天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (10): 1199-1203.doi: 10.11958/20160341

• 细胞与分子生物学 • 上一篇    下一篇

右旋-色氨酸对变异链球菌生物膜形成及离散的影响

杨晓月 廖晓辉 叶静 邵灿 王斌 刘颖   

  1. 1 天津医科大学口腔医院牙体牙髓科（邮编 300070）；2 浙江省口腔医院城西分院综合科；3 天津市天津医院口腔科
  • 收稿日期:2016-04-22 修回日期:2016-07-06 出版日期:2016-10-15 发布日期:2016-10-21
  • 通讯作者: 刘颖 E-mail:dentistmoon@163.com
  • 作者简介:杨晓月（1991）， 女， 硕士在读， 主要从事右旋氨基酸对变异链球菌生物膜形成及离散的研究

Effects of D-tryptophan on biofilm formation and dispersal in Streptococcus mutans

YANG Xiaoyue, LIAO Xiaohui, YE Jing, SHAO Can, WANG Bin, LIU Ying   

  1. 1 Department of Endodontics Dentistry, Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, China；2 General Department West Branch of Stomatology Hospital of Zhejiang Province; 3 Department of Stomatology, Tianjin Hospital
  • Received:2016-04-22 Revised:2016-07-06 Published:2016-10-15 Online:2016-10-21
  • Contact: LIU Ying E-mail:dentistmoon@163.com

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摘要： 目的 探讨右旋-色氨酸（D-Trp）对变异链球菌（S. mutans）生物膜形成及离散的影响， 以及在 D-Trp 作用下 S. mutans 对氯己定（CHX）药物敏感性的变化。 方法 吸光度法检测 5.0 mmol/L D-Trp 对悬浮 S. mutans 生长的影响， 非处理组不作 D-Trp 处理； 结晶紫染色法检测 1.0、2.5 及 5.0 mmol/L D-Trp 处理组 S. mutans 生物膜形成的变化， 非处理组不添加 D-Trp； 结晶紫染色法及激光扫描共聚焦显微镜（CLSM）观察 1.0、2.5 及 5.0 mmol/L D-Trp 处理组对 24 h S. mutans 生物膜的离散作用； 刃天青钠盐指示法检测 5.0 mmol/L D-Trp 处理（实验组）和阴性对照组的最小抑菌浓度（MIC）及最小生物膜抑菌浓度（MBIC）。 结果 单菌种悬浮 S. mutans 在 D-Trp 处理组与非处理组的作用下， 28 h 内生长趋势一致， 均从 4 h 开始进入对数期， 22 h 到达平台期。 1.0、2.5 及 5.0 mmol/L D-Trp 处理组与非处理组相比， S. mutans 生物膜在 0~72 h 内生物膜生物量均随时间推移而增加； 同一时间点， 各处理组各时点生物膜生物量均低于非处理组（P＜ 0.05）。 结晶紫染色法示 1.0、2.5 及 5.0 mmol/L D-Trp 处理组生物膜生物量（OD570）均低于非处理组（P＜ 0.01）。 激光扫描共聚焦显微镜观察结果显示， 1.0、2.5 及 5.0 mmol/L D-Trp 处理组均有细菌黏附于介质表面， 处理组生物膜生物量低于非处理组（P＜ 0.01）。 实验组和阴性对照组对 S. mutans 的 MIC 均为 0.073 mg/L，对 S. mutans 的 MBIC 分别为 0.293 mg/L 和 2.344 mg/L，添加 5.0 mmol/L D-Trp 后，CHX 对 S. mutans 的 MBIC 降至 1/8。结论 D-Trp 能够抑制生物膜形成， 促进已形成生物膜离散， 并提高 S. mutans 对 CHX 的敏感性。

关键词: 链球菌, 变异, 生物膜；氯己定, 微生物敏感性试验, 右旋色氨酸, 最小抑菌浓度

Abstract: Objective To investigate the effects of D-tryptophan (D-Trp) on the formation of Streptococcus mutans (S. mutans) biofilm and the dispersal of 24 h-old biofilm, and the drug susceptibility of S. mutans against chlorhexidine (CHX) under the role of D-Trp. Methods Optical density assay was used to evaluate the growth curve of S. mutans exposed to 5.0 mmol/L D-Trp for 28 h. The non-treated group was not added with D-Trp. After treatment with 1.0, 2.5 and 5.0 mmol/L D-Trp, crystal violet staining was used to observe the changes of S. mutans biofilm formation in treatment group and nontreatment group. Crystal violet staining and confocal laser scanning microscopy (CLSM) were applied to illustrate the effects of 1.0, 2.5 and 5.0 mmol/L D-Trp on the dispersal of 24 h-old S. mutans biofilm. Resazurin sodium was used to indicate the effect of 5.0 mmol/L D- Trp on the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) of treatment groups and negative control group. Results The growth curves of planktonic S. mutans within 28 h was consistent in treatment group and the non-treated group, both attained exponential phase after 4 h and reached stationary phase at 22 h. Notably, when compared with non-treated group, the biomass of S. mutans biofilm was increased with time from 0 to 72 h after treatment with 1.0, 2.5 and 5.0 mmol/L D-Trp. And at the same time point, the biomass was significantly less in each subgroup of treatment group than that of non-treated group (P＜ 0.05). Crystal violet staining demonstrated that values of biomass（OD570） were less in treatment groups treated with 1.0, 2.5 and 5.0 mmol/L DTrp than those of non-treated group (P＜ 0.01). CLSM also showed that bacteria was adhered to the surface of media in treatment groups treated with 1.0, 2.5 and 5.0 mmol/L D-Trp. The values of biomass were lower in treatment groups than those of non-treated group (P<0.01). The MIC against S. mutans was 0.073 mg/L in both experimental group and negative control group. The values of MBIC were 0.293 mg/L and 2.344 mg/L in experimental group and negative control group, respectively. Under the action of 5.0 mmol/L D-Trp, the MBIC of S. mutans was reduced to 1/8. Conclusion Results indicate that D-Trp may inhibit the formation of S. mutans biofilm and promote the dispersal of biofilm already formed. DTrp may further help CHX exert its bactericidal activity to S. mutans.

Key words: Streptococcus mutans, biofilms, Chlorhexidine, microbial sensitivity tests, D- tryptophan, the minimum inhibitory concentration