The rapid evolution of antibiotic resistance increasingly challenges the successful treatment of S. aureus infections. Here, we present an unconventional treatment approach by disassembly its membrane microdomains via pulsed laser photolysis of staphyloxanthin. After staphyloxanthin photolysis, membrane permeabilization, fluidification, and membrane protein detachment, were found the underlying mechanisms to malfunction its defense to several major classes of conventional antibiotics. Through resistance selection study, we found pulsed laser treatment completely depleted staphyloxanthin virulence. More importantly, laser treatment further inhibited development of resistance for several major classes of conventional antibiotics including fluoroquinolones, tetracyclines, aminoglycosides, and oxazolidinones. Collectively, this work highlights a novel platform to revive conventional antibiotics to treat S. aureus infections.
The World Health Organization (WHO) published a catalogue of 12 families of antibiotic-resistant bacteria which pose an alarming threat to human health in 2017. These bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), could cause a wide range of infections from minor subcutaneous infection to toxic shock syndrome, and bacteremia. As the body’s second line of host defense, phagocytosis could eliminate the majority of the invasive bacteria. However, the survival of microbial pathogens within the macrophage cells which act as ‘Trojan horses’ largely provides a reservoir relatively related protected from antibiotics, thus causing recurrent infections from the dissemination of intracellular bacteria. Moreover, the pace of antibiotic development can’t keep with the resistance acquisition from bacteria. Therefore, there is an unmet need for alternative approaches to eradicate multi-drug resistant intracellular bacteria. Here, we develop an effective photonic approach to assist macrophage cell (RAW 264.7) to efficiently eradicate intracellular MRSA, P. aeruginosa along with Salmonella enterica. This approach selectively targets intracellular bacteria without damaging macrophage cells through photoinactivation of a microbial detoxifying enzyme existing in most of the bacteria. Moreover, we utilize advanced nonlinear optical imaging methods to record the in situ photoinactivation process and to visualize the real-time phagocytosis difference with or without photoinactivation of this enzyme. Our findings and approach reported here could provide an effective method to eliminate multi-drug resistant intracellular bacteria, and also treat the clinical bacterial infection in the future.
The prevalence of antibiotic resistance and the presence of bacterial persisters increasingly challenge the successful treatment of Staphylococcus aureus infections, and thus poses a great threat to the global health. Here, we present a photonic approach to revive a broad spectrum of antibiotics for eradication of MRSA persisters via photo-disassembly of functional membrane microdomains. Membrane microdomains on MRSA cells are enriched in staphyloxanthin-derived lipids as constituent lipids with co-localized and oligomerized multimeric protein complexes including PBP2a to execute various cellular processes and cell virulence. We demonstrated that the membrane-bound staphyloxinthin is prone to photobleaching by blue light due to triplet-triplet annihilation and thus compromises the membrane integrity. Using high-intensity 460 nm pulsed laser (wide-field illumination, dosage far below human safety limit), we achieved strikingly high staphyloxanthin bleaching efficiency and depth when compared to low-level light sources (quantified by resonance Raman spectroscopy). More importantly, such efficient and selective photolysis of constituent lipids leads to catastrophic disassembly of membrane microdomains, yielding highly compromised cell membrane with nanometer-scale pores created and PBP2a unanchored from cell membrane or dispersed (proved and quantified by immunofluorescence, fluorescence assay, confocal, super-resolution imaging, and Western blotting). The disruption renders MRSA persisters highly traumatized, thus no longer in dormant state (verified by stimulated Raman scattering microscopy). Consequently, cells with compromised membrane are found highly susceptible to a broad spectrum of antibiotics: beta-lactam antibiotics, such as penicillin and cephalosporins, due to PBP2a disassembly; antibiotics that inhibit intracellular activities enabled by effective diffusion via nanometer-scale pores, such as quinolones, aminoglycosides and sulfonamides. These synergistic therapies are validated both in vitro and in clinically relative models including biofilm and mice skin infection model. Collective, our findings unveil the underlying mechanism of photo-disassembly of MRSA membrane microdomains and highlight this photonic approach as a novel platform to revive a broad spectrum of conventional antibiotics and guide the development of new antibiotics for treatment of MRSA infections.
Given that the dearth of new antibiotic development loads an existential burden on successful infectious disease therapy, health organizations are calling for alternative approaches to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report a drug-free photonic approach to eliminate MRSA through photobleaching of staphyloxanthin, an indispensable membrane-bound antioxidant of S. aureus. The photobleaching process, uncovered through a transient absorption imaging study and quantitated by absorption spectroscopy and mass spectrometry, decomposes staphyloxanthin, and sensitizes MRSA to reactive oxygen species attack. Consequently, staphyloxanthin bleaching by low-level blue light eradicates MRSA synergistically with external or internal reactive oxygen species. The effectiveness of this synergistic therapy is validated in MRSA culture, MRSAinfected macrophage cells. Collectively, these findings highlight broad applications of staphyloxanthin photobleaching for treatment of MRSA infections.
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