Bacterial diversity, viability and stability in lyophilised faecal microbiota capsules support ongoing clinical use

Lyophilised encapsulated faecal microbiota provides a practical and cost-effective treatment option to patients with recurrent Clostridioides difficile infection. This study focused on quality assurance of an enteric-coated capsule formulation of FMT as a medicinal product by evaluating bacterial composition, diversity and viability through manufacturing steps and upon product storage at a range of temperatures. Faecal donations from pre-screened healthy individuals (n = 5) were processed according to a published protocol into one or more treatments; 5 capsules = 1 treatment dose/patient. Culture-independent next-generation 16S rRNA gene sequencing was used to speciate and quantify bacteria using a live-dead cell separation method to discriminate the viable cell load. Species diversity in donor stools aligned with other healthy gut microbiome and remained unchanged through the manufacturing process and after storage at -80 °C for 36 weeks. While diversity indices were consistent, a notable difference was observed between viable and total microbiome, particularly in species richness, which decreased when non-viable or compromised cells were excluded from analysis. Anaerobic species exhibited minimal viability loss despite processing in an aerobic environment. Furthermore, capsules were stable with storage at -20 °C and 2-8 °C, with no significant reduction of total live bacterial load after 24 weeks. In summary, 'live-dead' culture-independent analysis was used to characterise the viable faecal microbiome, which retained a diversity of bacterial species, including anaerobes, through manufacture and after storage in capsules for up to 36 weeks. These data support the comparable effectiveness of lyophilised encapsulated FMT to other formulations and delivery methods.