3. METHODS OF STUDYING PHAGE-BIOFILM INTERACTION
3.研究噬菌体与生物膜相互作用的方法
Although numerous methods of biofilm formation have been described in the literature, there is still a lack of standardized and appropriate protocols to simulate real biofilms under laboratory conditions. The type of device used for biofilm formation, the culture media, and the presence of external stresses (e.g., shear forces) will directly influence the biofilm structure, which will have a major impact on the outcome of phage treatment. Below we present an overview of the experimental setups that are most commonly used to form biofilms, as well as the different methods that have been implemented to study phage-biofilm interactions (Figure 2).
尽管文献中描述了许多生物膜形成的方法，但仍然缺乏标准化的适当方案来模拟实验室条件下的真实生物膜。用于生物膜形成的设备类型、培养基以及外部应力（如剪切力）的存在都会直接影响生物膜的结构，从而对噬菌体处理的结果产生重大影响。下面我们将概述最常用于形成生物膜的实验装置，以及研究噬菌体与生物膜相互作用的不同方法（图 2）。
3.1. Experimental Setups for Biofilm Formation
3.1.生物膜形成的实验装置
The choice of an adequate platform for biofilm experiments can determine the outcome results. Numerous factors can influence biofilm formation, structure, and composition and consequently impact phage interaction with biofilm cells.
为生物膜实验选择合适的平台可以决定实验结果。许多因素都会影响生物膜的形成、结构和组成，进而影响噬菌体与生物膜细胞的相互作用。
3.1.1. In vitro models and the influence of biofilm formation conditions.
3.1.1.体外模型和生物膜形成条件的影响。
The majority of in vitro biofilm studies involve the use of microtiter plates as experimental setups (72). The main advantages of using these devices are their low price and the possibility of performing high-throughput studies (72). There are several studies reporting the efficacy of phages against biofilms formed in microtiter plates, and a compilation of them was previously reviewed (6). However, it is with high difficulty that the results obtained using different microtiter devices can be translated to the reality found in different biofilms of environmental, clinical, food industry, or veterinary contexts. The biofilms formed in real conditions face several stresses, namely shear forces under continuous liquid flow that static devices cannot mimic. Therefore, for a better understanding of phage-host interactions, the use of more sophisticated biofilm dynamic models is recommended. Examples of dynamic devices are flow cells, drip flow reactors, modified Robbins devices, and rotary biofilm devices (72). In a study by Rieu et al. (73), time-lapse CLSM was used to characterize the structural dynamics of Listeria monocytogenes growth in static (stainless steel chips on petri dishes) and dynamic (flow cell BST FC 81) conditions. In static conditions, thin unstructured biofilms were observed, while when biofilms were grown under dynamic conditions, they were highly organized with microcolonies surrounded by a network of knitted chains (73). More recently, Yang et al. (74) used nitrogen sparging to induce shear stress on biofilms formed on cubic dual-chamber air-cathode microbial fuel cells with a cation exchange membrane. Using electrochemical impedance, the authors observed that a shear stress–enriched anode biofilm showed a low-charge transfer resistance in comparison with the unperturbed enriched anode biofilm. Moreover, CLSM micrographs clearly indicated that the shear stress–enriched biofilms were entirely viable, in opposition to unperturbed biofilms that exhibited a viable outer layer with a high proportion of dead cells in the inner layers of the biofilm (74). Taken together, these results emphasize the importance of shear stress conditions on the biofilm formation outcome, which ultimately affects interaction with phages.
大多数体外生物膜研究都使用微孔板作为实验装置 ( 72)。使用这些装置的主要优点是价格低廉，而且可以进行高通量研究 ( 72)。有几项研究报告了噬菌体对微孔板中形成的生物膜的疗效，之前已对这些研究进行了综述 ( 6)。然而，使用不同微孔板设备获得的结果很难转化为环境、临床、食品工业或兽医环境中不同生物膜的实际情况。在实际条件下形成的生物膜面临着多种压力，即连续液体流动下的剪切力，而静态设备无法模拟这些压力。因此，为了更好地了解噬菌体与宿主的相互作用，建议使用更复杂的生物膜动态模型。动态装置的例子有流动细胞、滴流反应器、改良罗宾斯装置和旋转生物膜装置 ( 72)。在 Rieu 等人的研究中 ( 73) ，使用了延时 CLSM 来描述单核细胞增生李斯特菌在静态（培养皿上的不锈钢芯片）和动态（流动池 BST FC 81）条件下的生长结构动态。在静态条件下，观察到的是薄而无结构的生物膜，而在动态条件下生长的生物膜则具有高度组织性，微菌落被编织链网络包围 ( 73)。最近，Yang 等人（74）利用氮气喷射对阳离子交换膜立方双室空气阴极微生物燃料电池上形成的生物膜施加剪切应力。作者利用电化学阻抗观察到，与未受扰动的富集阳极生物膜相比，剪切应力富集阳极生物膜显示出较低的电荷转移电阻。此外，CLSM 显微照片清楚地表明，剪切应力富集的生物膜完全存活，而未扰动的生物膜则表现为外层存活，内层死细胞比例较高 ( 74)。综上所述，这些结果强调了剪切应力条件对生物膜形成结果的重要性，而生物膜形成结果最终会影响与噬菌体的相互作用。
Another important feature of biofilm studies is the effect of culture media on the biofilm structure and cells. Most biofilm studies are performed with bacteria growing in rich media. Jones et al. (75) used CLSM to compare the structure of Proteus mirabilis biofilms formed in Luria–Bertani broth and artificial urine. The authors observed that while biofilms formed on rich media displayed the typical mushroom structure with water/nutrient channels, biofilms formed using artificial urine exhibited a flat structure almost deprived of channels (75). Different phage-biofilm interaction studies were assessed on dynamic biofilms using simulated body fluids. In a study using microtiter plates, a phage cocktail containing two enterococci phages successfully reduced the bacterial load after three hours of infection in a medium simulating wound conditions (76). In two other studies (77, 78), phage cocktails were successfully applied on sections of Foley catheters to reduce biofilms grown in artificial urine.
