title: Molecular characterization of clinically important phenotypes of Staphylococcus aureus in a large cohort of bloodstream infections
Creators: name: Mapar, Maryam
Publisher: name: Graduate Studies
Year: 2024
Subjects: subject: Staphylococcus aureus; bacteremia
subject: Patient outcomes
subject: Metabolism
subject: Proteomics
subject: Genomics
subject: Multiomics analysis
subject: Cefazolin inoculum effect
subject: Small colony variants
subject: Microbiology
subject: FOS: Biological sciences
subject: Biochemistry
subject: Biology--Molecular
subject: Genetics
subject: Bioinformatics
subject: FOS: Computer and information sciences

description: Staphylococcus aureus is known for its ability to thrive in diverse host niches through various adaptive strategies. A key feature of this species is its ability to exhibit various phenotypes, such as the small colony variants (SCVs) and the inoculum effect, both of which are associated with persistent and difficult-to-treat infections. However, our current understanding of these phenotypes mostly comes from studies on relatively small cohorts, with findings that, while valuable, may not fully represent the complexity seen in real-world infections. To address this gap, I leveraged a collection of 7,604 S. aureus samples from a larger study cohort of over 38,000 bloodstream infections (BSIs) collected in the Calgary health zone from 2006 to 2020. Whole-genome sequencing on these isolates was completed at the Broad Institute of MIT and Harvard. Metabolomics and proteomics data were generated using ultra-high-performance liquid chromatography mass-spectrometry (UHPLC-MS) and tandem mass tag (TMT)-labeling LC-MS based analyses, respectively, at the Calgary Metabolomics Research Facility. This uniquely large cohort of isolates, integrated with multi-omics data and administrative health records, provided a great opportunity to reevaluate existing knowledge and deeply investigate the molecular underpinnings of these two clinically important phenotypes. Additionally, from my experience with working in the field of microbial metabolomics I learned that one of the key challenges in studying metabolism in the complex systems is the variability in metabolomic phenotypes across different batches of culture media. To address this, along the way of understanding the molecular underpinnings of SCVs and the cefazolin inoculum effect (CzIE) in S. aureus BSIs, I developed a chemically defined medium—Biomarker Enrichment Medium (BEM)—that not only supports the growth of BSI pathogens (including S. aureus), but also enables antibiotic susceptibility testing and restores key metabolic biomarkers used to differentiate six common BSI pathogens. My thesis has two main biological findings. First, my research on SCVs differs from previous reports regarding phenotypic instability and more severe infections while confirming prior findings related to SCV-linked protein expression patterns. Furthermore, this research has shown a greater molecular diversity underlying the SCV phenotype than previously appreciated, as my genomic analysis identified &gt;300 candidate mutations associated with SCVs. These findings suggest that characterization of SCVs as a single phenotype is a simplification of what they are in real-world infections. Second, my research on the β-lactamase-producing methicillin-susceptible Staphylococcus aureus (MSSA) isolates from the Calgary BSI cohort collected in 2012-2014 and 2019 study did not find a significant association between CzIE and higher mortality rates. However, specific molecular characteristics for the majority of CzIE isolates were identified: clonal complex 30, accessory gene regulatory group III, and type A β-lactamase. Additionally, a novel single nucleotide polymorphism in the bla operon was identified in MSSA isolates exhibiting the non-CzIE phenotype, which led to a disruption in the inducibility of the bla operon in these isolates. Collectively, these studies have provided a deeper understanding of epidemiologic, phenotypic, and molecular characteristics of the SCV phenotype and CzIE in S. aureus BSIs.