Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a major multidrug resistant pathogen responsible for several difficult-to-treat infections in humans. Clinical Hetero-resistant (HeR) MRSA strains, mostly associated with persistent infections, are composed of mixed cell populations that contain organisms with low levels of resistance (hetero-resistant HeR) and those that display high levels of drug resistance (homo-resistant HoR). However, the full understanding of β-lactam-mediated HeR/HoR selection remains to be completed. In previous studies we demonstrated that acquisition of the HoR phenotype during exposure to β-lactam antibiotics depended on two key elements: (1) activation of the SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, resulting in increased mutation rates, and (2) adaptive metabolic changes redirecting HeR-MRSA metabolism to the tricarboxylic acid (TCA) cycle in order to increase the energy supply for cell-wall synthesis. In the present work, we identified that both main mechanistic components are associated through TCA cycle-mediated reactive oxygen species (ROS) production, which temporally affects DNA integrity and triggers activation of the SOS response resulting in enhanced mutagenesis. The present work brings new insights into a role of ROS generation on the development of resistance to β-lactam antibiotics in a model of natural occurrence, emphasizing the cytoprotective role in HeR-MRSA survival mechanism.
Highlights
Staphylococcus aureus is an important pathogen responsible for a number of diseases ranging from skin and soft tissue infections to life-threatening endocarditis, both in hospitals and community settings [1,2,3]
The primary target of b-lactam antibiotics are penicillin binding proteins (PBPs), which are involved in the last stages of peptidoglycan biosynthesis [4]. b-lactam resistance in Methicillin-resistant Staphylococcus aureus (MRSA) involves the acquisition of PBP2a, a protein encoded by the mecA gene, that complements the four native staphylococcal PBPs (PBP1–4) once they have been inactivated [5,6]
To investigate whether reactive oxygen species (ROS) production may be involved in the process of b-lactam-mediated heterotypic resistance (HeR)/homotypic resistance (HoR) selection, sub-inhibitory concentrations of oxacillin (OXA; 0.5 mg/ml; minimal inhibitory concentrations (MICs): 2 mg/ml) were added to cultures of SA13011 (S. aureus heteroresistant strain; clinical MRSA strain SA13011-HeR and derivatives are shown in Table 1), after which ROS formation was measured at different time points (Figure 1)
Summary
Staphylococcus aureus is an important pathogen responsible for a number of diseases ranging from skin and soft tissue infections to life-threatening endocarditis, both in hospitals and community settings [1,2,3]. The primary target of b-lactam antibiotics are penicillin binding proteins (PBPs), which are involved in the last stages of peptidoglycan biosynthesis [4]. B-lactam resistance in MRSA involves the acquisition of PBP2a, a protein encoded by the mecA gene, that complements the four native staphylococcal PBPs (PBP1–4) once they have been inactivated [5,6]. Expression studies showed that high level of mecA expression not always correlates with higher level b-lactams resistance [7,8,9,10]. The main characteristic of the majority of clinical MRSA isolates associated with both hospital and community associated infections is their heterogeneous expression to b-lactam antibiotics. The evidence that sub-minimal inhibitory concentrations (MICs) of antibiotics can play an important role in the generation and dissemination of antibiotic resistance has begun to emerge in recent years [14,15]
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