beta-Lactam Resistance and Novel Therapeutics for Staphylococcus aureus.

摘要

Staphylococcus aureus is an important human pathogen capable of causing disease in otherwise healthy individuals. It causes mostly skin and soft tissue infections but can cause more invasive diseases. Treatment for S. aureus has become a problem due to increasing resistance and limited new therapeutics, particularly for more serious infections. Methicillin-resistant S. aureus (MRSA) displays class resistance to beta-lactam antibiotics through the presence of penicillin-binding protein 2a (PBP2a), encoded by mecA. Chapter 1 contains a thorough literature review on MRSA and antibiotic resistance, including epidemiology, therapeutic options, mechanisms of antibiotic resistance, modes of resistance acquisition and S. aureus animal models. MRSA has a great capacity to rapidly develop antibiotic resistance, defined as the ability of bacteria to resist a drug to which it was originally sensitive. Limited antibiotics are approved for severe MRSA infections including vancomycin, daptomycin, linezolid and ceftaroline. Resistance to these drugs almost always occurs in MRSA backgrounds rather than methicillin-sensitive S. aureus (MSSA) backgrounds. Invasive MRSA infections have been associated with treatment failure and increased risk of mortality. The development of new antibiotics is of utmost importance. This dissertation encompasses the following areas of research: i) ability of S. aureus to develop resistance to new beta-lactam antibiotics, ii) potential pathways to prevent antibiotic resistance and iii) efficacy studies of a new antibiotic as a potential alternative for MRSA treatment.;Ceftobiprole and ceftaroline, members of a new class of beta-lactams, target PBP2a with high affinity, the core component of beta-lactam resistance in MRSA strains. Ceftobiprole is in phase 3 clinical trials while ceftaroline has been FDA approved. The goal of Chapters 2 and 3 was to identify mechanisms of resistance and likely targets associated with resistance. Chapter 2 determined resistance could be generated to ceftobiprole and ceftaroline by passaging MRSA strains in increasing concentrations of antibiotic. The mechanism of resistance in these mutants was mutagenesis of mecA and mutations in other PBPs. Chapter 3 analyzed mecA-independent mechanisms of resistance to determine the affect of these antibiotics in the absence of PBP2a. MRSA strains cured of SCCmec were passaged in ceftaroline and ceftobiprole, resulting in mutants with high-level, broad-spectrum beta-lactam resistance. These mutants have mutations in pbp4 and other genes as well as upregulaed pbp4 mRNA levels in some mutants. Knowledge gained from these studies will provide information on novel mechanisms of beta-lactam resistance and will guide development of new antibiotics.;Chapter 4 is dedicated to exploring the role of the SOS stress response in antibiotic resistance. The SOS response is a stress response regulated by LexA, a transcriptional repressor, and RecA, activator of LexA. When bacteria are exposed to stimuli that break DNA (e.g. UV, antibiotics, etc.), RecA activates LexA, causing derepression of the SOS genes, including error-prone polymerases that increase mutational frequencies. beta-lactams have been shown to activate the SOS response in certain strains but not in a prevalent community-acquired MRSA (CA-MRSA) background. Given the increasing prevalence of CA-MRSA strains and beta-lactam resistance, exposure of MRSA to beta-lactams could potentially activate the SOS response resulting in increased antibiotic resistance. The goal of this chapter is to test the role of the SOS response in antibiotic resistance with beta-lactam induction in CA-MRSA. To test this hypothesis, a non-cleavable lexA mutant was created, which constitutively represses the SOS response. The results of this study indicate that, in the USA300 background, the SOS response was not solely responsible for increased antibiotic resistance and suggests that this pathway might not be a good therapeutic target to decrease emergence of resistance in CA-MRSA strains.;Chapter 5 explores recently discovered options in the presence of ceftaroline-resistance or beta-lactam intolerance. Given the emergence of strains resistant to antibiotics used for severe MRSA infections including vancomycin, daptomycin, linezolid and ceftaroline, new therapeutics are needed. Tedizolid phosphate is a second-generation oxazolidinone in late stage clinical development with activity against MRSA. The goal of this chapter is to test the efficacy of tedizolid phosphate in an animal model of invasive MRSA infection. Tedizolid phosphate was compared to standard of care antibiotics used to treat MRSA bacteremia, vancomycin and daptomycin, in a rabbit model of endocarditis. At high doses, tedizolid phosphate was non-inferior to vancomycin. At doses achieving serum concentrations similar to human doses, tedizolid phosphate was not as efficacious as vancomycin or daptomycin. Our results suggest tedizolid phosphate is ineffective at treating severe infections, such as MRSA endocarditis, compared to vancomycin.;The dissertation confirms the ability of S. aureus to develop resistance to new antibiotics and further describes the difficulty in developing efficacious new therapies. Discovering mechanisms of resistance will provide knowledge for potential new anti-MRSA therapeutics.