High-throughput screening of natural compounds for prophage induction in controlling pathogenic bacteria in food

Introduction: The clean label trend emphasizes the need for natural approaches to combat pathogenic bacteria in food. This study explores the potential of inducing prophages within bacterial genomes as a novel strategy to control pathogenic and spoilage bacterial growth.Methods: A luminescence-based high-throughput assay was developed to identify natural compounds capable of inducing prophages. Bioactive compounds from four chemical libraries were screened at a final concentration of 10 µM. The assay measured luminescence production in Escherichia coli BR513, a genetically modified strain producing β-galactosidase upon prophage λ induction. Luminescence values were normalized to cell concentration (OD600) and the interquartile mean of each 384-well plate. A cut-off for normalized luminescence values, set at 2.25 standard deviations above the mean, defined positive prophage induction.Results: Four naturally-derived compounds (osthol, roccellic acid, galanginee, and sclareol) exhibited positive prophage induction, along with previously identified inducers, rosemary, and gallic acid. Dose-response experiments were conducted to determine optimal concentrations for prophage induction. However, the results could not distinguish between prophage-induced cell death and other mechanisms, making it challenging to identify ideal concentrations.Discussion: The high-throughput luminescent prophage induction assay serves as a valuable tool for the initial screening of natural bioactive compounds that have the potential to enhance food safety and quality by inducing prophages. Further research is required to understand the mechanism of bacterial cell death and to establish optimal concentrations for prophage induction in a food preservation context

Genomic and Phenotypic Analysis of Salmonella enterica Bacteriophages Identifies Two Novel Phage Species

Bacteriophages (phages) are potential alternatives to chemical antimicrobials against pathogens of public health significance. Understanding the diversity and host specificity of phages is important for developing effective phage biocontrol approaches. Here, we assessed the host range, morphology, and genetic diversity of eight Salmonella enterica phages isolated from a wastewater treatment plant. The host range analysis revealed that six out of eight phages lysed more than 81% of the 43 Salmonella enterica isolates tested. The genomic sequences of all phages were determined. Whole-genome sequencing (WGS) data revealed that phage genome sizes ranged from 41 to 114 kb, with GC contents between 39.9 and 50.0%. Two of the phages SB13 and SB28 represent new species, Epseptimavirus SB13 and genera Macdonaldcampvirus, respectively, as designated by the International Committee for the Taxonomy of Viruses (ICTV) using genome-based taxonomic classification. One phage (SB18) belonged to the Myoviridae morphotype while the remaining phages belonged to the Siphoviridae morphotype. The gene content analyses showed that none of the phages possessed virulence, toxin, antibiotic resistance, type I–VI toxin–antitoxin modules, or lysogeny genes. Three (SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins. Whole-genome-based phylogeny of the eight phages with their 113 homologs revealed three clusters A, B, and C and seven subclusters (A1, A2, A3, B1, B2, C1, and C2). While cluster C1 phages were predominantly isolated from animal sources, cluster B contained phages from both wastewater and animal sources. The broad host range of these phages highlights their potential use for controlling the presence of S. enterica in foods