The role of bile-metabolising enzymes in the pathogenesis of Clostridium difficile infection, and the impact of faecal microbiota transplantation.

Our gut is full of billions of bacteria; whilst some may be harmful, many of these live there without problem, and actually perform important roles in keeping us healthy. Some of these 'good' gut bacteria in fact appear to act to stop other bacteria that could cause harmful gut infections from growing within the gut. Whilst antibiotics help us overcome chest, urine and other infections, doctors now realise that an unintended effect of their use is that they may also destroy some of the gut's 'good' bacteria, meaning that we lose the benefit of their protective roles. One example of this occurs in Clostridium difficile infection (CDI). Clostridium difficile is a form of bacteria that can grow within the human gut and cause disease ranging from mild diarrhoea up to severe bowel inflammation and even death. CDI is responsible for many hospital admissions and deaths worldwide every year. Whilst this infection rarely happens in healthy people, it occurs much more frequently in people who have had recent antibiotics. Doctors believe that this is because antibiotics destroy the 'good' bacteria in the gut that protect against CDI, and therefore allows Clostridium difficile bacteria to grow within the gut and cause disease. However, exactly which beneficial bacteria they destroy - and how these bacteria protect us normally - is not properly understood.

CDI is becoming more difficult to treat; the main reason for this is that the usual antibiotics used as treatment do not work as well as they used to. One unusual treatment that has been recently introduced is faecal microbiota transplantation (FMT), i.e. taking faeces from a healthy person (containing normal healthy gut bacteria), processing this in a laboratory to create a liquid suspension, and delivering this (via a tube up the nose and into the stomach, or via a colonoscopy) into the gut of people with CDI. Trials show that this appears to be a much more effective treatment for recurrent CDI than conventional antibiotic treatment. However, FMT is not without drawbacks; for instance, it may be unpleasant for a person with CDI to receive this, it can be difficult to administer, and there is a theoretical risk of transmitting infections from the donor to the recipient. Furthermore, exactly which 'good' bacteria in the transplant lead to treatment of CDI (and the means by which they do this) is still unknown.

We intend to identify which 'good' bacteria are killed by antibiotics with CDI; in addition, we will find which bacteria replaced into the gut by FMT cause people to get better from the infection, and how they do this. Recent research shows that certain components of bile (a liquid made by our livers and secreted into our guts) help Clostridium difficile grow under the microscope, whilst other components prevent it growing. Based on this, we suspect that FMT may work by replacing the gut bacteria that produce enzymes that alter the composition of bile (called bile salt hydrolases (BSH)). We think that FMT restoring BSH-producing bacteria may result in an increase in bile components that stop C. difficile growing, and reduction in those that help the bacteria divide.

To investigate this, we will take samples from healthy people and those with CDI (both pre- and post-FMT, both from people where FMT has worked and where it has not) to compare which bacteria and which bile components are present in the gut in these different situations, and to investigate how much BSH enzyme is present in all cases. We will then test adding bacteria that produce BSH to a simulated model of a gut suffering from CDI, to see if this is as effective as FMT, and also assess how these bacteria affect C difficile's survival. If our data support this hypothesis, we may in the future be able to move on from FMT and instead treat CDI (or people at risk of the condition) by giving a drink or pill specifically containing bacteria that produce BSH, or that just contain BSH alone.

Antibiotic use is the major risk factor for Clostridium difficile infection (CDI), although the exact pathogenesis of the condition is unclear. Conversely, whilst faecal microbiota transplantation (FMT) is an effective therapy for CDI, its mechanism of action is unexplained. In vitro, conjugated primary bile salts promote C. difficile's germination, whilst unconjugated secondary bile salts inhibit its vegetative growth. In vivo, bile salt hydrolases (BSHs) are enzymes secreted by selected bacteria in the healthy gut microbiota which deconjugate primary bile salts. This project hypothesises that antibiotic-related loss of BSH-secreting gut microbiota predisposes to CDI by enriching conjugated primary bile acids within the gut (and consequent germination and growth of C. difficile), and that FMT reverses this process.

