最近的流行病学研究表明,耐棘白菌素(ECR)光滑念珠菌血液分离株的患病率惊人地增加。已知ECR分离株来自克隆种群的次要亚群,称为棘白菌素坚持者。尽管人们认为具有较高的棘白菌素持久性(ECP)的分离株更有可能发展为ECR,需要更好地理解ECP的含义。此外,用快速,方便,可靠的工具对于促进我们在临床实践中对这一新兴概念的理解至关重要.在这里,使用广泛的离体和体内全身感染模型,我们表明,米卡芬净处理对高ECP分离株的清除效率较低,并且仅产生ECR菌落。此外,我们开发了一种基于流式细胞术的工具,该工具利用基于SYTOX的检测方法对ECP水平进行分层.一旦受到各种对棘白菌素敏感的血液分离物的挑战,与依赖集落形成单位计数的传统方法相比,我们的试验可靠地区分了体外ECP水平,并在离体和体内条件下实时预测了ECP水平.考虑到ECP的高预测值和低预测值分别为92.3%和82.3%,分别,我们的分析显示与传统方法高度一致。总的来说,我们的研究支持临床环境中ECP水平测定的概念,并为高通量环境提供了可扩展的强大工具.该工具的应用促进了突变体和药物库的询问,以进一步了解我们对持久性生物学的理解和设计抗持久性疗法。
目的:光滑念珠菌是一种流行的真菌病原体,能够在巨噬细胞内复制,并迅速产生对一线抗真菌棘白菌素的耐药性。多项研究表明,在致命浓度的棘白菌素中幸存下来的易感细胞的小亚群的存活促进了棘白菌素抗性。重要的是,表现出高抗生素持久性的细菌病原体也会带来很高的负担,并产生更多的抗生素抗性菌落。尽管如此,在光滑梭菌的临床分离株中,棘白菌素持久性(ECP)的含义尚未确定。此外,ECP水平的确定依赖于一种费力且耗时的方法,这很容易发生高度变化。通过利用体内全身感染和离体模型,我们表明,ECP较高的光滑梭菌分离株与较高的负担相关,并且在米卡芬净治疗后更可能产生棘白菌素耐药性。此外,我们开发了一种实时可靠测定ECP水平的方法.因此,我们的研究将表现出高ECP水平的光滑梭菌分离株确定为重要实体,并为测量棘白菌素的持久性提供了可靠和方便的工具。可扩展到其他真菌和细菌病原体。
Recent epidemiological studies documented an alarming increase in the prevalence of
echinocandin-resistant (ECR) Candida glabrata blood isolates. ECR isolates are known to arise from a minor subpopulation of a clonal population, termed
echinocandin persisters. Although it is believed that isolates with a higher
echinocandin persistence (ECP) are more likely to develop ECR, the implication of ECP needs to be better understood. Moreover, replacing laborious and time-consuming traditional approaches to determine ECP levels with rapid, convenient, and reliable tools is imperative to advance our understanding of this emerging concept in clinical practice. Herein, using extensive ex vivo and in vivo systemic infection models, we showed that high ECP isolates are less effectively cleared by micafungin treatment and exclusively give rise to ECR colonies. Additionally, we developed a flow cytometry-based tool that takes advantage of a SYTOX-based assay for the stratification of ECP levels. Once challenged with various collections of
echinocandin-susceptible blood isolates, our assay reliably differentiated ECP levels in vitro and predicted ECP levels in real time under ex vivo and in vivo conditions when compared to traditional methods relying on colony-forming unit counting. Given the high and low ECP predictive values of 92.3% and 82.3%, respectively, our assay showed a high agreement with traditional approach. Collectively, our study supports the concept of ECP level determination in clinical settings and provides a robust tool scalable for high-throughput settings. Application of this tool facilitates the interrogation of mutant and drug libraries to further our understanding of persister biology and designing anti-persister therapeutics.
OBJECTIVE: Candida glabrata is a prevalent fungal pathogen able to replicate inside macrophages and rapidly develop resistance against frontline antifungal echinocandins. Multiple studies have shown that echinocandin resistance is fueled by the survival of a small subpopulation of susceptible cells surviving lethal concentrations of echinocandins. Importantly, bacterial pathogens that exhibit high antibiotic persistence also impose a high burden and generate more antibiotic-resistant colonies. Nonetheless, the implications of
echinocandin persistence (ECP) among the clinical isolates of C. glabrata have not been defined. Additionally, ECP level determination relies on a laborious and time-consuming method, which is prone to high variation. By exploiting in vivo systemic infection and ex vivo models, we showed that C. glabrata isolates with a higher ECP are associated with a higher burden and more likely develop echinocandin resistance upon micafungin treatment. Additionally, we developed an assay that reliably determines ECP levels in real time. Therefore, our study identified C. glabrata isolates displaying high ECP levels as important entities and provided a reliable and convenient tool for measuring echinocandin persistence, which is extendable to other fungal and bacterial pathogens.