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

Analys av bildning av små kolonier och läkemedelsresistens hos 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.

Författare i fokus: Fotodynamisk terapi som ett nytt tillvägagångssätt för att inducera små kolonier i läkemedelsresistent candida för svampdödande forskning

Induktion av små kolonier i Candida glabrate via Rose Bengal-medierad 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 funktionsanalys i etidiumbromidinducerade små kolonier av Candida glabrata

För att börja odla Candida glabrata, ordna allt experimentellt material på arbetsplattformen. Plocka en enda koloni av C.glabrata från en jäst-pepton-, dextros- eller YPD-agarplatta. Och överför det till ett provrör av glas som innehåller 3 milliliter YPD-medium.Inkubera i 14 till 16 timmar vid 25 grader Celsius med skakning vid 155 varv per minut. Efter inkubation späds kulturen i färsk YPD för att erhålla en optisk densitet på 600 nanometer på 0,1. Inkubera den utspädda kulturen vid 25 grader Celsius i sex timmar med skakning vid 155 varv per minut.Förbered en jästsuspension till en optisk densitet vid 600 nanometer på 0,1 i YPD till 3 milliliter. Tillsätt 30 mikroliter etidbromidbuljong och inkubera jästsuspensionen över natten vid 25 grader Celsius vid 155 rpm vid 45 graders vinkel. Nästa dag, justera kulturens optiska densitet till 0,65 med nytt YPD-medium.I en platta med 96 brunnar, tillsätt 20 mikroliter av jästsuspensionen till 180 mikroliter PBS per brunn för en tiofaldig utspädning. Platta 20 mikroliter av varje utspädning på fyra kvadranter av YPD-agarplattan. Ruva plattan över natten vid 37 grader Celsius.Följande dag, välj kvadranter med fem till 80 kolonier och tillsätt 20 mikroliter 20% trifenyltetrazolium. Och tillsätt 20 mikroliter 20% trifenyltetrazoliumklorid direkt i mitten av varje koloni. Inkubera plattan vid 37 grader Celsius i 30 till 40 minuter.Skanna plattor med 1200 DPI med en 48-bitars optisk fullfärgsskanner. Det DNA-interkalerande medlet, Etidiumbromid, inducerar effektivt små 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

Mitokondriell funktionsanalys i flukonazolinducerade små kolonier av Candida glabrata

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

Mitokondriell funktionsanalys vid svampdödande fotodynamisk terapiinducerade små kolonier av Candida glabrata