Genetic and phenotypic characterization of small colony variant Staphylococcus aureus cystic fibrosis isolates

Abstract

Staphylococcus aureus small colony variants (SCVs) are recovered frequently from chronic, antibiotic refractory infections and are associated with worse disease outcomes and treatment failures. The defining SCV characteristic is slow growth in vitro resulting in small, atypical colony formation. SCVs arise following exposure to antimicrobials both in vitro and in vivo, however their unusual growth phenotype prevents the use of traditional laboratory diagnostics to guide treatment. In an effort to define SCV antibiotic resistance patterns, and provide empirical treatment options; we developed, optimized, and performed antibiotic susceptibility testing on a collection of clinical cystic fibrosis (CF) isolates. These findings are presented in Chapter 2. The SCV phenotype is caused by genetic changes in critical metabolic pathways that result in nutrient auxotrophies, most commonly for thymidine (THY), hemin, or menadione. THY–dependent SCVs have been associated with poor clinical outcomes in CF and we present evidence presented in Chapter 3 that the pro-inflammatory, small molecule cyclic-di-AMP may play a role in disease progression. Additionally, although genetic mechanisms contributing to SCV formation have been reported, only a limited number of genes and pathways have been described. Furthermore, we and other groups have found that SCVs of the same auxotrophy type display morphological and colony size variability, suggesting additional mutations. Thus, we examined the genetic mechanisms behind SCV formation using whole-genome sequencing for a collection of clinical SCVs and normal colony (NC) S. aureus and have identified novel mutations presented in Chapter 4. Lastly, the traditional SCV designation, ~1/10 the size of NC S. aureus, is somewhat vague and a more precise definition would benefit both clinical as well as research laboratories working to categorize atypical, slow growing S. aureus. In an effort to ultimately provide a more rigorous, statistically driven definition of what constitutes an SCV, we examined the distribution of colony sizes that clinical S. aureus isolates display in vitro and performed preliminary statistical analyses, presented in Chapter 5. The full body of work presented here addresses important topics including antibiotic resistance, diagnostic development, pathogenesis, phenotypic, and genomic analyses of SCVs in an effort to further our understanding of this unusual variant of S. aureus.