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Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in Escherichia coli.

Silva, Maria João LU ; Van Den Bossche, Tim ; Collin, Mattias LU orcid and Lood, Rolf LU (2025) In Antibiotics 14(7).
Abstract


Background/Objectives: 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.
Methods: This study utilized a
recA-
GFP construct in
... (More)


Background/Objectives: 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.
Methods: This study utilized a
recA-
GFP construct in
Escherichia coli to measure fluorescence as an indicator of SOS response activation. The experiments involved treating
E. coli 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
E. coli strain, allowing for correlation analysis between the two methods.
Results: The
recA-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.
Conclusions: The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based
E. coli 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.

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Please use this url to cite or link to this publication:
author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Antibiotics
volume
14
issue
7
article number
714
publisher
MDPI AG
external identifiers
  • scopus:105011641041
  • pmid:40724015
ISSN
2079-6382
DOI
10.3390/antibiotics14070714
language
English
LU publication?
yes
id
71969370-3898-4ce6-94c7-c15075c6b18f
date added to LUP
2025-08-05 12:50:22
date last changed
2025-08-06 04:03:11
@article{71969370-3898-4ce6-94c7-c15075c6b18f,
  abstract     = {{<p><br>
 Background/Objectives: 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.<br>
 Methods: This study utilized a <br>
 recA-<br>
 GFP construct in <br>
 Escherichia coli to measure fluorescence as an indicator of SOS response activation. The experiments involved treating <br>
 E. coli 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 <br>
 E. coli strain, allowing for correlation analysis between the two methods. <br>
 Results: The <br>
 recA-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 (&gt;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.<br>
 Conclusions: The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based <br>
 E. coli 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.<br>
 </p>}},
  author       = {{Silva, Maria João and Van Den Bossche, Tim and Collin, Mattias and Lood, Rolf}},
  issn         = {{2079-6382}},
  language     = {{eng}},
  month        = {{07}},
  number       = {{7}},
  publisher    = {{MDPI AG}},
  series       = {{Antibiotics}},
  title        = {{Illuminating the Invisible: Green Fluorescent Protein as a Beacon for Antibiotic-Induced Phage Activity in 
        Escherichia coli.}},
  url          = {{http://dx.doi.org/10.3390/antibiotics14070714}},
  doi          = {{10.3390/antibiotics14070714}},
  volume       = {{14}},
  year         = {{2025}},
}