(p)ppGpp介导饥饿状态粪肠球菌根管生物膜抗菌剂耐受机制研究

Enterococcus faecalis is frequently recovered from root-filled teeth with post treatment disease. It could survive in harsh environment, develop biofilm and be highly resistant to antimicrobial agents. Our previous study demonstrated that the 48h biofilm formed by starved E.faecalis was more resistant to 5.25% NaClO than 6h and 24h biofilm. However, the expression of genes related with drug resistance decreased in 48h biofilm, which indicated that there was no correlation between tolerance of NaClO and those genes. It has been confirmed that stringent response induced by (p)ppGpp leads to antibiotic tolerance of bacteria. We have found expression of relA which encoded a strong bifunctional synthetase/hydrolase in 48h biofilm was higher than that in 6h and 24h biofilm, which may suggested accumulation of (p)ppGpp in 48h biofilm and its potential role in NaClO tolerance of biofilm formed by starved E. faecalis, although the mechanism remains unclear. In this study, we are going to construct ΔrelA mutants to investigate the role of (p)ppGpp in NaClO tolerance of biofilm developed by starved E.faecalis and its mechanism for better control of persistent periapical infections.

粪肠球菌是口腔常见病根管治疗后疾病的优势致病菌，可长期饥饿生存并形成生物膜，对抗菌措施具有较强的抵抗能力。本课题组前期研究发现饥饿状态粪肠球菌48h生物膜较6h和24h生物膜对抗菌剂次氯酸钠敏感性降低，而此时抗药基因表达水平下调，表明其对次氯酸钠抵抗力的增强并非依赖于抗药基因的高表达。研究认为，(p)ppGpp介导的严谨反应是细菌抗生素耐受的重要调控机制。我们发现，(p)ppGpp合成/水解酶RelA编码基因relA在48h生物膜中表达显著增强，提示饥饿状态粪肠球菌可在生物膜状态下产生(p)ppGpp的积累，而后者与生物膜次氯酸钠耐受相关，但尚需证实并阐明机制。本项目拟构建粪肠球菌relA基因突变株，观察(p)ppGpp水平与生物膜次氯酸钠敏感性之间的关系，以期阐明(p)ppGpp介导饥饿状态粪肠球菌生物膜次氯酸钠耐受的机制，为寻找根管治疗药物新靶点、制定临床感染控制策略提供科学依据。

粪肠球菌是根管治疗后疾病的优势致病菌，可长期饥饿生存并形成生物膜，对抗菌措施具有较强抵抗能力。(p)ppGpp介导的严谨反应是细菌抗生素耐受的重要调控机制。粪肠球菌的(p)ppGpp代谢由双功能(p)ppGpp合成/水解酶RelA和小分子合成酶RelQ调控。本课题组前期研究发现饥饿状态粪肠球菌48h生物膜较6h和24h生物膜对次氯酸钠敏感性降低，relA基因表达显著增强，提示饥饿状态粪肠球菌可能在生物膜状态下产生(p)ppGpp的积累，而后者与次氯酸钠敏感性降低相关，但尚需证实。本项目观察了粪肠球菌OG1RF及其ΔrelA、ΔrelQ和ΔrelAΔrelQ突变株的饥饿生存能力、生物膜形成能力、(p)ppGpp水平与生物膜次氯酸钠敏感性之间的关系。发现经30天饥饿培养后，ΔrelA突变株的活菌量高于野生株，ΔrelAΔrelQ突变株活菌量低于野生株，差异具有统计学意义，提示(p)ppGpp与细菌的饥饿耐受相关。扫描电镜与CFU计数显示稳定期和饥饿期的三种突变株48h生物膜的活菌量与野生株相比无明显差别，但细菌间粘附聚集能力较差，排列分散，证明虽然(p)ppGpp不影响生物膜内活菌数量，但与生物膜形态相关。高效液相色谱检测结果显示，OG1RF饥饿诱导48h后生成(p)ppGpp，对数期的OG1RF也能生成少量的(p)ppGpp，而ΔrelAΔrelQ突变株不能形成(p)ppGpp，同时，0.25%次氯酸钠分别作用于稳定期和饥饿期细菌形成的48h生物膜后，三种突变株的活菌比例均显著低于野生株，表明(p)ppGpp在粪肠球菌对次氯酸钠的抵抗性中发挥作用。饥饿状态下生物膜内H2O2和HAQ水平增高，过氧化氢酶和超氧化物歧化酶水平降低，提示粪肠球菌可通过(p)ppGpp的调节作用减少氧化强化剂的产生而增加抗氧化防御能力。本项目进一步针对relA基因，合成ppGpp类似物Relacin，将其作用于内氏放线菌、具核梭杆菌，厌氧消化链球菌，发现其具有明显的杀菌作用。将其与次氯酸钠联合使用，发现Relacin的预处理可以增强次氯酸钠的对粪肠球菌生物膜的破坏作用。综上，本课题初步探讨了（p)ppGpp介导饥饿状态粪肠球菌生物膜次氯酸钠耐受的机制，提出以(p)ppGpp为根管治疗药物新靶点、将其类似物作为根管消毒药物，为制定临床感染控制措施提供了新的途径。