The role of bile-metabolising enzymes in the pathogenesis of Clostridioides difficile infection, and the impact of faecal microbiota transplantation
File(s)
Author(s)
Mullish, Benjamin H
Type
Thesis or dissertation
Abstract
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.
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.
Version
Open Access
Editor(s)
Marchesi, Julian R
Thursz, Mark R
Williams, Horace RT
Date Issued
2019-09-01
Date Awarded
2019-09
Citation
2019
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Marchesi, Julian
Thursz, Mark
Williams, Horace
Sponsor
Medical Research Council (Great Britain)
National Institute for Health Research (Great Britain)
Imperial College London
Identifier
https://spiral.imperial.ac.uk:8443/handle/10044/1/73899
Grant Number
MRC grant reference: MR/R000875/1
Publisher Department
Department of Surgery and Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)