Fingerprint Stimulated Raman Scattering Imaging Unveils Ergosteryl Ester as a Metabolic Signature of Azole-Resistant Candida albicans.

Candida albicans (C. albicans), a major fungal pathogen, causes life-threatening infections in immunocompromised individuals. Fluconazole (FLC) is recommended as first-line therapy for treatment of invasive fungal infections. However, the widespread use of FLC has resulted in increased antifungal resistance among different strains of Candida, especially C. albicans, which is a leading source of hospital-acquired infections. Here, by hyperspectral stimulated Raman scattering imaging of single fungal cells in the fingerprint window and pixel-wise spectral unmixing, we report aberrant ergosteryl ester accumulation in azole-resistant C. albicans compared to azole-susceptible species. This accumulation was a consequence of de novo lipogenesis. Lipid profiling by mass spectroscopy identified ergosterol oleate to be the major species stored in azole-resistant C. albicans. Blocking ergosterol esterification by oleate and suppressing sterol synthesis by FLC synergistically suppressed the viability of C. albicans in vitro and limited the growth of biofilm on mouse skin in vivo. Our findings highlight a metabolic marker and a new therapeutic strategy for targeting azole-resistant C. albicans by interrupting the esterified ergosterol biosynthetic pathway.

Catalase Deactivation Increases Dermatophyte Sensitivity to ROS Sources.

As the leading cause of fungal skin infections around the globe, dermatophytes are responsible for a multitude of skin ailments, ranging from athlete's foot to ringworm. Due to the combination of its growing prevalence and antifungal misuse, antifungal-resistant dermatophyte strains like Trichophyton indotineae have begun to emerge, posing a significant global health risk. The emergence of these resistant dermatophytes highlights a critical need to identify alternative methods of treating dermatophyte infections. In our study, we utilized a 405 nm LED to establish that blue light can effectively inactivate catalase within a variety of both susceptible and resistant dermatophytes. Through this catalase inactivation process, light-treated dermatophytes were found to exhibit increased sensitivity to reactive oxygen species (ROS)-producing agents, improving the performance of antimicrobial agents such as H2O2 and amphotericin B. Our findings further demonstrate that light-induced catalase inactivation can inhibit the formation and polarized growth of hyphae from dermatophytes, suppressing biomass formation. Thus, by increasing ROS sensitization and inhibiting hyphal development, catalase-deactivating blue light offers a potential non-invasive and non-drug-reliant method of managing dermatophyte infections, opening new avenues for the potential treatment of these common infections in conjunction with existing treatments.

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches.

Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017-2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Blue Light Improves Antimicrobial Efficiency of Silver Sulfadiazine Via Catalase Inactivation.

Background: Blue light exhibits the ability to deactivate catalase present in pathogens, significantly improving the antimicrobial performance of compounds such as hydrogen peroxide (H2O2). However, H2O2 is not used within clinical settings due to its short half-life, limiting its potential applications. In this study, we explore the usage of Food and Drug Administration-approved and clinically used silver sulfadiazine (SSD) as a potential alternative to H2O2, acting as a reactive oxygen species (ROS)-producing agent capable of synergizing with blue light exposure. Materials and methods: For in vitro studies, bacterial strains were exposed to a continuous wave 405 nm light-emitting diode (LED) followed by treatment with SSD for varying incubation times. For in vivo studies, bacteria-infected murine abrasion wounds were treated with daily treatments of 405 nm LED light and 1% SSD cream for up to 4 days. The surviving bacterial population was quantified through agar plating and colony-forming unit quantification. Results: Through a checkerboard assay, blue light and SSD demonstrated synergistic interactions. Against both gram-negative and gram-positive pathogens, blue light significantly improved the antimicrobial response of SSD within both phosphate-buffered saline and nutrient-rich conditions. Examination into the mechanisms reveals that the neutralization of catalase significantly improves the ROS-producing capabilities of SSD at the exterior of the bacterial cell, producing greater amounts of toxic ROS capable of exerting antimicrobial activity against the pathogen. Additional experiments reveal that the incorporation of light improves the antimicrobial performance of SSD within methicillin-resistant Staphylococcus aureus (MRSA)- and Pseudomonas aeruginosa strain 1 (PAO-1)-infected murine abrasion wounds. Conclusions: As an established, clinically used antibiotic, SSD can act as a suitable alternative to H2O2 in synergizing with catalase-deactivating blue light, allowing for better translation of this technology to more clinical settings and further implementation of this treatment to more complex animal models.

Chromophore-Targeting Precision Antimicrobial Phototherapy.

