变异链球菌绿色荧光蛋白报告株在双菌种生物膜研究的应用

Application of green fluorescent protein reporter system in Streptococcus mutans for study on dualspecies biofilms

目的探讨变异链球菌(S.mutans)绿色荧光蛋白(GFP)报告株在单、双菌种生物膜的形成、代谢和抗药性研究的应用。方法采用基因重组技术构建S.mutansGFP报告株C67-1pDM15和UA159pDM15;通过荧光显微镜和荧光光度计,评估报告株转化效率、生长能力和荧光表达规律;构建S.mutansGFP报告株与戈登链球菌(S.gordonii)的单、双菌种生物膜,比较单、双菌种生物膜中S.mutans的生物膜形态、糖代谢活力及氯已定(CHX)作用下的抗药性差异。数据运用单因素方差分析、Pearson相关性分析与独立样本t检验进行统计学分析。结果S.mutansGFP报告株可稳定地表达gfp基因,生长能力和生物膜形成能力与野生株相似;生物膜加入0.2%的葡萄糖后荧光量迅速增加,可检测细菌代谢改变,4h的相对荧光增长值(ΔRLU)可反映生物膜量,二者显著相关(r=0.9818~0.9985,P<0.001);S.gordonii改变S.mutansC67-1和UA159的生物膜结构,抑制UA159菌株生物膜的形成;剩余荧光增长率[ΔRLU(%)]反映耐药能力,单菌种生物膜C67-1和UA159的ΔRLU(%)相似,分别为(70.2±8.0)%和(72.3±7.9)%(t=-0.521,P=0.630),在双菌种生物膜中S.mutans的ΔRLU(%)发生改变,与各自单菌种生物膜相比差异有统计学意义:C67-1升高至(85.6±4.3)%(t=-2.872,P=0.045),UA159降低至(41.2±10.1)%(t=3.551,P=0.024)。结论S.gordonii对S.mutans生物膜形成能力和抗药性的改变具有亚型差异。GFP报告株可作为多菌种生物膜定量与空间结构研究的模式菌。

Objective To investigate the application of green fluorescent protein(GFP)reporter system in Streptococcus mutans(S.mutans)in the formation,metabolism and antimicrobial resistance of single-and dual-species biofilms. Methods The genomic reporter strains C67-1 pDM15 and UA159 pDM15 were constructed by gene recombination technique. The transformation efficiency,growth curve and fluorescence expression of the reporter strains were detected by fluorescent microscopy, spectrophotometer and fluorophotometer. Single-and dual-species biofilms were formed by GFP S.mutans and Streptococcus gordonii(S.gordonii). Glucose metabolism activity and chlorhexidine(CHX)resistance were evaluated by related light unit(RLU). Morphology of the biofilms was observed under fluorescent microscope. The data were analyzed by One-Way ANOVA,Pearson correlation analysis and independent samples t-test. Results The GFP plasmids were effectively expressed in S.mutans strains. Their growth ability and biofilm formation were similar to those of wild-type strains. Upon addition 0.2% of glucose,the fluorescence intensity in the biofilm increased significantly. GFP synthesis can be used as a metabolic activity indicator. A significant correlation was obtained between the relative fluorescence change value (ΔRLU)within 4 h and the biomass(r = 0.9818 ~ 0.9985,P<0.001). S.gordonii altered the biofilm structure of C67-1 and UA159 in the dual-species biofilm,significantly inhibited the biofilm formation of UA159. The residual fluorescence growth rate[ΔRLU(%)]indicating antimicrobial resistance showed that ΔRLU(%)of single-species biofilm C67-1 and UA159 were close,which were(70.2± 8.0)% and(72.3± 7.9)% respectively(t =-0.521,P = 0.630). Compared with the single-species biofilm,the ΔRLU(%)of S.mutans changed significantly in the dual-species biofilms:C67-1 increased to(85.6± 4.3)%(t =-2.872, P = 0.045),while UA159 reduced to(41.2 ± 10.1)%(t = 3.551,P = 0.024). Conclusions The biofilm formation and antimicrobial resistance of S.mutans displayed distinct strain dependence when co-cultured with S.gordonii. GFP reporter can be used as a strain model for quantification and spatial structure research in multi-species biofilms.