# Crude preparation of a phage-encoded biofilm-dispersing factor expressed in E. coli and its potential application in bacteriological analysis of environmental water samples

## TL;DR

A phage-encoded biofilm-dispersing factor produced in E. coli can enhance detection of waterborne bacteria like V. cholerae by breaking down biofilms.

## Contribution

A phage-encoded biofilm-degrading factor was cloned in E. coli and shown to improve detection of pathogens in water samples.

## Key findings

- The crude extract from recombinant E. coli dispersed biofilms of multiple bacterial species.
- Supplementing enrichment medium with the extract increased detection of V. cholerae O1 in water samples.
- The biofilm-dispersing activity was heat- and pH-stable but inactivated by proteinase-K.

## Abstract

A fundamental technical challenge in detecting pathogenic bacteria in aquatic reservoirs is the inability to accurately estimate biofilm-associated cells in water. Considering the role of biofilms in environmental persistence and waterborne transmission of bacterial pathogens, there is an increasing interest in substances that can effectively degrade bacterial biofilms. The biofilm-dispersing Vibrio cholerae phage JSF7 was analyzed by whole genome sequencing and found to carry a gene predicted to encode an enzyme for degrading complex polysaccharides. The gene was cloned in Escherichia coli DH5α, and crude extract from the recombinant E. coli enhanced the dispersion of diverse bacterial biofilms, including those of E. coli, Shigella dysenteriae, Pseudomonas aeruginosa, and V. cholerae. The crude extract was fully active at a temperature of 37°C and pH of 7.0 but was inactivated by proteinase-K treatment. Analysis of environmental water samples for the presence of V. cholerae O1 by enrichment culture detected significantly more V. cholerae O1-positive samples when the enrichment medium was supplemented with the extract, as compared with typical enrichment without the extract. These results suggest that a crude preparation of the phage-encoded biofilm-degrading factor expressed in E. coli has potential application in degrading bacterial biofilms and enhancing bacteriological analysis of water.

In their aquatic reservoirs, bacteria often exist as biofilms and are difficult to accurately detect by culturing water samples. Such biofilms have been implicated in waterborne transmission of pathogenic bacteria. We identified a bacteriophage that can disintegrate biofilms and disperse biofilm-associated bacteria. The putative phage gene responsible for this activity was cloned in an E. coli strain, and the crude cellular extract of the recombinant E. coli was found to promote dispersion of a variety of bacterial biofilms. Supplementation of bacterial growth medium with the crude extract also enhanced detection of V. cholerae O1, the causative agent of cholera in environmental water samples. The ability of a phage-derived biofilm-degrading factor to disperse diverse bacterial biofilms provides a novel approach for enhancing detection of waterborne bacterial pathogens in water beyond traditional enrichment methods.