Results 成果
Phage isolation 噬菌体分离
The first step of the optimized preparation pipeline for emergency phage therapy against Pseudomonas aeruginosa in our Center addresses phage isolation. Phage vB_PaeA_SB (hereafter abbreviated ΦSB) was environmentally sourced in New Haven, CT, USA, and was found to have lytic activity against the clinical strain PA4.6C. ΦSB was amplified on PAO1 and further processed as described (see Methods). Co-incubation of ΦSB and mid-log growth-phase PAO1 bacteria at an MOI of ~ 0.01, for 6–8 h shaking constantly at 200 rpm at 37 °C, resulted in a titer of 1e10 PFU/mL prior to purification and concentration.
本中心针对铜绿假单胞菌的应急噬菌体疗法优化制备流程的第一步是噬菌体分离。噬菌体 vB_PaeA_SB（以下简称 ΦSB）来自美国康涅狄格州纽黑文的环境，对临床菌株 PA4.6C 具有杀菌活性。ΦSB 在 PAO1 上扩增，并按所述方法进一步处理（见方法）。以约 0.01 的 MOI 将 ΦSB 与生长期中期的 PAO1 细菌共培养 6-8 小时，在 37 °C 下以 200 rpm 的转速不断振荡，在纯化和浓缩前可获得 1e10 PFU/mL 的滴度。
Quality tests of the primary cell banks
原始细胞库的质量检测
The second step of the pipeline entails the quality tests of the PCB. Colony picking, differential agar testing, and phenotypic morphology (detailed in Methods) ensured that strain PA4.6C was not contaminated. Strain identity and purity was confirmed by sequencing (data not shown).
第二步是对 PCB 进行质量检测。菌落挑拣、差异琼脂测试和表型形态学（详见方法）确保了菌株 PA4.6C 没有受到污染。菌株的身份和纯度通过测序得到确认（数据未显示）。
Quality tests of the primary virus stocks
原始病毒库存的质量检测
Phage identity 噬菌体特性
The third step of the pipeline relates to quality tests of the PVS beginning with phage identity. Analysis of the phage genome39,54 predicted dsDNA virus from the family Autographiviridae (43.1 kb, 62.2% GC content). The genome map of ΦSB (Fig. 1A) and the feature table (Supplementary Table S2) describe 49 open reading frames (ORFs) with 19 functionally assigned genes aside from hypothetical proteins. Structural, packaging, and scaffolding genes account for 9 of the 19 genes and 7 of the 19 genes were assigned to nucleic acid replication, recombination, regulation, and modification. PhageTerm55 suggested a headful packaging mechanism with a higher coverage obtained after the packaging site when aligning all raw reads (8,762,162 with 90% mapping reads) to the phage genome of ΦSB. The phages most closely related to ΦSB were phages of the genus Phikmvviruses. The assembled and annotated phage genome is deposited under the NCBI accession number OR208619. No virulence or lysogeny factors were identified.
第三步是对 PVS 进行质量检测，首先是噬菌体身份鉴定。对噬菌体基因组 39,54 的分析预测，dsDNA 病毒属于 Autographiviridae 科（43.1 kb，62.2% GC 含量）。ΦSB的基因组图谱（图1A）和特征表（补充表S2）描述了49个开放阅读框（ORF），除假定蛋白外，还有19个功能基因。结构、包装和支架基因占 19 个基因中的 9 个，核酸复制、重组、调节和修饰基因占 19 个基因中的 7 个。PhageTerm 55 将所有原始读数（8,762,162，90%的映射读数）与ΦSB的噬菌体基因组进行比对时，在包装位点之后获得了较高的覆盖率，表明了头状包装机制。与ΦSB关系最密切的噬菌体是Phikmvviruses属的噬菌体。组装和注释的噬菌体基因组保存在 NCBI 的登录号 OR208619 下。未发现毒力因子或溶菌因子。