Phototherapy, encompassing the utilization of both natural and artificial light, has emerged as a dependable and non-invasive strategy for addressing a diverse range of illnesses, diseases, and infections. This therapeutic approach, primarily known for its efficacy in treating skin infections, such as herpes and acne lesions, involves the synergistic use of specific light wavelengths and photosensitizers, like methylene blue. Photodynamic therapy, as it is termed, relies on the generation of antimicrobial reactive oxygen species (ROS) through the interaction between light and externally applied photosensitizers. Recent research, however, has highlighted the intrinsic antimicrobial properties of light itself, marking a paradigm shift in focus from exogenous agents to the inherent photosensitivity of molecules found naturally within pathogens. Chemical analyses have identified specific organic molecular structures and systems, including protoporphyrins and conjugated C=C bonds, as pivotal components in molecular photosensitivity. Given the prevalence of these systems in organic life forms, there is an urgent need to investigate the potential impact of phototherapy on individual molecules expressed within pathogens and discern their contributions to the antimicrobial effects of light. This review delves into the recently unveiled key molecular targets of phototherapy, offering insights into their potential downstream implications and therapeutic applications. By shedding light on these fundamental molecular mechanisms, we aim to advance our understanding of phototherapy's broader therapeutic potential and contribute to the development of innovative treatments for a wide array of microbial infections and diseases.

Blue Light Deactivation of Catalase Suppresses Candida Hyphae Development Through Lipogenesis Inhibition.

Hyphae formation is a key step for fungal penetration into epithelial cells and escaping from macrophages or neutrophils. We found that 405 nm light-induced catalase deactivation results in the inhibition of hyphae growth in Candida albicans. The treatment is capable of inhibiting hyphae growth across multiple hyphae-producing Candida species. Metabolic studies on light-treated C. albicans reveal that light treatment results in a strong reduction in both lipid and protein metabolism. A significant decrease in unsaturated and saturated fatty acids was detected through mass spectroscopy, indicating that the suppression of hyphae through light-induced catalase deactivation may occur through inhibition of lipid metabolism. Initial in vivo tests indicate that blue light treatment can suppress the hyphae forming capabilities of C. albicans within murine abrasion infections. Together, these findings open new avenues for the treatment of Candida fungal infections by targeting their dimorphism.

Photoinactivation of Catalase Sensitizes Candida albicans and Candida auris to ROS-Producing Agents and Immune Cells.

Microbes have developed their own specific strategies to cope with reactive oxygen species (ROS). Catalase, a heme-containing tetramer expressed in a broad range of aerobic fungi, shows remarkable efficiency in degrading hydrogen peroxide (H2 O2 ) for fungal survival and host invasion. Here, it is demonstrated that catalase inactivation by blue light renders fungal cells highly susceptible to ROS attack. To confirm catalase as a major molecular target of blue light, wild type Candida albicans are systematically compared with a catalase-deficient mutant strain regarding their susceptibility to ROS through 410 nm treatment. Upon testing a wide range of fungal species, it is found that intracellular catalase can be effectively and universally inactivated by 410 nm blue light. It is also found that photoinactivation of catalase in combination with ROS-generating agents is highly effective in total eradication of various fungal species, including multiple Candida auris strains, the causative agent of the global fungal epidemic. In addition, photoinactivation of catalase is shown to facilitate macrophage killing of intracellular Candida albicans. The antifungal efficacy of catalase photoinactivation is further validated using a C. albicans-induced mouse model of skin abrasion. Taken together, the findings offer a novel catalase-photoinactivation approach to address multidrug-resistant Candida infections.

Photoinactivation of catalase sensitizes a wide range of bacteria to ROS-producing agents and immune cells.

Bacteria have evolved to cope with the detrimental effects of ROS using their essential molecular components. Catalase, a heme-containing tetramer protein expressed universally in most aerobic bacteria, plays an indispensable role in scavenging excess hydrogen peroxide (H2O2). Here, through use of wild-type and catalase-deficient mutants, we identified catalase as an endogenous therapeutic target of 400-420 nm blue light. Catalase residing inside bacteria could be effectively inactivated by blue light, subsequently rendering the pathogens extremely vulnerable to H2O2 and H2O2-producing agents. As a result, photoinactivation of catalase and H2O2 synergistically eliminated a wide range of catalase-positive planktonic bacteria and P. aeruginosa inside biofilms. In addition, photoinactivation of catalase was shown to facilitate macrophage defense against intracellular pathogens. The antimicrobial efficacy of catalase photoinactivation was validated using a Pseudomonas aeruginosa-induced mouse abrasion model. Taken together, our findings offer a catalase-targeting phototherapy approach against multidrug-resistant bacterial infections.

