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中华口腔医学研究杂志(电子版) ›› 2015, Vol. 09 ›› Issue (06) : 442 -448. doi: 10.3877/cma.j.issn.1674-1366.2015.06.002

所属专题: 文献

基础研究

外排子抑制剂对变异链球菌致龋毒力因子的影响
曾荟荟1, 凌均棨1,(), 刘佳1   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2015-10-02 出版日期:2015-12-01
  • 通信作者: 凌均棨
  • 基金资助:
    国家自然科学基金(青年科学基金项目81500836); 广东省医学科学技术研究基金(A2015190)

Effect of an efflux pump inhibitor on cariogenic factors of Streptococcus mutans

Huihui Zeng1, Junqi Ling1,(), Jia Liu1   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2015-10-02 Published:2015-12-01
  • Corresponding author: Junqi Ling
  • About author:
    Corresponding author: Ling Junqi, Email:
引用本文:

曾荟荟, 凌均棨, 刘佳. 外排子抑制剂对变异链球菌致龋毒力因子的影响[J]. 中华口腔医学研究杂志(电子版), 2015, 09(06): 442-448.

Huihui Zeng, Junqi Ling, Jia Liu. Effect of an efflux pump inhibitor on cariogenic factors of Streptococcus mutans[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2015, 09(06): 442-448.

目的

研究外排子抑制剂(EPI)对变异链球菌(S.mutans)及其生物学特性的调控作用。

方法

通过溴化乙锭(EtBr)吸收和外排实验筛选变异链球菌EPI。检测变异链球菌外排子功能被抑制后,其药物敏感性、耐酸性、菌种间竞争能力和生物膜形成能力等生物学特性的改变。EPI筛选采用方差分析,生物学特性检测采用独立样本t检验进行比较,检验水准α = 0.05。

结果

128 μg/ml利血平显著抑制变异链球菌的外排功能且不抑制其生长代谢,可作为变异链球菌的EPI。添加128 μg/ml利血平后,洗必泰对变异链球菌的最小抑菌浓度(MIC)降低75%至0.25 μg/ml;在pH = 3的环境中35 min后无活菌生长,与对照组相比差异有统计学意义;对戈登链球菌的抑菌圈减小[(8.81 ± 0.33)mm VS(14.25 ± 0.51)mm,P<0.0001];代表12和24 h生物膜生物量的A575值分别从1.31 ± 0.01降低63.92%至0.47 ± 0.004(t = 227.267,P<0.0001)和1.94 ± 0.02降低73%至0.52 ± 0.003(t = 223.730,P<0.0001);12 h生物膜总菌量与对照组(73.39 ± 2.56)μm3/μm2相比降低了24.73%,为(55.24 ± 3.20)μm3/μm2t = 9.896,P<0.0001)。

结论

EPI能显著提高变异链球菌对洗必泰的敏感性,降低其耐酸性、菌种间竞争和生物膜形成能力,提示外排子可作为潜在的防治龋病的新靶点,EPI有望成为临床龋病防治的一类新药物。

Objective

To investigate the effect of an efflux pump inhibitor onthe biological characteristics of Streptococcus mutans (S.mutans) .

Methods

Screen efflux pump inhibitor (EPI) of S.mutans through EtBr uptaking and effluxingassays. The tested biological characteristics of S.mutans included MIC of chlorhexidine (CHX) , acid tolerance, competition ability against Streptococcus gordonii (S.gordonii) and the ability to form biofilms. The accumulation of EtBr and biological characteristics were evaluated by ANOVA and t test respectively. When α equaled to 0.05, it was considered to be significant.

Results

When the concentration is 128 μg/ml, reserpine inhibited the function of efflux pumps dramatically without affecting growth and metabolism of S.mutans. Therefore, reserpine can be regarded as a EPI at the concentration of 128 μg/ml. In thepresenceof 128 μg/ml reserpine, MIC of CHX decreased 75% to 0.25 μg/ml; under the environment of pH = 3 for 35 min, no viable bacteria was observed andthe results were statistically different from the results of control groups; inhibition zone against S.gordonii decreased [ (8.81 ± 0.33) mm VS (14.25 ± 0.51) mm, P<0.0001]; values of A575 represented the biomass of biofilm declined from 1.31 ± 0.01 by 63.92% to 0.47 ± 0.004 (t = 227.267, P<0.0001) and 1.94 ± 0.02 by 73% to 0.52 ± 0.003 (t = 223.730, P<0.0001) after 12 h and 24 h of incubation, respectively; the total quantity of bacteria dropped from (73.39 ± 2.56) μm3/μm2 by 24.73% to (55.24 ± 3.20) μm3/μm2 (t = 9.896, P<0.0001) .

Conclusions

EPI promoted Streptococcus mutans′s susceptibility to CHX notably, by weakening its acid tolerance, competition against other species, and its ability to form biofilm. These findings suggested that efflux pumps could be a new target forprevention and treatment of caries, thus EPI might be used as a new categoryof drugsfor combating caries in thefuture.

图1 利血平对变异链球菌吸收EtBr的影响
图2 利血平对变异链球菌外排EtBr的影响
图3 变异链球菌在pH 3.0的甘氨酸缓冲液中各时间点的细菌计数
图4 利血平对变异链球菌生物膜形成能力的影响
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