Biofilm production and eradication
生物膜的产生和消除
Biofilm assays were performed to estimate: (1) biofilm formation in the presence of phages, (2) the capability of phages to disrupt pre-formed biofilm, and (3) the virulence of ancestral and post-phage evolved bacterial strains. Biofilm assays were adapted from B. M. Coffey and G. G. Anderson42: 1.5 mL of fresh bacterial culture diluted to ~ 1e7 CFU/mL overnight was added to 24-well plate (n = 6) and incubated under static conditions at 37 °C for 24 h43. To assess biofilm formation (#1 above) phages were added to a final concentration of 1e9 PFU/mL in each well. To assess biofilm disruption (#2 above), plates were washed after 24 h to remove planktonic cells, and phages were added to a final concentration of 1e9 PFU/mL in each well. To investigate virulence (#3 above), the ancestral strain (PA4.6C) was compared to the evolved phage-resistant strain (PA4.6C-R) without the addition of phages. After 24 h, cultures were washed and stained with 125 µL 0.1% crystal violet stain per well, followed by 150 µL 30% glacial acetic acid addition and measured at OD550. CFUs were measured on separate plates by gently scratching each well after washing steps and counting on Pseudomonas isolation agar (Neogen, Lansing, MI, USA).
进行生物膜试验的目的是评估：(1) 在噬菌体存在的情况下生物膜的形成；(2) 噬菌体破坏已形成的生物膜的能力；(3) 祖先和噬菌体进化后细菌菌株的毒力。生物膜检测改编自 B. M. Coffey 和 G. G. Anderson 42 ：将 1.5 mL 稀释至 ~ 1e7 CFU/mL 的新鲜细菌培养液加入 24 孔板（n = 6）中过夜，并在 37 °C 静态条件下培养 24 小时 43 。为了评估生物膜的形成（上述 #1），在每个孔中加入最终浓度为 1e9 PFU/mL 的噬菌体。为了评估生物膜的破坏情况（上述 #2），24 小时后清洗平板以去除浮游细胞，然后在每个孔中加入最终浓度为 1e9 PFU/mL 的噬菌体。为研究毒力（上述第 3 项），将祖先菌株（PA4.6C）与不添加噬菌体的进化抗噬菌体菌株（PA4.6C-R）进行比较。24 小时后，清洗培养物，每孔用 125 µL 0.1% 水晶紫染色，然后加入 150 µL 30% 冰醋酸，并在 OD 550 时进行测量。清洗步骤结束后，轻轻刮擦每孔，在假单胞菌分离琼脂（Neogen，Lansing，MI，USA）上计数，在不同的平板上测量 CFU。
Evolution of a phage-resistant Pseudomonas aeruginosa strain
耐噬菌体铜绿假单胞菌菌株的进化
The phage-resistant Pseudomonas aeruginosa strain (PA4.6C-R) was evolved by liquid co-cultivation of PA4.6C with phage ΦSB overnight until the culture was not visibly clear, which equaled co-cultivation twice overnight34. PA4.6C-R was subsequently colony-purified in triplicate, and resistance to phage was confirmed by plaque assay and co-incubation growth curves in triplicate.
耐噬菌体铜绿假单胞菌菌株（PA4.6C-R）是通过 PA4.6C 与噬菌体 ΦSB 的液体共培养进化而来的，共培养过夜直至培养物不明显变清，相当于共培养两次过夜 34 。随后对 PA4.6C-R 进行了一式三份的菌落纯化，并通过斑块检测和一式三份的共培养生长曲线确认了其对噬菌体的抗性。
Antibiotic sensitivity testing
抗生素敏感性检测
Antibiotic sensitivity to cefiderocol (MedChemExpress, Monmouth Junction, NJ, USA) and minimum inhibitory concentrations (MIC) for piperacillin/tazobactam, ciprofloxacin, cefepime, meropenem, and imipenem (Liofilchem, Waltham, MA, USA) were obtained by recording the lowest concentration of antibiotic that inhibited growth of bacteria on Mueller Hinton agar (Neogen, Lansing, MI, USA), as described by the manufacturer’s protocol.
对头孢克洛（MedChemExpress，Monmouth Junction，NJ，USA）的抗生素敏感性以及哌拉西林/他唑巴坦、环丙沙星、头孢吡肟、美罗培南和亚胺培南（Liofilchem、美国马萨诸塞州沃尔瑟姆）上抑制细菌生长的最低抗生素浓度。
Bacterial stimulation of THP-1 cells to produce IL-8 and LDH
细菌刺激 THP-1 细胞产生 IL-8 和 LDH
PA4.6C and PA4.6C-R cell culture supernatants were obtained by centrifugation of fresh overnight cultures at 4000×g for 15 min. The pellet was washed twice with PBS, normalized to OD600 = 0.5, resuspended in LB, and centrifuged at 4000×g for 15 min. The supernatants were frozen at − 80 °C.
PA4.6C 和 PA4.6C-R 细胞培养上清经新鲜过夜培养物 4000×g 离心 15 分钟获得。沉淀用 PBS 冲洗两次，归一化至 OD 600 = 0.5，重悬于 LB 中，4000×g 离心 15 分钟。上清液冷冻于 – 80 °C。
Human monocytic THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, USA), 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere of 5% CO2. THP-1 cells were differentiated to macrophages by stimulation with 5 ng/mL phorbol-12-myristate-13 acetate (PMA, Sigma Aldrich, St. Louis, MO, USA) for 48 h in 24-well plates at a density of 1e6 cells/well in a volume of 1 mL/well44. Microscopy confirmed cell attachment to each well after washing.
人单核细胞 THP-1 细胞（American Type Culture Collection，Manassas，VA，USA）在 RPMI 1640 培养基中培养，培养基中添加 10% 热灭活胎牛血清（Gemini Bio-Products，West Sacramento，CA，USA）、2 mM L-谷氨酰胺和 100 U/mL 青霉素/链霉素（Sigma Aldrich，St. Louis，MO，USA），培养温度为 37 °C，湿度为 5% CO 2 。用 5 ng/mL phorbol-12-myristate-13 acetate（PMA，Sigma Aldrich，St. Louis，MO，USA）刺激 THP-1 细胞分化为巨噬细胞 48 小时，24 孔板中细胞密度为 1e6 个/孔，体积为 1 mL/ 孔 44 。清洗后，显微镜检查确认细胞附着在每个孔中。
THP-1 s were washed twice with PBS and treated with negative (RPMI, PBS, LB) and positive (lipopolysaccharide) controls, as well as the PA4.6C and PA4.6C-R supernatants, and the ΦSB at 1e11 PFU/mL, all in a 1:10 dilution in triplicate. After incubation for 24 h, cell culture supernatants were collected and stored at − 80 °C. IL-8 was measured by ELISA (R&D Systems, Minneapolis, MN). Cell cytotoxicity was measured using a lactate dehydrogenase (LDH) kit (Roche, Basel, Switzerland) as described previously45.
用 PBS 冲洗 THP-1 s 两次，然后用阴性（RPMI、PBS、LB）和阳性（脂多糖）对照组、PA4.6C 和 PA4.6C-R 上清液以及 1e11 PFU/mL 的 ΦSB 处理，均以 1:10 稀释，一式三份。培养 24 小时后，收集细胞培养上清并保存在 – 80 °C。IL-8 用 ELISA（R&D Systems，明尼苏达州明尼阿波利斯市）检测。细胞细胞毒性的测定采用乳酸脱氢酶（LDH）试剂盒（瑞士巴塞尔罗氏公司），如前所述 45 。