Models to study host-pathogen interactions in vitro are an important tool for investigating the infectious disease process and evaluating the efficacy of antimicrobial compounds. In these models, the viability of mammalian cells is often determined using the lactate dehydrogenase (LDH) cytotoxicity assay. In the present study we evaluated whether bacteria could interfere with the LDH assay. As a model for host-pathogen interactions, we co-cultured lung epithelial cells with eight bacteria encountered in the lower respiratory tract. We show that LDH activity is affected by Pseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Streptococcus pneumoniae, and that this depends on the density of the start inoculum and the duration of infection. Two different mechanisms were discovered through which bacteria interfered with LDH activity, i.e., acidification of the cell culture medium (by K. pneumoniae and S. pneumoniae) and protease production (by P. aeruginosa and S. maltophilia). In addition, we developed and validated a modified protocol to evaluate cytotoxicity using the LDH assay, where bacterial interference with LDH quantification is avoided.

研究宿主-病原体相互作用的模型 体外是研究传染病过程和评估抗菌化合物功效的重要工具。在这些模型中，通常使用乳酸脱氢酶（LDH）细胞毒性测定法确定哺乳动物细胞的生存力。在本研究中，我们评估了细菌是否会干扰LDH分析。作为宿主-病原体相互作用的模型，我们将肺上皮细胞与下呼吸道中遇到的八种细菌共培养。我们显示LDH活性受以下因素影响铜绿假单胞菌，肺炎克雷伯菌，嗜麦芽窄食单胞菌和 肺炎链球菌，这取决于起始接种物的密度和感染的持续时间。发现了两种不同的机制，细菌通过这种机制干扰LDH活性，即细胞培养基的酸化（通过肺炎克雷伯氏菌 和 肺炎链球菌）和蛋白酶生产（通过 铜绿假单胞菌 和 嗜麦芽孢杆菌）。此外，我们开发并验证了使用LDH分析评估细胞毒性的改良方案，从而避免了细菌对LDH定量的干扰。