Valdés Lorena, Gueimonde Miguel, Ruas-Madiedo Patricia
Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias - Consejo Superior de Investigaciones Científicas (IPLA-CSIC) Villaviciosa, Asturias, Spain.
Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias - Consejo Superior de Investigaciones Científicas (IPLA-CSIC) Villaviciosa, Asturias, Spain.
J Microbiol Methods. 2015 Dec;119:66-73. doi: 10.1016/j.mimet.2015.09.022. Epub 2015 Oct 5.
The incidence and severity of Clostridium difficile infections (CDI) has been increased not only among hospitalized patients, but also in healthy individuals traditionally considered as low risk population. Current treatment of CDI involves the use of antibiotics to eliminate the pathogen, although recurrent relapses have also been reported. For this reason, the search of new antimicrobials is a very active area of research. The strategy to use inhibitors of toxin's activity has however been less explored in spite of being a promising option. In this regard, the lack of fast and reliable in vitro screening methods to search for novel anti-toxin drugs has hampered this approach. The aim of the current study was to develop a method to monitor in real time the cytotoxicity of C. difficile upon the human colonocyte-like HT29 line, since epithelial intestinal cells are the primary targets of the toxins. The label-free, impedance based RCTA (real time cell analyser) technology was used to follow overtime the behaviour of HT29 in response to C. difficile LMG21717 producing both A and B toxins. Results obtained showed that the selection of the medium to grow the pathogen had a great influence in obtaining toxigenic supernatants, given that some culture media avoided the release of the toxins. A cytotoxic dose- and time-dependent effect of the supernatant obtained from GAM medium upon HT29 and Caco2 cells was detected. The sigmoid-curve fit of data obtained with HT29 allowed the calculation of different toxicological parameters, such as EC50 and LOAEL values. Finally, the modification in the behaviour of HT29 reordered in the RTCA was correlated with the cell rounding effect, typically induced by these toxins, visualized by time-lapsed captures using an optical microscope. Therefore, this RTCA method developed to test cytotoxicity kinetics of C. difficile supernatants upon IEC could be a valuable in vitro model for the screening of new anti-CDI agents.
艰难梭菌感染（CDI）的发病率和严重程度不仅在住院患者中有所增加，在传统上被视为低风险人群的健康个体中也有所上升。目前CDI的治疗方法包括使用抗生素来清除病原体，不过也有复发性复发的报道。因此，寻找新型抗菌药物是一个非常活跃的研究领域。尽管使用毒素活性抑制剂是一个有前景的选择，但这一策略的探索较少。在这方面，缺乏快速可靠的体外筛选方法来寻找新型抗毒素药物阻碍了这种方法的发展。本研究的目的是开发一种方法，实时监测艰难梭菌对人结肠癌细胞系HT29的细胞毒性，因为肠道上皮细胞是毒素的主要靶标。基于无标记、阻抗的实时细胞分析仪（RCTA）技术被用于随时间追踪HT29对产生A和B两种毒素的艰难梭菌LMG21717的反应。获得的结果表明，选择用于培养病原体的培养基对获得产毒上清液有很大影响，因为某些培养基可避免毒素的释放。检测到从GAM培养基获得的上清液对HT29和Caco2细胞具有细胞毒性剂量和时间依赖性效应。用HT29获得的数据进行S形曲线拟合，可计算不同的毒理学参数，如半数有效浓度（EC50）和最低观察到有害作用水平（LOAEL）值。最后，RTCA中HT29行为的改变与细胞变圆效应相关，这种效应通常由这些毒素诱导，通过光学显微镜的延时拍摄可见。因此，这种用于测试艰难梭菌上清液对肠上皮细胞细胞毒性动力学的RTCA方法可能是筛选新型抗CDI药物的有价值的体外模型。
