2. Phages as a Promising Alternative Therapy against MDR Bacteria
2.噬菌体作为一种有希望的抗MDR细菌的替代疗法
Phage diversity is enormous, being the most abundant entity in the biosphere [18]. They can be found in a wide range of environments, including extreme ones [19]. Despite the variation found in phages, they present two main biological cycles, with some modifications: lysogenic cycles (temperate phages), in which phage DNA integrates into the host genome as a prophage; and lytic cycles (lytic or virulent phages), in which phage multiplies inside the host bacteria and releases new phage particles by lysing the host cell [20,21]. Lytic phages are especially interesting for phage therapy due to two main reasons: firstly, because their cycle leads to bacterial death [22] and secondly, because they lack integrases and other related enzymes, thus avoiding horizontal gene transfer [23].
噬菌体多样性是巨大的，是生物圈中最丰富的实体[ 18]。它们可以在各种环境中找到，包括极端环境[ 19]。尽管在噬菌体中发现了变异，但它们呈现出两个主要的生物循环，并进行了一些修改：溶原性循环（温和性噬菌体），其中噬菌体DNA作为原噬菌体整合到宿主基因组中;和裂解性循环（裂解性或毒性噬菌体），其中噬菌体在宿主细菌内增殖并通过裂解宿主细胞释放新的噬菌体颗粒[ 20，21]。由于两个主要原因，裂解酶对于噬菌体治疗特别感兴趣：首先，因为它们的循环导致细菌死亡[ 22]，其次，因为它们缺乏整合酶和其他相关酶，从而避免了水平基因转移[ 23]。
2.1. Phage Therapy Overview
2.1.噬菌体治疗概述
Phages were discovered independently by the English military physician Fredrick W. Twort in 1915, and the Canadian microbiologist Félix H. d’Hérelle in 1917 [22,24]. Phages were successfully tested by d’Hérelle early on to treat dysentery and cholera. However, World War II and the discovery of antibiotics led to the abandonment of phages as a therapeutic tool in Western countries [25]. Nowadays, due to the emergence of MDR bacteria, phage therapy has re-emerged and is considered a potential therapeutic alternative to antibiotics [21,26].
噬菌体是由英国军事医生弗雷德里克·W. 1915年，Twort和加拿大微生物学家Félix H. d’Hérelle in 1917 [ 22，24].噬菌体在早期就被d’Hérelle成功地用于治疗痢疾和霍乱。然而，第二次世界大战和抗生素的发现导致了西方国家放弃将抗生素作为治疗工具[ 25]。如今，由于MDR细菌的出现，噬菌体疗法重新出现，并被认为是抗生素的潜在治疗替代品[ 21，26]。
Phage therapy presents some potential advantages for treating bacterial infections. They are able to kill bacteria regardless of whether the bacteria are resistant or not [27]. In fact, MDR Acinetobacter baumannii strains have been shown to be more susceptible to phage therapy than other non-MDR strains [26,28]. Furthermore, phages multiply at the site of infection (Figure 1), increasing in number during the infection process and making it likely that a single (or few) doses will be sufficient to have the desired effect compared to antibiotic treatment. This makes hard-to-reach infections more accessible to phages than to antibiotics if sufficient numbers of phages are applied initially [29]. Phages are also highly specific (Figure 1), implying that they may not affect the commensal bacteria of the host [30]. If a broader spectrum is required to treat an infection, it is possible to use combinations of phages, known as phage cocktails [31]. Phage cocktails are a growing strategy in phage therapy to improve the results of monophage therapy. In addition to extending the utility of phage formulations, phage cocktails may also be useful in preventing the development of phage-resistant strains during individual treatments [32]. Another advantage of phage therapy is that phages can be considered as “adaptive drugs” (Figure 1). Bacteria and phages have co-evolved in nature for millions of years. This may also occur in phage-treated infections, where this coevolution makes the treatment adaptive [26,33]. Furthermore, phage cocktails tend to decrease the emergence of bacterial resistance compared with the application of individual phages [21], as has been shown in other combination therapies [34,35].
