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

Nur Masirah Zain; Blair Merrick; Tom Martin-Lilley; Lindsey Ann Edwards; Daniëlle ter Linden; Sophia Tsoka; James Mason; Grace Hatton; Elizabeth Allen; Paul Royall; Andy Lilley; Kenneth Bruce; Debbie Shawcross; Simon Goldenberg; Ben Forbes

doi:10.1016/j.ijpharm.2025.125703

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

Nur Masirah Zain, Blair Merrick, Tom Martin-Lilley, Lindsey Ann Edwards, Daniëlle ter Linden, Sophia Tsoka, James Mason, Grace Hatton, Elizabeth Allen, Paul Royall, Andy Lilley, Kenneth Bruce, Debbie Shawcross, Simon Goldenberg, Ben Forbes^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Article number	125703
Journal	INTERNATIONAL JOURNAL OF PHARMACEUTICS
Volume	678
DOIs	https://doi.org/10.1016/j.ijpharm.2025.125703
Publication status	Published - 10 Jun 2025

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Zain, N. M., Merrick, B., Martin-Lilley, T., Edwards, L. A., ter Linden, D., Tsoka, S., Mason, J., Hatton, G., Allen, E., Royall, P., Lilley, A., Bruce, K., Shawcross, D., Goldenberg, S., & Forbes, B. (2025). Bacterial diversity, viability and stability in lyophilised faecal microbiota capsules support ongoing clinical use. INTERNATIONAL JOURNAL OF PHARMACEUTICS, 678, Article 125703. https://doi.org/10.1016/j.ijpharm.2025.125703

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

abstract = "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, {\textquoteleft}live-dead{\textquoteright} 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.",

author = "Zain, {Nur Masirah} and Blair Merrick and Tom Martin-Lilley and Edwards, {Lindsey Ann} and {ter Linden}, Dani{\"e}lle and Sophia Tsoka and James Mason and Grace Hatton and Elizabeth Allen and Paul Royall and Andy Lilley and Kenneth Bruce and Debbie Shawcross and Simon Goldenberg and Ben Forbes",

note = "Publisher Copyright: {\textcopyright} 2025",

year = "2025",

month = jun,

day = "10",

doi = "10.1016/j.ijpharm.2025.125703",

language = "English",

volume = "678",

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Zain, NM, Merrick, B, Martin-Lilley, T , Edwards, LA, ter Linden, D, Tsoka, S , Mason, J, Hatton, G, Allen, E, Royall, P , Lilley, A , Bruce, K , Shawcross, D , Goldenberg, S & Forbes, B 2025, 'Bacterial diversity, viability and stability in lyophilised faecal microbiota capsules support ongoing clinical use', INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 678, 125703. https://doi.org/10.1016/j.ijpharm.2025.125703

TY - JOUR

T1 - Bacterial diversity, viability and stability in lyophilised faecal microbiota capsules support ongoing clinical use

AU - Zain, Nur Masirah

AU - Merrick, Blair

AU - Martin-Lilley, Tom

AU - Edwards, Lindsey Ann

AU - ter Linden, Daniëlle

AU - Tsoka, Sophia

AU - Mason, James

AU - Hatton, Grace

AU - Allen, Elizabeth

AU - Royall, Paul

AU - Lilley, Andy

AU - Bruce, Kenneth

AU - Shawcross, Debbie

AU - Goldenberg, Simon

AU - Forbes, Ben

PY - 2025/6/10

Y1 - 2025/6/10

N2 - 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.

AB - 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.

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U2 - 10.1016/j.ijpharm.2025.125703

DO - 10.1016/j.ijpharm.2025.125703

M3 - Article

SN - 0378-5173

VL - 678

JO - INTERNATIONAL JOURNAL OF PHARMACEUTICS

JF - INTERNATIONAL JOURNAL OF PHARMACEUTICS

M1 - 125703

ER -

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

Abstract

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