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

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 <i>Vibrio cholerae</i> 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 <i>Escherichia coli</i> DH5α<i>,</i> and crude extract from the recombinant <i>E. coli</i> enhanced the dispersion of diverse bacterial biofilms, including those of <i>E. coli</i>, <i>Shigella dysenteriae</i>, <i>Pseudomonas aeruginosa</i>, and <i>V. cholerae</i>. 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 <i>V. cholerae</i> O1 by enrichment culture detected significantly more <i>V. cholerae</i> 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 <i>E. coli</i> has potential application in degrading bacterial biofilms and enhancing bacteriological analysis of water.IMPORTANCEIn 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 <i>E. coli</i> strain, and the crude cellular extract of the recombinant <i>E. coli</i> was found to promote dispersion of a variety of bacterial biofilms. Supplementation of bacterial growth medium with the crude extract also enhanced detection of <i>V. cholerae</i> 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.