Raman Study of Pathogenic Candida auris: Imaging Metabolic Machineries in Reaction to Antifungal Drugs.

The multidrug-resistant Candida auris often defies treatments and presently represents a worldwide public health threat. Currently, the ergosterol-targeting Amphotericin B (AmB) and the DNA/RNA-synthesis inhibitor 5-flucytosine (5-FC) are the two main drugs available for first-line defense against life-threatening Candida auris infections. However, important aspects of their mechanisms of action require further clarification, especially regarding metabolic reactions of yeast cells. Here, we applied Raman spectroscopy empowered with specifically tailored machine-learning algorithms to monitor and to image in situ the susceptibility of two Candida auris clades to different antifungal drugs (LSEM 0643 or JCM15448T, belonging to the East Asian Clade II; and, LSEM 3673 belonging to the South African Clade III). Raman characterizations provided new details on the mechanisms of action against Candida auris Clades II and III, while also unfolding differences in their metabolic reactions to different drugs. AmB treatment induced biofilm formation in both clades, but the formed biofilms showed different structures: a dense and continuous biofilm structure in Clade II, and an extra-cellular matrix with a "fluffy" and discontinuous structure in Clade III. Treatment with 5-FC caused no biofilm formation but yeast-to-hyphal or pseudo-hyphal morphogenesis in both clades. Clade III showed a superior capacity in reducing membrane permeability to the drug through chemically tailoring chitin structure with a high degree of acetylation and fatty acids networks with significantly elongated chains. This study shows the suitability of the in situ Raman method in characterizing susceptibility and stress response of different C. auris clades to antifungal drugs, thus opening a path to identifying novel clinical solutions counteracting the spread of these alarming pathogens.

Raman Imaging of Pathogenic Candida auris: Visualization of Structural Characteristics and Machine-Learning Identification.

Invasive fungal infections caused by yeasts of the genus Candida carry high morbidity and cause systemic infections with high mortality rate in both immunocompetent and immunosuppressed patients. Resistance rates against antifungal drugs vary among Candida species, the most concerning specie being Candida auris, which exhibits resistance to all major classes of available antifungal drugs. The presently available identification methods for Candida species face a severe trade-off between testing speed and accuracy. Here, we propose and validate a machine-learning approach adapted to Raman spectroscopy as a rapid, precise, and labor-efficient method of clinical microbiology for C. auris identification and drug efficacy assessments. This paper demonstrates that the combination of Raman spectroscopy and machine learning analyses can provide an insightful and flexible mycology diagnostic tool, easily applicable on-site in the clinical environment.

Antifungal activity of polymethyl methacrylate/Si3N4 composites against Candida albicans.

Previous studies using gram-positive and -negative bacteria demonstrated that hydrolysis of silicon nitride (Si3N4) in aqueous suspensions elutes nitrogen and produces gaseous ammonia while buffering pH. According to immunochemistry assays, fluorescence imaging, and in situ Raman spectroscopy, we demonstrate here that the antipathogenic surface chemistry of Si3N4 can be extended to polymethylmethacrylate (PMMA) by compounding it with a minor fraction (~8 vol.%) of Si3N4 particles without any tangible loss in bulk properties. The hydrolytic products, which were eluted from partly exposed Si3N4 particles at the composite surface, exhibited fungicidal action against Candida albicans. Using a specific nitrative stress sensing dye and highly resolved fluorescence micrographs, we observed in situ congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while these radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. These in situ observations suggest that the surface chemistry of Si3N4 mimics the antifungal activity of macrophages, which concurrently produce NO radicals and superoxide anions (O2•-) resulting in the formation of candidacidal ONOO-. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit the uncontrolled growth of Candida albicans and ensuing candidiasis while being synergic with chemoprophylaxis. STATEMENT OF SIGNIFICANCE: In a follow-up of previous studies of gram-positive and gram-negative bacteria, we demonstrate here that the antipathogenic surface chemistry of Si3N4 could be extended to polymethylmethacrylate (PMMA) containing a minor fraction (~8 vol.%) of Si3N4 particles without tangible loss in bulk properties. Hydrolytic products eluted from Si3N4 particles at the composite surface exhibited fungicidal action against Candida albicans. Highly resolved fluorescence microscopy revealed congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit uncontrolled growth of Candida albicans and ensuing candidiasis in synergy with chemoprophylaxis.

In situ molecular vibration insights into the antibacterial behavior of silicon nitride bioceramic versus gram-negative Escherichia coli.

Gram-negative bacteria represent a substantial fraction of pathogens responsible for periprosthetic infections. Given the increasing resistance of such bacteria to antibiotics, significant efforts are nowadays paid in developing new biomaterial surfaces, which offer resistance against bacterial adhesion and/or possess inherent antibacterial effects. Non-oxide silicon nitride (Si3N4) bioceramic in its polycrystalline form is a biomaterial with inherent antibacterial properties. Building upon previous phenomenological findings, the present study focuses on vibrational analyses of the metabolic response of Escherichia coli at the molecular level. A time-lapse study is conducted upon exposing the bacteria in vitro to Si3N4 bioceramic surfaces. A comparison is carried out with the as-cultured bacterial strain and with bacteria exposed to other commercially available biomaterials, namely, Ti-alloy (Ti6Al4V-ELI) and zirconia-toughened alumina (ZTA) oxide bioceramic tested under exactly the same experimental conditions. The metabolic pathways before and after exposure to different substrates were monitored by means of Raman and FTIR spectroscopies. Results indicated the development of significant osmotic stress in the bacterial strain and constant concentration decreases of its cellular compounds markers over time upon exposure to Si3N4. This ultimately led to bacterial lysis (also confirmed by conventional fluorescence microscopy assays). The main antibacterial effect was of chemical origin and driven by the elution of nitrogen ions from the Si3N4 surface, successively converted into ammonia (NH3) or ammonium (NH4)+ in aqueous solution, depending on environmental pH. The presence of these nitrogen species created osmotic stress in the cytoplasmic space. In answer to the osmotic stress, metabolic rates changed rapidly, the bacterial membrane was damaged, and lysis occurred almost completely within 48 h exposure. The antibacterial behavior exerted by the Si3N4 substrate on E. coli was more effective than that observed on the biomedical Ti6Al4V alloy. Conversely, no lysis but bacterial proliferation was recorded for E. coli exposed to ZTA bioceramic oxide substrates.