Abstract 摘要
Phage therapy is one of the alternatives to treat infections caused by both antibiotic-sensitive and antibiotic-resistant bacteria, with no or low toxicity to patients. It was started a century ago, although rapidly growing bacterial antimicrobial resistance, resulting in high levels of morbidity, mortality, and financial cost, has initiated the revival of phage therapy. It involves the use of live lytic, bioengineered, phage-encoded biological products, in combination with chemical antibiotics to treat bacterial infections. Importantly, phages will be removed from the body within seven days of clearing an infection. They target specific bacterial strains and cause minimal disruption to the microbial balance in humans. Phages for medication must be screened for the absence of resistant genes, virulent genes, cytotoxicity, and their interaction with the host tissue and organs. Since they are immunogenic, applying a high phage titer for therapy exposes them and activates the host immune system. To date, no serious side effects have been reported with human phage therapy. In this review, we describe phage–phagocyte interaction, bacterial resistance to phages, how phages conquer bacterial resistance, the role of genetic engineering and other technologies in phage therapy, and the therapeutic application of modified phages and phage-encoded products. We also highlight the comparison of antibiotics and lytic phage therapy, the pros and cons of phage therapy, determinants of human phage therapy trials, phage quality and safety requirements, phage storage and handling, and current challenges in phage therapy.
噬菌体疗法是治疗由抗生素敏感细菌和抗生素耐药细菌引起的感染的替代疗法之一，对患者无毒或低毒。噬菌体疗法始于一个世纪前，尽管细菌对抗生素的耐药性迅速增长，导致发病率、死亡率和经济成本居高不下，但噬菌体疗法仍在复兴。噬菌体疗法是指使用活体溶菌、生物工程、噬菌体编码的生物制品，结合化学抗生素来治疗细菌感染。重要的是，噬菌体会在清除感染后的七天内从体内清除。它们针对特定的细菌菌株，对人体微生物平衡的破坏极小。用于药物治疗的噬菌体必须经过筛选，以确定是否含有抗药性基因、毒性基因、细胞毒性以及与宿主组织和器官的相互作用。由于噬菌体具有免疫原性，使用高滴度的噬菌体进行治疗会暴露噬菌体并激活宿主的免疫系统。迄今为止，人类噬菌体疗法尚未出现严重的副作用。在这篇综述中，我们将介绍噬菌体与吞噬细胞的相互作用、细菌对噬菌体的抗药性、噬菌体如何战胜细菌的抗药性、基因工程和其他技术在噬菌体疗法中的作用，以及改良噬菌体和噬菌体编码产品的治疗应用。我们还重点介绍了抗生素和溶菌噬菌体疗法的比较、噬菌体疗法的利弊、人类噬菌体疗法试验的决定因素、噬菌体的质量和安全要求、噬菌体的储存和处理以及当前噬菌体疗法面临的挑战。
Keywords: lysogenization, lytic phage, modified phages, resistance to phages, CRISPR, immunity, conquering CRISPR, phage-encoded products
关键词： 溶酶原化 溶菌噬菌体 改性噬菌体 对噬菌体的抵抗力 CRISPR 免疫力 征服 CRISPR 噬菌体编码产物
Introduction 导言
Phages are obligate intracellular viruses that infect and kill bacteria. They exist everywhere that bacteria live and there are about 1029–1030 phages in the biosphere. They have high durability in natural systems and the inherent potential to reproduce rapidly in their appropriate host.1,2 Phages are made up of proteins or proteolipid capsids containing fragments of deoxynucleic acid (DNA) or ribonucleic acid (RNA) (Figure 1). Their genome size ranges from a few thousand to 498 kbs.3 Phages have no machinery to generate energy or ribosomes to make proteins, even though they carry the genetic information needed to replicate in the right host cell.
噬菌体是一种能感染和杀死细菌的细胞内病毒。细菌生活的任何地方都有噬菌体，生物圈中约有 10 29 -101 个噬菌体。噬菌体在自然系统中具有很高的耐久性，并具有在适当宿主体内快速繁殖的内在潜力。 1 2 噬菌体由含有脱氧核糖核酸（DNA）或核糖核酸（RNA）片段的蛋白质或蛋白脂囊体组成（图 1）。它们的基因组大小从几千到 498 kbs 不等。 3 尽管噬菌体携带在合适的宿主细胞中复制所需的遗传信息，但它们没有产生能量的机器，也没有制造蛋白质的核糖体。
Classification of phages.
