School of Dentistry, University of California at Los Angeles, CA 90095, USA.
Int J Oral Sci. 2011 Apr;3(2):90-7. doi: 10.4248/IJOS11033.
Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as "bait": Fusobacterium nucleatum (F. nucleatum) whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans (S. mutans) whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the "bait" oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The "prey" bacterial mixtures (including known species or natural saliva samples) were added, unbound cells were washed off after the incubation period and the remaining cells were eluted using 0.2 mol x L(-1) glycine. Genomic DNA was extracted, subjected to 16S rRNA PCR amplification and separation of the resulting PCR products by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F. nucleatum adhered to a variety of bacterial species including uncultivable and uncharacterized ones. This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.
关于不同口腔细菌种间共附着的信息对于理解口腔微生物群落内种间相互作用很重要。目前，该主题的知识主要基于已知可培养种间的成对聚集。在这项研究中，我们采用膜结合测定法与聚合酶链反应-变性梯度凝胶电泳（PCR-DGGE）相结合，系统地分析了口腔细菌种间的共附着谱，获得了超越成对聚集的更深入的知识。选择两种口腔细菌作为“诱饵”：已知具有广泛的种间相互作用研究的具核梭杆菌（Fusobacterium nucleatum），以及其相互作用伙伴大多未知的变形链球菌（Streptococcus mutans）。为了能够在细菌混合物中筛选相互作用的伙伴种，将“诱饵”口腔细菌的细胞固定在硝酸纤维素膜上，然后对膜进行洗涤和封闭以防止非特异性结合。添加“猎物”细菌混合物（包括已知种或天然唾液样本），孵育期后洗去未结合的细胞，然后用 0.2mol/L 甘氨酸洗脱剩余的细胞。提取基因组 DNA，进行 16S rRNA PCR 扩增，然后通过 DGGE 分离所得 PCR 产物。从凝胶中回收选定的条带，进行测序，并通过核苷酸 BLAST 搜索不同的数据库进行鉴定。虽然只有少数细菌物种与 S. mutans 结合，但与先前的发现一致，F. nucleatum 与多种细菌物种结合，包括不可培养和未鉴定的细菌物种。这种新方法可以更有效地分析口腔细菌的共附着谱，并有助于系统地研究口腔微生物种间的细菌结合。
