5. Driving Bacterial Evolution Toward Favorable Trade-Offs
5.推动细菌进化，实现有利权衡
Bacterial resistance to phage therapy may be inevitable, especially for large bacterial populations with rapid generation times. One possible strategy is to direct the evolution of bacterial resistance toward clinically useful outcomes (87–89) (Figure 4). Genetic trade-offs occur during evolution by natural selection when an advantageous trait evolves to solve an environmental challenge (e.g., phage attack), at the expense of worsened performance in some other unselected trait (90). For example, evolution of brightly colored plumage might help a male bird attract mates and sire progeny, but these colorful feathers also make the bird more easily spotted by possible predators and therefore increase predation risk (91). Similarly, evolution of phage resistance can protect against lethal virus attack but often can impose costs on bacterial fitness (89). In a classic example, competitive growth measurements by Lenski (92) revealed that E. coli mutants that developed resistance to phage T4 had lower fitness relative to the ancestral bacteria.
细菌对噬菌体疗法产生抗药性可能是不可避免的，尤其是对于繁殖速度快的大型细菌种群。一种可能的策略是将细菌耐药性的进化导向临床有用的结果（87- 89）（图 4）。在自然选择的进化过程中，当一个优势性状进化以解决环境挑战（如噬菌体攻击）时，就会出现遗传权衡，而代价是其他一些未被选择的性状性能下降（90）。例如，进化出色彩鲜艳的羽毛可能有助于雄鸟吸引配偶和生育后代，但这些色彩鲜艳的羽毛也会使鸟类更容易被可能的捕食者发现，从而增加被捕食的风险 ( 91)。同样，噬菌体抗性的进化可以抵御致命病毒的攻击，但往往也会给细菌的健康带来代价（89）。在一个经典的例子中，Lenski（92）通过竞争性生长测量发现，开发出对噬菌体T4有抗性的大肠杆菌突变体，相对于其祖先菌种而言，具有较低的适应性。
The frequent occurrence of evolutionary trade-offs can be leveraged to design more effective phage therapies. Therapeutic phages can be chosen based on their ability to steer bacterial evolution toward trade-offs of biomedical significance. Below, we discuss several examples of phage-driven favorable trade-offs, including evolution of phage resistance in bacteria that coincides with increased sensitivity to antibiotics and reduced virulence.
可以利用进化过程中经常出现的权衡来设计更有效的噬菌体疗法。可以根据噬菌体引导细菌进化的能力，选择具有生物医学意义的治疗性噬菌体。下面，我们将讨论噬菌体驱动有利权衡的几个例子，包括细菌中噬菌体抗性的进化与抗生素敏感性的提高和毒力的降低。
5.1. Driving Re-Sensitization to Antibiotics
5.1.推动抗生素的再敏感化
In some bacterial pathogens, a key mechanism of resistance to toxic molecules, such as antibiotics, is the active export of these antimicrobials out of the cell through transmembrane efflux pumps (93). This drug resistance mechanism may spread across bacterial populations via horizontal gene transfer, raising major concerns for evolution of MDR bacterial infections. However, numerous phages are known to use efflux pumps as host surface receptors. Thus, evolution of bacterial resistance to phage attack often should result in downregulation or even deletion of genes encoding efflux pumps. Such lytic phages can exert selection to re-sensitize bacteria to antibiotics because the evolved phage-resistant bacteria can be impaired in active transport of antibiotics out of the cell.
