激光共聚焦扫描显微镜观察变异链球菌高致龋力临床株与标准株合成胞外多糖能力差异

Confocal laser scanning microscopy for observing difference in synthesizing exopolysaccharide by highly cariogenic clinical and standard strains of biofilm Streptococcus mutans

目的了解变异链球菌(简称变链菌)593号高致龋力临床分离株在生物膜状态不同时期与标准株ATCC25175(均为血清型c)合成胞外多糖的能力差异。方法在聚苯乙烯塑料片上分别形成3、12、20 h变链菌生物膜标本,采用异硫氰酸荧光素标记的伴刀豆球蛋白A对合成的胞外多糖进行染色,用激光共聚焦扫描显微镜(CLSM)观察。采用静置法培养形成黏附生长的细菌,蒽酮法测定3～24 h其合成胞外多糖的量。结果胞外多糖与染料结合后发出绿色荧光,各时间段593号临床株绿色荧光的分布较ATCC 25175标准株更致密和广泛。3～20 h 593号临床株合成水溶性葡聚糖能力强于标准株(P<0.05);3～16 h 593号临床株合成水不溶性葡聚糖能力强于标准株(P<0.05);其他时间两者合成胞外多糖的能力差异无统计学意义(P>0.05)。结论在生物膜形成过程中两菌株合成胞外多糖的模式相同,均随时间延长而合成量增加,但在生物膜形成早期(3～16 h)高致龋力临床株表现出不同于标准株的胞外多糖合成模式,其更强的合成胞外多糖能力可能是其具高致龋力的原因之一,提示研究致龋机制时使用临床株作为研究样本较标准株更为敏感。

Objective To observe the different abilities in synthesizing exopolysaccharides between biofilm cells of Streptococcus mutans（S.mutans） clinical isolate 593 and standard stain S.mutans ATCC 25175（serotype c）.Methods S.mutans biofilm specimens were formed on the polystyrene plastic sheets for 3,12 and 20 h.The exopolysaccharides was stained with fluorescein isothiocyanate-conjugated concanavalin A and were visualized by confocal laser scanning microscopy.The amounts of exopolysaccharides produced by adhesive S.mutans in 3-24 h were determined by the anthrone method.Results The green fluorescence in strain 593 group was stronger and wider than that in the S.mutans ATCC 25175 group,with significant differences found for the amounts of water-soluble glucans during 3-20 h and water-insoluble glucans during 3-16 h（P0.05）.Conclusion The synthesis of exopolysaccharides during formation of biofilm increases with time in both clinical and standard strains of S.mutans.However,the stronger synthesizing ability of strain 593 in the early biofilm formation stage（3-16 h） may be the reason for its higher cariogenic ability,indicating that the clinical isolates may be more sensitive in studying the mechanism of caries pathogenesis.