Quantitative Analysis of Autoinducing Peptide I (AIP-I) from Staphylococcus aureus Cultures using Ultrahigh Performance Liquid Chromatography – High Resolving Power Mass Spectrometry

摘要

Staphylococcus aureusinfections acquired in hospitals now cause more deaths per annum in the US than does HIV/AIDS. Perhaps even more alarming is the rise in community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, which have spread out of hospital settings and are infecting otherwise healthy individuals. The mechanism of enhanced pathogenesis in CA-MRSA remains unclear, but it has been postulated that high activity in the agr quorum-sensing system could be a contributing factor. The purpose of this study was to develop a quantitative method for analysis of autoinducing peptide I (AIP-I), the activating signal for the agr system in S. aureus. An effective method was developed using ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization mass spectrometry with an LTQ Orbitrap mass spectrometer. Relying on the exceptional resolving power and mass accuracy of this instrument configuration, it was possible to quantify AIP-I directly from the complex growth media of S. aureus cultures with a limit of detection (LOD) of 0.25 μM and a linear dynamic range of 2.3 to 63 μM. The method was then employed to monitor time-dependent production of AIP-I by S. aureus cultures, and it was observed that AIP-I production reached a maximum and leveled off after approximately 16 hrs. Finally, it was determined that virulence of S. aureus was correlated with AIP-I production in some (but not all) strains analyzed.