Salmonella is a major causative agent of foodborne illness and rapid identification of this pathogen is essential to prevent disease. Currently most assays require high bacterial burdens or prolonged enrichment to achieve acceptable performance. A reduction in testing time without loss of sensitivity is critical to allow food processors to safely decrease product holding time. To meet this need, a method was developed to detect Salmonella using luciferase reporter bacteriophages. Bacteriophages were engineered to express NanoLuc, a novel optimized luciferase originating from the deep-sea shrimp Oplophorus gracilirostris. NanoLuc-expressing bacteriophages had a limit of detection of 10–100 CFU per mL in culture without enrichment. Luciferase reporters demonstrated a broad host range covering all Salmonella species with one reporter detecting 99.3% of 269 inclusivity strains. Cross-reactivity was limited and only observed with other members of the Enterobacteriaceae family. In food matrix studies, a cocktail of engineered bacteriophages accurately detected 1 CFU in either 25 g of ground turkey with a 7 h enrichment or 100 g of powdered infant formula with a 16 h enrichment. Use of the NanoLuc reporter assay described herein resulted in a considerable reduction in enrichment time without a loss of sensitivity.

Host range is a major determinant in the industrial utility of a bacteriophage. A model host range permits broad recognition across serovars of a target bacterium while avoiding cross-reactivity with commensal microbiota. Searching for a naturally occurring bacteriophage with ideal host ranges is challenging, time-consuming, and restrictive. To address this, SPTD1.NL, a previously published luciferase reporter bacteriophage for Salmonella, was used to investigate manipulation of host range through receptor-binding protein engineering. Similar to related members of the Ackermannviridae bacteriophage family, SPTD1.NL possessed a receptor-binding protein gene cluster encoding four tailspike proteins, TSP1-4. Investigation of the native gene cluster through chimeric proteins identified TSP3 as the tailspike protein responsible for Salmonella detection. Further analysis of chimeric phages revealed that TSP2 contributed off-target Citrobacter recognition, whereas TSP1 and TSP4 were not essential for activity against any known host. To improve the host range of SPTD1.NL, TSP1 and TSP2 were sequentially replaced with chimeric receptor-binding proteins targeting Salmonella. This engineered construct, called RBP-SPTD1-3, was a superior diagnostic reporter, sensitively detecting additional Salmonella serovars while also demonstrating improved specificity. For industrial applications, bacteriophages of the Ackermannviridae family are thus uniquely versatile and may be engineered with multiple chimeric receptor-binding proteins to achieve a custom-tailored host range.

The lack of bacteriophages capable of infecting the Listeria species, Listeria grayi, is academically intriguing and presents an obstacle to the development of bacteriophage-based technologies for Listeria. We describe the isolation and engineering of a novel L. grayi bacteriophage, LPJP1, isolated from farm silage. With a genome over 200,000 base pairs, LPJP1 is the first and only reported jumbo bacteriophage infecting the Listeria genus. Similar to other Gram-positive jumbo phages, LPJP1 appeared to contain modified base pairs, which complicated initial attempts to obtain genomic sequence using standard methods. Following successful sequencing with a modified approach, a recombinant of LPJP1 encoding the NanoLuc luciferase was engineered using homologous recombination. This luciferase reporter bacteriophage successfully detected 100 stationary phase colony forming units of both subspecies of L. grayi in four hours. A single log phase colony forming unit was also sufficient for positive detection in the same time period. The recombinant demonstrated complete specificity for this particular Listeria species and did not infect 150 non-L. grayi Listeria strains nor any other bacterial genus. LPJP1 is believed to be the first reported lytic bacteriophage of L. grayi as well as the only jumbo bacteriophage to be successfully engineered into a luciferase reporter.