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

Analisi della formazione di colonie minuscole e della resistenza ai farmaci nella 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.

Autore in primo piano: La terapia fotodinamica come nuovo approccio per indurre piccole colonie nella Candida resistente ai farmaci per la ricerca antifungina

Induzione di piccole colonie in Candida glabrato tramite terapia fotodinamica mediata dal Rose Bengala

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.

Analisi della funzione mitocondriale in piccole colonie di Candida glabrata indotte da bromuro di etidio

Per iniziare a coltivare la Candida glabrata, disporre tutti i materiali sperimentali sulla piattaforma di lavoro. Scegli una singola colonia di C. glabrata da una piastra di peptone destrosio di lievito o YPD Agar. E trasferiscilo in una provetta di vetro contenente 3 millilitri di terreno YPD.Incubare per 14-16 ore a 25 gradi Celsius agitando a 155 giri/min. Dopo l'incubazione, diluire la coltura in YPD fresco per ottenere una densità ottica di 600 nanometri di 0,1. Incubare la coltura diluita a 25 gradi Celsius per sei ore agitando a 155 giri/min.Preparare una sospensione di lievito a una densità ottica a 600 nanometri di 0,1 in YPD a 3 millilitri. Aggiungere 30 microlitri di bromuro di etidio e incubare la sospensione di lievito per una notte a 25 gradi Celsius a 155 giri/min con un angolo di 45 gradi. Il giorno successivo, regolare la densità ottica della coltura a 0,65 con terreno YPD fresco.In una piastra da 96 pozzetti, aggiungere 20 microlitri della sospensione di lievito a 180 microlitri di PBS per pozzetto per una diluizione decuplicata. Piastra 20 microlitri di ciascuna diluizione su quattro quadranti della piastra YPD Agar. Incubare la piastra per una notte a 37 gradi Celsius.Il giorno seguente, selezionare i quadranti da cinque a 80 colonie e aggiungere 20 microlitri di trifenil tetrazolio al 20%. E aggiungi 20 microlitri di cloruro di trifenil tetrazolio al 20% direttamente al centro di ogni colonia. Incubare la piastra a 37 gradi Celsius per 30-40 minuti.Scansiona lastre a 1200 DPI utilizzando uno scanner ottico a colori a 48 bit. L'agente intercalante del DNA, il bromuro di etidio, induce efficacemente piccoli mutanti di 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 ...

Ricerca

Biologia

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

Analisi della funzione mitocondriale in piccole colonie di Candida glabrata indotte da fluconazolo

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

Analisi della funzione mitocondriale nella terapia fotodinamica antifungina indotta da piccole colonie di Candida glabrata