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

Abstract

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.

Author Summary

The emergence and rapid dissemination of new bacterial pathogens presents multiple challenges to healthcare systems, including the need for rapid detection, precise diagnostics, effective transmission control and effective treatment. E. coli ST131 is an example of a recently emerged multidrug resistant pathogen that is capable of causing urinary tract and bloodstream infections with limited available treatment options. In order to increase our molecular understanding of E. coli ST131, we developed a high-throughput transposon mutagenesis system in combination with next generation sequencing to test every gene for its essential role in growth and for its contribution to serum resistance. We identified 315 essential genes, 270 of which were conserved among all currently available complete E. coli genomes. Fifty-six genes that define the serum resistome of E. coli ST131 were identified, including genes encoding membrane proteins, proteins involved in LPS biosynthesis, regulators and several novel proteins with previously unknown function. This study therefore provides an inventory of essential and serum resistance genes that could form a framework for the future development of targeted therapeutics to prevent disease caused by multidrug-resistant E. coli ST131 strains.