Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms.
Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms.
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DOI：
10.1186/1471-2180-11-14
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发表时间：
2011-01-19
期刊：
影响因子：
4.2
通讯作者：
Gmür R
中科院分区：
文献类型：
--
作者：
Quevedo B;Giertsen E;Zijnge V;Lüthi-Schaller H;Guggenheim B;Thurnheer T;Gmür R
本研究的目的是设计和评价荧光原位杂交(FISH)探针，用于乳酸菌的单细胞检测和计数，特别是属于主要系统发育类群和种类的口腔乳杆菌和无菌/颗粒菌。众所周知，乳酸菌对探针穿透的抵抗力是众所周知的，因此，通过实验开发了探针特异的检测方案，以提供最大的细胞壁渗透性、探针可及性、杂交严格性和荧光强度。然后，新的分析方法被应用于三个生物膜样本的试点研究中，这些样本来自不同的志愿者在原位携带了10天的不同脱矿的牛釉质盘。溶菌酶和脱色素酶联合处理后，再经脂肪酶处理，可获得最佳的探针穿透和荧光标记。必须为每个探针建立严格的杂交严格性。此后，所有探针都显示了与参考菌株的预期特异性，并标记了牙菌斑中预期的形态类型。将这组探针应用于原位生长的生物膜，只检测到发酵乳杆菌和干酪乳杆菌群的细菌。最具致龋性的生物膜所含发酵乳杆菌细胞数比其他生物膜高两个数量级。在MITS组的所有样本中都发现了高水平的厌氧/颗粒菌和链球菌，而变形链球菌只在一个样本中检出的数量很少。将这些新的组和物种特异的FISH探针应用于形成口腔生物膜的乳酸菌，将使我们能够更清楚地了解牙龈上的生物群，它的空间结构，以及在菌斑稳态和龋齿发展过程中所涉及的结构-功能关系。这些探针应该被证明具有远远超出口腔微生物学领域的价值，因为它们中的许多探针检测到了在各种医疗条件和食品工业中具有重要意义的非口腔物种和系统发育群体。
The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.
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影响因子：
14.9
作者：
Ludwig, W;Strunk, O;Schleifer, KH
通讯作者：
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影响因子：
4.4
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影响因子：
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作者：
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影响因子：
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作者：
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Kinane, Denis F.
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