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.

Engineered bacteriophages (phages) can be effective diagnostic reporters for detecting a variety of bacterial pathogens. Although a promising biotechnology, the large-scale use of these reporters may result in the unintentional release of genetically modified viruses. In order to limit the potential environmental impact, the ability of these phages to propagate outside the laboratory was targeted. The phage SEA1 has been previously engineered to facilitate food safety as an accurate and sensitive reporter for Salmonella contamination. In this study, homologous recombination was used to replace the expression of an essential baseplate wedge subunit (gp141) in SEA1 with a luciferase, NanoLuc^®. This reporter, referred to as SEA1Δgp141.NL, demonstrated a loss of plaque formation and a failure to increase in titer following infection of Salmonella. SEA1Δgp141.NL was thus incapable of producing infectious progeny in the absence of gp141. In contrast, production of high titer stocks was possible when gp141 was artificially supplied in trans during infection. As a reporter, SEA1Δgp141.NL facilitated rapid, sensitive, and robust detection of Salmonella despite an inability to replicate. These results suggest that replication-deficient reporter phages are an effective method to obtain improved containment without sacrificing significant performance or the ease of production associated with many phage-based diagnostic methods.

The PhageDx™ Salmonella Assay is based on the infection of Salmonella spp. by specific bacteriophages and expression of a luciferase reporter gene. Results are generated in as little as 9.5 h for raw ground turkey and 18.5 h for milk-based powdered infant formula (PIF). An AOAC Performance Tested Methods ^SM (PTM) study was conducted to validate the PhageDx™ Salmonella Assay for the detection of Salmonella in 25 g raw ground turkey and 100 g Powdered Infant Formula (PIF) test portions. The performance of the PhageDx™ Salmonella Assay was compared to that of the USDA/FSIS MLG 4.10 for raw ground turkey and FDA/BAM Chapter 5 for PIF. Inclusivity/exclusivity, product consistency and stability, and robustness testing were conducted. There was no significant difference between the 25 g raw ground turkey and 100 g PIF PhageDx™ Salmonella Assay and the USDA/FSIS MLG 4.10 and FDA/BAM Chapter 5, respectively. The reporter bacteriophages were specific for Salmonella and infected 108 strains in inclusivity testing. They did not infect 30 non-Salmonella bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least six months. The data collected in the validation study demonstrate that the PhageDx™ Salmonella Assay meets the qualifications for PTM status.

The PhageDx™ Cronobacter Assay is based on the infection of Cronobacter spp. by specific bacteriophages and expression of a luciferase reporter gene. Results are generated in as little as 18.5 h for powdered infant formula (PIF). An AOAC Performance Tested Methods^SM (PTM) study was conducted to validate the PhageDx™ Cronobacter Assay for the detection of Cronobacter in 10, 100 and 300 g milk- and soy-based PIF test portions. The performance of the PhageDx™ method was compared to the ISO 22964:2006 Microbiology of the food chain – Horizontal method for the detection of Cronobacter spp. and U.S. FDA Bacteriological Analytical Manual (BAM) Ch. 29 Cronobacter: 2012. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted. There was no significant difference between the 10 g, 100 g, or 300 g test portions for the milk and soy PIF matrixes between the PhageDx™ Cronobacter Assay, the ISO 22964:2017 and the FDA BAM Ch. 29 Cronobacter:2012 methods. The reporter bacteriophages were specific for Cronobacter and infected 75 strains in inclusivity testing. They did not infect 35 non-Cronobacter bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least three months. Work in the submitting and independent laboratories demonstrated that the PhageDx™ Cronobacter Assay meets the qualifications for PTM status.

The PhageDx™ E. coli O157: H7 Assay originally required a 5 h enrichment period and a 10 mL test sample for 25 g ground beef. The proposed method modifications seek to include, beef trim (375 g) matrix and a new method procedure for 25 g ground beef comprised of 6–7 h enrichment and 1 mL test sample. To validate the method modifications for PhageDx™ E. coli O157: H7 Assay to include beef trim (375 g) matrix and modify enrichment time and test sample size for 25 g ground beef. For both matrixes, pre-warmed TSB (42 ± 1 °C) was added in a 3:1 (media: sample size) ratio, blended, and enriched for 9–10 h (beef trim, 375 g) or 6 h (ground beef, 25 g) at 42 ± 1 °C. One milliliter samples were transferred to a microfuge tube, centrifuged, and the supernatant removed. The pellet was resuspended in 0.2 mL of TSB and phage reagent added. Samples were incubated for 2 h at 37 ± 1 °C. After infection, the sample was centrifuged, and 150 µl of the supernatant was transferred to a 96- well plate. Then, lysis buffer and luciferase substrate were added and the sample read on a luminometer to determine the presence or absence of E. coli O157: H7. No significant differences were found between the PhageDx™ E. coli O157: H7 Assay method modifications and the USDA-FSIS MLG reference method. The independent study demonstrated that the PhageDx™ E. coli O157: H7 Assay method modifications meet the qualifications for PTM status.

The PhageDx™ Listeria Assay is a simple, specific, and sensitive assay based on the infection of Listeria spp. by selected bacteriophages and the resultant expression of a luciferase reporter gene. Results are generated in as little as 24.5 h for stainless steel and ceramic environmental surfaces. An AOAC Performance Tested Methods ^SM (PTM) study was conducted to validate the PhageDx™ Listeria Assay for the detection of Listeria on stainless steel and ceramic surfaces. The performance of the PhageDx™ method was compared to that of the FDA/BAM Ch. 10. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted. Inclusivity testing demonstrated that the reporter bacteriophages were specific for Listeria ssp. and detected 58/61 Listeria strains tested, including all 34 Listeria monocytogenes strains. The reporter bacteriophage also was shown to not detect 46/47 non-Listeria bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least 6 months. Matrix studies on stainless steel and ceramic surfaces showed that there was no significant difference between the PhageDx™ Listeria Assay and the FDA/BAM Chapter 10 reference methods. The validation study demonstrates that the PhageDx™ Listeria Assay is an effective method for the detection of Listeria spp. on stainless steel and ceramic environmental surfaces and meets the qualifications for AOAC PTM status.