3.2. Methods of Studying Phage-Biofilm Interactions
3.2.研究噬菌体与生物膜相互作用的方法
Phage-biofilm interactions can be studied by a set of approaches that assess biofilm biomass and/or cell viability. These approaches can be divided into culture-based, molecular, physical, chemical, microscopy, and computational and mathematical models (Figure 2). The advantages and disadvantages of the majority of these methods have been thoroughly reviewed (72).
噬菌体与生物膜之间的相互作用可以通过一系列评估生物膜生物量和/或细胞活力的方法进行研究。这些方法可分为培养法、分子法、物理法、化学法、显微镜法以及计算和数学模型（图 2）。大多数这些方法的优缺点都已经过深入研究 ( 72)。
3.2.1. Culture-based methods.
3.2.1.基于文化的方法。
The determination of the number of colony-forming units (CFUs) is the most widely used technique to assess the efficacy of phage killing in biofilms. This technique is based on serial dilutions of bacterial suspensions; it is a straightforward and universally used method. But despite these advantages, CFU determination usually underestimates the number of biofilm cells. Biofilms are composed of a subpopulation of viable but nonculturable cells that normally are not detected by CFUs (84). In addition, the presence of biofilm aggregates also dramatically interferes with cell counting (85).
菌落形成单位（CFU）数量的测定是评估噬菌体在生物膜中杀灭效果的最广泛使用的技术。这种技术基于细菌悬浮液的系列稀释，是一种直接且普遍使用的方法。但尽管有这些优点，CFU 测定通常会低估生物膜细胞的数量。生物膜由可存活但不可培养的细胞亚群组成，这些细胞通常无法被 CFU 检测到 ( 84)。此外，生物膜聚集体的存在也会严重干扰细胞计数 ( 85)。
3.2.2. Molecular methods.
3.2.2.分子方法。
In an alternative to culture-based methods, PCR- or molecular-based methods can be used to study biofilm communities. These approaches allow the quantification of the number of viable cells, usually assessed by quantitative (q) PCR. Unlike CFU determination, regular qPCR frequently overestimates the number of viable cells, as the results are influenced by the presence of eDNA and dead cells (86). To overcome this limitation, Magin et al. (87) used PEMAX® dye, which detects only metabolically active cells, to study the effect of phages against P. aeruginosa biofilms.
除了基于培养的方法外，还可以使用基于 PCR 或分子的方法来研究生物膜群落。这些方法可以量化存活细胞的数量，通常通过定量（q）PCR 进行评估。与测定 CFU 不同，常规 qPCR 经常会高估存活细胞的数量，因为结果会受到 eDNA 和死细胞的影响 ( 86)。为了克服这一局限性，Magin 等人（87）使用 PEMAX ® 染料来研究噬菌体对铜绿微囊桿菌生物膜的影响。
Whole-transcriptome analysis has also been used to study the effect of phages on biofilm cells. Fernández et al. (61) showed that when S. aureus biofilms were exposed to low doses of phage vB_SauM_phiIPLA-RODI (phiIPLA-RODI), the cells entered a unique physiological state that can benefit both prey and predator. This happens because, under phage predation, biofilms are thicker and have higher amounts of eDNA. In addition, RNA sequencing data evidenced that infected biofilms activate a stringent response that can delay phage infection progression, helping both populations tend to an equilibrium (61).
全转录组分析也被用于研究噬菌体对生物膜细胞的影响。Fernández 等人（61）的研究表明，当金黄色葡萄球菌生物膜暴露于低剂量的噬菌体 vB_SauM_phiIPLA-RODI（phiIPLA-RODI）时，细胞会进入一种独特的生理状态，这种状态对猎物和捕食者都有利。这是因为，在噬菌体捕食下，生物膜更厚，eDNA 含量更高。此外，RNA 测序数据证明，受感染的生物膜会激活一种严格的反应，从而延缓噬菌体感染的进程，帮助两个种群趋于平衡 ( 61)。