Optimized preparation pipeline for emergency phage therapy against Pseudomonas aeruginosa at Yale University耶鲁大学针对铜绿假单胞菌的紧急噬菌体疗法的优化制备流水线

Motility assays 运动试验

Twitching motility was assessed for PAO1 using a type IV pilus knockout strain of the transposon mutant library (ΔpilQ), PA4.6C and PA4.6C-R, diluted to OD₆₀₀ in LB as described previously⁴⁹ with the following modifications: to avoid superficial growth, a pipette was used to inoculate 1 µL of the bacterial suspension underneath 1% agar in a sterile petri dish agar plate. After 24 h, the petri dish was inverted over a waste receptacle and the agar was gently removed by an inoculation stick without touching the bacteria. The bottoms of the petri dishes were soaked in 3 mL 0.1% crystal violet stain for 10 min before gently washing twice with water, which was then quantified using ImageJ⁵⁰. A variation of this twitching motility assay was assessed by inoculating the middle layer of an agar plate with 0.8, 1.0 and 1.2% agar and measuring the maximum diameter of the middle layer growth areas after 36 h. Swimming motility⁵¹ (middle of the agar plate, 0.25% agar) and swarming motility⁵² (surface of the agar plate, 0.75% agar) followed the adapted twitching motility assay described above, aside from the varied agar percentage and inoculation layer.
使用转座子突变体库（ΔpilQ）中的IV型柔毛敲除菌株PA4.6C和PA4.6C-R来评估PAO1的抽动运动性，按照之前的描述 ⁴⁹ ，在LB中稀释至OD值 ₆₀₀ ，并做了以下修改：为避免表层生长，用移液管将1 µL的细菌悬浮液接种到无菌培养皿琼脂平板中的1%琼脂下。24 小时后，将培养皿倒置在废物容器上，用接种棒轻轻去除琼脂，但不接触细菌。将培养皿底部浸泡在 3 mL 0.1% 水晶紫染色剂中 10 分钟，然后用水轻轻清洗两次，再用 ImageJ ⁵⁰ 进行量化。在琼脂平板的中间层接种 0.8%、1.0% 和 1.2%的琼脂，36 小时后测量中间层生长区的最大直径，以此评估抽动运动试验的变体。(琼脂平板中间，0.25%琼脂）和蜂群运动 ⁵² （琼脂平板表面，0.25%琼脂）。(琼脂平板表面，0.75%琼脂）除琼脂比例和接种层不同外，均沿用了上述适应性抽动运动试验。

