An in vitro biofilm model system maintaining a highly reproducible species and metabolic diversity approaching that of the human oral microbiome.
An in vitro biofilm model system maintaining a highly reproducible species and metabolic diversity approaching that of the human oral microbiome.
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DOI：
10.1186/2049-2618-1-25
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发表时间：
2013-10-02
期刊：
影响因子：
15.5
通讯作者：
McLean JS
中科院分区：
文献类型：
--
作者：
Edlund A;Yang Y;Hall AP;Guo L;Lux R;He X;Nelson KE;Nealson KH;Yooseph S;Shi W;McLean JS
在过去二十年的研究中，我们对人类口腔中微生物多样性的了解已经大大扩展。然而，迄今为止，关于口腔物种行为的大部分知识来自纯培养方法和结合几种栽培物种的研究，这可能并不能完全反映它们在复杂微生物群落中的功能。在有限数量的培养物种的研究中已经表明，早期口腔生物膜的发育以连续的方式发生，并且持续的低pH值可导致耐酸物种的富集。观察到体外生长的菌斑生物膜微宇宙可以保持类似的pH值曲线，以响应碳水化合物的添加，作为菌斑在体内表明，可以在实验室中建立一个复杂的微生物群落。有鉴于此，我们的主要目标是从合并的唾液接种物中开发一种稳健的体外生物膜模型系统，以研究口腔微生物组的稳定性、再现性和发育，以及其对环境变化的动态响应，从群落到分子水平。比较宏基因组分析证实了生物膜中的代谢潜力与最近可获得的来自健康受试者的口腔宏基因组的高度相似性，作为人类微生物组计划的一部分。生物膜中的分类群落组成的时间序列宏基因组学分析显示，在生物膜发育的48小时期间，生长3小时的主要物种的比例得以维持。通过采用16 S rRNA基因的深度焦磷酸测序来研究该生物膜模型的细菌分类学多样性，我们显示了分类学携带和比例之间的高度再现性：1）单个生物膜样品; 2）在不同日期生长的生物膜批次; 3）DNA提取技术和4）研究实验室。我们的研究表明，我们现在有能力生长稳定的口腔微生物体外生物膜，其含有超过100个操作分类单位（OTU），其代表原始接种物OTU丰富度的60-80%。在生物膜中鉴定了以前未培养的人类口腔分类群（HOT），并贡献了约三分之一的总捕获的16 S rRNA基因多样性。据我们所知，这代表了迄今为止体外模型系统报告的最高口腔细菌多样性。这个强大的模型将有助于调查目前未培养的物种和已知的毒力特性，许多口腔病原体不仅限于纯培养系统，但在多物种的生物膜。
Our knowledge of microbial diversity in the human oral cavity has vastly expanded during the last two decades of research. However, much of what is known about the behavior of oral species to date derives from pure culture approaches and the studies combining several cultivated species, which likely does not fully reflect their function in complex microbial communities. It has been shown in studies with a limited number of cultivated species that early oral biofilm development occurs in a successional manner and that continuous low pH can lead to an enrichment of aciduric species. Observations that in vitro grown plaque biofilm microcosms can maintain similar pH profiles in response to carbohydrate addition as plaque in vivo suggests a complex microbial community can be established in the laboratory. In light of this, our primary goal was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum in order to study the stability, reproducibility, and development of the oral microbiome, and its dynamic response to environmental changes from the community to the molecular level. Comparative metagenomic analyses confirmed a high similarity of metabolic potential in biofilms to recently available oral metagenomes from healthy subjects as part of the Human Microbiome Project. A time-series metagenomic analysis of the taxonomic community composition in biofilms revealed that the proportions of major species at 3 hours of growth are maintained during 48 hours of biofilm development. By employing deep pyrosequencing of the 16S rRNA gene to investigate this biofilm model with regards to bacterial taxonomic diversity, we show a high reproducibility of the taxonomic carriage and proportions between: 1) individual biofilm samples; 2) biofilm batches grown at different dates; 3) DNA extraction techniques and 4) research laboratories. Our study demonstrates that we now have the capability to grow stable oral microbial in vitro biofilms containing more than one hundred operational taxonomic units (OTU) which represent 60-80% of the original inoculum OTU richness. Previously uncultivated Human Oral Taxa (HOT) were identified in the biofilms and contributed to approximately one-third of the totally captured 16S rRNA gene diversity. To our knowledge, this represents the highest oral bacterial diversity reported for an in vitro model system so far. This robust model will help investigate currently uncultivated species and the known virulence properties for many oral pathogens not solely restricted to pure culture systems, but within multi-species biofilms.
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