生物膜研究的另一个重要特点是培养基对生物膜结构和细胞的影响。大多数生物膜研究都是在富含培养基的情况下进行的。Jones 等人（75）使用 CLSM 比较了在 Luria-Bertani 肉汤和人工尿液中形成的 mirabilis 变形杆菌生物膜的结构。作者观察到，在富含培养基上形成的生物膜显示出典型的蘑菇结构，具有水/营养通道，而使用人工尿液形成的生物膜则显示出几乎没有通道的扁平结构 ( 75)。使用模拟体液对动态生物膜进行了不同的噬菌体-生物膜相互作用研究评估。在一项使用微孔板的研究中，含有两种肠球菌噬菌体的鸡尾酒噬菌体在模拟伤口条件的培养基中感染三小时后，成功减少了细菌负荷（76）。在另外两项研究中（77、78），噬菌体鸡尾酒被成功应用于福里导尿管的切片上，以减少在人工尿液中生长的生物膜。
Both biofilm-formation devices and conditions used, such as culture media, highly interfere with biofilm structure and composition. As previously discussed, this has a huge influence on the way phages interact with biofilms.
生物膜形成装置和所使用的条件（如培养基）都会对生物膜的结构和组成产生严重干扰。如前所述，这对噬菌体与生物膜相互作用的方式有很大影响。
3.1.2. Ex vivo and in vivo models.
3.1.2.体内外模型。
The majority of studies mimicking real conditions are usually performed to simulate phage therapy against infectious biofilms. Lebeaux et al. (79) discussed the applicability of ex vivo models as an interesting alternative approach to the use of in vivo models. Ex vivo models have reduced alterations of natural conditions, as they involve the use of tissue derived from a living organism in an artificial environment. In comparison to in vivo models, they also allow a more controlled experimental setup, with reduced ethical concerns. For example, phages were applied in porcine skin to simulate wound treatment of infections caused by different pathogens (80, 81). Despite the advantages of using this type of model, the lack of host (human or animal) response and the short duration of experiments are still some hurdles to the widespread implementation of ex vivo models. In that sense, in vivo models are the best choice for studies that intend to understand the pathology of infection. Recently, a comprehensive review of the most relevant in vivo studies accomplished in the past decade was published, and different routes of phage administration, dosage effect, and different animal models of distinct types of infections were compared (82). It is important to highlight that the in vivo studies performed in biofilm models usually represent acute infections, in opposition to real biofilm infections that are usually characterized by their chronicity and recalcitrance (83).
大多数模拟真实条件的研究通常是为了模拟针对传染性生物膜的噬菌体疗法。Lebeaux 等人 ( 79) 讨论了体内外模型的适用性，认为这是一种替代体内模型的有趣方法。体外模型减少了对自然条件的改变，因为它们涉及在人工环境中使用从活生物体中提取的组织。与体内模型相比，体外模型的实验设置更加可控，减少了伦理方面的担忧。例如，噬菌体被应用于猪皮肤，模拟由不同病原体引起的伤口感染治疗 ( 80, 81)。尽管使用这类模型有很多优点，但缺乏宿主（人类或动物）的反应和实验持续时间短仍然是广泛使用体外模型的一些障碍。从这个意义上说，体内模型是了解感染病理学研究的最佳选择。最近，一篇关于过去十年中完成的最相关体内研究的综述发表了，其中比较了不同的噬菌体给药途径、剂量效应以及不同类型感染的不同动物模型（82）。需要强调的是，在生物膜模型中进行的体内研究通常代表急性感染，而真实的生物膜感染通常以慢性和顽固性为特征（83）。