Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known public health concern. However, numerous aspects concerning the natural transfer and stability of the methicillin resistance genes and staphylococcal cassette chromosome mec (SCCmec) carrying them remains unknown. Older scientific literature suggests transduction as a means of mecA transfer, but the optimal conditions in these reports are conflicting and are suggested to require plasmids and potentially a lysogenic phage. Additionally, older scientific literature also suggests that methicillin resistance is unstable. However, these reports preceded discovery of SCCmec elements. Furthermore, stability studies did not fully characterize the loss of methicillin resistance by molecular means. |Thus, I undertook the studies found in Chapter 2 to confirm and clarify the conditions promoting transduction of SCCmec in S. aureus populations using well-characterized donor and recipient strains, primarily of the USA300 lineage. Both bacteriophages 80α and 29 were capable of transducing SCCmec type IV and SCCmec type I to recipient strains of S. aureus. Pulse-field gel electrophoresis and mec-associated dru typing were used to confirm the identity of the transductants. Transfer of mecA via transduction occurred at low frequency and required extended selection times for mecA gene expression as well as the presence of a penicillinase plasmid in the recipient. However, interference with the process by β-lactamase inhibitors and the necessity of lysogeny with ϕ11 in the recipient or the presence of a small (4-Kb) tetracycline resistance plasmid, as previously reported were not confirmed. SCCmec transduction was occasionally associated with substantial deletions or truncation of SCCmec and the arginine catabolic mobile element in USA300 recipients. |Additionally, I carried out a longitudinal study found in Chapter 3 to understand the stability of methicillin resistance and SCCmec using a variety of MRSA lineages. I found that methicillin resistance is indeed unstable in MRSA isolates in stationary phase longitudinal cultures. However, loss of methicillin resistance gave rise to three distinct phenotypes: SCCmec-negative methicillin-susceptible S. aureus (MSSA), SCCmec remnant MSSA, and oxacillin-susceptible, mecA-positive, PBP 2a-positive (OS) MRSA. The deletions which occurred in SCCmec remnants all occurred site-specifically at the 5 ́ end of IS431 insertion sequence elements, but randomly at the other end. The explanation of the OS-MRSA isolates remains obscured, but likely relates to altered post-translational mechanisms or mutations in accessory proteins that have yet to be linked to a role in regulating methicillin resistance. |Overall, my work has helped clarify the conditions required for SCCmec transduction. It has also helped explain the natural stability of methicillin resistance and SCCmec. The data presented in chapter 2 of this dissertation show that methicillin resistance transfer does occur to a limited degree between S. aureus populations. Furthermore, the data presented in chapter 3 of this dissertation suggest that methicillin resistance is unstable and that loss of methicillin resistance leads to three distinct phenotypes. Both sets of data document that rearrangements of SCCmec may occur during the transfer process and under non-antibiotic selective pressures in S. aureus. These rearrangements and, in particular SCCmec remnant and OS-MRSA strains, are of clinical concern as they may affect tests used in the clinical lab to determine antibiotic susceptibility.