Watching Bacteria Adapt: investigating how pathogens evolve to cause disease

Abstract

Evolution experiments are indispensable for exploring the abilities of bacteria to adapt and evolve, and to predict some mechanisms by which bacteria can adapt to new challenges. We propose to use Citrobacter rodentium to investigate natural adaptation to its host, the laboratory mouse, following natural transmission. C. rodentium is a murine pathogen which has been shown to be in a state of genome flux, and is a model of attaching-effacing pathogens, such as the clinically relevant human pathogens enterohaemorrhagic and enteropathogenic Escherichia coli. This work aims to provide the groundwork for the future evolution experiments by the creation of two novel bioluminescent C. rodentium strains, enabling competition experiments, and by investigating a novel wax moth larvae virulence assay, which will both be used as a means to assess changes between ancestral and adapted bacteria. C. rodentium was tagged with red- and green-shifted thermostable firefly luciferase genes, in order to enable monitoring of infection processes using in vivo bioluminescent imaging techniques and to reduce the number of mice required, thereby following the principles of the Three R’s. The bioluminescent C. rodentium are not attenuated in growth or virulence, and can be transmitted between C57BL/6 cage-mates. Integration of the luciferase genes appears to be stable over at least 8 days. The wax moth larvae virulence assay was investigated as a means to assess C. rodentium virulence while reducing the number of mice required for the experiments and was shown to be an effective means of assessing relative virulence. We have also developed the Caterpillar Health Index scoring system as a novel method of monitoring symptoms in larvae, the scores of which have been shown to positively correlate with both bacterial counts (R2 = 0.7425) and bioluminescence (R2 = 0.8907). We have successfully created two bioluminescent strains of C. rodentium (CR-Red and CR-Green) which will allow competition experiments between ancestral and adapted strains during the future evolution experiments. The two strains may be suitable for use in the proposed evolution experiments which will investigate bacterial adaptation following natural transmission over multiple generations.