Granadaene Photobleaching Reduces the Virulence and Increases Antimicrobial Susceptibility of Streptococcus agalactiae.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is increasingly recognized as a major cause of soft tissue and invasive diseases in the elderly and diabetic populations. Antibiotics like penicillin are used with great frequency to treat these infections, although antimicrobial resistance is increasing among GBS strains and underlines a need for alternative methods not reliant on traditional antibiotics. GBS granadaene pigment is related to the hemolysin/cytolysin of GBS, which is critical for the pathogenesis of GBS diseases. Here, we show that photobleaching granadaene dampens the hemolytic activity of GBS. Furthermore, photobleaching of this antioxidant was found to increase GBS susceptibility to killing by reactive oxygen species like hydrogen peroxide. Treatment with light was also shown to affect GBS membrane permeability and contribute to increased susceptibility to the cell membrane-targeting antibiotic daptomycin. Overall, our study demonstrates dual effects of photobleaching on the virulence and antimicrobial susceptibility of GBS and suggests a novel approach for the treatment of GBS infection.

Photo-Disassembly of Membrane Microdomains Revives Conventional Antibiotics against MRSA.

Confronted with the rapid evolution and dissemination of antibiotic resistance, there is an urgent need to develop alternative treatment strategies for drug-resistant pathogens. Here, an unconventional approach is presented to restore the susceptibility of methicillin-resistant S. aureus (MRSA) to a broad spectrum of conventional antibiotics via photo-disassembly of functional membrane microdomains. The photo-disassembly of microdomains is based on effective photolysis of staphyloxanthin, the golden carotenoid pigment that gives its name. Upon pulsed laser treatment, cell membranes are found severely disorganized and malfunctioned to defense antibiotics, as unveiled by membrane permeabilization, membrane fluidification, and detachment of membrane protein, PBP2a. Consequently, the photolysis approach increases susceptibility and inhibits development of resistance to a broad spectrum of antibiotics including penicillins, quinolones, tetracyclines, aminoglycosides, lipopeptides, and oxazolidinones. The synergistic therapy, without phototoxicity to the host, is effective in combating MRSA both in vitro and in vivo in a mice skin infection model. Collectively, this endogenous chromophore-targeted phototherapy concept paves a novel platform to revive conventional antibiotics to combat drug-resistant S. aureus infections as well as to screen new lead compounds.

Nanobiotechnology for the Environment: Innovative Solutions for the Management of Harmful Algal Blooms.

Nanobiotechnology has played important roles in solving contemporary health problems, including cancer and diabetes, but has not yet been widely exploited for problems in food security and environmental protection. Water scarcity is an emerging worldwide problem as a result of climate change and population increase. Current methods of managing water resources are not efficient or sustainable. In this perspective, we focus on harmful algal blooms to demonstrate how nanobiotechnology can be explored to understand microbe-environment interactions and allow for toxin/pollutant detection with significantly improved sensitivity. These capabilities hold potential for future development of sustainable solutions for drinking water management.

Oxygen Tension and Riboflavin Gradients Cooperatively Regulate the Migration of Shewanella oneidensis MR-1 Revealed by a Hydrogel-Based Microfluidic Device.

Shewanella oneidensis is a model bacterial strain for studies of bioelectrochemical systems (BESs). It has two extracellular electron transfer pathways: (1) shuttling electrons via an excreted mediator riboflavin; and (2) direct contact between the c-type cytochromes at the cell membrane and the electrode. Despite the extensive use of S. oneidensis in BESs such as microbial fuel cells and biosensors, many basic microbiology questions about S. oneidensis in the context of BES remain unanswered. Here, we present studies of motility and chemotaxis of S. oneidensis under well controlled concentration gradients of two electron acceptors, oxygen and oxidized form of riboflavin (flavin+), using a newly developed microfluidic platform. Experimental results demonstrate that either oxygen or flavin+ is a chemoattractant to S. oneidensis. The chemotactic tendency of S. oneidensis in a flavin+ concentration gradient is significantly enhanced in an anaerobic in contrast to an aerobic condition. Furthermore, either a low oxygen tension or a high flavin+ concentration considerably enhances the speed of S. oneidensis. This work presents a robust microfluidic platform for generating oxygen and/or flavin+ gradients in an aqueous environment, and demonstrates that two important electron acceptors, oxygen and oxidized riboflavin, cooperatively regulate S. oneidensis migration patterns. The microfluidic tools presented as well as the knowledge gained in this work can be used to guide the future design of BESs for efficient electron production.