Discovery of Polyvalent Myovirus (vB_STM-2) Phage as a Natural Antimicrobial System to Lysis and Biofilm Removal of Salmonella Typhimurium Isolates from Various Food Sources

New and natural antimicrobials as an alternative control system are now an urgent need to overcome stubborn bacterial infections. Salmonella Typhimurium has become the most frequent serovar responsible for salmonellosis in humans around the world. The high antimicrobial resistance and biofilm production make this pathogen more dangerous. We aimed to isolate a broad lytic phage to prevent Salmonella infection and reduce its biofilms. Using Salmonella Typhimurium (ST-4) as a host, seven phages were isolated, and only three phages showed clear lytic plaques, two members of the Siphoviridae family (vB_STS-1 and vB_STS-3) and one of the Myoviridae family (vB_STM-2). The vB_STM-2 phage was the most potent broad lytic phage, infecting 100% of tested Salmonella Typhimurium serovars and non- Salmonella strains. Additionally, the vB_STM-2 phage was thermostable at −20 to 55 °C up to 24 h, while at 65 and 75 °C, a significant ( p < 0.05) titer reduction was observed after 7 days. Moreover, the phage seemed to be stable at different pHs (4–11) after one to twelve hours (hrs), while increasing the time made the phage more sensitive to the alkaline medium rather than the acidic medium. Interestingly, the vB_STM-2 phage had the capacity to diminish or eradicate the biofilms of tested Salmonella Typhimurium, e.g., ST-4, ST-19, ST-30, ST-37, ST-45 and ST-49 by 81.2%, 76.4%, 43.6%, 41%, 39.8% and 93.4%, respectively, at a titer concentration of 10 6 PFU/mL. Eventually, the vB_STM-2 phage showed significant ( p < 0.05) efficacy in the elimination of Salmonella Typhimurium (ST-4) from contaminated chicken breasts at both storage periods with high titer stability. The Salmonella count showed a severe decline from 7.00 ± 0.63 log 10 CFU/cm 2 to 0.88 ± 0.17 log 10 CFU/cm 2 on the seventh day of the short-term storage, and from 5.13 ± 0.44 log 10 CFU/cm 2 to 1.10 ± 0.12 log 10 CFU/cm 2 on day 27 of the long-term assay. In both periods, the phage titers remained stable, with insignificant ( p < 0.05) loss. Therefore, this phage is considered a prime candidate to combat multi-drug-resistant Salmonella Typhimurium and its biofilms.