Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a β-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to β-lactam (HeR) in which only a small portion (≤0.1%) of the population expresses resistance to oxacillin (OXA) ≥10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of β-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with β-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished β-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.
Highlights
S. aureus is a main pathogen responsible for a number of diseases ranging from skin and soft tissue infections to life-threatening endocarditis in hospitals and community settings [1]
In an attempt to determine differentially expressed genes associated with b-lactam-mediated heterogeneous expression of resistance to b-lactam (HeR)/HoR selection, we performed gene expression analysis using spotted DNA microarrays as previously described [13]
Oxidative DNA Damage Another difference between SA13011-HeR/HoR strains corresponded to a marked decreased in the levels of glutathione (Fig. S2), which may be associated in these cells with both increased oxidative stress and activation of a DNA damage response, as we previously reported during SA13011-HeR/HoR selection [5]
Summary
S. aureus is a main pathogen responsible for a number of diseases ranging from skin and soft tissue infections to life-threatening endocarditis in hospitals and community settings [1]. The mechanism has been explored in detail, less is known about the cellular physiology of HeR-MRSA strains during the process of HeR/HoR selection by sub-inhibitory concentrations of b-lactams. Recent studies suggest that the basic physiology of S. aureus determines growth and survival and pathogenicity and adaptation to stress conditions, including antibiotic pressure [7]. In this sense, it has been shown that S
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