Environmental stress often causes phenotypic changes among pathogenic cryptococci, such as altered antifungal susceptibility, changes in capsule and melanin formation, as well as altered levels of the membrane sterol and antifungal target, ergosterol. We therefore hypothesised that nitrogen limitation, a prevalent environmental stress in the natural habitat of these yeasts, might affect virulence and antifungal susceptibility. We tested the effect of different nitrogen concentrations on capsule, melanin and ergosterol biosynthesis, as well as amphotericin B (AmB) and fluconazole (FLU) susceptibility. This was achieved by culturing cryptococcal strains representing Cryptococcus neoformans and Cryptococcus gattii in media with high (0.53 g/l), control (0.42 g/l) and low (0.21 g/l) NH4Cl concentrations. India ink staining was used to determine capsule thickness microscopically, while melanin and ergosterol content were determined spectrophotometrically. We found that lower nitrogen concentrations enhanced both ergosterol and capsule biosynthesis, while a variable effect was observed on melanisation. Evaluation of drug tolerance using time-kill methodology, as well as tests for FLU heteroresistance, revealed that the low nitrogen cultures had the highest survival percentages in the presence of both AmB and FLU, and showed the highest frequency of FLU heteroresistance, suggesting that nitrogen concentration may indeed influence drug tolerance.

中文翻译：

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氮浓度影响致病隐球菌的两性霉素B和氟康唑的耐受性。

环境压力通常会导致致病隐球菌的表型改变，例如抗真菌药性改变，胶囊和黑色素形成的改变，以及膜固醇和抗真菌靶标麦角固醇的水平改变。因此，我们假设氮限制是这些酵母自然栖息地中普遍存在的环境压力，它可能会影响毒力和抗真菌药性。我们测试了不同氮浓度对胶囊，黑色素和麦角固醇生物合成以及两性霉素B（AmB）和氟康唑（FLU）敏感性的影响。这是通过在高（0.53 g / l），对照（0.42 g / l）和低（0.21 g / l）NH4Cl浓度的培养基中培养代表新型隐球菌和gattii隐球菌的菌株实现的。印度墨水染色用于显微镜下测定胶囊厚度，而黑色素和麦角固醇含量则通过分光光度法测定。我们发现较低的氮浓度会增强麦角固醇和胶囊的生物合成，同时在黑色素化方面观察到不同的作用。使用时间杀灭方法进行的药物耐受性评估以及FLU异抗性测试表明，在AmB和FLU均存在的情况下，低氮培养物的存活率最高，并且显示出FLU异抗性的频率最高，这表明氮浓度确实可能影响药物耐受性。而观察到的黑色素变化效果不一。使用时间杀灭方法进行的药物耐受性评估以及FLU异抗性测试表明，在AmB和FLU均存在的情况下，低氮培养物的存活率最高，并且显示出FLU异抗性的频率最高，这表明氮浓度确实可能影响药物耐受性。而观察到的黑色素变化效果不一。使用时间杀灭方法进行的药物耐受性评估以及FLU异抗性测试表明，在AmB和FLU均存在的情况下，低氮培养物的存活率最高，并且显示出FLU异抗性的频率最高，这表明氮浓度确实可能影响药物耐受性。