Understanding the Molecular Basis of Virulence in Clostridium difficile.

There are currently heightened public concerns over infection rates in UK hospitals, and in particular those caused by so called ?superbugs? that have become resistant to available antibiotics. One such bug is Clostridium difficile. It causes debilitating diarrhoea, which in extreme cases can kill. It mainly affects the elderly. As this proportion of the population is increasing, the disease is becoming more common. Worryingly, a new, even more deadly variant has now arrived in Europe from North America. Aside from the human suffering, it costs the NHS over #402 million per year, and now is responsible for more deaths per year than MRSA. To control infections, we need to understand how an organism causes disease. Under Wellcome Trust sponsorship, the complete genome sequence of the organism (ie., its genetic blueprint) has been determined. However, whilst we now know the sequences of every gene in the C. difficile chromosome, we do not understand what they are doing. The best way of working out what genes do, is to mutate them (make them non-functional) and assess the consequences. Until now this has not been possible. We have now developed the tools needed, and wish to use them to better understand how this bug causes disease. This should eventually lead to better ways of controlling the disease.

Clostridium difficile-associated diarrhoea (CDAD) has become the most frequent nosocomial infection in many European hospitals, causing twice as many deaths in the UK during 2003 than MRSA. Its impact is considerable, both in terms of human suffering and costs of disease management. Taking the latest incidence figures (51,690 in 2005), it can be estimated to be costing England & Wales over #400 million per annum. A number of factors have contributed to this escalation. The proportion of the population in the main high-risk group, the elderly, is rapidly rising (over 65?s will double by 2050). More worrying has been the steady increase in strains exhibiting increased virulence. In 2004, the situation was exacerbated by the arrival in Europe of a new hypervirulent strain (ribotype 027) from N. America, where it has caused a large increase in CDAD incidence and associated deaths. In Stoke Mandeville Hospital, where first reported in the UK, two outbreaks of C difficile have affected 334 patients, killing at least 33. The inexorable rise in C. difficile incidence shows no sign of abating. It is imperative that more effect countermeasures are devised. However, pathogenesis is extremely poorly understood. As a result of our efforts, all of the elements needed to make significant progress in better understanding virulence in C. difficile are now in place. Thus, we have developed: (i) reproducible methods for the introduction DNA into C. difficile; (ii) a universal, clostridial, gene knock-out system, the ClosTron, which allows, for the first time, the rapid generation of stable, directed genes knock-outs, and; (iii) a transposon mutagenesis system with which random mutants may, for the first time, be readily generated. Moreover, at the projects initiation we will have in place: (iv) the animal model; (v) a new microarray for studies with both strain 630 AND the 027 strain, and; (vi) the genome sequences of CD 630 and a 027 strain. The overall aim of this project is, therefore, to capitalise on our unique expertise and resource to embark on a programme of work designed to unequivocally identify those C. difficile factors that are important in disease and to begin to determine both how they are regulated and how they contribute to virulence. Our programme of mutagenesis will lead to the identification of those factors (structural and regulatory) that are most important in disease, allowing a subsequent more in depth analysis of their role in pathogenesis to be initiated.