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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 investigate the expression of genes associated with the biofilm formation in Staphylococcus epidermidis, such as intercellular adhesion A (ica A) gene, fibrinogen binding protein (fbe) gene, accumulation-associated protein aap) gene in mixed biofilm formation of Staphylococcus epidermidis and Candida albicans. Methods The experiment was divided into three groups: Staphylococcus epidermidis, white leave, white staphylococcus aureus and white staphylococcus aureus were cultured and mixed culture (mixed group) respectively with Staphylococcus epidermidis ATCC35984 (Table group) and Candida albicans ATCC10231 Candida albicans and their in vitro biofilm model. The biofilm formation ability was detected semi-quantitatively by crystal violet staining at 2,4,6,8,12,24,48,72 hrs after culture. The biofilm formation ability of 2,3 bis (2-methoxy-4- nitro-5-sulfophenyl) 5 (phenylamino) Carbonyl] 2H-tetrazolium hydroxide assay, XTT] colorimetric method was used to evaluate the in vitro growth kinetics of biofilms and the ultrastructure of biofilms was observed by scanning electron microscopy for 24 and 72 h. The results of crystal violet staining biomembrane semiquantitative test showed that the biofilm thickening was observed in the mixed group and the epiglottic group at 12 h after culture, and the biofilm at 72 h was mixed Group than in the group of epigallocatechin, compared with the group except 72 h, the rest of the time points were significantly different between the two groups (P <0.05); Bai Nian group 12 h appeared biofilm growth throughout the training cycle Bai Nian read The biofilm thickness of the mixed group was lower than that of the mixed group (P <0.05) .XTT colorimetric growth kinetics test showed that the growth rate of the mixed group was faster than that of the Bai-nian group and exceeded that of the epigallocatechin- There was no significant difference between the two groups at all time points (P> 0.05) except for 12 h (P <0.05) , A value was lower than that of Baimnian group at 4 hours (P> 0.05), and A value at 6 hours was significantly higher than that of Baimin group (P <0.05) , The biofilm with complex structure and maturation was formed in all the groups with the prolongation of culture time.The quantitative expression of fbe, ica A and aap in the mixed group increased by 1.93 and 1.52, 1.46-fold, respectively (all P <0.05) .Conclusion Staphylococcus epidermidis and Candida albicans can form a mixed biofilm more complex than that of single microorganism. The biofilm with mixed biofilm is thicker than single biofilm , Which may be related to the increased expression of ica A, aap and fbe genes of Staphylococcus epidermidis.