2.4. How Phages Can Modulate and Trigger Biofilm Formation
2.4.噬菌体如何调节和触发生物膜的形成
Although several studies have highlighted the potential of phages for biofilm control, not all phages have this ability, and studies have shown evidence that some phages can modulate biofilm formation and even increase biofilm levels (44). This can be explained by the selective pressure caused by phages that results in fast propagation of phage-resistant cells or by the induction of prophages that contributes to a release of biofilm-promoting molecules.
尽管一些研究强调了噬菌体在控制生物膜方面的潜力，但并非所有噬菌体都具有这种能力，有研究表明，一些噬菌体可以调节生物膜的形成，甚至增加生物膜水平（44）。这可能是由于噬菌体造成的选择性压力导致了抗噬菌体细胞的快速繁殖，或者是噬菌体诱导释放了促进生物膜形成的分子。
Hosseinidoust et al. (58) studied whether a phage treatment can lead to enhanced biofilm formation in consequence of resistant cells or spatial refuges. To address this question, the authors exposed single-species biofilms (P. aeruginosa, Salmonella enterica, and S. aureus) to specific phages (as a pretreatment or post-treatment) and observed that some phage treatments resulted in increased biofilm formation with levels above the control (58). In a study by Henriksen et al. (59), where different phage treatments against P. aeruginosa flow-cell biofilms were evaluated, the authors observed that repeated phage treatments (three phage doses every 24 h) did not improve the antibiofilm efficacy of phages, resulting in a significant increase of microcolonies, which provide protection from phages, as well as increased biofilm thickness. Tan et al. (60) studied the effect of two vibriophages in the biofilm formation of V. anguillarum and observed different effects depending on the phage used: While one of the phages was able to control biofilm formation, the other one stimulated biofilm development. The authors of the study explained the different behaviors of the phages by the presence of spatial refuges formed by some strains, which can promote the coexistence of phages and bacteria, as already mentioned above. The authors also highlighted the diversity of phage-host interactions even within the same bacterial species (60). Similarly, Fernández et al. (61) showed that the exposure of S. aureus biofilms to subinhibitory doses of phages can promote biofilm formation and protect cells from complete eradication.
Hosseinidoust 等人（58）研究了噬菌体处理是否会导致抗性细胞或空间庇护所增强生物膜的形成。为了解决这个问题，作者将单种生物膜（铜绿假单胞菌、肠炎沙门氏菌和金黄色葡萄球菌）暴露于特定的噬菌体（作为预处理或后处理）中，观察到一些噬菌体处理导致生物膜形成增加，其水平高于对照组（58）。Henriksen 等人的研究（59）评估了针对铜绿假单胞菌流动细胞生物膜的不同噬菌体处理方法，作者观察到，重复噬菌体处理（每 24 小时三次噬菌体剂量）并不能提高噬菌体的抗生物膜功效，反而会导致微菌落显著增加（微菌落可提供噬菌体保护）以及生物膜厚度增加。Tan 等人（60）研究了两种噬菌体对鳗鲡生物膜形成的影响，观察到不同的噬菌体有不同的效果：其中一种噬菌体能够控制生物膜的形成，而另一种噬菌体则刺激生物膜的发展。该研究的作者解释说，噬菌体的不同行为是由于一些菌株形成了空间庇护所，如上所述，这可以促进噬菌体和细菌的共存。作者还强调了噬菌体-宿主相互作用的多样性，即使在同一细菌物种中也是如此（60）。同样，Fernández 等人（61）的研究表明，将金黄色葡萄球菌生物膜暴露于亚抑制剂量的噬菌体可促进生物膜的形成，保护细胞不被彻底清除。
Although these studies were performed with lytic phages, prophages are also known to directly affect biofilm formation. In fact, prophage induction during biofilm development might mediate a release of biofilm-promoting components as observed by Carrolo et al. (62). In this study, the authors reported that the lysis of Streptococcus pneumoniae host cells mediated by spontaneous induction of prophages into the lytic cycle contributed to extracellular DNA (eDNA) release, which favored biofilm formation by the remaining pneumococcal population (62). This is not surprising because eDNA is a key component of the biofilm matrix of most bacterial species, and it is known to have a major role in biofilm development by promoting adhesion to surfaces and maintenance of the structural integrity of biofilms (reviewed in 63). The enhanced biofilm formation in consequence of phage-induced lysis was also reported by Gödeke et al. (64). While the cell lysis mediated by three prophages harbored in the genome of Shewanella oneidensis MR-1 promoted biofilm formation, a bacterial mutant devoid of prophages revealed impaired biofilm formation ability (64). Similar observations related to the ability of prophages to trigger biofilm formation were also reported for Actinomyces odontolycus (65). In addition to these studies, it is important to highlight that the P. aeruginosa filamentous phages (Pf-like) have also been revealed to play an important role in the life cycle and structural integrity of P. aeruginosa biofilms (66, 67). Another interesting example of how phages can modulate biofilm formation was reported by Ojha et al. in Mycobacterium (68). In this study, the authors observed that the integration of the Mycobacterium smegmatis temperate phage Bxb1 led to the inactivation of gene groEL1, which contains the attB site for phage integration. Although Bxb1 integration did not affect the planktonic growth of bacteria, it prevented biofilm maturation, as the groEL1 gene is involved in the synthesis of mycolic acids, namely during biofilm formation.
