Silva Maria João, Van Den Bossche Tim, Collin Mattias, Lood Rolf
Division of Infection Medicine, Department of Clinical Sciences, Faculty of Medicine, Lund University, SE-22184 Lund, Sweden.
VIB-UGent Center for Medical Biotechnology, VIB, 9052 Ghent, Belgium.
Antibiotics (Basel). 2025 Jul 16;14(7):714. doi: 10.3390/antibiotics14070714.
Antibiotic resistance presents an urgent public health threat. By developing a streamlined and effective method for studying bacteriophage induction, this research marks a step further in understanding how antibiotic-resistant genes might spread across different environments. This knowledge is essential for creating strategies to reduce the spread of antimicrobial resistance (AMR), particularly from a One Health perspective. In this study, we develop and validate a Green Fluorescent Protein (GFP)-based method as a proxy for bacteriophage induction. This method screens compounds for their potential to promote bacteriophage induction. This study utilized a - construct in to measure fluorescence as an indicator of SOS response activation. The experiments involved treating cultures with varying concentrations of the DNA-damaging chemical mitomycin C and measuring fluorescence over time. Additionally, droplet digital PCR (ddPCR) quantified bacteriophage induction in a lambda phage-carrying strain, allowing for correlation analysis between the two methods. The -driven SOS response depended on both dose and time, with increasing concentrations of mitomycin C leading to higher fluorescence. ddPCR analysis confirmed that mitomycin C induced prophage activation, with gene ratios increasing at higher drug concentrations over time. A strong Spearman correlation (>0.7) was noted between fluorescence and ddPCR results at elevated concentrations and relevant time points, indicating the validity of the GFP-based model as a proxy for bacteriophage induction. The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based model is a reliable, cost-effective, and efficient tool for studying phage induction and its potential role in AMR spread. This method could facilitate the screening of environmental samples and specific drugs to evaluate their impact on bacteriophage induction, which opens the door for applications such as screening for antibiotic resistance dissemination.
抗生素耐药性对公共卫生构成了紧迫威胁。通过开发一种精简且有效的噬菌体诱导研究方法，这项研究在理解抗生素耐药基因如何在不同环境中传播方面又迈出了一步。这些知识对于制定减少抗菌药物耐药性（AMR）传播的策略至关重要，特别是从“同一健康”的角度来看。在本研究中，我们开发并验证了一种基于绿色荧光蛋白（GFP）的方法作为噬菌体诱导的替代指标。该方法筛选化合物促进噬菌体诱导的潜力。本研究利用一个构建体来测量荧光，作为SOS反应激活的指标。实验包括用不同浓度的DNA损伤化学物质丝裂霉素C处理培养物，并随时间测量荧光。此外，液滴数字PCR（ddPCR）对携带λ噬菌体的菌株中的噬菌体诱导进行了定量，从而能够对这两种方法进行相关性分析。由诱导的SOS反应取决于剂量和时间，丝裂霉素C浓度的增加导致荧光增强。ddPCR分析证实丝裂霉素C诱导了前噬菌体激活，随着时间的推移，在较高药物浓度下基因比例增加。在较高浓度和相关时间点，荧光与ddPCR结果之间存在很强的斯皮尔曼相关性（>0.7），表明基于GFP的模型作为噬菌体诱导替代指标的有效性。研究结果表明，两种测量噬菌体诱导的方法之间存在很强的关联，这表明基于GFP的模型是一种可靠、经济高效的工具，可用于研究噬菌体诱导及其在AMR传播中的潜在作用。该方法有助于筛选环境样品和特定药物，以评估它们对噬菌体诱导的影响，这为诸如筛选抗生素耐药性传播等应用打开了大门。
