Department of Molecular Biology, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
Laboratory of Phage Therapy, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kładki 24, 80-822 Gdansk, Poland.
Toxins (Basel). 2021 Sep 11;13(9):644. doi: 10.3390/toxins13090644.
Shiga toxin-producing (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) contaminated with cow feces (often due to the use of natural fertilizers in agriculture). Since the use of antibiotics against STEC is controversial, other methods for protection of food against contaminations by these bacteria are required. Here, we propose a validation system for selection of bacteriophages against STEC contamination. As a model system, we have employed a STEC-specific bacteriophage vB_Eco4M-7 and the O157:H7 strain no. 86-24, bearing Shiga toxin-converting prophage ST2-8624 (Δ::). When these bacteria were administered on the surface of sliced cucumber (as a model vegetable), significant decrease in number viable cells was observed after 6 h of incubation. No toxicity of vB_Eco4M-7 against mammalian cells (using the Balb/3T3 cell line as a model) was detected. A rapid decrease of optical density of STEC culture was demonstrated following addition of a vB_Eco4M-7 lysate. However, longer incubation of susceptible bacteria with this bacteriophage resulted in the appearance of phage-resistant cells which predominated in the culture after 24 h incubation. Interestingly, efficiency of selection of bacteria resistant to vB_Eco4M-7 was higher at higher multiplicity of infection (MOI); the highest efficiency was evident at MOI 10, while the lowest occurred at MOI 0.001. A similar phenomenon of selection of the phage-resistant bacteria was also observed in the experiment with the STEC-contaminated cucumber after 24 h incubation with phage lysate. On the other hand, bacteriophage vB_Eco4M-7 could efficiently develop in host bacterial cells, giving plaques at similar efficiency of plating at 37, 25 and 12 °C, indicating that it can destroy STEC cells at the range of temperatures commonly used for vegetable short-term storage. These results indicate that bacteriophage vB_Eco4M-7 may be considered for its use in food protection against STEC contamination; however, caution should be taken due to the phenomenon of the appearance of phage-resistant bacteria.
产志贺毒素大肠杆菌（STEC）可引起人类严重感染，导致严重疾病和危险并发症，如溶血性尿毒综合征。尽管牛是 STEC 的主要宿主，但人类感染的最常见来源是受牛粪便污染的食品产品（例如蔬菜）（通常是由于农业中使用天然肥料）。由于对抗 STEC 使用抗生素存在争议，因此需要其他方法来保护食品免受这些细菌的污染。在这里，我们提出了一种针对 STEC 污染的噬菌体选择验证系统。作为模型系统，我们使用了一种 STEC 特异性噬菌体 vB_Eco4M-7 和 O157:H7 菌株 86-24，其携带志贺毒素转化噬菌体 ST2-8624（Δ::）。当这些细菌被施用于切片黄瓜表面（作为模型蔬菜）时，在孵育 6 小时后，观察到活菌数明显减少。未检测到 vB_Eco4M-7 对哺乳动物细胞（使用 Balb/3T3 细胞系作为模型）的毒性。在添加 vB_Eco4M-7 裂解物后，观察到 STEC 培养物的光密度迅速下降。然而，在更长时间的孵育后，易感细菌与这种噬菌体接触会导致出现噬菌体抗性细胞，这些细胞在 24 小时孵育后在培养物中占优势。有趣的是，在更高的感染复数（MOI）下，选择对 vB_Eco4M-7 具有抗性的细菌的效率更高；在 MOI 10 时效率最高，而在 MOI 0.001 时效率最低。在用噬菌体裂解物孵育 24 小时后，在受 STEC 污染的黄瓜上进行的实验中也观察到了类似的选择噬菌体抗性细菌的现象。另一方面，噬菌体 vB_Eco4M-7 可以在宿主细菌细胞中有效地发育，在 37、25 和 12°C 下的平板效率相似，表明它可以在蔬菜短期储存中常用的温度范围内破坏 STEC 细胞。这些结果表明，噬菌体 vB_Eco4M-7 可考虑用于食品保护，以防 STEC 污染；然而，由于出现噬菌体抗性细菌的现象，应谨慎使用。
