Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria
Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria
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针对抗菌药物耐药临床分离细菌开发新型噬菌体疗法鸡尾酒的选择方法分析
DOI：
10.1101/2020.10.13.337345
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Haines M
中科院分区：
文献类型：
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作者：
Haines M
抗菌素耐药性（AMR）是全球面临的一个主要问题。与尿路感染（UTI）相关脓毒症相关的主要细菌是大肠杆菌。大肠杆菌和克雷伯氏菌以及肠杆菌属。这些细菌都含有被称为 ESBL（超广谱β-内酰胺酶）的 AMR 菌株，它们在世界卫生组织优先病原体清单中被列为对研究“至关重要”。噬菌体作为可以感染和杀死细菌的病毒，可以提供有效的工具来应对这些 AMR 菌株。目前尚无开发噬菌体鸡尾酒的“黄金标准”。在这里，我们描述了一种针对一组产 ESBL 的大肠杆菌开发有效噬菌体混合物的新方法。大肠杆菌和克雷伯氏菌主要从英国医院的患者中分离出来。通过比较不同的噬菌体功效测量方法，我们展示了哪种方法最有效，并提出了一种有效的筛选级联，可用于开发针对其他 AMR 细菌物种的噬菌体混合物。对从尿样中分离出的 38 种产生 ESBL 的临床菌株进行了整理，并用于测试噬菌体功效。在对 68 种噬菌体进行初步筛选后，鉴定出 6 种噬菌体，并针对这 38 种菌株进行测试，以确定它们的临床覆盖率和杀灭效率。为了实现这一目标，我们评估了四种不同的方法来评估这些细菌分离株的噬菌体毒力。这些是直接斑点测试（DST）、电镀效率（EOP）测定、浮游杀灭测定（PKA）和生物膜测定。最终的六种噬菌体的 ESBL 混合物可以有效杀死 23/38 株菌株 (61%)，其中克雷伯氏菌 13/19 (68%) 和大肠杆菌。 coli10/19 (53%) 基于 PKA 数据。 ESBLE。大肠杆菌集合中有 6 个来自流行的 UTI 相关 ST131 序列类型的分离株，其中 5 个是最终鸡尾酒有效靶向的。在用于评估噬菌体毒力的四种方法中，数据表明 PKA 与耗时得多的 EOP 一样有效，并且两种测定的数据具有良好的相关性。这表明浮游杀灭是确定鸡尾酒中应使用哪些噬菌体的良好指标。该测定与毒力指数相结合还允许“噬菌体协同作用”为鸡尾酒设计提供信息。
Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis areE. coliandKlebsiellaalong withEnterobacterspecies. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as “critical” for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no “gold standard” for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producingE. coliandKlebsiellalargely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%), forKlebsiella13/19 (68%) and forE. coli10/19 (53%) based on the PKA data. The ESBLE. colicollection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows “phage synergy” to inform cocktail design.