Institut für Mikrobiologie und Hygiene, Charité-Universitätsmedizin Berlin, Berlin, Germany.
PLoS One. 2012;7(5):e37583. doi: 10.1371/journal.pone.0037583. Epub 2012 May 24.
The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.
牙周病的多微生物性质反映在龈下菌斑中检测到的生物型多样性以及发现与疾病发展相关的是可疑病原体的联合体而不是单一物种。许多这些微生物已在体外证明相互作用并增强生物膜的整合、存活甚至致病特性。为了研究这些拟议相互作用的体内相关性，我们扩展了软件 daime（微生物生态学中的数字图像分析）的空间排列分析工具。这种修改使我们能够对龈下生物膜物种图像中的微生物共定位进行定量分析，其中生物量局限于整个图像区域的分数，这种情况在医学样本中很常见。先前在体外相互作用的疾病相关红橙色复合体的选定代表（Tannerella forsythia 与 Fusobacterium nucleatum 和 Porphyromonas gingivalis 与 Prevotella intermedia）被选择用于分析，并通过荧光原位杂交用特定荧光探针进行标记。体内生长生物膜的对交叉相关分析显示，F. nucleatum/periodonticum 和 T. forsythia 在短距离（高达 6 µm）紧密聚集，在 1.5 µm 处有明显峰值。虽然这些结果证实了之前在体外对 F. nucleatum 和 T. forsythia 的观察结果，但在体内样本中，P. gingivalis 和 P. intermedia 之间检测到随机的空间分布。总之，我们成功地在临床相关医学样本中对单细胞水平进行了空间排列分析，并通过分析统计上相关数量的不同患者，证明了这种方法用于体内验证体外观察的实用性。更重要的是，这种方法的非培养依赖性使类似的定量分析能够用于“尚未培养”的生物型，这些生物型无法在体外进行特征化。
