Optimisation of a propidium monoazide based method to determine the viability of microbes in faecal slurries for...

The efficacy of faecal microbiota transplantation (FMT) as a therapeutic intervention may depend on the viability of the microorganisms in faecal slurries (FS) prepared from donor stool. However, determining the viability of these organisms is challenging. Most microorganisms in stool are refractory to culture using standard techniques, and culture-independent PCR-based methods derive signal from both viable and non-viable cells. Propidium monoazide (PMA) treatment has been shown to be effective in preventing PCR amplification of DNA from non-viable bacteria in a range of contexts. However, this methodology can be sensitive to factors such as bacterial load and sample turbidity. We describe the optimisation of a PMA treatment methodology for FS that restricts quantitative PCR-based bacterial enumeration to viable cells. When applied to concentrated FS (10-25% stool content), PMA treatment at 100 μM concentration was ineffective in preventing DNA amplification from heat-killed cells. Efficacy was not significantly improved by doubling the PMA concentration. However, PMA treatment efficacy was improved markedly following 10-fold sample dilution, and was found to be optimal at 100-fold dilution. Substantial reductions in viable bacterial load could be observed following both freeze-thaw and heat-treatment of FS. This method successfully prevented DNA amplification of heat-killed Pseudomonas and Staphylococcus spiked into stool and could reliably determine the proportion of live bacteria and viable E. coli counts present in fresh and heat-treated stool. With appropriate sample dilution, PMA treatment excluded >97% of non-viable cells from amplification in all assays, without significantly affecting the amplification of DNA from viable cells. This method can be applied to optimise sample processing of FMT donor material, and to characterise bacterial viability within faecal samples more widely.