Watching Bacteria Adapt: Investigating How Pathogens Evolve to Cause Disease

Abstract

Experimental evolution has provided us with many insights into varied evolutionary processes, however there has been a lack of studies seeking to follow natural infection and transmission of a pathogen through its natural host. I have followed the adaptation of the murine enteropathogen Citrobacter rodentium as it infects laboratory mice. Ten independent lineages were started, with mice from five of the lineages receiving a low dose of the quinolone nalidixic acid in their drinking water to alter their normal microbiome, and mice from the remaining five lineages receiving untreated drinking water. Mice were orally inoculated with the bioluminescent C. rodentium derivative ICC180 and individually housed animals allowed to infect naïve animals through tightly controlled mouse-to-mouse exposure, a process which was repeated weekly over a period of five months, by which time at least 20 transmissions had occurred. The in vivo-adapted C. rodentium were isolated from the mice and compared and competed with the ancestral strain. Multiple phenotypic and genotypic differences were observed, including the evolution of a ‘hypertransmissible’ isolate which preferentially transmits to naïve animals when in competition with the wildtype strain, an isolate which gained the ability to form aggregates in rich media, and a hypermutatable isolate. In addition to the in vivo evolution experiment, a complementary in vitro evolution experiment was performed, with C. rodentium passaging through laboratory media using the traditionally established method of serial transfer. These in vitro-adapted isolates adapted to a restricted media with 1% glucose supplementation, and half of the isolates exhibited a trade-off of reduced growth on rich media accompanying improved growth on the restricted media. Such a trade-off was not observed in the in vivo-adapted C. rodentium. In conclusion, a five month period of experimental evolution was sufficient for observable adaptation to occur, with separate evolutionary trajectories for C. rodentium adapting in vivo versus in vitro. Populations have been stored for future analyses, and the bank of samples will be an important resource for evolutionary biologists. The work detailed in this thesis extends existing flask-based experimental evolution with an in vivo model following the entire infectious process.