Development of a high throughput comparative microbiomics platform applied to Clostdridium difficile-associated disease.

Cdiff is an abbreviation that has recently received much public attention as the bacterium it denotes is responsible for a hospital acquired infection that now rivals MRSA in terms of illness and deaths in the UK. The diarrhoeal disease due to Cdiff follows on from antibiotic treatment in which the large number of microbes normally resident in the human gut have been substantially disturbed. It is likely that many other human conditions are either caused by or affected by such disturbances. These collections of microbes normally resident in the gut have recently been referred to as the microbiome (the totality of microbes present at a particular location). It would be highly desirable to determine the composition of different microbiomes as this could enable us to understand and control the conditions that they influence. However, this is not possible by classical laboratory growth methods as many microbes simply cannot be cultured. DNA based analyses have recently surmounted this problem but these are largely confined to small scale studies applied to samples from a few individuals as they demand enormous financial and computing resources. To fully recognize the influence of different microbiomes on human health and disease we need to develop means of determining and comparing their compositions on a large scale involving samples from many individuals. We have established an economic high throughput approach that will provide microbiomic signatures from substantial sample sets. Through this project we aim to complete the development process and establish our approach for large scale application to clinical samples. Within the programme we will make initial analyses of the microbiome associated with Cdiff infection. These have the potential to identify organisms that may be necessary for humans to resist Cdiff infection and therefore a potential means of protection. A more certain outcome is the validation of an approach that will be a resource for all researchers wishing to make large scale microbiomic determinations and comparisons. Such analyses have clear potential to impact on Cdiff disease and many other infective and non-infective conditions.

Clostridium difficile associated diarrhoea (CDAD) is one of many human conditions in which the complex assembly of microorganisms at a particular location in the body appears to be a key determinant of susceptibility and robust response to treatment. Such assemblies or microbiomes occupy particular niches in the body and can be viewed as equivalent to accessory organs in the body with a remarkably diverse and powerful metabolic processing capacity. The advent of molecular techniques has rendered the hitherto insurmountable task of characterizing of microbiomes feasible. However, in human population studies, both the clone library sequencing and direct metagenomics approaches to achieving this are too resource and computing intensive to be applied to sample sets of sufficient size to achieve statistical validity. Here we aim to concert and validate our preliminary studies on a reverse microarray methodology based around arraying 16S rDNA clone libraries derived from clinical samples as probes and interrogating them with a set of tester oligonucleotides. In one step the approach achieves a comparison between the sample sets and provides an immediate readout of the taxa that are constant and distinct between the test groups. Moreover, the approach also reveals non-hybridising clones that may contain sequences from previously unknown organisms. In contrast to the established methodologies in which much resource is consumed by repetitive analysis of numerically dominant organisms, this approach allows sequencing to be focussed on key areas of the microbiome that reflect important constant and variable components relating to the human populations to be studied. We will synergistically combine our array strategy with state of the art pyro-sequencing to enable strategy development and more detailed microbiome determinations by refined iteration. Through this intensive technical development we will compare the faecal microbiomes of healthy individuals with those from patients with and recovered from CDAD and non CD antibiotic associated diarrhoea. While this small scale analysis has the potential to reveal major differences between these groups, our primary aim is to establish and validate an array platform for major clinical studies in this area. This outcome will not only contribute to our understanding of CDAD but will also provide a generic tool for microbiomic studies within and beyond biomedical applications.