噬菌体的分类。
Phages are generally specific and their specificity is determined by phage–host receptor surface, genetic and host physical defense mechanisms, the nature of the phage(s), and their co-evolution. Phage lytic enzymes (endolysins) have broader specificity at the genus and/or species level. However, their specificity varies from infecting many bacteria to infecting a single strain. Limitations in sensitivity to a single phage therapy during polymicrobial infection are solved by applying phage cocktails.4
噬菌体一般具有特异性，其特异性由噬菌体-宿主受体表面、基因和宿主物理防御机制、噬菌体的性质以及它们的共同进化决定。噬菌体溶解酶（内溶酶）在属和/或种一级具有更广泛的特异性。不过，它们的特异性也不尽相同，有的能感染多种细菌，有的只能感染单一菌株。在多微生物感染过程中，单一噬菌体疗法的敏感性局限性可通过应用噬菌体鸡尾酒来解决。 4
Phage therapy is a way of delivering virulent phages to a clinically ill patient to rapidly kill pathogenic bacteria.5 It involves the use of lytic phages, bioengineered phages, and purified lytic proteins of phages to infect and lyse bacteria at the site of infection. Phages and their lytic proteins can be used specifically to treat multidrug-resistant (MDR) bacteria, either alone or supplemented with antibiotics. Therapeutic approaches using phages are rapidly increasing, although there is still no adequate knowledge on phage–phage, phage–bacteria, or phage–human interactions, mainly because of safety and efficacy concerns.6 Novel concepts in phage therapy involve direct treatment of bacterial infections, phage-mediated prevention of bacterial infection, and the exploration of phage diversity in environmental and human ecological niches (Figure 2).5 Currently, human phage therapy trials are being undertaken, although therapeutic use of phages is limited to Georgia, Poland, and Russia.7 Phage therapy is a promising approach to fighting bacterial infections, as phages have unique bacteria-killing mechanisms and life cycles; either lytic or lysogenic growth cycles (Figure 3). Only lytic phages are used for therapeutic purposes. They inhibit the emergence of resistant bacteria by killing the bacteria that they infect and are preferable to antibiotics as they cause less damage to the general microbiome.8 Lytic phage therapy involves the replication of phages in phage-infected bacteria; the phages disrupt bacterial metabolism and kill the bacteria.
噬菌体疗法是一种向临床病人输送毒性噬菌体以快速杀死致病细菌的方法。 5 它包括使用溶菌噬菌体、生物工程噬菌体和纯化的噬菌体溶菌蛋白来感染和裂解感染部位的细菌。噬菌体及其溶菌蛋白可专门用于治疗耐多药（MDR）细菌，既可单独使用，也可与抗生素一起使用。使用噬菌体的治疗方法正在迅速增加，尽管人们对噬菌体-噬菌体、噬菌体-细菌或噬菌体-人之间的相互作用仍缺乏足够的了解，这主要是出于安全性和有效性方面的考虑。 6 噬菌体疗法的新概念包括直接治疗细菌感染、噬菌体介导的细菌感染预防以及探索环境和人类生态位中的噬菌体多样性（图 2）。 5 目前，人类正在进行噬菌体疗法试验，但噬菌体的治疗应用仅限于格鲁吉亚、波兰和俄罗斯。 7 噬菌体疗法是对抗细菌感染的一种很有前景的方法，因为噬菌体具有独特的杀灭细菌机制和生命周期；要么是溶菌性生长周期，要么是溶解性生长周期（图 3）。只有溶解性噬菌体可用于治疗目的。噬菌体通过杀死其感染的细菌来抑制抗药性细菌的出现，由于对整个微生物群造成的破坏较小，因此比抗生素更可取。 8 吞噬噬菌体疗法包括在受噬菌体感染的细菌中复制噬菌体；噬菌体破坏细菌的新陈代谢并杀死细菌。
Novel concepts of phage therapy.
噬菌体疗法的新概念。
Phage lytic life cycle. 噬菌体的生命周期
Notes: Adapted from Adesanya O, Oduselu T, Akin-Ajani O, Adewumi OM, Ademowo OG. An exegesis of bacteriophage therapy: An emerging player in the fight against anti-microbial resistance. AIMS Microbiol. 2020;6(3):204–230 (https://creativecommons.org/licenses/by/4.0/).104
注释：改编自 Adesanya O, Oduselu T, Akin-Ajani O, Adewumi OM, Ademowo OG.噬菌体疗法释义：抗微生物耐药性斗争中的新兴参与者。AIMS Microbiol.2020；6（3）：204-230 ( https://creativecommons.org/licenses/by/4.0/). 104
The ability of most lytic phages to encode the enzymes, holins and endolysins, that degrade bacterial structures (cell membrane and cell wall) make them a potential new weapon in the fight against bacterial infections. This property makes them efficacious against both antibiotic-sensitive and antibiotic-resistant bacteria. However, some lytic phages use only endolysins. Indeed, holins degrade the bacterial cytoplasm, allowing endolysins to access bacterial glycoproteins. Holins control the exact point in time for endolysins to access the bacterial murein and synchronize the holin–endolysin system to the late stage of viral replication. The synergetic holin–endolysin system causes cell lysis and the release of mature lytic phage progeny.9 Thereby, 50–200 mature phage progenies will be released from the lysis of a bacterium.11,45
