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Title: The role of bile-metabolising enzymes in the pathogenesis of Clostridioides difficile infection, and the impact of faecal microbiota transplantation
Author: Mullish, Benjamin Harvey
ISNI:       0000 0004 8499 5358
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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The pathogenesis of Clostridioides difficile infection (CDI), and mechanisms of efficacy of faecal microbiota transplant (FMT) in treating recurrent CDI (rCDI), remain poorly-understood. Certain bile acids affect the ability of C. difficile to undergo germination or vegetative growth. Loss of gut microbiota-derived bile-metabolising enzymes may predispose to CDI via perturbation of bile metabolism, and restitution of gut bile-metabolising functionality could mediate FMT's efficacy. Initially, human samples were analysed, i.e.: 1) biofluids collected from rCDI patients pre- and post-FMT (and their donors), and 2) stool samples from primary CDI patients, including both recurrers and non-recurrers. Analysis included: 16S rRNA gene sequencing; liquid chromatography-mass spectrometry for bile acid profiling; gas chromatography-mass spectrometry for short chain fatty acid (SCFA) quantification; bile salt hydrolase (BSH) enzyme activity; and qPCR of bsh/ baiCD genes involved in bile metabolism. Human results were validated in C. difficile batch cultures and a rCDI mouse model. A reduced proportion of the stool microbiota of rCDI patients pre-FMT contained BSH-producing bacteria compared to donors or post-FMT. Pre-FMT stool was enriched in taurocholic acid (TCA; a potent trigger to C. difficile germination); TCA levels negatively correlated with bacterial genera containing BSH-producing organisms. Post-FMT stool demonstrated recovered BSH activity and microbial bsh/ baiCD gene copy number compared with pre-treatment (p < 0.05), and recovery of SCFA including valerate (p < 0.001). Dynamics of stool bile acids/ BSH activity differed in primary CDI patients with and without disease recurrence. In batch cultures, culture supernatant from engineered bsh-expressing E. coli reduced TCA-mediated C. difficile germination relative to supernatant from BSH-negative E. coli. C. difficile total viable counts were ~70% reduced in a rCDI mouse model after administration of BSH-expressing E. coli relative to mice receiving BSH-negative E. coli (p < 0.05). These data demonstrate that gut microbiota BSH functionality is a key mechanism influencing vulnerability to CDI and efficacy of FMT.
Supervisor: Marchesi, Julian ; Thursz, Mark ; Williams, Horace Sponsor: Medical Research Council ; National Institute for Health Research ; Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral