基于RNA的qPCR作为定量和表征双物种生物膜的工具。,Scientific Reports

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基于RNA的qPCR作为定量和表征双物种生物膜的工具。
Scientific Reports ( IF 3.8 ) Pub Date : 2019-09-20 , DOI: 10.1038/s41598-019-50094-3
Andreia Patrícia Magalhães ₁ , Ângela França ₁ , Maria Olívia Pereira ₁ , Nuno Cerca ₁

Affiliation

尽管大量研究集中在研究个体物种上，但我们现在面临着确定物种间相互作用如何改变细菌行为和发病机理的挑战。经常发现铜绿假单胞菌和金黄色葡萄球菌可共同感染囊性纤维化患者。奇怪的是，据报道它们的相互作用在实验室条件下具有竞争性。因此，选择适当的方法对于分析多物种群落至关重要。在本文中，我们证明了与细菌种群的qPCR定量相关的主要偏见，并优化了基于RNA的qPCR，不仅能够定量而且能够表征铜绿假单胞菌和金黄色葡萄球菌组成的双物种生物膜内的微生物相互作用，基因表达定量。将qPCR定量与流式细胞仪和基于培养的定量进行了比较。不依赖培养物的方法和依赖培养物的方法之间的差异可能是由于生物膜内存在可存活但不可培养的细菌的结果。荧光显微镜证实了这一点。检测活细胞的更高灵敏度进一步凸显了qPCR方法定量生物膜群落的潜力。通过使用细菌RNA和外源mRNA对照，还可以表征细菌转录组谱，这是该方法的主要优势。检测活细胞的更高灵敏度进一步凸显了qPCR方法定量生物膜群落的潜力。通过使用细菌RNA和外源mRNA对照，还可以表征细菌转录组谱，这是该方法的主要优势。检测活细胞的更高灵敏度进一步凸显了qPCR方法定量生物膜群落的潜力。通过使用细菌RNA和外源mRNA对照，还可以表征细菌转录组谱，这是该方法的主要优势。

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RNA-based qPCR as a tool to quantify and to characterize dual-species biofilms.

While considerable research has focused on studying individual-species, we now face the challenge of determining how interspecies interactions alter bacterial behaviours and pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are often found to co-infect cystic-fibrosis patients. Curiously, their interaction is reported as competitive under laboratory conditions. Selecting appropriate methodologies is therefore critical to analyse multi-species communities. Herein, we demonstrated the major biases associated with qPCR quantification of bacterial populations and optimized a RNA-based qPCR able not only to quantify but also to characterize microbial interactions within dual-species biofilms composed by P. aeruginosa and S. aureus, as assessed by gene expression quantification. qPCR quantification was compared with flow-cytometry and culture-based quantification. Discrepancies between culture independent and culture dependent methods could be the result of the presence of viable but not-cultivable bacteria within the biofilm. Fluorescence microscopy confirmed this. A higher sensitivity to detect viable cells further highlights the potentialities of qPCR approach to quantify biofilm communities. By using bacterial RNA and an exogenous mRNA control, it was also possible to characterize bacterial transcriptomic profile, being this a major advantage of this method.

更新日期：2019-09-21

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