3. Occurrence of Bacterial Resistance In Vitro, In Vivo, and In the Clinic
3.细菌耐药性在体外、体内和临床中的出现
Abundant evidence shows that bacteria tend to rapidly evolve resistance to phages in laboratory conditions. Typically, in vitro phage-host experiments are conducted in nutrient-rich, well-mixed environments where harmful effects of mutations can be buffered (36–40). In these conditions, the most frequently observed mechanism of phage resistance is modification of cell-surface receptors, such as LPS, outer membrane proteins, capsules, and flagella (20, 41), to prevent or reduce phage adsorption. Bacterial resistance due to new genetic innovations or via adaptive-immune mechanisms such as CRISPR-Cas constitutes a gain-of-function change that is unlikely to occur in the simple microcosms and short timescales of typical in vitro studies. Thus, the annotation and characterization of most bacterial defense systems and their implications in phage therapy remain largely underexplored through laboratory studies.
大量证据表明，细菌往往会在实验室条件下迅速进化出对噬菌体的抗性。通常，体外噬菌体-宿主实验是在营养丰富、混合良好的环境中进行的，在这种环境中，突变的有害影响可以得到缓冲（36- 40）。在这些条件下，最常观察到的噬菌体抗性机理是细胞表面受体（如 LPS、外膜蛋白、噬菌体囊和鞭毛）的修饰 ( 20, 41)，以阻止或减少噬菌体的吸附。新的基因创新或通过适应性免疫机制（如 CRISPR-Cas）产生的细菌抗药性是一种功能增益变化，不太可能在简单的微观环境和典型的体外研究的短时标中发生。因此，大多数细菌防御系统的注释和特征及其对噬菌体疗法的影响在很大程度上仍未通过实验室研究得到充分探索。
Fewer studies have looked at evolution of bacterial resistance to phages in animal models of bacterial infections, but these are environmental settings where bacterial resistance mechanisms may have a higher cost relative to laboratory habitats. In these studies, evolution of phage-resistant bacteria is observed in 50–80% of experiments and is dependent on the type of infection and animal model employed (20). Similar to observations in vitro, evolved phage resistance in animal models is typically linked to alteration of cell-surface features. While providing phage resistance, these changes often come at the expense of attenuated virulence, re-sensitization to antibiotics, easier recognition by immune system cells, and/or inferior competitive ability relative to native microbiome species. Although potentially inconsequential for growth in vitro, any of these trade-offs would hamper the ability of phage-resistant mutants to survive in animal models, as shown by Oechslin et al. (42) and in the classic studies by Smith and Huggins and colleagues (43–45). The occurrence of such evolutionary trade-offs has been leveraged to design phage therapies that address emergence of bacterial resistance, as we discuss later in this review.
较少研究关注细菌感染动物模型中细菌对噬菌体抗性的进化，但与实验室环境相比，这些环境中细菌抗性机制的成本可能更高。在这些研究中，50%-80%的实验观察到噬菌体抗性细菌的进化，这取决于所采用的感染类型和动物模型（20）。与体外观察类似，动物模型中噬菌体抗性的进化通常与细胞表面特征的改变有关。在提供噬菌体抗性的同时，这些变化往往以削弱毒力、对抗生素重新敏感、更容易被免疫系统细胞识别和/或相对于本地微生物群物种的低劣竞争能力为代价。虽然对体外生长可能无关紧要，但正如 Oechslin 等人 ( 42) 以及 Smith 和 Huggins 及其同事 ( 43-45) 的经典研究所示，任何这些权衡都会妨碍抗噬菌体突变体在动物模型中的生存能力。这种进化权衡的出现被用来设计噬菌体疗法，以解决细菌耐药性的出现，我们将在本综述的后面讨论。
In the clinic, findings to date are either conflicting or inconclusive (20). Some compassionate-use cases and pilot studies of phage therapy in humans report the emergence of phage-resistant bacteria (46, 47), while in others no phage-resistant bacteria were detected (48, 49). Interestingly, one of these case studies reported bacterial re-sensitization to antibiotics (46), which is in line with the virulence trade-offs observed in some animal studies. In the majority of clinical trials on phage therapy [e.g., PhagoBurn (50) and an acute pediatric Escherichia coli diarrhea trial (51)], evolution of bacterial resistance to phage therapy has been poorly studied and documented, leaving a clear need to better understand the mechanisms of bacterial resistance to phage therapy and their clinical significance. Recent efforts in compassionate-use cases of personalized phage therapy shed light on understanding phage-resistance phenotypes in clinical settings (52).
在临床上，迄今为止的研究结果要么相互矛盾，要么没有定论（20）。一些人类噬菌体疗法的同情使用病例和试点研究报告称出现了噬菌体抗性细菌（46，47），而在其他病例中则未检测到噬菌体抗性细菌（48，49）。有趣的是，其中一项病例研究报告称细菌对抗生素重新敏感（46），这与一些动物研究中观察到的毒力权衡一致。在大多数噬菌体疗法临床试验中（如 PhagoBurn 试验（50）和急性小儿大肠埃希氏菌腹泻试验（51）），细菌对噬菌体疗法耐药性的演变研究和记录很少，因此显然需要更好地了解细菌对噬菌体疗法的耐药性机制及其临床意义。最近在个性化噬菌体疗法的同情使用病例方面所做的努力为了解临床环境中的噬菌体耐药性表型提供了启示（52）。
Fortunately, it seems unlikely that widespread development of phage therapy would recapitulate the antibiotic resistance crisis because bacteria do not seem to easily acquire phage defense mechanisms via horizontal genetic transfer, unlike multi-drug resistance genes that easily spread via plasmid conjugation (53). Nonetheless, evolved bacterial resistance remains a major concern for the success of phage therapy. Mounting evidence in the literature suggests that this concern can be addressed (54). In the remainder of this review, we discuss the two major complementary approaches to mitigate bacterial resistance in phage therapy: minimizing the evolution of bacterial resistance and driving evolution of phage-resistant bacteria toward clinically favorable outcomes (Figure 2).
幸运的是，噬菌体疗法的广泛发展似乎不太可能重现抗生素耐药性危机，因为细菌似乎不容易通过水平基因转移获得噬菌体防御机制，而不像耐多药基因那样容易通过质粒共轭传播 ( 53)。尽管如此，细菌耐药性的演变仍然是噬菌体疗法能否成功的一个主要问题。文献中越来越多的证据表明，这一问题是可以解决的 ( 54)。在本综述的其余部分，我们将讨论在噬菌体疗法中减轻细菌耐药性的两种主要互补方法：最大限度地减少细菌耐药性的进化和推动耐噬菌体细菌向临床有利结果进化（图 2）。