在某些细菌病原体中，对抗生素等有毒分子产生耐药性的一个关键机制是通过跨膜外排泵将这些抗菌素主动排出细胞（93）。这种耐药机制可能会通过水平基因转移在细菌种群中传播，从而引发对多重耐药细菌感染进化的严重关切。然而，已知有许多噬菌体使用外排泵作为宿主表面受体。因此，细菌对噬菌体攻击的抗性进化往往会导致编码外排泵的基因下调甚至删除。由于进化后的细菌对噬菌体产生了抗药性，其主动将抗生素转运出细胞的能力可能会受到损害，因此这类溶解性噬菌体可以通过选择使细菌重新对抗生素敏感。
This idea was popularized in a study using the P. aeruginosa–targeting phage OMKO1. This phage likely interacts either directly or indirectly with the outer membrane porin M (OprM), which is a component of the multi-drug efflux systems MexAB and MexXY. The evolution of P. aeruginosa resistance to this phage may change OprM expression or cause gene deletions that modify efflux pump function, generally compromising antibiotic export from the cell. Clinical isolates of P. aeruginosa that developed resistance to OMKO1 had up to 50-fold higher sensitivity to ciprofloxacin, tetracycline, ceftazidime, and erythromycin antibiotics, observed in vitro and in experiments where phage protected against lethal bacterial infection of Galleria mellonella moth larvae (94, 95). By this logic, phage OMKO1 was predicted correctly to synergize with ceftazidime antibiotic in emergency therapy against MDR P. aeruginosa when treating a patient with an infected aortic arch replacement; the phage killed the bacteria while also steering phage resistance that coincided with re-sensitization to the ordinarily useless antibiotic (96).
这项研究利用铜绿假单胞菌靶向噬菌体 OMKO1 推广了这一观点。这种噬菌体可能会直接或间接地与外膜孔蛋白 M（OprM）相互作用，而后者是多重药物外排系统 MexAB 和 MexXY 的组成部分。铜绿假单胞菌对这种噬菌体产生抗药性的进化过程可能会改变 OprM 的表达或导致基因缺失，从而改变外排泵的功能，一般会影响抗生素从细胞中排出。对 OMKO1 产生抗药性的铜绿假单胞菌临床分离株对环丙沙星、四环素、头孢他啶和红霉素等抗生素的敏感性提高了 50 倍。根据这一逻辑，噬菌体 OMKO1 在治疗一名主动脉弓置换术后感染的患者时，可与头孢他啶类抗生素协同作用，对 MDR 铜绿假单胞菌进行紧急治疗；噬菌体在杀死细菌的同时，还能引导噬菌体产生抗药性，而这种抗药性与对通常无用的抗生素的重新敏感性相吻合 ( 96)。
Evolution of resistance against some phages may indirectly affect efflux pump function, seemingly without phage binding to pump proteins. ØS12-3, another lytic phage of P. aeruginosa, targets the O-antigen on the polysaccharide. Bacterial mutants resistant to this phage carry a deletion in gene galU, resulting in removal of the O-antigen (97). Nakamura and coworkers (98) recently investigated ØS12-3-resistant mutants of Pa12 host bacteria, a veterinary isolate of P. aeruginosa. They discovered that as a consequence of large chromosomal deletions in the region surrounding the galU gene, multi-drug efflux genes mexXY were also deleted. Because MexXY mediates quinolone efflux removal, the phage-resistant mutants evolved to be more sensitive to fluoroquinolones (98). Thus, phages that do not use efflux pumps as receptors per se can still indirectly affect the function of these protein complexes and may be potentially useful in re-sensitizing MDR bacteria to antibiotics.
对某些噬菌体的抗药性进化可能会间接影响外排泵的功能，但噬菌体似乎不会与泵蛋白结合。ØS12-3是铜绿假单胞菌的另一种溶菌噬菌体，以多糖上的O抗原为目标。对这种噬菌体有抗性的细菌突变体基因 galU 缺失，导致 O 抗原被去除 ( 97)。中村（Nakamura）和同事（98）最近研究了Pa12宿主细菌（一种铜绿假单胞菌的兽医分离物）的抗ØS12-3突变体。他们发现，由于 galU 基因周围区域的染色体大缺失，多种药物外流基因 mexXY 也被缺失。由于 MexXY 介导喹诺酮类药物的外排清除，噬菌体抗性突变体进化为对氟喹诺酮类药物更加敏感（98）。因此，不使用外排泵本身作为受体的噬菌体仍然可以间接影响这些蛋白复合物的功能，并有可能使 MDR 细菌对抗生素重新敏感。
Components of the bacterial membrane (e.g., polysaccharide layers) provide a frontline barricade to foreign molecules such as antibiotics. Mutations in these components may also render bacterial cells sensitive to certain antibiotics. An example comes from phage U136B, which uses both the multi-drug efflux pump protein TolC and LPS components as coreceptors on host E. coli cells. Several U136B-resistant bacteria presented mutations in tolC and as a result became re-sensitized to various antibiotics such as tetracycline, which is effluxed by TolC (39). However, some phage-resistant bacteria harbored mutations in the genes encoding the LPS, rather than in tolC. These changes in LPS made the phage-resistant cells sensitive to antibiotics such as colistin, which are not removed by TolC efflux pumps. Therefore, phage U136B drives separate trade-offs in the same E. coli bacterial host: Cells harboring mutations in tolC were more sensitive to tetracycline, while LPS mutants became more sensitive to colistin.
