Women & Infants Hospital of Rhode Island and Warren Alpert Medical School of Brown University;
Women & Infants Hospital of Rhode Island and Warren Alpert Medical School of Brown University.
J Vis Exp. 2021 May 13(171). doi: 10.3791/62648.
C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.
近平滑假丝酵母(C. parapsilosis,Cp)是某些人群中血流感染的一种新兴病原体。假丝酵母菌属(Candida),包括 Cp,对一线和二线抗真菌药物的耐药性日益增加。Cp 常从手部和皮肤表面以及胃肠道中分离出来。念珠菌定植使个体易发生侵袭性血流感染。为了成功定植或侵袭宿主,酵母必须能够快速黏附于体表面,以防止被宿主防御机制清除。在这里,我们描述了一种在生理流体剪切下测量 Cp 与固定化蛋白黏附的方法,使用商业上可获得的多通道微流控装置中的终点黏附测定法。该方法经过优化,以提高重现性、降低主观性,并允许对单个分离株进行荧光定量。我们还证明,一些临床分离株的 Cp 在模拟哺乳动物宿主的条件下生长时表现出增强的黏附性,而常用的实验室菌株 CDC317 在流体剪切下不具有黏附性。