大多数溶菌噬菌体都能编码降解细菌结构（细胞膜和细胞壁）的酶–冬青酶和内溶酶，这使它们成为抗击细菌感染的潜在新武器。这种特性使它们对抗生素敏感细菌和抗生素耐药细菌都有效。不过，有些溶菌噬菌体只使用内溶菌素。事实上，溶菌酶能降解细菌的细胞质，让内溶菌酶进入细菌的糖蛋白。内溶酶体控制着内溶酶体进入细菌糖蛋白的确切时间点，并使内溶酶体-内溶酶体系统与病毒复制的后期阶段同步。协同作用的冬青肽-内溶素系统会导致细胞裂解，并释放出成熟的裂解噬菌体后代。因此，细菌裂解后会释放出 50-200 个成熟的噬菌体后代。 11 45
Temperate phages are not used for therapeutic purposes because they integrate their genome into the host chromosome or sometimes maintain it as a plasmid to be transmitted to daughter cells during cell division or horizontally across the bacterial community. They may undergo a typical lytic cycle or lysogenization. Temperate phages enter the lytic life cycle when host conditions are weakened, maybe due to a scarcity of nutrients; then, prophages become active. At this stage, they promote the reproductive cycle, resulting in lysis of the bacterial cell. In the lysogenic life cycle, the virus continues to replicate as the bacterium continues to reproduce, and is found in all bacterial offspring. Example, phage lambda of E. coli is a common phage that has both a lysogenic cycle and a lytic cycle.10 Surprisingly, temperate phages may increase the pathogenicity of the host bacteria because bacterial virulent genes are identified from their genome.11
温带噬菌体不用于治疗目的，因为它们会将基因组整合到宿主染色体中，有时也会将基因组作为质粒保留下来，在细胞分裂过程中或在细菌群落中水平传播给子细胞。它们可能会经历一个典型的溶菌周期或溶原过程。温带噬菌体在宿主条件变弱（可能是由于缺乏营养）时进入裂解生命周期；然后，原生噬菌体开始活跃。在这一阶段，它们会促进繁殖循环，导致细菌细胞裂解。在溶解性生命周期中，病毒随着细菌的不断繁殖而继续复制，并在所有细菌后代中发现。例如，大肠杆菌的λ噬菌体是一种常见的噬菌体，它既有溶解周期，也有溶解周期。 10 令人惊讶的是，温带噬菌体可能会增加宿主细菌的致病性，因为从它们的基因组中可以鉴定出细菌的毒力基因。 11
Phage cocktails are used for phage therapy owing to rapidly emerging bacterial resistance, because many types of phages infect the same species or strain of bacterium. Thus, phage cocktails could be used to target different structural sites and metabolic activities of a bacterium. It may be argued that only a single specific phage should be used against a pathogen to prevent the emergence of resistant bacteria, since the extensive use of phages may promote resistance to phage cocktails.12 Some of the challenges in applying phage cocktail therapy are the inability to predict the effect of mass use of phages, the very high cost of phage treatment, the issue of efficacy, and the high specificity of phages.13 The highly increased emergence and spread of resistant pathogens and the lack of new drug production have directed many institutions and commercial companies to become engaged in phage therapy.14 Antibiotic-resistant opportunistic pathogens are a threat, especially for immune-compromised and immune-incompetent patients in healthcare settings. These are serious problems in medicine, to which phage therapy may provide a solution.15
噬菌体鸡尾酒用于噬菌体疗法，是因为细菌的抗药性正在迅速出现，因为许多类型的噬菌体会感染同一种类或菌株的细菌。因此，鸡尾酒噬菌体可用于针对细菌的不同结构位点和代谢活动。有人可能会说，为了防止出现抗药性细菌，只应针对病原体使用单一的特定噬菌体，因为广泛使用噬菌体可能会增强对噬菌体鸡尾酒的抗药性。 12 采用噬菌体鸡尾酒疗法面临的一些挑战包括：无法预测大量使用噬菌体的效果、噬菌体治疗的成本非常高、疗效问题以及噬菌体的高度特异性。 13 由于抗药性病原体的出现和传播日益频繁，加上缺乏新药的生产，许多机构和商业公司开始涉足噬菌体疗法。 14 耐抗生素的机会性病原体是一种威胁，尤其是对医疗机构中免疫力低下和免疫功能不全的病人。这些都是医学界面临的严重问题，而噬菌体疗法或许可以提供一种解决方案。 15
Although the use of phage therapy against bacterial infections is very promising, with plenty of advantages, many advances will be needed to implement phage therapy on a large enough scale for therapeutic purposes, owing to emerging issues on the safety, quality, and stability of phages, and the lack of sufficient evidence for their use in human medication.16
尽管使用噬菌体疗法治疗细菌感染前景广阔，优势多多，但由于噬菌体的安全性、质量和稳定性等问题不断出现，而且缺乏足够的证据证明噬菌体可用于人类治疗，因此要想大规模地将噬菌体疗法用于治疗目的，还需要取得许多进展。 16