表皮葡萄球菌生物膜形成相关基因在表皮葡萄球菌和白假丝酵母菌混合生物膜形成中的作用研究

FUNCTION OF INTERCELLULAR ADHESION A,FIBRINOGEN BINDING PROTEIN,AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS

目的探讨表皮葡萄球菌生物膜形成相关基因——胞间黏附素A(intercellular adhesion A,ica A)基因、纤维蛋白原结合蛋白(fibrinogen binding protein,fbe)基因、聚集相关蛋白(accumulation-associated protein,aap)基因在表皮葡萄球菌和白假丝酵母菌混合生物膜形成中的作用。方法实验分为3组,用表皮葡萄球菌标准株ATCC35984(表葡组)及白假丝酵母菌标准株ATCC10231(白念组)分别培养及混合培养(混合组),建立表皮葡萄球菌、白假丝酵母菌及二者混合生长的体外生物膜模型。于培养2、4、6、8、12、24、48、72 h,采用结晶紫染色法半定量检测生物膜形成能力,二甲氧唑黄[(2,3 bis(2-methoxy-4-nitro-5-sulfophenyl)5〔(phenylamino)Carbonyl〕2H-tetrazolium hydroxide assay,XTT]比色法评价生物膜体外生长动力学;24、72 h扫描电镜观察生物膜超微结构。荧光定量PCR分析培养72 h表葡组及混合组ica A、fbe、aap基因表达情况。结果结晶紫染色法生物膜半定量检测示,混合组和表葡组均在培养12 h生物膜明显增厚,72 h混合组超过表葡组,组间比较除72 h外,其余各时间点两组比较差异均有统计学意义(P<0.05);白念组12 h出现生物膜的生长,在整个培养周期白念组生物膜厚度均低于混合组(P<0.05)。XTT比色法生长动力学检测示,混合组整体生长速度快于白念组,且48 h后超过表葡组;混合组与表葡组除12 h差异有统计学意义(P<0.05)外,其余各时间点比较差异均无统计学意义(P>0.05);混合组培养2、4 h时A值低于白念组,但比较差异无统计学意义(P>0.05);6 h后各时间点A值均显著高于白念组(P<0.05)。扫描电镜观察示,随培养时间延长各组均形成结构复杂、成熟的生物膜。培养72 h荧光定量PCR检测示,与表葡组相比,混合组fbe、ica A、aap基因表达量分别增高1.93、1.52、1.46倍,差异均有统计学意义(P<0.05)。结论表皮葡萄球菌和白假丝酵母菌混合生长能形成比单一微生物结构更复杂的混合生物膜;混合生物膜较单一微生物生物膜更厚,可能与表皮葡萄球菌ica A、aap、fbe基因表达增加有关。

Objective To explore the function of intercellular adhesion A（ica A）,fibrinogen binding protein（fbe）,and accumulation-associated protein（aap） genes in formation of Staphylococcus epidermidis-Candida albicans mixed species biofilms. Methods The experiment was divided into 3 groups： single culture of Staphylococcus epidermidis ATCC35984（S. epidermidis group） or Candida albicans ATCC10231（C. albicans group）,and co-culture of two strains（mixed group） to build in vitro biofilm model. Biofilm mass was detected by crystal violet semi-quantitative adherence assay at 2,4,6,8,12,24,48,and 72 hours after incubation. XTT assay was performed to determine the growth kinetics in the same time. Scanning electron microscopy（SEM） was used to observe the ultrastructure of the biofilms after 24 and 72 hours of incubation. The expressions of ica A,fbe,and aap genes were analyzed by real-time fluorescent quantitative PCR. Results Crystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 hours in the S. epidermidis and mixed groups; after co-cultured for 72 hours the thickness of biofilm in mixed group was more than that in the S. epidermidis group,and there was significant difference between 2 groups at the other time（P〈0.05） except at 72 hours（P〉0.05）. In C. albicans group,the biofilm started to grow at 12 hours of cultivation,but the thickness of the biofilm was significantly lower than that in the mixed group in all the time points（P〈0.05）. XTT assay showed that the overall growth speed in the mixed group was greater than that in the C. albicans group,and it was greater than that in the S. epidermidis group at 48 hours; there was no significant difference in the growth speed between the mixed groups and the S. epidermidis group in the other time points（P〉0.05） except at 12 hours（P〈0.05）. The absorbance（A） value in the mixed group was lower than that in the S. epidermidis group at 2 and 4 hours,but no significant difference was shown（P〉0.05）; the A value of mixed group was significantly higher than that of the C. albicans group after 6 hours（P〈0.05）. SEM observation showed that mature biofilms with complex structure formed in all groups. The real-time fluorescent quantitative PCR showed the expressions of fbe,ica A,and aap genes in mixed group increased 1.93,1.52,and 1.46 times respectively at 72 hours compared with the S. epidermidis group（P〈0.05）. Conclusion Mixed species biofilms have more complex structure and are thicker than single species biofilms of Staphylococcus epidermidis or Candida albicans,which is related to increased expressions of the ica A,fbe,and aap genes of Staphylococcus epidermidis.