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

Analyse av dannelse av små kolonier og medikamentresistens i 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.

Forfatter Spotlight: Fotodynamisk terapi som en ny tilnærming for å indusere små kolonier i medikamentresistent Candida for soppdrepende forskning

Induksjon av petite kolonier i Candida glabrate via Rose Bengal-mediert fotodynamisk terapi

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.

Mitokondriell funksjonsanalyse i ethidiumbromidinduserte petitekolonier av Candida glabrata

For å begynne å dyrke Candida glabrata, ordne alle eksperimentelle materialer på arbeidsplattformen. Velg en enkelt koloni av C.glabrata fra en gjær peptone druesukker eller YPD Agar plate. Og overfør det til et glassreagensrør som inneholder 3 ml YPD-medium.Inkuber i 14 til 16 timer ved 25 grader Celsius med risting ved 155 RPM. Etter inkubering, fortynn kulturen i fersk YPD for å oppnå en optisk tetthet på 600 nanometer på 0,1. Inkuber den fortynnede kulturen ved 25 grader Celsius i seks timer med risting ved 155 RPM.Forbered en gjærsuspensjon til en optisk tetthet ved 600 nanometer på 0,1 i YPD til 3 milliliter. Tilsett 30 mikroliter Ethidiumbromid-lager, og inkuber gjærsuspensjonen over natten ved 25 grader Celsius ved 155 o / min i en 45 graders vinkel. Neste dag, juster kulturens optiske tetthet til 0,65 med friskt YPD-medium.I en 96 brønnplate, tilsett 20 mikroliter av gjærsuspensjonen til 180 mikroliter PBS per brønn for en ti ganger fortynning. Plate 20 mikroliter av hver fortynning på fire kvadranter av YPD Agar-platen. Inkuber platen over natten ved 37 grader Celsius.Neste dag, velg kvadranter med fem til 80 kolonier og tilsett 20 mikroliter 20% trifenyltetrazolium. Og tilsett 20 mikroliter 20% trifenyltetrazoliumklorid direkte på midten av hver koloni. Inkuber platen ved 37 grader Celsius i 30 til 40 minutter.Skann plater ved 1200 DPI ved hjelp av en 48-biters fullfarge optisk skanner. DNA-interkaleringsmiddelet, Ethidiumbromid, induserer effektivt petite mutanter av 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 ...

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

Mitokondriefunksjonsanalyse i flukonazolinduserte petittkolonier av Candida glabrata

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

Mitokondriell funksjonsanalyse i antifungal fotodynamisk terapi induserte petite kolonier av Candida glabrata