Utilising Next-Generation Sequencing to investigate **Staphylococcus aureus** infections and associated alternative phenotypes

Over the past thirty years, Next-Generation Sequencing (NGS) has enabled the fields of clinical microbiology and infectious diseases to investigate the genetic context of human pathogens. Its many applications such as whole genome sequencing (WGS), transcriptomics and metagenomics have deepened our understanding of human pathogens and their infection mechanisms, thereby improving patient care. Staphylococcus aureus is a Gram-positive bacterium which is carried commensally by a significant proportion of the human population. However, it is also a notorious, opportunistic pathogen capable of inflicting several severe, and sometimes fatal, healthcare-associated infections (HAIs). Throughout history it has acquired high levels of antimicrobial resistance (AMR) against many clinically used antibiotic classes, impeding treatment and increasing medical costs and burdens on healthcare systems. Best known examples are methicillin-resistant S. aureus (MRSA) and, more recently, vancomycin-resistant S. aureus (VRSA). Moreover, S. aureus has developed several alternative phenotypes, such as shielding biofilm and antibiotic tolerant persisters, further complicating eradication of this pathogen in case of infection. All these factors make that the World Health Organization (WHO) classified S. aureus among the ESKAPE pathogens, a list of six highly virulent and antibiotic resistant bacterial pathogens which require urgent research and development of new targeted therapeutics. Therefore, in this thesis, we utilise NGS to investigate S. aureus and aim to identify (genomic) determinants to characterise it as infectious pathogen. Firstly, we investigate the current molecular epidemiology of commensal S. aureus in the European surgical community by WGS and found that this is still highly diverse with no apparent dominant pan-European clone or feature. This is followed by results from a study where we elucidated the effect of colonisation on the development of S. aureus pneumonia (SAP) in critically ill, mechanically ventilated intensive care unit (ICU) patients. The data shows that lower respiratory tract screening enhances the early detection of potential SAP, in addition to the standard nasal screening. We also investigate the genomes of S. aureus isolated at ICU admission and SAP infection to identify genomic determinants causative of SAP but conclude that SAP is usually caused by the initial colonising isolate. Both studies were imbedded in two pan-European, multicentre clinical trials conducted within COMBACTE-NET by the COMBACTE consortium. In the second part of this thesis, we investigate gene expression variation in S. aureus biofilm and persisters by transcriptomics to learn more about these alternative phenotypes and potential therapeutic targets. The obtained data shows differential gene expression in both alternative phenotypes compared to normal, free floating cells with arginine-related pathways found to play an important role. Furthermore, gene expression of young biofilms was found to vary drastically between the studied pandemic clones. In conclusion, this thesis utilises state-of-the-art sequencing technology to deepen our understanding of the S. aureus genome and its relation with the development of SAP and the formation of biofilm and persisters. This in order to identify current knowledge gaps and novel potential therapeutic targets to combat the many different mechanisms that make S. aureus a potent infectious pathogen.

Publication year:2022

Keywords:Doctoral thesis

Accessibility:Closed