Cronobacter sakazakii is an opportunistic pathogenic bacterium found in powdered infant formula and is fatal to neonates. Antibiotic resistance has emerged owing to overuse of antibiotics. Therefore, demand for high-yield bacteriophages as an alternative to antibiotics has increased. Accordingly, we developed a modified mass-production method for bacteriophages by introducing a two-stage self-cycling (TSSC) process, which yielded high-concentration bacteriophage solutions by replenishing the nutritional medium at the beginning of each process, without additional challenge. pH of the culture medium was monitored in real-time during C. sakazakii growth and bacteriophage CS01 propagation, and the changes in various parameters were assessed. The pH of the culture medium dropped to 5.8 when the host bacteria reached the early log phase (OD₅₄₀ = 0.3). After challenge, it decreased to 4.65 and then recovered to 4.94; therefore, we set the optimum pH to challenge the phage at 5.8 and that to harvest the phage at 4.94. We then compared phage production during the TSSC process in jar-type bioreactors and the batch culture process in shaker flasks. In the same volume of LB medium, the concentration of the phage titer solution obtained with the TSSC process was 24 times higher than that obtained with the batch culture process. Moreover, we stably obtained high concentrations of bacteriophage solutions for three cycles with the TSSC process. Overall, this modified TSSC process could simplify large-scale production of bacteriophage CS01 and reduce the unit cost of phage titer solution. These results could contribute to curing infants infected with antibiotic-resistant C. sakazakii.

中文翻译：

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通过两阶段自循环过程在生物反应器中大规模生产阪崎肠杆菌噬菌体 Φ CS01。

Cronobacter sakazakii是一种在婴儿配方奶粉中发现的机会性致病菌，对新生儿是致命的。由于过度使用抗生素，抗生素耐药性已经出现。因此，对高产量噬菌体作为抗生素替代品的需求增加了。因此，我们通过引入两阶段自循环 (TSSC) 过程开发了一种改进的噬菌体大规模生产方法，该方法通过在每个过程开始时补充营养培养基来产生高浓度噬菌体溶液，而没有额外的挑战。在C. sakazakii期间实时监测培养基的 pH 值生长和噬菌体 CS01 繁殖，并评估了各种参数的变化。当宿主细菌达到早期对数期（OD ₅₄₀= 0.3）。挑战后下降至4.65，随后回升至4.94；因此，我们将挑战噬菌体的最佳 pH 值设置为 5.8，将噬菌体收获的最佳 pH 值设置为 4.94。然后，我们比较了罐式生物反应器中 TSSC 过程中的噬菌体生产和摇瓶中的分批培养过程。在相同体积的LB培养基中，TSSC工艺获得的噬菌体滴度溶液浓度比分批培养工艺高24倍。此外，我们通过 TSSC 工艺稳定地获得了三个循环的高浓度噬菌体溶液。总体而言，这种改进的 TSSC 工艺可以简化噬菌体 CS01 的大规模生产并降低噬菌体滴度溶液的单位成本。这些结果可能有助于治愈感染抗生素耐药性的婴儿C. sakazakii .