mitochondrial function analysis in ethidium bromide&#45;induced petite colonies of candida glabrata

Análisis de la formación de pequeñas colonias y la resistencia a los medicamentos en Candida glabrata

Fungal infections, particularly those caused by Candida albicans and increasingly drug-resistant Candida glabrata, pose a serious global threat1. These infections can be deadly, especially for hospitalized patients and those with weakened immune systems. Rising antifungal resistance threatens the control of invasive candidiasis, a severe fungal infection with high mortality, especially from Candida albicans2. Resistant strains hinder effective treatment, potentially increasing both complexity and death rates. In Alameda County, California, USA, C. glabrata has become the most prevalent invasive species3. This shift in the prevalence and distribution of Candida species may be influenced by local healthcare practices, patient demographics, the utilization of antifungal agents, and the prevalence of risk factors for Candida infections.
Petite mutants in Candida, lacking functional mitochondria, reveal how this organelle affects drug response, virulence, and stress resistance4,5. C. glabrata readily forms these colonies, gaining sensitivity to polyenes while losing it to azoles6. Azole sensitivity and respiratory function are intricately linked, with diminished respiration leading to resistance via mitochondrial DNA loss7. Petite colonies of C. glabrata with azole resistance have been isolated from human stool samples from a bone marrow transplant recipient undergoing fluconazole treatment8 and from blood culture bottles of patients with bloodstream infections9. Their potential implications in drug resistance, virulence, and stress response highlight their clinical significance. Additionally, their distinct properties make them valuable tools for investigating fundamental questions in mitochondrial biology5. As research into petite mutants continues, their applications in both clinical and basic research are likely to expand.
This study discovered that photodynamic therapy (PDT) can induce petite colonies in C. glabrata, expanding the range of methods beyond the traditional techniques of exposing C. glabrata to ethidium bromide or fluconazole.

Autor destacado: Terapia fotodinámica como un enfoque novedoso para inducir pequeñas colonias en Candida resistente a los medicamentos para la investigación antifúngica

Inducción de pequeñas colonias en Candida glabrate a través de la terapia fotodinámica mediada por Rose Bengal

The research aims to use photodynamic therapy, also known as PDT, to fight against drug-resistant Candida infection. In this study, we are trying to answer how effectively PDT kills the fungi, and how the virulence of the fungi changes after the treatment. We found petite colonies from the after PDT.Present methods for inducing petite colonies rely on ethidium bromide or fluconazole, which may work through different pathways on drug resistance and the stress responses, compared to PDT. Recently, petite colonies of Candida have become a significant concern in the clinic because they may resist medication and increase mortality rates. Photodynamic therapy has become one of the antifungal treatments and our finding helps breach the gap between traditional methods used to study petite colonies.Our findings provide evidence that PDT can induce petite colonies in drug resistant Candida in-vitro, presenting a groundbreaking method to study petite colonies. We'll definitely delve deeper into this field. There are many questions to be answered.How and why do petite colonies form after PDT stress? Do different Candida species respond differently? I believe it's a never ending story to be explored.

Análisis de la función mitocondrial en pequeñas colonias de Candida glabrata inducidas por bromuro de etidio

Para comenzar el cultivo de Candida glabrata, coloque todos los materiales experimentales en la plataforma de trabajo. Elija una sola colonia de C. glabrata de una placa de levadura, peptona dextrosa o agar YPD. Y transfiéralo a un tubo de ensayo de vidrio que contenga 3 mililitros de medio YPD.Incubar durante 14 a 16 horas a 25 grados centígrados con agitación a 155 RPM. Después de la incubación, diluir el cultivo en YPD fresco para obtener una densidad óptica de 600 nanómetros de 0,1. Incubar el cultivo diluido a 25 grados centígrados durante seis horas agitando a 155 RPM.Prepare una suspensión de levadura a una densidad óptica de 600 nanómetros de 0,1 pulgadas YPD a 3 mililitros. Agregue 30 microlitros de caldo de bromuro de etidio e incube la suspensión de levadura durante la noche a 25 grados Celsius a 155 RPM en un ángulo de 45 grados. Al día siguiente, ajuste la densidad óptica del cultivo a 0,65 con medio YPD fresco.En una placa de 96 pocillos, agregue 20 microlitros de la suspensión de levadura a 180 microlitros de PBS por pocillo para una dilución diez veces mayor. Coloque 20 microlitros de cada dilución en cuatro cuadrantes de la placa de agar YPD. Incubar la placa durante la noche a 37 grados centígrados.Al día siguiente, seleccione cuadrantes con cinco a 80 colonias y agregue 20 microlitros de trifenil tetrazolio al 20%. Y agregue 20 microlitros de cloruro de trifenil tetrazolio al 20% directamente en el centro de cada colonia. Incuba la placa a 37 grados centígrados durante 30 a 40 minutos.Escanee placas a 1200 DPI con un escáner óptico a todo color de 48 bits. El agente intercalante del ADN, el bromuro de etidio, induce eficazmente mutantes pequeños de C. glabrata.

Research

JoVE Journal

Biology

Mitochondrial Function Analysis in Ethidium Bromide&#45;Induced Petite Colonies of Candida glabrata

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

Investigación

Biología

Mitochondrial Function Analysis in Fluconazole&#45;Induced Petite Colonies of Candida glabrata

Análisis de la función mitocondrial en pequeñas colonias de Candida glabrata inducidas por fluconazol

Mitochondrial Function Analysis in Antifungal Photodynamic Therapy Induced Petite Colonies of Candida glabrata

Análisis de la función mitocondrial en terapia fotodinámica antifúngica inducida por pequeñas colonias de Candida glabrata