虽然这些研究是用溶菌噬菌体进行的，但噬菌体也会直接影响生物膜的形成。事实上，在生物膜形成过程中，噬菌体诱导可能会介导生物膜促进成分的释放，正如 Carrolo 等人所观察到的那样 ( 62)。在这项研究中，作者报告说，噬菌体自发诱导肺炎链球菌宿主细胞进入溶菌循环，从而导致细胞外 DNA（eDNA）释放，这有利于剩余的肺炎球菌形成生物膜（62）。这并不奇怪，因为 eDNA 是大多数细菌物种生物膜基质的关键成分，而且已知 eDNA 在生物膜的形成过程中起着重要作用，它能促进细菌对表面的粘附并维持生物膜结构的完整性（综述见 63）。Gödeke 等人也报道了噬菌体诱导裂解后生物膜形成增强的情况（64）。Shewanella oneidensis MR-1 基因组中的三种噬菌体介导的细胞裂解促进了生物膜的形成，而不含噬菌体的细菌突变体则显示生物膜形成能力受损 ( 64)。关于噬菌体引发生物膜形成的能力，奥氏放线菌（Actinomyces odontolycus）也报道了类似的观察结果（65）。除了这些研究之外，有必要强调的是，铜绿假单胞菌丝状噬菌体（Pf-like）也被发现在铜绿假单胞菌生物膜的生命周期和结构完整性中发挥着重要作用 ( 66, 67)。Ojha 等人在分枝杆菌（68）中报道了噬菌体如何调节生物膜形成的另一个有趣例子。在这项研究中，作者观察到烟肉分枝杆菌温带噬菌体 Bxb1 的整合导致基因 groEL1 失活，而 groEL1 包含噬菌体整合的 attB 位点。虽然 Bxb1 整合并不影响细菌的浮游生长，但却阻止了生物膜的成熟，因为 groEL1 基因参与了霉菌醇酸的合成，即在生物膜形成过程中的合成。
Although some of the studies described above established a link between prophage induction and biofilm formation, the cell lysis mediated by spontaneously induced prophages may also lead to biofilm dispersion. For instance, Rossmann et al. (69) demonstrated that high levels of the QS molecule Al-2 produced by Enterococcus faecalis induced the dispersal of bacterial cells from established biofilms due to prophage release. In a recent study by Tan et al. (70), the authors also highlighted the role of QS signaling in coordinating phage-host interaction and biofilm formation in V. anguillarum; however, in this study an H2O-like prophage stimulated the host’s biofilm formation, although its induction was repressed by QS. In a study using P. aeruginosa PA14, Zegans et al. (71) observed that lysogeny by phage DMS3 inhibited biofilm formation and swarming motility of the strain. According to the authors, this inhibition was explained by a concerted action of the phage and the CRISPR system of the host (71).
尽管上述一些研究确定了噬菌体诱导与生物膜形成之间的联系，但自发诱导的噬菌体介导的细胞裂解也可能导致生物膜的分散。例如，Rossmann 等人 ( 69) 证实，粪肠球菌产生的高浓度 QS 分子 Al-2 可诱导细菌细胞从已形成的生物膜中因噬菌体释放而分散。在 Tan 等人最近的一项研究（70）中，作者也强调了 QS 信号在协调鳗鲡体内噬菌体-宿主相互作用和生物膜形成中的作用。O 样噬菌体刺激了宿主生物膜的形成，尽管 QS 抑制了其诱导作用。在一项使用铜绿假单胞菌 PA14 的研究中，Zegans 等人（71）观察到噬菌体 DMS3 的溶菌作用抑制了该菌株生物膜的形成和蜂拥运动。据作者解释，这种抑制作用是噬菌体和宿主的 CRISPR 系统共同作用的结果 ( 71)。