Laboratorio de Inmunoquímica y Biotecnología, Departamento SAMP, Fac. Cs. Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil Buenos Aires, Argentina; CONICET, Buenos Aires, Argentina.
Front Cell Infect Microbiol. 2012 Jun 15;2:82. doi: 10.3389/fcimb.2012.00082. eCollection 2012.
Shiga toxin-producing Escherichia coli (STEC) are characterized by the production of Shiga toxins (Stx) encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness. The variant named stx(2g) was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx(2g)-positive strains isolated from humans, animals, and environmental sources have shown that they have a close relationship. In this study, stx(2g)-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity. The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62) hybridized with a stx(2)-probe. Furthermore, the production of Stx was evaluated by enzyme immunoassay (EIA) and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal for induced than uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx(2g)-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected. Taking into account all the results, several differences could be found among stx(2g)-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx(2)-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx(2g)-positive strains Stx production is phage-regulated.
产志贺毒素大肠杆菌（STEC）的特征是产生由温和噬菌体编码的志贺毒素（Stx）。Stx 的产生与噬菌体裂解周期的诱导有关。已经描述了几种 Stx 变体，并且它们与发生严重疾病的风险相关。命名为 stx(2g)的变体最初是在从健康牛粪便中分离的 STEC 菌株中发现的。对从人类、动物和环境来源分离的 stx(2g)-阳性菌株进行分析表明，它们之间存在密切关系。在这项研究中，分析了从牛中分离的 stx(2g)-阳性 STEC 的噬菌体和 Stx 产生情况，目的是将结果与观察到的细胞毒性差异联系起来。通过分析细菌生长/裂解曲线和噬菌斑分析来评估可诱导噬菌体的存在。所有分离株在无诱导条件下的细菌生长曲线相似，但在诱导培养物中明显不同。在这种条件下明显表现出细菌裂解的两种菌株在噬菌斑分析中也显示出更高的噬菌体滴度。然而，只有这些菌株之一（FB 62）产生的噬菌体斑与 stx(2)-探针杂交。此外，通过酶免疫分析（EIA）和过夜上清液的 Western 免疫印迹评估 Stx 的产生。通过 EIA，我们仅在 FB 62 的上清液中检测到 Stx，诱导培养物的信号高于未诱导培养物。通过免疫印迹，在所有 stx(2g)-阳性分离株中都可以在诱导后检测到 Stx2，但在无法检测到噬菌体的那些上清液中，Stx2B 亚基的量较低。考虑到所有结果，stx(2g)-阳性菌株之间存在几个差异。具有最高细胞毒性滴度的菌株显示出更高水平的 stx(2)-噬菌体和 EIA 产生的毒素，而先前显示低细胞毒性滴度的菌株则相反，这证实了在 stx(2g)-阳性菌株中，Stx 的产生是受噬菌体调节的。
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