Defining the physiology of E. coli O157:H7 in cattle to develop phage-based interventions

Escherichia coli O157:H7 (O157) is a zoonotic bacterial pathogen which colonises cattle but does not cause disease in the bovine host. However, if the bacteria passes in to the food chain and infects a human host then it can result in severe health complications and sometimes death. If we can prevent the bacteria from colonising and persisting in cattle then we would reduce the burden of this pathogen entering the food chain. This is especially important in the case of food that won't be cooked before consumption such as unpasteurised cheese.

Interventions, particularly vaccines, have been trialled but with limited commercial success. We propose to develop a bacteriophage (phage) treatment that will selectively remove O157 bacteria from the cattle. Phage are viruses that only infect and kill bacteria. They are very specific and their life cycle is dependent on recognising a receptor, often a protein, on the outside of their bacterial host. Once they have found the receptor they can attach, inject their DNA, replicate inside the bacterial cell and then burst out killing the bacterial cell in the process. Phage and bacteria are continually at war with each other in nature and so both continually evolve to be able to resist infection (bacteria) and to be able to reinfect (phage). We can select phage in the laboratory that can infect and kill O157 strains with lab culture conditions. However, in the host the bacteria will be in a different physiological state to that in nutrient rich media in the laboratory. This means the specific receptors on their surface may be different in these different nutrient limited conditions and when the bacteria are attached to epithelial cells and growing more slowly. Phage intervention has previously been attempted as a control for O157 in cattle but with limited success. We have evidence this is because the physiological state of the bacteria in the host is too different from the lab conditions that the phage were selected in.

We propose that by understanding which receptors are expressed by the bacteria on their surface when they are in the bovine host we can select phage that will be active when used as an intervention strategy to remove/reduce O157 from cattle and therefore protect the food chain. With a greater understanding of the physiology of the bacteria in its host we can develop models in the laboratory that better represent the 'in host' conditions. We can then use these lab models to test phage activity and create phage combinations that can be tested in cattle colonised with O157. This will not only address an unmet need in O157 control but will be an exemplar for other bacterial infections that could be targeted by phage therapy including antibiotic resistant infections.

E. coli O157 is part of a unique group of bacterial pathogens that intimately attach to the outside of host cells as their main colonisation strategy. They employ a type 3 secretion system to inject effector proteins that result in remodelling of the actin cytoskeleton and silencing of innate cellular responses. The bacteria also inhibit host cell apoptosis to keep these 'base camp' cells alive and this is enabled by Shiga toxin which limits local proliferation of new epithelial cells. T3S and intimate attachment are essential for colonisation of the bovine reservoir host and excretion from cattle directly or indirectly can lead to severe human infections. Our grouping has been studying the biology of this zoonotic pathogen in the bovine host for the last two decades and we now want to dissect E. coli O157 gene expression at the main site of colonisation in cattle, the terminal rectum, to advance phage-based interventions. We will carry out RNAseq on O157 sampled from experimentally infected cattle. We will compare this expression with bacteria grown in mucus and intimately attached to cultured bovine epithelial cells. We propose that by understanding the expression 'states' of the bacteria in vivo, it will facilitate selecting bacteriophages active on E. coli O157 when colonising cattle. The second main aim of the research will be defining the bacterial surface receptors used by phage active on E. coli O157 under these specific growth conditions. By combining the expression and phage activity information we will be able to select phage combinations likely to be effective in vivo. The final part of the research will be to test phage formulations in cattle colonised by E. coli O157. An understanding of the in vivo of physiology of this public health threat is important for the design of any cattle-based intervention. The approach being taken to optimise phage treatments in this study can be applied to other important bacterial infections.