Department of Otorhinolaryngology-Head and Neck Surgery, Dongguk University Ilsan Hospital, 814 Siksa-Dong, Goyang, Gyeonggi 410-773, South Korea.
Microb Pathog. 2012 Nov-Dec;53(5-6):219-26. doi: 10.1016/j.micpath.2012.08.003. Epub 2012 Aug 29.
Streptococcus pneumoniae is a gram-positive bacterium that causes otitis media, pneumonia, meningitis and sepsis in young children and the elderly. Previous studies reported that pneumococci in different diseases do not behave as planktonic cells, but predominantly show characteristics of a biofilm. In this study we examine the effect of 5-azacytidine on S. pneumoniae, particularly on biofilm formation and investigate the gene expression involved in synthesis of autoinducer-2, competence and DNA repair. The effect of 5-aza on in vitro biofilm formation was studied by the crystal violet microtiter plate method. The S. pneumoniae biofilms were grown with different concentration of 5-azacytidine (15-500 μm), at variable time intervals and the inhibition percentages were calculated. The effects of 5-aza on the morphology of biofilms were analyzed by scanning electron microscope. The relative quantification of 11 genes of biofilms grown with 5-aza involved in autoinducer-2 synthesis, competence and DNA repair was carried out by real-time RT-PCR with respect to biofilms grown without 5-aza. The crystal violet microtiter assay detected a significant inhibitory effect of 5-aza on in vitro biofilm formation, at concentration that did not inhibited planktonic cell growth. The SEM analysis demonstrated thin and disrupted biofilms, without micro-colonies in the samples treated with 5-aza, while these structures were present in the biofilms grown without 5-aza. The relative quantification of gene expression of 5-aza biofilms showed a significant down regulation of genes involved in the methionine and homocysteine recycling pathway which produces quorum sensing molecule autoinducer-2 as by-products. A significant decrease in the expressions of luxS, metK, pfs and cmK was detected. In conclusion, 5-aza inhibits in vitro biofilm formation and decreases the expression of luxS, pfs and metK, which are involved in the synthesis of autoinducer-2 as by-products of the methionine recycling pathway. The inhibitory effect of 5-aza may be either due to down regulation of pfs, luxS and metK or due to accumulation of the toxic substrate of pfs, luxS and metK genes.
肺炎链球菌是一种革兰氏阳性菌，可导致婴幼儿和老年人中耳炎、肺炎、脑膜炎和败血症。先前的研究报告表明，不同疾病中的肺炎球菌并非以浮游细胞的形式存在，而是主要表现为生物膜的特征。在这项研究中，我们研究了 5-氮杂胞苷对肺炎链球菌的影响，特别是对生物膜形成的影响，并研究了参与合成自诱导物-2、感受态和 DNA 修复的基因表达。通过结晶紫微量平板法研究了 5-氮杂胞苷对体外生物膜形成的影响。用不同浓度的 5-氮杂胞苷（15-500μm）在不同时间间隔培养肺炎链球菌生物膜，并计算抑制百分比。用扫描电子显微镜分析 5-氮杂胞苷对生物膜形态的影响。用实时 RT-PCR 相对于无 5-氮杂胞苷生长的生物膜，对生长有 5-氮杂胞苷的生物膜中涉及自诱导物-2 合成、感受态和 DNA 修复的 11 个基因的相对定量进行分析。结晶紫微量平板法检测到 5-氮杂胞苷在不抑制浮游细胞生长的浓度下对体外生物膜形成有显著的抑制作用。SEM 分析表明，用 5-氮杂胞苷处理的样品中生物膜变薄且破裂，没有微集落，而在无 5-氮杂胞苷生长的生物膜中存在这些结构。5-氮杂胞苷生物膜基因表达的相对定量显示，涉及产生群体感应分子自诱导物-2 作为副产物的蛋氨酸和同型半胱氨酸循环途径的基因表达显著下调。luxS、metK、pfs 和 cmK 的表达显著降低。总之，5-氮杂胞苷抑制体外生物膜形成，并降低作为蛋氨酸循环途径副产物的自诱导物-2 合成相关基因 luxS、pfs 和 metK 的表达。5-氮杂胞苷的抑制作用可能是由于 pfs、luxS 和 metK 的下调，也可能是由于 pfs、luxS 和 metK 基因的毒性底物积累。
