Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, 08826, Republic of Korea.
Microb Cell Fact. 2021 Mar 2;20(1):56. doi: 10.1186/s12934-021-01549-8.
Antibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria-bacteriophage co-cultures, are required.
In the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 10 to 2 × 10 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 10 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 10 plaque forming unit (PFU)/mL). With this combination of phage-bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic Staphylococcus phage was 8.63 × 10 ± 9.71 × 10 PFU/mL (5.13-fold increase).
Therefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.
抗生素耐药菌已成为一个严重的问题；因此，噬菌体已被提议作为抗生素的替代治疗方法。一些权威机构，如药典、FDA，已经确认了它们的安全性，并且一些噬菌体在全球范围内都有商业供应。未来对噬菌体的需求预计将呈指数级增长；因此，迫切需要经济地大规模生产噬菌体。与非溶菌性噬菌体的复制不同，溶菌性噬菌体通过裂解宿主细菌进行复制，这导致噬菌体生产终止；因此，需要能够延长细菌-噬菌体共培养物中宿主细菌裂解的策略。
在本研究中，我们操纵金黄色葡萄球菌和噬菌体 pSa-3 的接种浓度（感染复数，MOI）及其能量来源，以延迟杀菌作用，同时优化噬菌体的产生。我们检查了一系列逐渐增加的细菌接种浓度（2×10 到 2×10 CFU/mL）以缩短迟滞期，同时逐渐降低噬菌体接种浓度（从 MOI 0.01 到 0.00000001）以延迟宿主的裂解。细菌浓度为 2×10 CFU/mL 和噬菌体 MOI 为 0.0001 显示出最大的最终噬菌体生产速率（1.68×10 噬菌斑形成单位（PFU）/mL）。使用这种噬菌体-细菌接种组合，我们分别选择甘油、甘氨酸和钙作为噬菌体生产的碳、氮和二价离子源。使用响应面法进行优化后，溶菌性金黄色葡萄球菌噬菌体的最终浓度为 8.63×10±9.71×10 PFU/mL（增加了 5.13 倍）。
因此，通过增加细菌接种浓度和降低种子噬菌体 MOI，可以最大化金黄色葡萄球菌噬菌体 pSa-3 的生产，这种组合策略可以缩短噬菌体生产时间。此外，我们建议响应面法具有优化溶菌性噬菌体大规模生产的潜力。
