2.1. How Biofilm Composition and Architecture Affect Phage Infection
2.1.生物膜的组成和结构如何影响噬菌体感染
It is well known that biofilm structure and composition can pose limitations on phage predation (6). In addition to the biofilm matrix that contributes to impairment of the diffusion of phages and their propagation, other factors such as the low metabolic activity of biofilm cells, the presence of secreted molecules that may act as phage decoys, or even the presence of more than one microbial species in the biofilm may also contribute to an inefficient phage infection (6, 14).
众所周知，生物膜的结构和组成会限制噬菌体的捕食（6）。除了生物膜基质会阻碍噬菌体的扩散和传播外，生物膜细胞代谢活性低、存在可充当噬菌体诱饵的分泌分子，甚至生物膜中存在多个微生物物种等其他因素也可能导致噬菌体感染效率低下 ( 6, 14)。
Flemming & Wingender (15) estimated that in most biofilms, the EPS matrix accounts for more than 90% of the biofilm dry mass, whereas the microbial cells account for less than 10%. This EPS matrix—composed of polymeric substances and other secreted products including enzymes, proteins, lipids, or nucleic acids—contributes to the cohesion of biofilms (15) and can cause phage entrapment, acting as a physical barrier to phage diffusion and access to the bacterial cells, and consequently preventing an efficient infection (16).
Flemming 和 Wingender（15）估计，在大多数生物膜中，EPS 基质占生物膜干物质的 90% 以上，而微生物细胞只占不到 10%。EPS 基质由高分子物质和其他分泌物（包括酶、蛋白质、脂质或核酸）组成，有助于生物膜的凝聚（15），并可导致噬菌体被困，成为噬菌体扩散和进入细菌细胞的物理屏障，从而阻止有效感染（16）。
González et al. (17) studied the parameters that affected the diffusion and propagation of two phages in Staphylococcus spp. biofilms. Although the authors confirmed that both phages could diffuse through all the different biofilms tested, their data suggested that the diffusion rates of phages within biofilms were influenced by several factors: the amount of biofilm biomass, the susceptibility of the bacterial strains to the phages, the phage concentration, and the composition of the biofilm matrix that might contain phage-inactivating enzymes or components able to anchor the phages (17). For instance, it is known that the outer membrane vesicles (OMVs) secreted by some bacterial species can mediate phage entrapment in biofilms. These OMVs may contain phage receptors, as observed for both Escherichia coli and Vibrio cholerae species (18, 19), which may contribute to an irreversible binding of phages that will not be available to infect the biofilm cells.
González 等人（17）研究了影响两种噬菌体在葡萄球菌生物膜中扩散和传播的参数。虽然作者证实这两种噬菌体都能在所有不同的生物膜中扩散，但他们的数据表明，噬菌体在生物膜中的扩散速率受多种因素影响：生物膜生物量、细菌菌株对噬菌体的敏感性、噬菌体浓度以及生物膜基质的组成，其中可能含有噬菌体失活酶或能够固定噬菌体的成分（17）。例如，众所周知，某些细菌物种分泌的外膜囊泡 (OMV) 可以介导生物膜中的噬菌体吸附。正如在大肠杆菌和霍乱弧菌中观察到的那样，这些外膜囊泡可能含有噬菌体受体（18、19），这可能有助于不可逆地结合噬菌体，使其无法感染生物膜细胞。
The protective role of the biofilm matrix to phage predation was clearly demonstrated in a recent study by Melo et al. (20) that assessed the interaction of a Staphylococcus epidermidis phage with different biofilm-associated host cell populations. The observations of this study were corroborated by confocal laser scanning microscopy (CLSM) data, which demonstrated that phage-infected cells appeared only in certain regions of the biofilm where lower amounts of matrix were present, evidencing that the biofilm matrix can serve as a shield to protect the embedded bacteria from viral attack (20). In fact, the spatial organization of the biofilm can be a determinant to the success of phage infection, as it may lead to limited mobility of cells that tend to organize in localized niches with different nutrient availability (21). While the proximity of the cells in these clusters might contribute to a decreased number of progeny phages as a result of multiple phages infecting the same host cell (22), the nutrient gradients often lead to cells under different metabolic states, including dormant or persister cells (6). It is known that phages require an active machinery of the host to propagate, and consequently, their replication is strongly influenced by the physiological state of the host cell (23, 24). Therefore, so far only a few phages were reported to have the capacity of infecting stationary-phase cells (25, 26). The number of biofilm cells with reduced metabolic activity is expected to increase with biofilm age; consequently, older biofilms (frequently found in nature) will be less susceptible to phages than younger biofilms (6).
Melo 等人最近的一项研究（20）清楚地证明了生物膜基质对噬菌体捕食的保护作用，该研究评估了表皮葡萄球菌噬菌体与不同生物膜相关宿主细胞群的相互作用。共聚焦激光扫描显微镜（CLSM）的数据证实了这项研究的观察结果，这些数据表明，被噬菌体感染的细胞只出现在生物膜中基质含量较低的特定区域，这证明生物膜基质可以充当保护罩，保护嵌入的细菌免受病毒攻击（20）。事实上，生物膜的空间组织可能是噬菌体感染成功与否的决定性因素，因为它可能导致细胞的流动性受到限制，而这些细胞倾向于在营养供应不同的局部壁龛中组织起来（21）。由于多个噬菌体感染同一个宿主细胞，这些细胞簇中的细胞距离较近可能会导致后代噬菌体数量减少（22），但营养梯度往往会导致细胞处于不同的代谢状态，包括休眠或宿主细胞（6）。众所周知，噬菌体需要宿主的活性机制才能繁殖，因此，宿主细胞的生理状态对噬菌体的复制有很大影响（23、24）。因此，迄今为止，只有少数噬菌体有能力感染静止期细胞（25、26）。新陈代谢活性降低的生物膜细胞数量预计会随着生物膜年龄的增长而增加；因此，较老的生物膜（经常在自然界中发现）对噬菌体的敏感性会低于较年轻的生物膜（6）。
Another important feature that also affects phage diffusion through the biofilm structure is the presence of more than one microbial strain or species. It is estimated that most biofilm communities found in nature are composed of a variety of microorganisms instead of a single one (27). Testa et al. (28) demonstrated that the outcome of phage infection is influenced by both the spatial structure of the biofilm and the presence of more than one strain. The interaction of phages with multispecies biofilms is a rather complex process due to the higher diversity of polymeric substances and heterogeneity of the biofilm (14). Although these biofilms are expected to be less susceptible to phage predation, more studies are needed to understand their interaction with phages in real habitats.
影响噬菌体在生物膜结构中扩散的另一个重要特征是存在多个微生物菌株或物种。据估计，自然界中发现的大多数生物膜群落都是由多种微生物而不是单一微生物组成的（27）。Testa 等人（28）证实，噬菌体感染的结果受到生物膜空间结构和存在多个菌株的影响。噬菌体与多菌种生物膜的相互作用是一个相当复杂的过程，因为生物膜的聚合物质和异质性较高 ( 14)。虽然预计这些生物膜不太容易被噬菌体捕食，但还需要更多的研究来了解它们在真实生境中与噬菌体的相互作用。