Incidence of infections caused by multi- and pan-drug resistant Enterobacteriaceae, such as Escherichia coli and Klebsiella, is increasing worldwide. Consequently, colistin, an old and out-of-use antibiotic, had to be re-introduced into clinical practice as a last resort therapeutic option as it is the only antibiotic to which such bacteria still remain susceptible. However, increasing use has inevitably led to emergence of colistin resistance (CR) among Gram-negative bacteria. The main goal of this project is to examine key elements of colistin resistant Enterobacteriaceae (CRE) that have hitherto not been fully investigated. We have established a unique collection of CR E. coli and Klebsiella spp., originating from hospitalized patients and sick animals as well as generated in vivo in a Galleria mellonella moth model, that will form the basis of this project. With these isolates and their colistin sensitive counterparts, we plan to undertake the following: 1, Strain typing; 2, Selection of CR in-vitro; 3, Genome sequencing and comparative genome analysis; 4, Targeted gene sequencing and protein expression; 5, Stability studies; 6, Fitness studies and 7, Assessment of mortality and pathogenicity. While strain typing has been done on few Klebsiella, we will type other CRE to examine if CR is associated with certain strain types. Continuous culture experiments in a morbidostat set-up will allow in vitro selection of CRE under continuous drug pressure that will be studied temporally at various stages of resistance development. Whole genome sequencing of in-vivo and in-vitro CRE isogenic strains will identify the sites of mutations potentially responsible for CR. Gene targets identified by genome analysis will be re-sequenced to confirm the changes conferring CR and those will be further characterized by real-time PCR and proteomics. The stability of CR and fitness of such strains will be assessed either by passaging in colistin-free medium, or under constant colistin pressure on the morbidostat model. The effects of CR on mortality will be investigated in the high-throughput reproducible in vivo Galleria mellonella model. Finally, the impact of CR on virulence and pathogenicity will be studied in a higher animal model i.e., a ventilator-associated pneumonia rat model that is already established in our laboratory.