Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i>
<b>Background/Objectives:</b> 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 diffe...
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MDPI AG
2025-07-01
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| Series: | Antibiotics |
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| Online Access: | https://www.mdpi.com/2079-6382/14/7/714 |
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| author | Maria João Silva Tim Van Den Bossche Mattias Collin Rolf Lood |
| author_facet | Maria João Silva Tim Van Den Bossche Mattias Collin Rolf Lood |
| author_sort | Maria João Silva |
| collection | DOAJ |
| description | <b>Background/Objectives:</b> 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. <b>Methods:</b> This study utilized a <i>recA</i>-<i>GFP</i> construct in <i>Escherichia coli</i> to measure fluorescence as an indicator of SOS response activation. The experiments involved treating <i>E. coli</i> 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 <i>E. coli</i> strain, allowing for correlation analysis between the two methods. <b>Results:</b> The <i>recA</i>-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. <b>Conclusions:</b> The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based <i>E. coli</i> 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. |
| format | Article |
| id | doaj-art-8d0d16f882df4e2eae946168d90edd03 |
| institution | DOAJ |
| issn | 2079-6382 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj-art-8d0d16f882df4e2eae946168d90edd032025-08-20T03:13:42ZengMDPI AGAntibiotics2079-63822025-07-0114771410.3390/antibiotics14070714Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i>Maria João Silva0Tim Van Den Bossche1Mattias Collin2Rolf Lood3Division of Infection Medicine, Department of Clinical Sciences, Faculty of Medicine, Lund University, SE-22184 Lund, SwedenVIB-UGent Center for Medical Biotechnology, VIB, 9052 Ghent, BelgiumDivision of Infection Medicine, Department of Clinical Sciences, Faculty of Medicine, Lund University, SE-22184 Lund, SwedenDivision of Infection Medicine, Department of Clinical Sciences, Faculty of Medicine, Lund University, SE-22184 Lund, Sweden<b>Background/Objectives:</b> 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. <b>Methods:</b> This study utilized a <i>recA</i>-<i>GFP</i> construct in <i>Escherichia coli</i> to measure fluorescence as an indicator of SOS response activation. The experiments involved treating <i>E. coli</i> 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 <i>E. coli</i> strain, allowing for correlation analysis between the two methods. <b>Results:</b> The <i>recA</i>-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. <b>Conclusions:</b> The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based <i>E. coli</i> 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.https://www.mdpi.com/2079-6382/14/7/714bacteriophagesantibiotic resistancetransductionfluorescenceddPCR |
| spellingShingle | Maria João Silva Tim Van Den Bossche Mattias Collin Rolf Lood Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i> Antibiotics bacteriophages antibiotic resistance transduction fluorescence ddPCR |
| title | Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i> |
| title_full | Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i> |
| title_fullStr | Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i> |
| title_full_unstemmed | Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i> |
| title_short | Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in <i>Escherichia coli</i> |
| title_sort | illuminating the invisible green fluorescent protein as a beacon for antibiotic induced phage activity in i escherichia coli i |
| topic | bacteriophages antibiotic resistance transduction fluorescence ddPCR |
| url | https://www.mdpi.com/2079-6382/14/7/714 |
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