Centre of Biological Engineering, LIBRO - Laboratório de Investigação em Biofilmes Rosário Oliveira, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
Sci Rep. 2019 Sep 20;9(1):13639. doi: 10.1038/s41598-019-50094-3.
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.
虽然已经有大量研究集中在研究单一物种上，但我们现在面临的挑战是确定种间相互作用如何改变细菌的行为和发病机制。铜绿假单胞菌和金黄色葡萄球菌经常被发现同时感染囊性纤维化患者。奇怪的是，在实验室条件下，它们的相互作用被报告为竞争关系。因此，选择适当的方法对于分析多物种群落至关重要。在这里，我们展示了与 qPCR 定量细菌种群相关的主要偏差，并优化了一种基于 RNA 的 qPCR，不仅能够定量，而且能够通过基因表达定量来表征由铜绿假单胞菌和金黄色葡萄球菌组成的双物种生物膜中的微生物相互作用。qPCR 定量与流式细胞术和基于培养的定量进行了比较。与培养无关和依赖于培养的方法之间的差异可能是生物膜中存在有活力但不可培养细菌的结果。荧光显微镜证实了这一点。qPCR 方法检测活细胞的灵敏度更高，进一步凸显了其定量生物膜群落的潜力。通过使用细菌 RNA 和外源性 mRNA 对照，还可以表征细菌转录组谱，这是该方法的主要优势。
