Combining bacteriophages and essential oils for the elimination of monophasic Salmonella Typhimurium and evaluation of phage persistence in poultry farm environments

Salmonella enterica serovar Typhimurium, monophasic variant (mST) and non-target species, such as E. coli, remain a major challenge for poultry production systems, particularly due to their ability to form biofilms and persist in farm environments and their increasing resistance to conventional chemical disinfectants. The present study aimed to develop and evaluate an innovative, sustainable, and eco-friendly cleaning and disinfection protocol using a combination of an essential oil (EO) and a bacteriophage (BP) cocktail under conditions simulating a poultry farming environment to eliminate persistent bacteria. Preliminary in vitro assays were conducted to identify the most effective EO against a target strain, selecting carvacrol as the disinfectant. In addition, three bacteriophages were selected based on their lytic spectrum against mST. The cleaning and disinfection (C&D) protocol was applied in an experimental poultry house following natural environmental contamination generated after experimental infection of the animals with mST. The protocol consisted of five sequential steps, ranging from mechanical removal of organic matter and detergent application to the disinfection phases (EO + BP). The results showed a progressive reduction of Salmonella loads on all surfaces, with complete elimination after the second bacteriophage application (p < 0.001). A marked reduction in non-target species counts was also observed, indicating an overall improvement in environmental hygiene. Analysis of bacteriophage environmental persistence revealed detectable levels up to one week after application, suggesting short-term environmental stability. Furthermore, the combined use of carvacrol and bacteriophages proved to be an effective and sustainable alternative to conventional chemical disinfectants, representing a promising green strategy for Salmonella control in poultry farm environments.