摘要
目的研究真菌密度感应分子对羟基苯乙醇(Tyrosol)在白色假丝酵母菌和表皮葡萄球菌混合生物膜形成中的作用,为临床生物材料相关混合微生物感染的预防治疗提供依据。方法 2014年5月将表皮葡萄球菌标准株ATCC 35984及白色假丝酵母菌标准株ATCC 10231混合培养,建立体外生物膜模型,按照是否用Tyrosol处理混合生物膜分为处理组和对照组,于孵育2、4、6、8、12、24、48、72、96h,用结晶紫染色法半定量检测生物膜形成能力;二甲氧唑黄(XTT)比色法评价生物膜体外生长动力学;电子显微镜观察生物膜超微结构;荧光定量PCR分析两组生物膜形成相关基因的表达。结果结晶紫染色法生物膜半定量检测示培养2、4、6h处理组生物膜厚度大于对照组,且差异有统计学意义(P<0.05);XTT法生长动力学检测示培养12、72h处理组生长动力学大于对照组,12h组间比较,差异有统计学意义(P<0.05);扫描电子显微镜显示培养72h,处理组及对照组混合生物膜结构中可见大量表皮葡萄球菌和白色假丝酵母菌孢子、菌丝密集生长形成具有三维立体结构的混合生物膜;荧光定量PCR结果示,处理组在培养6、24h时表皮葡萄球菌生物膜形成相关icaA、fbe、aap基因表达上调,白假丝酵母菌生物膜形成相关als3、hwp1、efg1基因表达下调。结论在真菌密度感应分子Tyrosol的干预下,处理组形成更厚的混合生物膜,可能与该组中表皮葡萄球菌生物膜形成相关icaA、fbe、aap基因的表达上调相关性更密切。
OBJECTIVE To explore the function of fungal quorum sensing molecule tyrosol in formation of Staphy‐lococcus epidermidis‐Candida albicans mixed biofilm ,so as to provide new ideas for prevention and treatment of mixed microorganism infection related to biomaterials .METHODS The strains of S .epidermidis ATCC 35984 and C .albicans ATCC10231 were co‐cultured in May 2014 to form the model of in vitro mixed biofilms which were di‐vided into the treatment group and the control group by tyrosol treatment .Biofilm mass was semi‐quantified by crystal violet semi‐quantitative adherence assay after 2 ,4 ,6 ,8 ,12 ,24 ,48 ,72 ,96 hours of incubation .XTT as‐say was performed to determine the growth kinetics at the same time .Scanning by electron microscopy was used to observe the ultrastructure of biofilms .The real‐time PCR was used to analyze the expression of icaA ,fbe ,aap , hwp1 ,als3 ,and ef g1 genes .RESULTS Crystal violet semi‐quantitative adherence assay showed that the biofilm thickness was significently larger in the treatment group than in the control group after 2 ,4 ,6 hours of incubation （P〈0 .05） .XTT assay for determination of biofilm growth kinetics showed that the growth kinetics was higher in the treatment group than in the control group after 12 ,72 hours of incubation ,there was significant difference in 12 h inter‐group comparison（P〈 0 .05） .The scanning by electron microscopy revealed the three‐dimensional structure of mix‐biofilm made by large amount of S .epidermidis and C .albicans spores and hypha that densely grew .The real‐time PCR showed that the expression of icaA ,fbe ,aap genes related to formation of S.epider‐midisbiofilminthetreatmentgroupwasup‐regulatedandtheexpressionofhwp1,als3,efg1genesrelatedtoC. albicans biofilm was down‐regulated .CONCLUSION With intervention of the fungal quorum sensing molecule ty‐rosol ,the biofilm in the treatment group was thicker ,which may be more closely correlated to the up‐regulation of the expression of icaA , fbe ,aap genes related to formation of S .epidermidis biofilm .
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2015年第19期4321-4324,共4页
Chinese Journal of Nosocomiology
基金
国家自然科学基金资助项目(81260228)
云南省科技厅专项基金资助项目(2011FB208)
