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Comparative analysis of phage cocktail activity against uropathogenic E. coli strains using spot overlay assay

Steven Arcidiacono1, Robert Stote1

  • 1DEVCOM Soldier Center, 10 General Greene Avenue, Natick, MA 01760 USA.

Methodsx
|September 25, 2025
PubMed

Insights

Spot overlay assays can now provide semi-quantitative phage activity data. This method enhances phage cocktail comparison beyond just host range, offering insights into overall efficacy.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Spot overlay assays are standard for measuring phage activity and host range against microorganisms.
  • These assays yield semi-quantitative results, making direct comparison of phage cocktail efficacy challenging.
  • Variations in clearing zones (complete, incomplete, or cloudy) limit precise activity assessment.

Purpose of the Study:

  • To develop a method for analyzing spot overlay assay results to derive semi-quantitative data.
  • To expand the information obtained from spot overlay assays beyond host range determination.
  • To enable comparison of overall phage cocktail strength and activity across multiple bacterial strains.

Main Methods:

  • Utilizing standard laboratory protocols for spot overlay assays.
  • Analyzing phage dilutions and clearing zone characteristics.
  • Consolidating activity data from all tested bacterial strains.

Main Results:

  • Demonstrated a novel method to analyze qualitative spot overlay data for semi-quantitative insights.
  • Identified differences in overall activity among three phage cocktails, despite similar high host range percentages (92-96%).
  • The method successfully converts spot overlay results into comparable metrics for overall cocktail strength.

Conclusions:

  • The developed method provides semi-quantitative information from qualitative spot overlay assays.
  • This approach allows for a more robust comparison of phage cocktail efficacy.
  • The findings offer valuable insights for phage therapy development and application.
Keywords:
Cocktail activityComparative analysisHost rangePhage cocktailsSpot overlay assayZones of clearing

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