Elucidating the success of Enterococcus faecium as a nosocomial pathogen through functional genomics

Abstract

Enterococcus faecium is a commensal of the gastrointestinal tract. However, E. faecium can also cause a large number of hospital-acquired enterococcal infections in humans and has rapidly acquired resistance to several classes of antibiotics. The ubiquitous nature of E. faecium, the ability to prosper in complex microbial communities, the great flexibility of its genome and the global use of antibiotics, are important factors driving E. faecium’s current emergence as a nosocomial pathogen around the world. The rise of vancomycin resistant enterococci (VRE), complicate treatment of infections caused by multi-drug resistant isolates. The species E. faecium can be divided into three distinct sub-populations that include the majority of clinical isolates, strains from healthy humans and animal isolates. In this thesis, we continue to expand the use of high-throughput approaches to characterize the genetic determinants of E. faecium that can explain its success as a nosocomial pathogen. In chapter 2 we performed comparative transcriptome profiling of an ampicillin-resistant E. faecium isolate grown at mammalian (37°C) and room temperature (25°C) and identified a thermo-regulated surface-exposed protein, termed PrpA. We showed that its functional N-terminal domain interacts with fibrinogen and fibronectin, which are components of the ECM present in mammalian hosts. The same domain was also able to bind to platelets. This thermo-regulated surface-exposed protein was found to be produced at 37°C in clinical and animal strains but not at room temperature or in commensal isolates. Chapter 3 describes a two-component system, identified through a microarray-based transposon mutant screening method, implicated in tolerance to the disinfectant chlorhexidine. In addition, we showed that this putative two-component system is also implicated in resistance to the antibiotic bacitracin, which may be used in the treatment of VRE colonization and infection. In chapter 4 we sequenced the genome of the vancomycin-resistant clinical E. faecium isolate E745 and implemented a high-throughput transposon mutant sequencing approach, to perform a sequencing-based genome-wide functional profiling of this strain. We identified the genes that contribute to survival and growth of E. faecium E745 in human serum, showing that genes involved in carbohydrate uptake and genes involved in the biosynthesis of purine and pyrimidine nucleotides, had a role in this phenotype. Transposon mutants in selected genes were isolated from the mutant library and were shown to have a growth defect in human serum and found to be attenuated in virulence in a zebrafish infection model. In chapter 5, we describe a novel resistance mechanism against the recently introduced antibiotic tigecycline. Resistance was caused by mutations in the rpsJ gene which encodes a ribosomal protein. This mechanism was identified using a combination of comparative genomic analysis of paired susceptible and resistant isolates, and functional genetic complementation. The widespread use of antibiotics in human and veterinary medicine, is a crucial factor in the emergence of succesful E. faecium clones, that have adapted to live in the hospital. However, other traits, like the ability of the bacteria to colonize the intestinal tract and cause infections, are also important to explain the transition of E. faecium from gut commensal to nosocomial pathogen. The research described in this thesis contributes to our understanding of the underlying mechanisms that have made E. Faecium, an important nosocomial pathogen and may contribute to the development of new therapies aimed against this bacterium.