Saccharibacteria Nanosynbacter lyticus type strain TM7x exhibits a remarkably compact genome and an extraordinarily small cell size. This obligate epibiotic parasite forms a symbiotic relationship with its bacterial host, Actinomyces odontolyticus, strain XH001. Due to its limited genome size, TM7x possesses restrained metabolic capacities, predominantly staying outside its host cells to sustain this symbiotic lifestyle. To comprehend the evolutionary strategies of Saccharibacteria, a thorough understanding of the physical interaction between TM7x and XH001 is imperative. Unexpectedly, we serendipitously found the formation of intrabacterial lipid droplets, a feature scarcely observed inside prokaryotic cells. Additionally, the binding with TM7x led to a substantial alteration in the membrane fluidity of the host cells. Employing label-free non-invasive Raman spectroscopy, we unveiled numerous phenotypical differences between the host cells alone and the TM7x/XH001 co-culture, especially in regard to saturated fatty acids.
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.
This work demonstrates a rapid platform that can determine the antimicrobial susceptibility testing (AST) in cation-adjusted Mueller-Hinton Broth medium, urine and blood by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) incorporation at a single bacterium level. The total AST assay time with the value of the single-cell metabolism inactivation concentration (SC-MIC) obtained is less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples were systematically validated by MIC determined by the Clinical and Laboratory Standards Institute criteria, with a category agreement of 94.6% and 5.4% minor error. Furthermore, SRS imaging of D2O metabolic incorporation can rapidly determine SC-MIC directly in clinical samples for urinary tract infection or septicemia blood infection.
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.
KEYWORDS: Crystals, Microscopy, Absorption, Raman spectroscopy, Blood, Scanning electron microscopy, Signal to noise ratio, In vitro testing, Chemical analysis, Imaging spectroscopy
Hemozoin, the heme detoxification end product in malaria parasites during their growth in the red blood cells (RBCs), serves as an important marker for diagnosis and treatment target of malaria disease. However, the current method for hemozoin-targeted drug screening mainly relies on in-vitro β-hematin inhibition assays, which may lead to false-positive events due to under-representation of the real hemozoin crystal. Quantitative in-situ imaging of hemozoin is highly desired for high-throughput screening of antimalarial drugs and for elucidating the mechanisms of antimalarial drugs. We present transient absorption (TA) imaging as a high-speed single-cell analysis platform with chemical selectivity to hemozoin. We first demonstrated that TA microscopy is able to identify β-hematin, the artificial form of hemozoin, from the RBCs. We further utilized time-resolved TA imaging to in situ discern hemozoin from malaria-infected RBCs with optimized imaging conditions. Finally, we quantitatively analyzed the hemozoin amount in RBCs at different infection stages by single-shot TA imaging. These results highlight the potential of TA imaging for efficient antimalarial drug screening and drug mechanism investigation.
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.
KEYWORDS: In vivo imaging, Skin, In vitro testing, Raman spectroscopy, Tissues, Bacteria, Pathogens, Scanning electron microscopy, Laser therapeutics, Resistance
With the effectiveness of antimicrobials waning because of antimicrobial resistance, it is imperative that novel strategies are investigated for the treatment of infections. Antimicrobial blue light (aBL) is an innovative strategy that has proven efficacy against an array of pathogens, albeit, with different species having variable susceptibilities to the therapy. Quinine was discovered during the mid-17th century as a plant-derived potent antimalarial. More recently, its bactericidal properties were revealed, illustrating its potential as an antimicrobial adjuvant. Here we report a novel combination therapy, aBL+quinine hydrochloride (Q-HCL) for the treatment of multi-drug resistant infections. QHCL successfully potentiated the antimicrobial effects of aBL in numerous microbial pathogens of different etiologies, in vitro and in vivo. In addition, it synergistically improved the antimicrobial effects of aBL against bacterial biofilms. Raman spectroscopy revealed that concurrent exposure of aBL and Q-HCL improved uptake of Q-HCL into bacterial cells, when compared to the non aBL exposed sample. In addition, ultra-pure liquid chromatography (UPLC) revealed that Q-HCL increased the relative abundance of porphyrins in bacteria, suggesting the mechanism of this synergistic interaction is through increased production of intermediate photosensitizing porphyrins arising through perturbation of the heme biosynthesis pathway by Q-HCL. Genotoxic potential of the combination therapy against mouse skin tissue, was evaluated using the TUNEL assay, where it was revealed that a high dose exposure of aBL+Q-HCL (<3x the therapeutic dose) was not genotoxic to mouse skin tissue. In conclusion, the findings strongly suggest the potential of aBL+Q-HCL combination therapy as an alternative to traditional antibiotics for the treatment of localized infections.
Type 2 diabetes is an increasingly prevalent disease, with more than 400 million people worldwide diagnosed in 2016. As a stable and accurate biomarker, glycated hemoglobin (HbA1c) is clinically used to diagnose type 2 diabetes with a threshold of 6.5% HbA1c among total hemoglobin (Hb). Current methods such as boronate affinity chromatography or enzymatic assay involve complex processing of large-volume blood samples, which inhibits real-time measurement in clinic. Moreover, these methods cannot measure the HbA1c fraction at single red blood cell level, thus unable to separate the contribution by diabetes from other factors such as diseases related to lifetime of red blood cells. Here, we demonstrate a transient absorption imaging approach that is able to differentiate HbA1c from Hb based on the excited state dynamics measurement. HbA1c fraction inside a single red blood cell is derived quantitatively through phasor analysis. HbA1c fraction distribution for diabetic blood is found apparently different from that for healthy blood. A mathematical model is developed to derive the long-term glucose concentration in the blood. Our technology provides a new way to study heme modification and to derive clinically important information avoid of glucose fluctuation in the bloodstream.
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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