Genetic engineering of bacteriophage S16 for Salmonella separation, concentration, and detection.

Salmonella contamination in food and water poses a major global health risk, creating an urgent need for rapid, reliable, and cost-effective detection methods. Conventional approaches are often expensive, labor-intensive, and time-consuming, and they frequently yield inconclusive or presumptive positive results. A significant bottleneck to rapid detection is the need to separate the target bacteria into smaller, clean, and concentrated samples. Bacteriophages can recognize, bind, and infect specific bacterial hosts. This study presents a genetically engineered S16 Salmonella-specific bacteriophage as a biosensor, optimized for enhanced sensitivity and efficiency in detecting Salmonella. Using CRISPR-Cas12a, the phages were engineered to include a gene for a NanoLuc luciferase reporter and a monomeric streptavidin (mSA) affinity tag fused to the gene for the capsid protein Soc. This design enabled conjugation of the phages to magnetic nanoparticles, facilitating the capture, concentration, and detection of Salmonella from 10 mL water samples. The modified S16 phage exhibited a detection limit of fewer than 10 CFU of Salmonella in 10 mL of water within a typical work shift. This innovative phage-based detection method offers a promising tool for enhancing food and water safety by providing a faster, more sensitive, and cost-effective approach to pathogen monitoring of Salmonella enterica subsp. enterica serovar Typhimurium.