目的:肺炎克雷伯菌,医院感染的重要机会病原体,以其形成生物膜的能力而闻名。本研究的目的是通过使用聚酯无纺布恒化器和Caco-2细胞系,评估共培养或单一培养的益生菌如何影响肺炎克雷伯菌产生生物膜的能力,并研究潜在的机制。
结果:与鼠李糖乳杆菌和清酒乳杆菌的纯培养物相比,鼠李糖乳杆菌的混合物显著抑制了肺炎克雷伯菌生物膜的形成,L.清酒,通过qPCR和FISH测定,以5:5:1的比例和枯草芽孢杆菌。此外,乳酸菌与枯草芽孢杆菌的组合可以通过使用抑制显著降低肺炎克雷伯菌对Caco-2细胞的粘附,竞争,和置换测定。根据RT-PCR检测,共培养的益生菌能有效抑制肺炎克雷伯菌对Caco-2细胞的吸附,导致肺炎克雷伯菌诱导的促炎细胞因子表达显著降低。此外,HPLC和RT-PCR分析表明,共培养的益生菌能够通过分泌大量有机酸和第二信号分子(c-di-GMP)成功阻止肺炎克雷伯菌生物膜相关基因的表达,导致对生物膜形成的抑制。
结论:清酒的共培养,L.鼠李糖,和枯草芽孢杆菌以5:5:1的比例可以通过下调生物膜相关基因的表达而对致病性肺炎克雷伯菌的定植产生拮抗作用。同时,共培养的益生菌能有效抑制肺炎克雷伯菌对Caco-2细胞的粘附,阻断肺炎克雷伯菌诱导的促炎细胞因子的表达。
OBJECTIVE: Klebsiella pneumoniae, an important opportunistic pathogen of nosocomial inflection, is known for its ability to form biofilm. The purpose of the current study is to assess how co- or mono-cultured probiotics affect K. pneumoniae\'s ability to produce biofilms and investigate the potential mechanisms by using a polyester nonwoven chemostat and a Caco-2 cell line.
RESULTS: Compared with pure cultures of Lactobacillus rhamnosus and Lactobacillus sake, the formation of K. pneumoniae biofilm was remarkably inhibited by the mixture of L. rhamnosus, L. sake, and Bacillus subtilis at a ratio of 5:5:1 by means of qPCR and FISH assays. In addition, Lactobacillus in combination with B. subtilis could considerably reduce the adherence of K. pneumoniae to Caco-2 cells by using inhibition, competition, and displacement assays. According to the RT-PCR assay, the adsorption of K. pneumoniae to Caco-2 cells was effectively inhibited by the co-cultured probiotics, leading to significant reduction in the expression of proinflammatory cytokines induced by K. pneumoniae. Furthermore, the HPLC and RT-PCR analyses showed that the co-cultured probiotics were able to successfully prevent the expression of the biofilm-related genes of K. pneumoniae by secreting plenty of organic acids as well as the second signal molecule (c-di-GMP), resulting in inhibition on biofilm formation.
CONCLUSIONS: Co-culture of L. sake, L. rhamnosus, and B. subtilis at a ratio of 5:5:1 could exert an antagonistic effect on the colonization of pathogenic K. pneumoniae by down-regulating the expression of biofilm-related genes. At the same time, the co-cultured probiotics could effectively inhibit the adhesion of K. pneumoniae to Caco-2 cells and block the expression of proinflammatory cytokines induced by K. pneumoniae.