The rapid rise of superbugs - bacteria that are resistant to all available antibiotics - is an increasing threat to health with the potential to overwhelm the healthcare system. However, our understanding of how bacteria resist antibiotics is far from complete. During infection the superbug Pseudomonas aeruginosa undergoes multiple mutations and evolves very high antibiotic resistance but only a subset of the mutations causing resistance have been identified. In this research we will obtain the first full overview of mutational events that result in high-level resistance. We will do so by developing highly resistant mutants of P. aeruginosa under controlled laboratory conditions, comparing the underlying mutations with those that occur during infection, and determining the effects of key mutations in a mouse model of infection. The results will provide a major step forward in understanding how bacteria resist antibiotics and may lead to improved treatment regimens for P. aeruginosa and other superbugs.