UC San Diego

Antibiotic-resistant Staphylococcus aureus is one of the most common causes of bacterial infections in humans and is responsible for a wide range of infections in healthcare and community settings. Methicillin-resistant S. aureus, in particular, has proven to be resistant to β-lactams and many other classes of antibiotics. The standard for evaluation of antibiotic efficacy involves testing on cation-adjusted Mueller Hinton broth or CA-MHB. However, this is not reflective of the human host environment. Changes in media conditions and environment have led to differences in antibiotic efficacy outcomes and have major implications in treatment. To further elucidate differential antibiotic resistance mechanisms, adaptive laboratory evolution was utilized to generate strains of the S. aureus clinical isolate, USA300 TCH1516, under an increasing antibiotic pressure of nafcillin in CA-MHB as well as Roswell Park Memorial Institute medium (RPMI). Evolutionary paths for strains were characterized at the physiological and genetic level utilizing whole genome sequencing to understand the genetic and metabolic basis of antibiotic resistance. Improvements in growth rate were observed for media adaptation to RPMI but not CA-MHB. Improved fitness in stressful conditions were identified and linked to mutated copies of apt. Key reproducibly occurring mutations were compared between the two environments after exposure to nafcillin showing mutations in common regions within the vraRST operon, a regulator of cell wall metabolism genes, and in mgt, a nonessential transglycosylase. Unique susceptible phenotypes to gentamicin and azithromycin were identified after tolerance to nafcillin. Mutated genes yybT and ybbP, codY, and oatA related to regulation of nucleotide and branched chain amino acid metabolism as well as peptidoglycan biosynthesis and modification were identified for tolerance to nafcillin in RPMI only. Mutations were subsequently compared across literature and presented here.