Santigli Elisabeth, Trajanoski Slave, Eberhard Katharina, Klug Barbara
Division of Oral Surgery and Orthodontics, Department of Dental Medicine and Oral Health, Medical University of Graz Graz, Austria.
Center for Medical Research, Medical University of Graz Graz, Austria.
Front Microbiol. 2017 Jan 18;7:2142. doi: 10.3389/fmicb.2016.02142. eCollection 2016.
Oral microbiota are considered major players in the development of periodontal diseases. Thorough knowledge of intact subgingival microbiomes is required to elucidate microbial shifts from health to disease. This comparative study investigated the subgingival microbiome of healthy children, possible inter- and intra-individual effects of modified sampling, and basic comparability of subgingival microprints. In five 10-year-old children, biofilm was collected from the upper first premolars and first molars using sterilized, UV-treated paper-points inserted into the subgingival sulcus at eight sites. After supragingival cleaning using an electric toothbrush and water, sampling was performed, firstly, excluding (Mode A) and, secondly, including (Mode B) cleansing with sterile cotton pellets. DNA was extracted from the pooled samples, and primers targeting 16S rRNA hypervariable regions V5 and V6 were used for 454-pyrosequencing. Wilcoxon signed rank test and -test were applied to compare sampling modes. Principal coordinate analysis (PCoA) and average agglomerative hierarchical clustering were calculated with unweighted UniFrac distance matrices. Sample grouping was tested with permutational MANOVA (Adonis). Data filtering and quality control yielded 67,218 sequences with an average sequence length of 243bp (SD 6.52; range 231-255). (2.8-24.6%), (9.2-25.1%), (4.9-50.6%), (16.5-57.4%), and (2.2-17.1%) were the five major phyla found in all samples. Differences in microbial abundances between sampling modes were not evident. High sampling numbers are needed to achieve significance for rare bacterial phyla. Samples taken from one individual using different sampling modes were more similar to each other than to other individuals' samples. PCoA and hierarchical clustering showed a grouping of the paired samples. Permutational MANOVA did not reveal sample grouping by sampling modes ( = 0.914 by = 0.09). A slight modification of sampling mode has minor effects corresponding to a natural variability in the microbiome profiles of healthy children. The inter-individual variability in subgingival microprints is greater than intra-individual differences. Statistical analyses of microbial populations should consider this baseline variability and move beyond mere quantification with input from visual analytics. Comparative results are difficult to summarize as methods for studying huge datasets are still evolving. Advanced approaches are needed for sample size calculations in clinical settings.
口腔微生物群被认为是牙周疾病发展的主要因素。要阐明从健康到疾病过程中的微生物变化，需要深入了解完整的龈下微生物群。这项比较研究调查了健康儿童的龈下微生物群、改良采样方法可能产生的个体间和个体内影响，以及龈下微量印片的基本可比性。在五名10岁儿童中，使用经过消毒和紫外线处理的纸尖插入上颌第一前磨牙和第一磨牙的龈下沟八个部位采集生物膜。在使用电动牙刷和水进行龈上清洁后，首先进行采样（模式A），排除用无菌棉球清洁；其次进行采样（模式B），包括用无菌棉球清洁。从合并样本中提取DNA，并使用靶向16S rRNA高变区V5和V6的引物进行454焦磷酸测序。应用Wilcoxon符号秩检验和t检验比较采样模式。使用未加权UniFrac距离矩阵计算主坐标分析（PCoA）和平均凝聚层次聚类。用置换多变量方差分析（Adonis）检验样本分组。数据过滤和质量控制产生了67218条序列，平均序列长度为243bp（标准差6.52；范围231 - 255）。所有样本中发现的五个主要菌门是厚壁菌门（2.8 - 24.6%）、放线菌门（9.2 - 25.1%）、拟杆菌门（4.9 - 50.6%）、变形菌门（16.5 - 57.4%）和梭杆菌门（2.2 - 17.1%）。采样模式之间微生物丰度的差异不明显。对于罕见细菌门，需要大量采样才能达到显著差异。使用不同采样模式从同一个体采集的样本彼此之间比与其他个体的样本更相似。PCoA和层次聚类显示了配对样本的分组。置换多变量方差分析未揭示按采样模式的样本分组（Adonis检验，F = 0.914，P = 0.09）。采样模式的轻微改变产生的影响较小，这与健康儿童微生物组谱的自然变异性相对应。龈下微量印片的个体间变异性大于个体内差异。微生物群体的统计分析应考虑这种基线变异性，并且不能仅仅局限于定量分析，还需要视觉分析的输入。由于研究庞大数据集的方法仍在不断发展，比较结果难以总结。在临床环境中进行样本量计算需要先进的方法。
