Commercial bacteriophage cocktail treatment reduces Salmonella Infantis levels in pre-harvest agricultural water

Authors: GUTIERREZ, ALLEN - Orise Fellow; XIONG, ZIRUI - University Of Delaware; JOHNSON, SHAYLA - Oak Ridge Institute For Science And Education (ORISE); BROOKS, AJANI - Alabama A & M University; GABRIEL, ELLEN - Oak Ridge Institute For Science And Education (ORISE); ADNAN, ADIB - Oak Ridge Institute For Science And Education (ORISE); VIRKRAM, AMIT - Intralytix, Inc; CALLAHAN, MARY - Intralytix, Inc; Sharma, Manan

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/23/2025
Publication Date: 8/25/2025
Citation: Gutierrez, A., Xiong, Z.R., Johnson, S.B., Brooks, A.A., Gabriel, E., Adnan, A., Virkram, A., Callahan, M.T., Sharma, M. 2025. Commercial bacteriophage cocktail treatment reduces Salmonella Infantis levels in pre-harvest agricultural water. Journal of Food Protection. 88(11). Article e100605. https://doi.org/10.1016/j.jfp.2025.100605.
DOI: https://doi.org/10.1016/j.jfp.2025.100605

Interpretive Summary: Lytic bacteriophages are bacterial viruses that can be used to infect and kill bacterial pathogens that cause foodborne illness. Bacteriophages have been used in various food production settings to reduce levels of the pathogen Salmonella. Salmonella is one of the leading causes of foodborne illness in the U.S., and has been associated with contaminated produce (fruits and vegetables). Produce can become contaminated from surface (river or pond) water containing Salmonella. Many farmers use chemical sanitizers to reduce foodborne pathogen levels in surface irrigation water. Our research evaluated the ability of lytic bacteriophages as an alternative to chemical sanitizers to reduce levels of Salmonella in surface water with different commonly measured characteristics, like turbidity (cloudiness), pH, and temperature. In some cases, these characteristics reduce the ability of chemical sanitizers in surface water to reduce pathogen levels, but their effect on lytic bacteriophages are unknown. Our work showed that in test agricultural water (TAW, a synthetic laboratory formulation) when lytic bacteriophages were applied at a level that was 100 times more than the Salmonella levels, they reduced the levels of the pathogen. Our work also showed that in natural pond water, lytic bacteriophages needed to be applied at a level that was 10,000 times greater than Salmonella levels to effectively reduce levels. Overall, lytic bacteriophages were effective across a variety of turbidity, pH and temperature characteristics. This work shows that lytic bacteriophages can reduce Salmonella in surface water but requires higher levels of bacteriophages to be applied compared to laboratory conditions. Our intention in this research is to show that bacteriophages can be optimized for use in combination with other filtration-based technologies or chemical sanitizers to reduce the level of bacterial foodborne pathogens in surface water. This research benefits farmers by providing alternatives to chemical sanitizers to reduce the burden of foodborne pathogens in irrigation water.

Technical Abstract: Contaminated irrigation water in the pre-harvest environment can lead to outbreaks associated with fruits and vegetables. The potential of microbial contamination in pre-harvest waters with bacterial pathogens has created an ongoing demand for effective water treatment methods to mitigate this risk. This study sought to evaluate the efficacy of a commercial bacteriophage cocktail against Salmonella Infantis in agricultural water. A test agricultural water (TAW) medium prepared at different pH (6.5 and 8.4) and turbidity levels (4, 20, 50, and 100 NTU) was inoculated with S. Infantis (~6 log CFU/mL), in triplicate trials, and treated with a bacteriophage cocktail (SalmoFresh™) at a phage titer of 8 log PFU/mL. Water samples were taken after a 5-minute contact time, with shaking (250 rpm), at 25oC. Additionally, collected pond water – natural (non-sterile) and autoclaved - was inoculated with S. Infantis (~4 log CFU/mL) and treated with bacteriophage at either 12°C or 32°C for 5, 10, or 30 min and at phage titer levels of 7 log PFU/mL and 8 log PFU/mL in triplicate trials. In both experiments, samples were enumerated by plating onto XLD or TSA supplemented with 50 µg/mL Nalidixic acid during the pond water trials. In TAW, S. Infantis levels were reduced by an average of 1.0 log CFU/mL after the 5-minute phage treatment, with no significant differences in reductions across all pH and turbidity levels tested (p > 0.05). In pond water, S. Infantis reductions only occurred when the phage titer was 8 log PFU/mL, with average reductions of 1.04, 1.50, and 1.67 log CFU/mL after 5, 10, and 30 min, respectively, in pond water (natural and autoclaved) at 32°C. At 12°C, average reductions were 0.90, 1.15, and 1.36 log CFU/mL after 5, 10, and 30 min, respectively. These results demonstrate that lytic phage specific for Salmonella are effective across various conditions in water (pH, turbidity, temperature) and may be optimally considered with other technologies to reduce Salmonella levels in agricultural water.