(1) Background: Formation of biofilms on food-contact surfaces by Shiga-toxigenic Escherichia coli (STEC) can pose a significant challenge to the food industry, making conventional control-methods insufficient. Targeted use of bacteriophages to disrupt these biofilms could reduce this problem. Previously isolated and characterized bacteriophages (n=52) were evaluated against STEC biofilms in-vitro and on food-contact surfaces. (2) Methods: Phage-treatments (9 logs PFU/ml), in phosphate-buffered-saline, were used individually or as cocktails. Biofilms of STEC (O157, O26, O45, O103, O111, O121, and O145) were formed in 96-well micro-titer plates (7 logs CFU/ml; 24 h) or on stainless-steel (SS) and high-density-polyethylene (HDPE) coupons (9 logs CFU/cm2; 7 h), followed by phage-treatment. Biofilm-disruption was measured in-vitro at 0, 3, and 6 h as a change in optical-density (A595). Coupons were treated with STEC-serotype-specific phage-cocktails or a 21-phage cocktail (3 phages/serotype) for 0, 3, 6 and 16 h and surviving STEC populations enumerated. (3) Results: Of the 52 pages, 77% showed STEC-biofilm disruption in-vitro. Serotype-specific phage-treatments reduced pathogen population within the biofilms by 1.9-4.1 and 2.3-5.6 logs CFU/cm2, while the 21-pahge cocktail reduced it by 4.0 and 4.8 logs CFU/cm2 on SS and HDPE, respectively. (4) Conclusions: Bacteriophages can be used to reduce STEC and their biofilms.

Cholera is a severe diarrheal disease caused by Vibrio cholerae, a natural inhabitant of brackish water. Effective control of cholera outbreaks depends on prompt detection of the pathogen from clinical specimens and tracking its source in the environment. Although the epidemiology of chol-era is well studied, rapid detection of V. cholerae remains a challenge, and data on its abundance in environmental sources are limited. Here, we describe a sensitive molecular quantification as-say by qPCR, which can be used on-site in low resource settings directly on water without the need for DNA extraction. This newly optimized method exhibited 100% specificity for total V. cholerae as well as V. cholerae O1 and allowed detection of as few as three target genome copies per reaction. The limit of detection is as low as 5 × 10E3 genome copies/L of water after concentrat-ing biomass from the sample. The ability to perform qPCR directly on water samples, portable features of the equipment, stability of the reagents at 4°C and user-friendly online software facili-tate fast quantitative analysis of V. cholerae. These characteristics make this assay extremely use-ful for field research in resource-poor settings and could support continuous monitoring in cholera endemic areas.

Gastroenteritis cases associated with non-toxigenic strains of Vibrio parahaemolyticus have been reported in many countries, suggesting the contribution of novel virulence factors. One candidate is zonula occludens toxin (Zot), which increases the intestinal permeability by other bacteria. Recently we identified prophages belonging to the Inoviridae family encoding putative Zot-like toxins in Chilean strains. Based on this information we performed sequence-based analyses of these toxins, followed by phylogenetic and structural analyses using computational tools. Our results showed that Zots found in Chilean V. parahaemolyticus strains are grouped into three different phylogenetic clusters, sharing two conserved motifs (Walker A and B) in their N-terminal region. These motifs are also conserved in Zots from the human pathogens Vibrio cholerae, Neisseria meningitidis and Campylobacter concisus. Although Zots of V. parahaemolyticus do not possess the FCIGRL sequence responsible for the effects produced by V. cholerae, they do possess a conserved secondary structure within their C-terminal region with Zots proteins able to disrupt the intestinal barrier, which is interesting since it has been suggested that the structure and not the Zot sequence would be responsible for the biological effects. This preliminary study provides the basis to study the function of Zots found in V. parahaemolyticus on the intestinal barrier and their possible role as a virulence factor.