We will firstly compare stool and urine samples collected from people treated successfully and unsuccessfully for CDI with FMT both pre- and post-transplant. Assays will be performed for gut microbial profiling/ metataxonomics (via 16S rRNA sequencing), bile acid/ general metabolite profiles (via mass spectrometry/ NMR), and for BSH expression and activity (via qPCR, and a plate-based functional assay respectively). Analyses of these data will identify culprit BSH-producing organisms responsible for restoration of normal bile acid metabolism post-FMT.

The next phase of work will be a functional analysis of these BSH-producing bacteria. Co-culture experiments between BSH-producing organisms and C. difficile spores (in the presence of a bile pool milieu similar to that found in vivo) will define the effect of these bacteria upon C. difficile's germination. Finally, CDI will be modelled with an in vitro distal gut system (chemostat); the 'real time' effect of addition of a defined microbial community of BSH-secreting organisms upon this system will be examined, exploring changes in microbial and bile acid profiles, and in C. difficile viability.

1. Academia:
The methods used to perform the study and the results it generates will be of direct interest to those interested in basic and clinical aspects of research into the gut microbiota and its manipulation, as well as those with an interest in novel treatments to tackle antimicrobial resistance, as already described.

2. Commercial:
i. Diagnostics: This study assesses gut microbiota structure/ metabolic function based on samples taken from people with Clostridium difficile infection (CDI) who have been successfully treated with faecal microbiota transplantation (FMT), and those who have not responded to FMT. Success of FMT may reflect patient factors or donor factors. This study may allow identification of a gut microbial/ metabolic 'signature' that predicts likelihood of response to FMT, and/ or allow a threshold gut BSH expression/ activity to be defined as a 'screening test' before acceptance as a stool donor. If so, we would aim to develop simplified assays to allow application on a larger scale. Working with Imperial Innovations, we would then look to protect the intellectual property and commercialise the tests using our extensive network of commercial diagnostics companies.

ii. Pharmaceutical: The major predicted outcome from this project is identification and in vitro demonstration of a defined microbial community of BSH-producing organisms that has the potential to serve as a novel, targeted therapy for CDI which could replace FMT in its current form. The expectation would be that this would be administered in the format of a capsule of lysophilised bacteria, or a tablet containing BSH. We would then liaise with pharmaceutical companies to aid formulation of such treatments, before seeking instigation of animal studies and/or clinical trials. Our expectation is that liaison with commercial partners may be able to start immediately after completion of this project. In addition to realising benefit for commercial partners, achieving this aim would represent a significant success for the host institution and the MRC.

3. Clinical benefit:
Given the concerns that exist regarding FMT (including unpalatability, an invasive means of administration, and potential infectious complications), refinement from FMT to a novel targeted therapy for CDI would clearly be of direct benefit to patients with CDI. It may also have broader benefits upon health systems - for instance, given the significant impact of CDI on hospital bed days and health expenditure within the UK, a novel therapy for CDI would also likely result in earlier hospital discharges and reduced health spending on CDI.

4. Public health and health policymakers:
One of the key clinical concerns about CDI is the increasing recognition of failure of metronidazole, tying in with the concerns more generally about increasing antimicrobial resistance within the UK and beyond. This has been made a high profile priority area for research by the Department of Health/ Public Health England and research charities including the MRC. It is hoped that the outcome from this project is a novel non-antibiotic therapy that treats a bacterial pathogen, which has no capacity for the development of antimicrobial resistance. If this outcome is met, then this would raise the profile of the host institution and the MRC, and would be of great interest internationally. This would influence public health policy and subsequent research spending, and could also serve as a launchpad for other studies on the theme of novel approaches to tackling antimicrobial resistance. For instance, a number of recent case studies have reported successful gut decontamination of multiple antibiotic resistant bacteria (including ESBL and CPE) through the use of FMT from donor without these bacteria within their native gut microbiota; this would be one obvious area in which further research of potential public health benefit could be pursued.