Antibiotic resistance has posed a grand and rising threat to the global health. Blue light, specifically 400-430 nm range, has been shown as an attractive antimicrobial alternative considering its drug/agent-free nature, broad-spectrum antimicrobial effect, and no reported resistance. However, its clinical applications have been hampered by several major bottlenecks. Here, we present our translational development towards clinical application of blue light for managing wound infections via innovations in establishing a safe, effective treatment regimen built upon bacteriostatic and long-term illumination strategy (with therapeutical window identified between minimum inhibitory irradiance, MII, and maximum permissible irradiance, MPI), a wearable LED array-based device prototype, an in vivo testbed of free-moving rats with skin wound infections, and its integration with standard wound care procedures. These concepts, devices, safety, and effectiveness have been validated in vitro, ex vivo, and in vivo. This work paves a solid yet encouraging foundation for our follow-up clinical study on contaminated/infected wounds.
Catalase plays an essential role in degrading hydrogen peroxide (H2O2), which is one of the major enzymatic ROS scavenging mechanisms. Here, using wild-type Candida albicans along with its catalase-deficient mutant, we report that catalase inside fungi could be effectively and universally inactivated by blue light 410 nm, subsequently rendering these pathogens extremely sensitive to H2O2 and ROS-generating agents. This strategy could also significantly eradicate multiple notorious clinical Candida strains, including Candida auris. The antimicrobial efficacy of catalase photoinactivation is further validated using immune cell co-culturing system and a Candida albicans-induced mouse model of skin abrasion. Taken together, our findings offer a novel catalase-targeting approach against multidrug-resistant fungal infections.
Candida auris, the deadly infectious fungus, was reported to infest nearly 60 hospitals and more than 90 nursing homes in New York City. Moreover, these fungal species have developed resistance to all three major anti-fungal drugs. Drug-resistant Candida spp. and other non-albicans have developed multi-drug resistance around the world. Here, we show that, through efficient photoinactivation of an essential detoxifying enzyme which exists in most of the fungal strains, we could achieve significant eradication of those pathogens by subsequent administration of low-concentration of hydrogen peroxide and antifungal drugs. Noteworthy, hydrogen peroxide or antifungal alone is not effective to eradicate them.
Candidemia remains the fourth most common cause of nosocomial bloodstream infections. For more than a half-century, amphotericin B (Amp B) has been the last line of defense in the treatment of life-threatening fungal infections. However, during the past several years, severe infections due to Amp B-resistant Candida spp. isolates have been increasingly reported. Here, through polarization stimulated Raman scattering microscopy, Amp B was found to accumulate largely in the cell membrane of Candida spp. in a highly orientated approach, and the interaction between Amp B and ergosterol was investigated as well. Moreover, we found that the correlation between Amp B and ergosterol in Amp B-susceptible Candida spp. is different from that of Amp B-resistant Candida spp., which provides us important information to understand the working mechanism of Amp B, and to achieve fast determination of the Amp B susceptibility of Candida spp.
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.
KEYWORDS: Image resolution, In vivo imaging, Fiber optics, Ultrasonography, Mirrors, Imaging systems, Real time imaging, Photoacoustic imaging, Absorption, Image processing, High speed imaging
Intravascular photoacoustic-ultrasound (IVPA-US) imaging is an emerging hybrid modality for the detection of lipidladen plaques by providing simultaneous morphological and lipid-specific chemical information of an artery wall. The clinical utility of IVPA-US technology requires real-time imaging and display at speed of video-rate level. Here, we demonstrate a compact and portable IVPA-US system capable of imaging at up to 25 frames per second in real-time display mode. This unprecedented imaging speed was achieved by concurrent innovations in excitation laser source, rotary joint assembly, 1 mm IVPA-US catheter, differentiated A-line strategy, and real-time image processing and display algorithms. By imaging pulsatile motion at different imaging speeds, 16 frames per second was deemed to be adequate to suppress motion artifacts from cardiac pulsation for in vivo applications. Our lateral resolution results further verified the number of A-lines used for a cross-sectional IVPA image reconstruction. The translational capability of this system for the detection of lipid-laden plaques was validated by ex vivo imaging of an atherosclerotic human coronary artery at 16 frames per second, which showed strong correlation to gold-standard histopathology.
Lipid deposition inside the arterial wall is a hallmark of plaque vulnerability. Overtone absorption-based intravascular photoacoustic (IVPA) catheter is a promising technology for quantifying the amount of lipid and its spatial distribution inside the arterial wall. Thus far, the clinical translation of IVPA technology is limited by its slow imaging speed due to lack of a high-power and high-repetition-rate laser source for lipid-specific excitation at 1.7 μm. Here, we demonstrate a potassium titanyl phosphate-based optical parametric oscillator (OPO) with output pulse energy up to 2 mJ at a wavelength of 1724 nm and with a repetition rate of 500 Hz. This OPO enabled IVPA imaging at 1 frame per sec, which is about 50-fold faster than previously reported IVPA systems. The IVPA imaging system was characterized by a pencil lead and a lipid-mimicking phantom for its imaging resolution, sensitivity, and specificity, respectively. Its performance was further validated by ex vivo study of an atherosclerotic human femoral artery and comparison to gold standard histology.
Atherosclerotic plaque at the carotid bifurcation is the underlying cause of the majority of ischemic strokes. Noninvasive imaging and quantification of the compositional changes preceding gross anatomic changes within the arterial wall is essential for diagnosis of disease. Current imaging modalities such as duplex ultrasound, computed tomography, positron emission tomography are limited by the lack of compositional contrast and the detection of flow-limiting lesions. Although high-resolution magnetic resonance imaging has been developed to characterize atherosclerotic plaque composition, its accessibility for wide clinical use is limited. Here, we demonstrate a fiber-based multispectral photoacoustic tomography system for excitation of lipids and external acoustic detection of the generated ultrasound. Using sequential ultrasound imaging of ex vivo preparations we achieved ~2 cm imaging depth and chemical selectivity for assessment of human arterial plaques. A multivariate curve resolution alternating least squares analysis method was applied to resolve the major chemical components, including intravascular lipid, intramuscular fat, and blood. These results show the promise of detecting carotid plaque in vivo through esophageal fiber-optic excitation of lipids and external acoustic detection of the generated ultrasound. This imaging system has great potential for serving as a point-ofcare device for early diagnosis of carotid artery disease in the clinic.
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