不同培养基中耐药株及标准株白念珠菌生物膜法尼醇分泌的比较研究

Production of farnesol in Candida albicans biofilms of resistant and standard strains in different media

目的 比较不同培养基中白念珠菌耐药株及标准株生物膜法尼醇分泌量的差异，了解生物膜各时相法尼醇分泌变化趋势，探讨耐药株法尼醇产生的特点。方法 用氟康唑诱导形成白念珠菌耐药株。将白念珠菌标准株及耐药株分别接种到RPMI 1640、酵母浸出粉蛋白胨葡萄糖（YPD）、无氨基酵母氮源（YNB） ＋ 0.5%葡萄糖（Glu）和RPMI 1640 ＋ 10%胎牛血清（FCS）4种培养基中培养，构建白念珠菌生物膜。倒置显微镜下观察培养24 h时白念珠菌生物膜形态，气相色谱-质谱联用（GC/MS）法检测培养1.5、3、6、12、24、36、48 h法尼醇分泌量。结果 在相同培养基中，耐药株及标准株生物膜形态没有明显差异，但不同培养基中生物膜形态各不相同。三因素重复测量方差分析显示，白念珠菌生物膜法尼醇的分泌有随时间变化的趋势（F = 70.628，P 〈 0.001），耐药株和标准株生物膜形成过程中，法尼醇分泌量在RPMI 1640、YPD及YNB ＋ 0.5% Glu培养基中均先逐渐增加，在生物膜形成后期至成熟期达到高峰值，随后降低。两种菌株在各培养基中法尼醇浓度达到高峰时间不同。两种菌株分泌法尼醇浓度在RPMI 1640 ＋ 10% FCS培养基中均在12 ~ 48 h呈现缓慢上升趋势。培养基不同会影响法尼醇的分泌（F = 176.665，P 〈 0.001），标准株及耐药株生物膜法尼醇分泌量在YNB ＋ 0.5% Glu培养基中较高。此外，在RPMI 1640培养基（36 h：1.157 ± 0.064比0.250 ± 0.075，P 〈 0.05）及YPD培养基（6 h：0.262 ± 0.036比0.055 ± 0.062；12 h：0.730 ± 0.030比0.482 ± 0.024；均P 〈 0.05）中耐药株生物膜法尼醇分泌量显著高于标准株，但在YNB ＋ 0.5% Glu培养基中（36 h）标准株法尼醇分泌量显著高于耐药株（2.950 ± 0.677比0.523 ± 0.266，P = 0.020）。结论 培养基不同可影响白念珠菌生物膜法尼醇的分泌，且耐药株与标准株之间法尼醇分泌有一定差异。

Objective To compare the production of farnesol between Candida albicans （C. albicans） biofilms formed by resistant and standard strains in different media, and to investigate the changing trend of farnesol production in different phases of biofilm formation and the features of farnesol production by resistant C. albicans. Methods Fluconazole-resistant C. albicans strains were induced in vitro. Standard strains and fluconazole-resistant strains of C. albicans were separately inoculated onto different media, including RPMI 1640 medium, yeast extract peptone dextrose （YPD） medium, yeast nitrogen base （YNB） ＋ 0.5% glucose medium, RPMI 1640 ＋ 10% fetal calf serum （FCS）, so as to form C. albicans biofilms. Morphological changes of C. albicans biofilms at 24 hours were observed under an inverted microscope, and gas chromatography-mass spectrometry（GC/MS）was performed to detect the level of farnesol at 1.5, 3, 6, 12, 24, 36 and 48 hours. Results There were no obvious differences in the morphology of C. albicans biofilms between the resistant and standard strains when they were cultured in the same medium, while the morphology of C. albicans biofilms markedly differed between the 2 kinds of strains in the different media. Three-factor analysis of variance showed that the production of farnesol in the C. albicans biofilms changed over time （F = 70.628, P 〈 0.001）. Concretely speaking, during the formation of resistant and standard C. albicans biofilms, the production of farnesol gradually increased in the RPMI 1640, YPD and YNB ＋ 0.5% glucose media until the biofilms matured, then showed a decreasing trend. However, the time to peak levels of farnesol was different between the 2 kinds of strains in these media. Moreover, the levels of farnesol in the 2 kinds of strains both slowly increased in the RPMI 1640 ＋ 10% FCS medium within 12 - 48 hours. Culture media also significantly affected the production of farnesol（F = 176.665, P 〈 0.001）, and the levels of farnesol in the resistant and standard C. albicans biofilms were both higher in the YNB ＋ 0.5% glucose medium. When resistant and standard strains were separately cultured in the RPMI 1640 media and the YPD media, the level of farnesol was significantly higher in the resistant strains than in the standard stains （RPMI 1640 media at 36 hours： 1.157 ± 0.064 vs. 0.250 ± 0.075, P 〈 0.05; YPD media at 6 hours： 0.262 ± 0.036 vs. 0.055 ± 0.062, P 〈 0.05; YPD media at 12 hours： 0.730 ± 0.030 vs. 0.482 ± 0.024, P 〈 0.05）. However, when they were separately cultured in the YNB ＋ 0.5% glucose media, the farnesol level was significantly higher in the standard stains than in the resistant strains （36 hours： 2.950 ± 0.677 vs. 0.523 ± 0.266, P = 0.020）. Conclusion The media markedly affect the production of farnesol in the C. albicans biofilms, and there is a certain difference in the production of farnesol between resistant and standard C. albicans strains.