Bioinformatic analysis 生物信息分析

High-titer sterile filtered phage lysates were treated with DNAse I reaction buffer (New England Biolabs, Ipswich, MA, USA), DNAse I (~ 40 U/mL, New England Biolabs, Ipswich, MA, USA), and RNAse A (~ 0.1 mg/mL, Thermo Fisher, Waltham, MA) for 1–2 h at 37 °C prior to heat inactivation and subsequent incubation with 2X Buffer A (200 mM NaCl, 200 mM Tris, and 20 mM EDTA), proteinase K (~ 0.2 mg/mL, Thermo Fisher, Waltham, MA) and 20% (wt/vol) SDS (AmericanBio, Canton, MA, USA) for 1 h at 56 °C. Phage and bacteria genomes were obtained by Phenol–Chloroform-DNA extraction (Sigma Aldrich, Burlington, MA, USA) and isopropanol precipitation (Sigma Aldrich, Burlington, MA, USA). The Yale Center for Genome Analysis performed library preparations with the IDT EZ Kit Cat#10009821 (IDT, Skokie, IL, USA) and ran samples on the Novaseq 6000 with the sequencing reagents S4 Reagent Kit v1.5 and NovaSeq XP 4-Lane Kit v1.5 (Illumina, San Diego, CA, USA). The first contig revealed the phage genome, as confirmed by Phaster⁵³ and NCBI Blast³⁹. Trimmed, error-corrected and normalized reads were de novo assembled by Geneious (Geneious version 2022.2 created by Biomatters, Auckland, New Zealand). The PAP Structural Workflow v2021.02 and the PAP Functional Workflow v2022.01 of CPT Phage Galaxy was used for structural (Glimmer3, MetageneAnnotator, Sixpack) and functional (Canonical Annotation, SwissProt, NR) annotation, respectively⁵⁴. PhageTerm was used to predict the packaging mode⁵⁵. A phage genome map was prepared with Geneious version 2022.2 (Biomatters, Auckland, New Zealand). The most similar phage genomes were identified using NCBI Blast³⁹. ABRicate and AMRFinder were used to screen for antimicrobial resistance genes in phage genomes^56,57 (last accession date June 19, 2023). The Virulence Factor Database was used to identify potential virulence factors⁵⁸ (last accession date June 20, 2023). The contigs of the strains PA4.6C and PA4.6C-R were checked with NCBI Blast³⁹, confirming Pseudomonas aeruginosa. The analysis involved in determining single nucleotide polymorphism differences between PA4.6C-R (sequence read archive SRS19816778) and the parent clinical strain PA4.6C (sequence read archive SRS19816777) was performed with the Breseq pipeline⁵⁹ using PAO1 (accession number ASM676v1) as a reference.
用 DNAse I 反应缓冲液（New England Biolabs, Ipswich, MA, USA）、DNAse I（~ 40 U/mL，New England Biolabs, Ipswich, MA, USA）和 RNAse A（~ 0.1 mg/mL，Thermo Fisher，Waltham，MA）在 37 °C加热灭活 1-2 小时，然后用 2X 缓冲液 A（200 mM NaCl、200 mM Tris 和 20 mM EDTA）、蛋白酶 K（~ 0.2 mg/mL，Thermo Fisher，Waltham，MA）和 20%（重量/体积）SDS（AmericanBio，Canton，MA，USA）在 56 °C孵育 1 小时。噬菌体和细菌基因组通过苯酚-氯仿-DNA 提取（Sigma Aldrich, Burlington, MA, USA）和异丙醇沉淀（Sigma Aldrich, Burlington, MA, USA）获得。耶鲁大学基因组分析中心使用 IDT EZ Kit Cat#10009821 （IDT，Skokie，IL，USA）进行文库制备，并使用测序试剂 S4 Reagent Kit v1.5 和 NovaSeq XP 4-Lane Kit v1.5 （Illumina，San Diego，CA，USA）在 Novaseq 6000 上运行样本。经 Phaster ⁵³ 和 NCBI Blast ³⁹ 确认，第一个等位组显示了噬菌体基因组。经修剪、误差校正和归一化的读数由 Geneious（新西兰奥克兰 Biomatters 公司创建的 Geneious 2022.2 版）重新组装。CPT Phage Galaxy 的 PAP Structural Workflow v2021.02 和 PAP Functional Workflow v2022.01 分别用于结构注释（Glimmer3、MetageneAnnotator、Sixpack）和功能注释（Canonical Annotation、SwissProt、NR） ⁵⁴ 。PhageTerm 用于预测包装模式 ⁵⁵ 。噬菌体基因组图谱由 Geneious 2022.2 版（Biomatters, Auckland, New Zealand）绘制。使用 NCBI Blast ³⁹ 确定了最相似的噬菌体基因组。ABRicate 和 AMRFinder 用于筛选噬菌体基因组中的抗菌药耐药性基因 ^56,57 。(最后加入日期为 2023 年 6 月 19 日）。毒力因子数据库（Virulence Factor Database）用于识别潜在的毒力因子 ⁵⁸ （最后登录日期：2023 年 6 月 19 日）。(最后登录日期：2023 年 6 月 20 日）。用 NCBI Blast {{7} 检查了 PA4.6C 和 PA4.6C-R 菌株的等位基因 {{8} ，确认为铜绿假单胞菌。确认为铜绿假单胞菌。以 PAO1（登录号：ASM676v1）为参照物，利用 Breseq pipeline {{8} 进行分析，确定 PA4.6C-R（序列读取档案 SRS19816778）与母本临床菌株 PA4.6C（序列读取档案 SRS19816777）之间的单核苷酸多态性差异。

Statistical analyses 统计分析

All statistical analyses were performed in R Studio (version 2022.07.1 + 554, R version 4.2.1, Wilcoxon rank sum test). Data were plotted using GraphPad Prism (San Diego, CA, USA, version 9.3.1).
所有统计分析均在 R Studio（版本 2022.07.1 + 554，R 版本 4.2.1，Wilcoxon 秩和检验）中进行。数据用 GraphPad Prism（美国加利福尼亚州圣地亚哥，9.3.1 版）绘制。