propidium monoazide (PMA) to differentiate between living and dead fecal microbes, and we created an analysis pipeline to identify individual bacteria that change in abundance between samples. We found that oxygen exposure degraded fecal bacterial communities, whereas freeze-thaw cycles and lag time between donor defecation and transplant preparation had much smaller effects. Notably, the abundance of Faecalibacterium prausnitzii—an anti-inflammatory commensal bacterium whose absence is linked to inflammatory bowel disease—decreased with oxygen exposure. Our results indicate that some current practices for preparing microbiota transplant material adversely affect living fecal microbial content and highlight PMA-seq as a valuable tool to inform best practices and evaluate the suitability of clinical fecal material.
Fecal microbiota transplantation—the transfer of fecal microbes from a donor to a patient—has emerged as an extremely effective therapy for recurrent infections of Clostridium difficile (~90% cure rate), a common hospital infection that kills nearly 30,000 patients each year [1,2]. Fecal transplants also hold promise for treating other gastrointestinal diseases, like inflammatory bowel disease and irritable bowel syndrome, and even systemic diseases linked to the gut microbiota, like obesity . To date, however, fecal transplants have been proven effective only for recurrent C. difficile infections, and clinical trials using fecal microbiota transplants to treat inflammatory bowel disease [4–9], irritable bowel syndrome [10,11], and insulin resistance  have produced mixed results.
To understand how transplant handling might alter fecal microbial communities—which may affect therapeutic efficacy—we investigated three potential sources of degradation: oxygen exposure during homogenization, freeze-thaw cycles during transplant storage and transport, and lag time between defecation and transplant preparation. For each experiment, we prepared two separate stool samples from the same donor and divided each sample into subsamples for analysis under different transplant preparation methods, thus controlling for variance across fecal samples. After transplant preparation, we then further divided each subsample into three technical replicates. We used qPCR to estimate total 16S rRNA abundance. We then evaluated the replicates’ resulting microbial composition using standard 16S rRNA sequencing [21,22] and PMA-seq, which selectively sequences DNA from bacteria with intact cell membranes—a proxy for living cells [23–25].
Although previous studies have evaluated the effects of various collection and storage methods on 16S rRNA extraction [34–37], to our knowledge, ours is the first analysis of how these variables alter the representation of living bacteria. Our results confirm a common assumption that oxygen exposure compromises the composition of viable fecal microbes [3,38,39]; they also support the notion that moderate numbers of freeze-thaw cycles and lag-time may only minimally damage the microbial community. Oxygen exposure disproportionately affected bacteria from the phylum Firmicutes—particularly the genera Megamonas and Faecalibacterium—while bacteria from the genus Bacteroides appeared to be more oxygen-tolerant (Fig 2). Members of all three genera are generally thought to be strictly anaerobic [28,32,40], suggesting that information on growth conditions alone may be insufficient to predict how bacterial taxa in complex communities will respond to stress.