Characterization of two novel Salmonella phages having biocontrol potential against Salmonella spp. in gastrointestinal conditions

Authors: Zhang, Yujie; CHU, MACKENNA - Hispanic Association Of Colleges & Universities (HACU); Liao, Yen-Te; Salvador, Alexandra; Wu, Vivian

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2024
Publication Date: 5/29/2024
Citation: Zhang, Y., Chu, M., Liao, Y., Salvador, A., Wu, V.C. 2024. Characterization of two novel Salmonella phages having biocontrol potential against Salmonella spp. in gastrointestinal conditions. Scientific Reports. 14. Article 12294. https://doi.org/10.1038/s41598-024-59502-9.
DOI: https://doi.org/10.1038/s41598-024-59502-9

Interpretive Summary: The poultry industry has grown drastically worldwide due to the increasing demand for the consumption of poultry and other related food products. However, a common pathogen—Salmonella—has been widely found in poultry gut environments and contributed to several foodborne outbreaks, posing food safety risks to human health. Lytic bacteriophage (or phage) is a type of virus that can infect and kill bacteria after infection as natural predators, and thus has become a promising antimicrobial agent in combating bacterial pathogens. This study was to isolate and characterize two lytic phages—S4lw and D5lw—for controlling Salmonella in a simulated gut environment. These two phages have no harmful genes in their genomes and are safe for phage application. Both phages have a broad host range and can render strong lytic activities against various Salmonella types. Additionally, alginate was used to encapsulate the mixture of the phages S4lw and D5lw and provided extra protection for the phages to sustain acidic stress in a simulated gut environment. The findings of this study demonstrated that phages S4lw and D5lw are promising biocontrol agents and could be further developed for oral phage therapy to combat Salmonella pathogens in poultry gut environments.

Technical Abstract: Salmonella is the primary enteric pathogen related to the contamination of poultry and other food products in numerous foodborne outbreaks. The continuous emergence of multidrug-resistant bacteria has become a serious food safety issue due to the overuse of antibiotics in the poultry industry. Hence, lytic phages are considered an alternative biocontrol method against these bacterial superbugs. This study aimed to characterize and encapsulate a two-phage cocktail for the biocontrol of Salmonella spp in a simulated gastrointestinal environment. Two novel Salmonella phages—S4lw and D5lw—were subjected to whole-genome sequencing and biological characterization, including morphology, antimicrobial activity, and acid stability tests. The encapsulation was used to improve the viability of the two-phage cocktail under in vitro gastrointestinal conditions. S4lw had a capsid with approximate 64.5 ± 0.5 nm in diameter and a long non-contractile tail of 132.3 ± 0.5 nm in length. D5lw contained a capsid of approximately 96.7 ± 0.5 nm in diameter and a long contractile tail of about 135.5 ± 0.5 nm in length. Phages S4lw and D5lw taxonomically belonged to unclassified species under the Guernseyvirinae and Ackermannviridae families, respectively. No virulent, antibiotic resistance or lysogenic genes were detected in the two lytic phage genomes. Each phage showed antimicrobial activities against diverse Salmonella spp, such as S. Enteritidis and S. Typhimurium, achieving 1.7 to 3.4 log reduction after 2-6 hours of treatment. Moreover, the phage cocktail at an MOI of 100 or 1000 completely inhibited these Salmonella strains for at least 14 hours at 25 °C. In addition, phage S4lw was more stable in acid environments than D5lw. The bead-encapsulated phage cocktail could withstand a pH of 3 or higher levels for at least 1 hour, while the free phages (without encapsulation) were mostly inactivated. Moreover, bead-encapsulated phages had significantly higher viability than the free phages in different simulated gut environments after 1-hour treatment, demonstrating the protective effect of encapsulation from the acidic stress. These findings provide valuable insights into a biocontrol agent—the two-phage cocktail (S4lw and D5lw)—in mitigating Salmonella spp within the poultry gastrointestinal environments via oral administration.