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Escherichia coli  ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of  E. coli  ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of  E. coli  ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for  in vitro  growth and the serum resistome (i.e. genes required for resistance to human serum) of  E. coli  EC958, a representative of the predominant  E. coli  ST131 clonal lineage. We identified 315 essential genes in  E. coli  EC958, 231 (73%) of which were also essential in  E. coli  K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for  E. coli  EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes,  hyxA  and  hyxR , as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of  E. coli  ST131, and provides a framework for future research on  E. coli  and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

The Serum Resistome of a Globally Disseminated Multidrug Resistant Uropathogenic Escherichia coli Clone