HtrA对乳牙高致龋性变异链球菌生物膜形成的影响

Effect of HtrA on the formation of biofilm of Streptococcus mutans with high cariogenicity in primary teeth

目的研究乳牙高致龋性变异链球菌(S.mutans)高温需要蛋白A(HtrA)基因缺陷株和HtrA高毒力株在体外非应激环境中生物膜形成的差异。方法用分光光度计比浊法测定前期获得的乳牙高致龋性S.mutans HtrA高毒力株、基因缺陷株和S.mutans国际标准株UA159的生长情况;体外构建三菌株生物膜模型,扫描电镜下观察生物膜形态结构;结晶紫染色实验评估形成生物膜的量。结果3组S.mutans增殖的各个阶段,与HtrA基因缺陷株相比,HtrA高毒力株与UA159标准株的细菌浓度(OD_(600)值)更高(P<0.05)。扫描电镜下观察HtrA高毒力株密度最大,UA159标准株次之,HtrA缺陷株密度最低。与HtrA基因缺陷株相比,生物膜结晶紫半定量测定4、8、12、24 h HtrA高毒力株OD_(600)值差异均有统计学意义(P<0.001)。结论S.mutans生物膜的形成受到HtrA的影响,HtrA基因的缺失会降低细菌的黏附和减缓生物膜的形成。

Objective To study the difference between the high temperature requirement serine proteinase A(HtrA)gene-deficient Streptococcus strains and high-virulent strains of Streptococcus mutans(S.mutans)in the for-mation of biofilm in vitro on non-stressful environment.Methods Taking the high virulence strain of HtrA screened from high caries-sensitive deciduous teeth obtained in the early stage and the high cariogenic HtrA gene defective strain and the international standard strain UA159 as the research objects,and the growth curve of them was evaluated by spectrophotometer turbidimetry.The biofilm model of the three strains was constructed in vitro,the morphology and structure of the biofilm were observed under scanning electron microscope(SEM),and the amount of biofilm formation was evaluated by crystal purple staining.Results Compared with HtrA gene-deficient strains,the bacterial concentration(OD600 value)of HtrA high virulent strains and UA159 standard strains was higher than that of HtrA gene deficient strain in each stage of S.mutans proliferation in the three groups,and the differences were statistically significant(P<0.05).Under scanning electron microscope,the density of HtrA high virulence strain was the highest,followed by UA159 standard strain,and the density of HtrA deficient strain was the lowest.Semi-quantitative determination of biofilm crystal purple for 4,8,12,24 h,there was significant difference in OD600 value between HtrA high virulence strain and HtrA gene deficient strain(P<0.001).Conclusion The for-mation of S.mutans biofilm is affected by HtrA,and the deletion of HtrA gene reduces bacterial adhesion and slows biofilm formation.