Materials and methods 材料和方法
Bacterial culture conditions
细菌培养条件
The laboratory strain Pseudomonas aeruginosa PAO1 and the PAO1 transposon mutant library were kindly provided by B. Kazmierczak (Yale University) and C. Manoil (University of Washington, Seattle), respectively. The mutants were previously created as detailed by Jacobs et al.36. Mutants of this library were selected for this study based on 26 candidate surface phage receptors. De-identified clinical bacterial strains were collected from clinical microbiology laboratory after informed consent was obtained. All strains of this study are listed in Supplementary Table S1. All laboratory experiments were performed in accordance with Yale University’s approved laboratory protocols. For subjects that proceed to bacteriophage therapy, FDA eaIND and Yale University Human Investigation Committee/Institutional Review Board approval is obtained.
实验室菌株铜绿假单胞菌 PAO1 和 PAO1 转座子突变体文库分别由 B. Kazmierczak（耶鲁大学）和 C. Manoil（华盛顿大学西雅图分校）友情提供。突变体的创建过程详见 Jacobs 等人的研究 36 。本研究根据 26 个候选表面噬菌体受体选择了该文库中的突变体。在获得知情同意后，从临床微生物实验室收集了去鉴定的临床细菌菌株。本研究的所有菌株列于补充表 S1。所有实验室实验均按照耶鲁大学批准的实验室方案进行。对于进行噬菌体治疗的受试者，需获得美国食品药物管理局 eaIND 和耶鲁大学人类调查委员会/机构审查委员会的批准。
All bacteria for this study were cultured with shaking at 37 °C in Luria Bertani broth (LB; 1.5% w/v bottom agar, 0.75% w/v soft agar). The Primary Cell Banks (PCB) of bacteria were aliquoted as homogenous collections and stored at − 80 °C in glycerol. Working PCB stocks were obtained from culturing one vial of the homogeneous PCB at 37 °C in LB overnight. For phage host range testing 52 Pseudomonas aeruginosa and 6 E. coli were included in this study.
本研究中的所有细菌均在 37 °C、振荡条件下于 Luria Bertani 肉汤（LB；1.5% w/v 底琼脂，0.75% w/v 软琼脂）中培养。细菌的原代细胞库（PCB）以等分的方式均质收集，并在 – 80 °C 的温度下保存在甘油中。在 37 ℃ 的 LB 培养液中培养一小瓶均质 PCB 过夜，即可获得工作 PCB 储存液。本研究包括 52 个铜绿假单胞菌和 6 个大肠杆菌的噬菌体宿主范围测试。
Master virus stocks 主病毒库
Phage vB_PaeA_SB (hereafter abbreviated: ΦSB) was isolated from local wastewater (New Haven, CT, USA), amplified on PAO1, plaque-purified three times and re-amplified on PAO1 with a multiplicity of infection (MOI) of ~ 0.01 in Tryptic Soy broth of non-animal origin (Merck, Rahway, NJ, USA) by co-incubation for 6–8 h with shaking at 37 °C, followed by centrifugation, sterile filtration (0.22 µm), and centrifugation with Amicon Ultra-15 Centrifugal Filter Units (100 kDa, Millipore Sigma, St. Louis, MO, USA). Purification by CsCl step density gradient was performed as previously described37 at 38,000 rpm for 3 h with four dialysis steps using Amicon Ultra-15 Centrifugal Filter Units (100 kDa, Millipore Sigma, St. Louis, MO, USA) at 4000×g for 15 min38 and subsequent sterile filtration. The virus family for each phage was predicted using BLASTn search39 and confirmed by transmission electron microscopy. Primary Virus Stocks (PVS) aliquoted as a homogenous collection were stored at 4 °C in PBS. For phage amplification (Step 4; Fig. 1), a vial of PVS was amplified on PAO1 (MOI of ~ 0.01) in Tryptic Soy broth of non-animal origin as described above, using Centricon plus-70 concentrators (100 kDa, Millipore/Sigma, Burlington, MA, USA). After completing isolation, characterization, amplification, and purification (Steps 1–4; Fig. 1), the solution was ready for preparation for phage therapy, which is beyond the scope of this manuscript, and includes: dilution to the appropriate titer and buffer, release tests, and formulation for the particular application (e.g., inhaled, intravenous, or topical administration). Endotoxin testing and sterility are confirmed via external testing according to U.S. Pharmacopeia (USP < 71 >)40, and while these methodologies are not included, they are discussed below because they are an integral part of the pipeline for phage therapy SPIND eaINDs.
从当地废水（美国康涅狄格州纽黑文）中分离出噬菌体 vB_PaeA_SB（以下简称：ΦSB），在 PAO1 上扩增，进行三次斑块纯化，然后在 PAO1 上再次扩增，感染倍数（MOI）约为 0.01 在非动物源胰蛋白酶大豆肉汤（Merck, Rahway, NJ, USA）中共同培养 6-8 小时，37 °C振荡，然后离心，无菌过滤（0.22 µm），用 Amicon Ultra-15 离心过滤器单元（100 kDa，Millipore Sigma, St.）按照之前所述的方法 37 ，使用 Amicon Ultra-15 Centrifugal Filter Units（100 kDa，Millipore Sigma，St. Louis，MO，USA）在 38,000 rpm 的转速下进行四步透析，15 分钟 38 ，然后进行无菌过滤。使用 BLASTn 搜索 39 预测每种噬菌体的病毒科，并通过透射电子显微镜进行确认。将等分的原始病毒储存液（PVS）均匀地保存在 4 °C 的 PBS 中。为了进行噬菌体扩增（步骤 4；图 1），如上所述，使用 Centricon plus-70 浓缩器（100 kDa，Millipore/Sigma，Burlington，MA，USA）在非动物来源的胰蛋白酶大豆肉汤中对一小瓶 PVS 进行 PAO1 扩增（MOI 约为 0.01）。在完成分离、表征、扩增和纯化（步骤 1-4；图 1）后，溶液就可以准备用于噬菌体治疗了，这超出了本手稿的范围，包括：稀释到适当的滴度和缓冲液、释放测试和特定应用的配制（如吸入、静脉注射或局部用药）。内毒素测试和无菌性根据《美国药典》（USP < 71 >）{{3}通过外部测试确认。｝虽然这些方法未包括在内，但由于它们是噬菌体疗法SPIND eaIND的一个组成部分，因此在下文中将进行讨论。
Quality tests for primary cell banks (PCB) and primary virus stocks (PVS)
原始细胞库（PCB）和原始病毒库（PVS）的质量检测
Identity and purity of PCB was performed by colony-purification on Pseudomonas isolation agar (Neogen, Lansing, MI, USA) and sequencing. PCB potency was confirmed by CFU/mL. PVS was confirmed by genetic sequencing and phage characterization. Phage amplification was performed on PAO1, and titer was measured by PFU/mL.
通过在假单胞菌分离琼脂（Neogen，Lansing，MI，USA）上进行菌落纯化和测序，确定多氯联苯的特性和纯度。多氯联苯的效力通过 CFU/mL 来确认。通过基因测序和噬菌体鉴定确认了 PVS。噬菌体扩增在 PAO1 上进行，滴度按 PFU/mL 测定。