Cell wall metabolite levels as a function of the growth phase in Staphylococcus aureus and library screening for synergistic combinations of FDA approved drugs against methicillin resistant S. aureus
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methods for quantifying cell wall biosynthesis intermediates. These include LC-MS/MS methods for quantifying cytoplasmic UDP-linked peptidoglycan biosynthesis intermediates. Retention and separation of these polar intermediates on C18 media were significantly improved using N, N-dimethylhexylamine as an ion-pairing agent. We also have developed Marfey’s reagent-based LC-MS/MS methods to stereospecifically resolve D- and L- amino acids and the key D-alanine-D-alanine intermediate involved in CWB on a reverse-phase analytical column. In the first part of this study, we examined how CWB intermediates and energy-rich nucleotide (ATP, etc.) levels change over the whole growth curve of S. aureus, from senescence to logarithmic growth and back to senescence. We first extended our established ion-pairing agent-based LC-MS/MS method for detecting UDP-linked intermediates, including energy-rich nucleotides (ATP, etc.) (unpublished). In this study, a culture of Methicillin-resistant Staphylococcus aureus (MRSA) American Type Culture Collection (ATCC 43300) is grown to senescence for 48 hours in Muller Hinton (MH) broth. From senescence to the exponential phase, the levels of UDP-linked intermediates increase significantly, and the levels of energy-rich nucleotides are low during these phases of the growth curve. Hence, UDP-linked intermediates appear to be the high-priority biomolecules required for maintaining bacterial physiology, where nucleotides with high energy are rapidly assimilated by UDP-linked intermediates. Additionally, the accumulation of energy-rich nucleotides coincided with the decline in cell growth rates, suggesting a concomitant reduction in their utilization. Retention and separation of glucosamine-1-phosphate (GlcN-1-P) and glucosamine-6-phosphate (GlcN-6-P) on a reverse-phase column is not feasible due to their high polarity. The second part of this study tested and demonstrated several derivatization procedures to improve the retention of GlcN-1-P and GlcN-6-P on a reverse-phase column. Derivatization with isobutyric anhydride, hexanoic anhydride, octanoic anhydride, and decanoic anhydride combined with multichannel LC-MS/MS detection demonstrated resolution and quantification of these three analytes. MRSA, vancomycin-resistant enterococcus (VRE), and vancomycin-resistant enterococcus (VSE) extracts were prepared and derivatized in this study. The LC-MS/MS analysis of these samples demonstrated peaks of GlcN-1-P and alpha and beta anomers of GlcN-6-P that were clearly resolved on a reverse-phase analytical column. Therefore, we performed a high-throughput screening of FDA-approved compounds in the presence and absence of ceftobiprole, identifying intrinsically active agents and synergistic combinations with ceftobiprole. A checkerboard analysis was then performed on potential ceftobiprole synergistic agents. Strongly synergistic agents included β-lactamase resistant β-lactams (cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, and cefotaxime). There was also evidence of synergy between ceftobiprole and both meropenem and imipenem. Vancomycin, balofloxacin, and floxuridine showed weak synergy. A similar antagonistic effect of mupirocin on ceftobiprole was also observed in other studies showing that it led to β-lactam resistance. We also performed a high-throughput library screening of FDA-approved drugs in the presence and absence of floxuridine against MRSA with the aim of surveying the full scope of floxuridine antibacterial synergy. This study examined joint MIC determination of the FDA library hits in combination with floxuridine, a high-resolution method to depict the fractional inhibitory concentration of √2. This way, several strong synergistic compounds (FICmin ≤ 0.5) like imipenem and biapenem were identified. Moreover, many agents showed weak synergy (0.5 < FICmin ≤ 0.75), such as nafcillin, doripenem, meropenem, and moxalactam.
Table of Contents
Cell wall metabolite levels as a function of growth phase in S. aureus -- Perform LC-MS/MS-based method development and quantification of Glucosamine-1-Phosphate (GlcN-1-P) and Glucosamine 6-Phosphate (GlcN-6-P) which are involved in the early steps of cell wall biosynthesis -- Library screening for synergistic combinations of FDA-approved drugs with Ceftobiprole against Methicillin-resistant Staphylococcus Aureus -- A high-throughput library screening of FDA-approved drugs with Floxuridine against MRSA -- General conclusion
Ph.D (Doctor of Philosophy)