聚氯乙烯材料表面白色念珠菌-表皮葡萄球菌混合生物膜结构观察

STRUCTURE OF CANDIDA ALBICANS-STAPHYLOCOCCUS EPIDERMIDIS MIXED SPECIES BIOFILM ON POLYVINYL CHLORIDE BIOMATERIAL

目的建立聚氯乙烯(polyvinyl chloride,PVC)材料表面白色念珠菌-表皮葡萄球菌混合生物膜体外模型,观察混合生物膜的形成与微观结构。方法取表皮葡萄球菌(ATCC35984)与白色念珠菌(ATCC10231),分别制备浓度为1×106 CFU/m L的悬液并混合后,与直径0.5 cm PVC膜在胰蛋白胨大豆肉汤(tryptic soy broth,TSB)培养基中共培养形成混合生物膜(实验组)。培养2、6、12、24、48、72 h时取PVC膜,激光共聚焦显微镜检测生物膜厚度、单位视野菌落数,并于48 h时测量生物膜内活菌百分比、三维重建PVC膜表面生物膜图像;扫描电镜观察各时间点混合生物膜结构。以单纯PVC膜置于TSB培养基中培养作为对照组。结果对照组各时间点PVC材料表面无病原菌黏附。实验组激光共聚焦显微镜观察示,培养6 h时可见菌落及生物膜形成,随时间延长均逐渐增加,24 h时菌落达高峰,48 h时生物膜厚度达峰值。实验组PVC膜表面菌落数比较,2、6、24 h间以及2、6 h与48、72 h间比较差异有统计学意义(P<0.05),24、48、72 h间比较差异无统计学意义(P>0.05);生物膜厚度除48、72 h间比较差异无统计学意义外(P>0.05),其余各时间点间比较差异均有统计学意义(P<0.05)。培养48 h时,混合生物膜外层活菌百分比明显高于内层及中间层(P<0.05)。三维重建显示混合生物膜表面凹凸不平,突起部分活菌数量较多。扫描电镜观察示,实验组随培养时间增加,PVC膜表面白色念珠菌由孢子状逐渐伸长出现假丝状及菌丝状,表皮葡萄球菌黏附于白色念珠菌周围,逐渐形成复杂的多层次网状混合生物膜。结论白色念珠菌-葡萄球菌混合培养可在PVC材料表面形成结构复杂的混合生物膜,激光共聚焦显微镜、扫描电镜与三维图像重建技术的结合是研究混合生物膜的理想方法。

Objective To establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm on polyvinyl chloride（PVC） material,and to observe mixed species biofilm formation and its microstructure.Methods Staphylococcus epidermidis bacteria（ATCC35984） and Candida albicans fungal（ATCC10231）were co-incubated with 0.5 cm diameter PVC pieces in tryptic soy broth（TSB） to form mixed specie biofilms（experimental group）.At 2,6,12,24,48,and 72 hours,the thicknesses of the biofilms,the number of bacteria per sight,and the percentage of viable cells in biofilms were measured,and three-dimensional images of biofilms were obtained using confocal laser scanning microscope（CLSM） and scanning electron microscope（SEM） at 48 hours.PVC material cultured in the TSB medium served as control group.Results In control group,there was no pathogenic bacteria adhesion on the PVC material surface.In experimental group,CLSM showed that colonies and biofilm formation were found at 6 hours after co-culture,and gradually increased with time.The pathogenic bacteria colonies reached the peak at 24 hours,and biofilm thickness attained peak value at 48 hours.In experimental group,the number of colony was significantly different among 2,6,and 24 hours,and between 2,6 hours and 48,72 hours（P〈0.05）,but no significant difference was found among 24,48,and 72 hours（P〉0.05）.The biofilm thickness showed significant difference between the other time points（P〈0.05） except between 48 and 72 hours（P〉0.05）.The percentage of viable cells in the outer layers of the biofilm was significantly higher than that in inner and middle layers at 48 hours（P〈0.05）.Three-dimensional reconstruction displayed that the surface of mixd species was uneven;living bacterium mainly located at the protuberance,and dead bacteria mainly located at the concaves.SEM image showed that Staphylococcus epidermidis attached to various forms of Candida albicans（spores,pseudohyphae,hyphae） gradually,and formed multilayer reticulate sophisticated structure on the surface of PVC with time.Conclusion Candida albicans-Staphylococcus epidermidis mixed species biofilm is sophisticated in structure.The combination of CLSM,SEM,and three-dimensional image reconstruction technology is ideal for investigation of mixed species biofilm on PVC material.