噬菌体疗法在治疗细菌感染方面具有潜在优势。它们能够杀死耐药菌和非耐药菌 [27]。事实上，研究表明，多重耐药性鲍曼不动杆菌菌株比其他非多重耐药性菌株更容易受到噬菌体治疗的影响 [26,28]。此外，噬菌体可以在感染部位繁殖（图 1），并在感染过程中数量增加，这使得只需要单次（或几次）剂量就可能达到预期效果，相比于抗生素治疗更具优势。如果一开始应用足够数量的噬菌体，那么噬菌体疗法比抗生素疗法更容易治疗难以到达的感染部位 [29]。噬菌体还具有高度特异性（图 1），这意味着它们可能不会影响宿主体的共生菌 [30]。如果治疗感染需要更广谱的噬菌体，则可以使用噬菌体混合物，称为噬菌体鸡尾酒疗法 [31]。噬菌体鸡尾酒疗法是噬菌体治疗领域的一项新兴策略，旨在改善单噬菌体疗法的效果。除了扩展噬菌体制剂的用途外，噬菌体鸡尾酒疗法还可以帮助预防个体治疗过程中耐噬菌体菌株的产生 [32]。噬菌体治疗的另一个优势是，噬菌体可以被视为“适应性药物”（图 1）。细菌和噬菌体已经在自然界中共同进化了数百万年。这种共同进化也可能发生在噬菌体治疗的感染中，使治疗具有适应性 [26,33]。此外，与单独使用噬菌体相比，噬菌体鸡尾酒疗法往往会降低细菌耐药性的出现，这一点在其他联合治疗方法中也得到了证实 [34,35]。
Main differences between phages’ and antibiotics’ action against pathogenic bacteria; Phages are able to multiply in the site of infection, unlike antibiotics. Phages are able to evolve with bacteria while antibiotics are static structures. Phages are highly specific in their targets while antibiotics are generalists.
噬菌体和抗生素对致病菌作用的主要区别;噬菌体能够在感染部位繁殖，不像抗生素。噬菌体能够与细菌一起进化，而抗生素是静态结构。噬菌体在其靶点上具有高度特异性，而抗生素则是通才。
An important remark in phage therapy is that the pharmacokinetics of phages is more complex than that of traditional antibiotics, and there may be differences between phages in terms of their persistence and ability to replicate. They may also interact differently with the immune system, and their efficacy would also depend on the characteristics of the infection, such as its location, abundance, and bacterial composition. Their interaction with many plasma proteins is also largely unknown. Thus, pharmacodynamics and pharmacokinetics considerations become more complex with phages or phage-derived combinations [36] and should be especially important in defining the administration process and dosing.
噬菌体治疗中的一个重要注意事项是，抗生素的药代动力学比传统抗生素更复杂，并且就其持久性和复制能力而言，抗生素之间可能存在差异。它们也可能与免疫系统发生不同的相互作用，它们的疗效也取决于感染的特征，如感染的位置、丰度和细菌组成。它们与许多血浆蛋白的相互作用也在很大程度上是未知的。因此，药效学和药代动力学的考虑变得更加复杂与噬菌体衍生的组合[ 36]，并应在定义管理过程和剂量特别重要。
Regarding phage delivery strategies, oral administration has limitations due to the poor stability of phages in acidic environments such as the stomach. Many authors have described this problem, proposing as a solution the administration of phages with buffering compounds. This would significantly increase phage survival [16]. Other strategies such as nano- and microencapsulation allow controlled or sustained release of phages at the site of infection or increase the time of phage circulation [37]. Another alternative suggested by some authors is rectal application. It has been successfully tested in rabbits using a nonionic surfactant to increase the presence of phages in the blood [38]. In another study, the authors tested a form of phage suppository. This method ensured the presence of phage particles in the foci of infection directly in contact with the preparation [39]. For the treatment of pulmonary infections, phage inhalation has been proposed as an alternative and seems particularly promising, but more research on this method of application is still needed [40]. To treat skin and wound infections, the application of phage therapy directly on the skin has been successfully tested. One example is the treatment of Klebsiella pneumoniae, a pathogen predominantly associated with burn-wound infections. In one study, both a phage therapy and a phage cocktail have been tested in a murine model. Both groups showed a significant reduction in bacterial load compared to the untreated group of mice. The growth that received the phage cocktail showed the maximum reduction (p < 0.01) [41].
关于噬菌体递送策略，口服给药具有局限性，这是由于噬菌体在酸性环境如胃中的稳定性差。许多作者已经描述了这个问题，提出了一种解决方案，即用缓冲化合物给药。这将显著增加噬菌体存活[ 16]。其他策略，如纳米和微胶囊化，允许在感染部位控制或持续释放Escherichia coli，或增加噬菌体循环的时间[ 37]。一些作者建议的另一种替代方法是直肠应用。已在家兔中成功进行了试验，使用非离子表面活性剂增加血液中的肌酐[ 38]。在另一项研究中，作者测试了一种形式的噬菌体栓剂。该方法确保了直接与制剂接触的感染灶中存在噬菌体颗粒[ 39]。 对于肺部感染的治疗，噬菌体吸入已被提出作为一种替代方案，似乎特别有前途，但仍需要对这种应用方法进行更多的研究[ 40]。为了治疗皮肤和伤口感染，已经成功地测试了直接在皮肤上应用噬菌体疗法。一个例子是治疗肺炎克雷伯氏菌，一种主要与烧伤伤口感染相关的病原体。在一项研究中，噬菌体疗法和噬菌体混合物都已在鼠模型中进行了测试。与未处理的小鼠组相比，两组均显示出细菌负荷的显著减少。接受噬菌体混合物的生长显示出最大的降低（p < 0.01）[ 41]。