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

Analyse af dannelse af petite koloni og lægemiddelresistens 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 tilgang til at inducere små kolonier i lægemiddelresistent Candida til svampedræbende forskning

Induktion af små kolonier i Candida-glabrat via rosenbengalmedieret 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.

Mitokondriefunktionsanalyse i ethidiumbromidinducerede petitekolonier af Candida glabrata

For at begynde at dyrke Candida glabrata skal du arrangere alle eksperimentelle materialer på arbejdsplatformen. Vælg en enkelt koloni af C.glabrata fra en gærpeptondextrose eller YPD agarplade. Og overfør det til et glas reagensglas indeholdende 3 ml YPD-medium.Inkuber i 14 til 16 timer ved 25 grader Celsius med omrystning ved 155 RPM. Efter inkubation fortyndes kulturen i frisk YPD for at opnå en optisk densitet på 600 nanometer på 0,1. Den fortyndede kultur inkuberes ved 25 grader Celsius i seks timer under omrystning ved 155 omdr./min.Forbered en gærsuspension til en optisk densitet ved 600 nanometer 0,1 i YPD til 3 ml. Tilsæt 30 mikroliter ethidiumbromid, og inkuber gærsuspensionen natten over ved 25 grader Celsius ved 155 RPM i en 45 graders vinkel. Den næste dag justeres kulturens optiske tæthed til 0,65 med frisk YPD-medium.I en 96 brøndplade tilsættes 20 mikroliter gærsuspension til 180 mikroliter PBS pr. Brønd for en ti gange fortynding. Plade 20 mikroliter af hver fortynding på fire kvadranter af YPD agarpladen. Inkuber pladen natten over ved 37 grader Celsius.Den følgende dag vælges kvadranter med fem til 80 kolonier og tilsættes 20 mikroliter 20% triphenyltetrazolium. Og tilsæt 20 mikroliter 20% triphenyltetrazoliumchlorid direkte på midten af hver koloni. Inkuber pladen ved 37 grader Celsius i 30 til 40 minutter.Scan plader ved 1200 DPI ved hjælp af en 48 bit optisk farvescanner. DNA-interkaleringsmidlet, ethidiumbromid, inducerer effektivt små mutanter af 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

Mitokondriefunktionsanalyse i fluconazol-inducerede petite kolonier af Candida glabrata

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

Mitokondriefunktionsanalyse i svampedræbende fotodynamisk terapi inducerede petite kolonier af Candida glabrata