基于茶多酚（TP）的广谱抗菌特性,提出其可能抑制变异链球菌（Streptococcus mutans）与白色念珠菌（Candida albicans）双菌种生物膜形成的假说,并通过体外试验初步验证。通过二倍稀释法测定TP对双菌种的最低抑菌浓度（MIC）,利用生长曲线分析TP对浮游菌生长动力学的影响,采用噻唑蓝溴化四唑比色法、结晶紫染色法及菌落形成单位计数法分别检测生物膜代谢活性、生物量、活菌数量及生物膜黏附力,同时测定培养上清液pH并采用实时荧光定量PCR分析TP对双菌种生物膜中毒力相关基因表达的影响。结果表明,TP对S. mutans-C. albicans双菌种的MIC为2.0 mg·mL-1,且浓度依赖性抑制浮游菌生长;TP显著降低生物膜代谢活性、生物量和活菌数;在质量浓度为0.5、1.0 mg·mL-1和2.0 mg·mL-1时,黏附率分别下降至约60%、20%和10%;TP处理组24 h培养上清液pH显著高于对照组,并随浓度升高而升高;分子机制研究表明,2.0 mg·mL-1 TP处理可显著下调双菌种生物膜中S. mutans的gtfB、gtfD、gbpA、ftf、glgC以及C. albicans的als1、hwp1、ras1的表达。本研究初步证实TP在体外能有效抑制双菌种生物膜形成,提示其有望成为调控口腔微生态失衡的天然成分。
Abstract
Based on the broad-spectrum antibacterial activity of tea polyphenols (TP), it was hypothesized that TP could suppress the formation of Streptococcus mutans-Candida albicans dual-species biofilms . Preliminary in vitro validation was conducted. The minimum inhibitory concentration (MIC) against the mixed biofilms was determined by two-fold serial dilution. Planktonic growth kinetics were monitored by optical-density curves. The MTT colorimetric assay, crystal violet staining, and colony-forming unit were used to determine biofilm metabolic activity, biomass, viable cell number, and adhesion strength, respectively. The expression of virulence-related genes in the biofilms of the two species was analyzed by real-time fluorescent quantitative PCR. TP exhibited a MIC of 2.0 mg·mL-1 against the S. mutans-C. albicans dual-species consortium and inhibited planktonic growth in a concentration-dependent manner. TP significantly reduced biofilm metabolic activity, biomass, and viable cell count; At concentrations of 0.5, 1.0, and 2.0 mg·mL-1, adhesion rates dropped to approximately 60%, 20%, and 10%, respectively. The pH of the 24-hour culture supernatant in the TP-treated groups was markedly higher than that of the control and increased with concentration. Mechanistically, TP of 2.0 mg·mL-1 downregulated key virulence gene expression in both species (gtfB, gtfD, gbpA, ftf, glgC in S. mutans and als1, hwp1, ras1 in C. albicans). These results provide initial evidence that TP can disrupt and inhibit mixed S. mutans-C. albicans biofilms in vitro, indicating its potential as a natural agent for modulating oral microecological dysbiosis.
关键词
茶多酚 /
变异链球菌 /
白色念珠菌 /
双菌种生物膜 /
口腔微生态
Key words
tea polyphenols /
Streptococcus mutans /
Candida albicans /
dual-species biofilms /
oral microbiome
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