Fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum

Enterococcus faecium is a common commensal of the mammalian gastrointestinal tract. However, an E. faecium sub-population, termed clade A-1, has recently emerged that is responsible for an increasing number of nosocomial infections. Clade A-1 E. faecium strains can colonize indwelling medical devices, gaining access to the human bloodstream, thereby causing bacteremia. Bloodstream infections with E. faecium frequently pose a therapeutic challenge, as multi-drug resistance is common in this species.In this study, we elucidate the mechanisms by which E. faecium can grow in human serum. To this aim, we first sequenced the genome of E. faecium E745, a vancomycin-resistant, clinical isolate from clade A-1, to completion, after which a transposon mutant library was generated in this strain. We then utilized a high-throughput transposon sequencing (Tn-seq) approach to identify conditionally essential genes in E. faecium E745. Genes involved in de novo nucleotide biosysnthesis (pyrK_2, pyrF, purD, purH) and a gene that encodes for a subunit of a predicted mannose-specific phosphotransferase system (manY_2) were thus identified to be contributing to E. faecium growth in human serum. RNA-Seq revealed major shifts in the gene expression profile of E. faecium during growth in human serum, including the up-regulation of nucleotide biosynthesis genes, which were also identified to be essential for growth in serum by Tn-seq. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants also exhibited significantly attenuated virulence in an intravenous zebrafish infection model. Our analyses indicate that the relatively large repertoire of genes involved in carbohydrate and nucleotide metabolism of E. faecium contributes to its ability to cause bloodstream infections.