摘要
采用微量稀释法测定月桂酸单甘油酯(GML)对致病性念珠菌的最小抑菌浓度(MIC)和最小杀菌浓度(MFC),利用时间-抑菌曲线分析GML对念珠菌的动态抑菌作用;通过XTT法测定GML对生物被膜形成早期1~4 h黏附阶段以及成熟生物被膜的抑制作用;采用Sport assay结合激光共聚焦显微镜检测其对念珠菌生物被膜细胞活性和形态结构的影响,进而显微镜观察不同浓度GML作用不同时间抑制菌丝和芽管生成的程度,以及对生物被膜细胞表面疏水性的影响;最后用分子生物学qRT-PCR检测白色念珠菌生物被膜形成相关基因ALS3、HWP1、ECE1、ERG11及克柔念珠菌ERG11在不同处理组中表达水平的变化。结果显示,GML对克柔念珠菌的MIC为15.625 mg/L,MFC为31.25 mg/L,对白色念珠菌的MIC为250 mg/L,MFC为500 mg/L;GML作用有效延长了真菌生长的延滞期;对不同生长阶段(前、中、成熟期)生物被膜均有显著抑制作用,克柔念珠菌生物被膜的最低黏附抑菌质量浓度(SMIC50)为62.5 mg/L,白色念珠菌SMIC50>1000 mg/L;GML不仅可以抑制生物被膜的形成,还可以破坏成熟生物被膜的维持,有效降低生物被膜的厚度和活力;并且对菌丝的形成及芽管长度具有明显的抑制作用,显著降低生物被膜细胞表面疏水性;白色念株菌ALS3、HWP1、ECE1、ERG11和克柔念珠菌ERG11基因均出现了不同倍数的下调趋势。结果表明,GML对浮游态和生物被膜态白色念珠菌和克柔念珠菌具有较好的抑制作用,尤其对克柔念珠菌抑菌效果明显。GML可以通过降低黏附、形态转换和杀真菌作用来抑制致病性念珠菌生物被膜的形成,还可以破坏成熟生物被膜的维持,有效降低生物被膜的厚度和活力。
The minimal inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of glycerol monolaurate(GML)against pathogenic Candida were measured by microdilution method,and the dynamic bacteriostatic effect of GML on Candida was analyzed by time-inhibitory curve,and XTT method assay the inhibitory effect of GML on the adhesion phase and mature biofilm in the early 1-4 h biofilm formation,in order to detect the effect on Candida biofilm cell activity and morphological structure by using a Sport assay combined with laser confocal microscopy,further,the extent of inhibition of mycelial and germ tube formation by different concentrations of GML was observed by microscopy,and the effect of GML on the hydrophobicity and phospholipase activity of biofilm cells,finally,molecular biology techniques qRT-PCR were used to detect the level of biofilm formation-related genes,such as HWP1,ALS3,ERG11 of C.albicans,and ERG11 of C.krusei in the treatment group.The MIC of GML against C.krusei is 15.625 mg/L,the MFC is 31.25 mg/L,the MIC of C.albicans is 250 mg/L,and the MFC is 500 mg/L;GML effectively extended the lag phase of fungal growth;it can inhibit biofilm formation in different growth periods(pre,middle,and mature),The minimum adhesion inhibitory concentration(SMIC50)of C.krusei biofilm is 62.5 mg/L,SMIC50 of C.albicans>1000 mg/L;GML can not only inhibit the formation of biofilm but also destroy the maintenance of mature biofilm,effectively reducing the thickness and vigor of biofilm;It also has obvious inhibit effect on the formation of hyphae,the length of germ tube,and effectively reduce the surface hydrophobicity of biofilm cell;The ALS3,HWP1,ERG11 of C.albicans and ERG11 of C.krusei showed the downward trend of different multiples.GML has a good inhibitory effect on the C.albicans and C.krusei in planktonic and biofilm states,especially for the antibacterial effect on C.krusei.
作者
马生妍
周学章
MA Sheng-yan;ZHOU Xue-zhang(Ministry of Education Key Laboratory of Conservation and U-tilization of Western Characteristic Biological Resources,Ningxia University,Yinchuan 750021,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2020年第8期1536-1545,共10页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31660728)。