细菌膜的成分（如多糖层）为抗生素等外来分子提供了前沿屏障。这些成分的突变也可能使细菌细胞对某些抗生素敏感。噬菌体 U136B 就是一个例子，它利用多种药物外排泵蛋白 TolC 和 LPS 成分作为宿主大肠杆菌细胞的核心受体。一些对 U136B 产生抗药性的细菌出现了 TolC 突变，因此对四环素等多种抗生素重新敏感，而四环素是由 TolC 外排的（39）。然而，一些噬菌体抗性细菌的 LPS 编码基因而不是 TolC 发生了突变。LPS 的这些变化使噬菌体抗性细胞对可乐定等抗生素敏感，而 TolC 外排泵无法清除这些抗生素。因此，噬菌体 U136B 在同一大肠杆菌宿主中驱动了不同的权衡：携带 TolC 突变的细胞对四环素更敏感，而 LPS 突变体对可乐定更敏感。
In the previous section, we mentioned the potential of combining phages and antibiotics to expand overall host range and minimize bacterial resistance. Evolutionary trade-offs between increased phage resistance and antibiotic re-sensitivity suggest another benefit of these combinations: In clinical settings, phages that drive antibiotic sensitization can be delivered together with currently ineffective antibiotics. During therapy, phages will kill the target bacteria while selecting for phage-resistant mutants in the bacterial population that become re-sensitized to the antibiotic, increasing the possibility for reduced bacterial load and infection clearance. This type of interaction has been termed phage-antibiotic synergy. A recent example comes from murine models of Acinetobacter baumannii bacteremia, where Gordillo Altamirano et al. (99) reported that phages targeting the bacterial capsule resulted in emergence of phage resistance in 96% of test animals. All phage-resistant bacterial isolates lost their ability to synthesize the capsule, which is the identical phenotypic outcome of evolved phage resistance previously observed by these researchers in vitro (100). Capsule-deficient mutants became susceptible to other antimicrobial agents, including two antibiotics (and one alternative phage) (99).
在上一节中，我们提到了噬菌体与抗生素结合的潜力，可以扩大宿主的总体范围，并最大限度地减少细菌的抗药性。噬菌体抗药性增强与抗生素再敏感性之间的进化权衡表明了这些组合的另一个好处：在临床环境中，可将抗生素致敏的噬菌体与目前无效的抗生素一起投放。在治疗过程中，噬菌体将杀死目标细菌，同时在细菌群中选择对抗生素重新敏感的噬菌体抗性突变体，从而增加减少细菌负荷和清除感染的可能性。这种相互作用被称为噬菌体-抗生素协同作用。最近的一个例子来自鲍曼不动杆菌菌血症的小鼠模型，据 Gordillo Altamirano 等人 ( 99) 报道，针对细菌囊的噬菌体导致 96% 的试验动物出现噬菌体抗药性。所有抗噬菌体的细菌分离物都失去了合成胶囊的能力，这与这些研究人员之前在体外观察到的噬菌体抗性进化的表型结果相同（100）。缺乏胶囊的突变体对其他抗菌剂也变得敏感，包括两种抗生素（和一种替代噬菌体）（99）。