The interactions between Clostridium difficile, intestinal microbiota and the host response in hospitalised patients

Clostridium difficile is a bacterium that causes chronic diarrhoea and sometimes life-threatening disease (referred to as C. difficile infection or CDI) mainly in elderly and hospitalized patients. C. difficile is now the most significant hospital acquired infection with over 34,000 UK cases last year and an estimated cost to be £500 million per annum. The reported incidence of CDI has risen dramatically over the last decade and is related to the emergence of aggressive strains. Given the ability of C. difficile to evolve to be highly virulent coupled with the ageing population and increasing rates of hospitalization, the problem of CDI is likely to continue. It is a current NHS imperative to reduce the burden of CDI.

CDI is unique among infections in that in almost all cases, disease follows the administration of antibiotics that disrupt the "normal" bacteria (called microbiota) present in the gut. It provides C. difficile the opportunity to overgrow, produce toxins and cause disease. It has become apparent that the balance of the microbiota plays a key role in disease. One of the most perplexing questions concerning CDI is the diversity of clinical symptoms. For example, C. difficile colonization can result in a spectrum of clinical outcomes ranging from asymptomatic carriage to death. The relative virulence of C. difficile organism clearly plays a role in determining clinical outcome, but the nature of this role has not been identified. Up to 30% of patients treated successfully for symptomatic CDI succumb to recurrent disease, but it is unclear why only some patients relapse. In addition to the pathogen, the host immune response and/or host microbiota are also likely important determinants of clinical outcome, but remain largely unexplored. Our central hypothesis is that variations in C. difficile genetics, the composition of the resident intestinal microbiota and the host immune response to the bacterium shape the clinical outcome.

We will investigate the three-way interaction between pathogen, host and microbiota by monitoring a unique group of patients with and without CDI. C. difficile serum and faecal samples will be studied in unprecedented detail to provide a platform for follow up studies to understand how C. difficile causes disease. We have established a high quality prospectively collected bioarchive comprising bacterial, blood and serum samples from 300 patients with CDI and full clinical history. These samples are linked to detailed clinical data and a disease severity-scoring index along with 150 control samples from hospitalised patients who have been exposed to antibiotics but did not develop CDI. We will use the existing cohort to validate methodologies and then also recruit another cohort of 150 confirmed CDI cases, 100 diarrhoea controls and 100 healthy volunteers (e.g. patient's spouses) to confirm our findings. This unique resource will be used to monitor the host response (including how the immune system responds to the infection), determine the complete genome sequence of infecting C. difficile and identify the intestinal microbiota by sequencing and culturing. The studies will also allow us to distinguish between re-infection (infection caused by another strain) and relapse (re-infection caused by the same strain).

Robust statistical analysis comparing disease severity, host response, infecting pathogen and the composition of intestinal microbiota will facilitate subsequent studies on C. difficile to determine how and why it causes disease. This will be achieved by selecting C. difficile genes and constructing defined mutants and studying these alongside the parent strain in a range of assays established in the applicants' laboratories.

A comprehensive understanding of C. difficile, the host response and the intestinal microbiota will strengthen our understanding of the microorganism and to develop novel strategies to reduce the burden of CDI.

Reduction in C. difficile infection (CDI) related morbidity and mortality is a current healthcare imperative. To develop appropriate sustained interventions, a holistic integrated view of the pathogen, the host response and the intestinal microbiota during infection is required. Our central hypothesis is that variation in C. difficile genetics, the composition of the resident intestinal microbiota and the repertoire of host factors, including the humoral immune response, shape the clinical outcome of CDI. To this end we will evaluate in unprecendented detail the pathogen genome sequence, intestinal microbial composition and the host response in a case-control study of CDI hospitalised patients. Rigorous statistical comparisons of data from the organism, intestinal microbiota and host response in relation to a patient disease severity index will enable selected genes/proteins to be chosen for functional analysis. To determine their role, defined/complemented mutants and wild type strains will be compared in several relevant in vivo-relevant assays. Potential biomarkers of disease severity will be assessed based on the combined clinical and molecular profile. Additionally, we will identify key bacterial groups important in re-establishing healthy microbiota during infection. Such analysis will investigate the role of the microbiota in providing colonisation resistance against CDI and will give new information on the bacterial species important in the re-establishment of healthy microbiota after infection, with applications in the rational design of microbial replacement therapy. This integrated clinical case control study will provide for the first time a comprehensive comparison of the pathogen, host response and the role of the intestinal microbiota in CDI that will afford new clues to pathogenesis with practical implications in terms of diagnosis and therapeutic interventions.

C. difficile is now known to be the most frequent cause of hospital acquired infections in developed countries. Given the continued use of broad-spectrum antibiotics, other drugs and the rising numbers of immuno-compromised and elderly patients, the problems associated with CDI will remain a challenge to healthcare delivery for the foreseeable future. To date, most research on C. difficile has been piecemeal and has not taken into account the role of the microbiota and the host. This study will uniquely follow infection in a cohort of patients and be compared to appropriate controls. This multidisciplinary research programme fits squarely into several priority research areas identified by the MRC, in terms of reducing the burden of hospital acquired infections, health in the ageing population, the application of genomics, to the identification of biomarkers, drug and vaccine candidate and the design of novel treatments.

Given the mortality and morbidity caused by CDI and the enormous cost to the UK economy (estimated to be £500 million per annum), the benefits of improved methods to detect, prevent and understand CDI to health and wealth of society are self-evident. The commercial private sector will potentially benefit from the research in several areas. Specific examples include the investigation of the role of the healthy microbiota in providing colonisation resistance against CDI and will give new information on the bacterial species important in the re-establishment of a healthy microbiota after infection, with applications in the rational design of microbial replacement therapy. The identification of immunodominant antigens from samples of patient serum with the pan-C.difficile protein microarray may identify useful immunodiagnostic markers and provide information on potential vaccine candidates. A deeper understanding of the factors important during relapse may provide clues to novel strategies to curtail the effect of CDI. Given the healthcare issues surrounding hospital-acquired infections and the human microbiota, the project will also engage the public

Additionally, through these studies and our deeper understanding of CDI and recurrent disease, this is likely to influence policy makers and regulators as to the optimum treatment for CDI. C. difficile and CDI are very much in the public and political eye, the co-ordinated program of work described is unique and promises to break new ground in both basic and translational research. The research program should give the UK a significant boost in this important and topical health care issue and will contribute significantly to the UK science base.