Foodborne diseases are a major challenge in the global food industry, especially those caused by multidrug-resistant (MDR) bacteria. Bacteria capable of biofilm formation, in addition to MDR strains, reduce the treatment efficacy, posing a significant threat to bacterial control. Bacteriophages, which are viruses that infect and kill bacteria, are considered a promising alternative in combating MDR bacteria, both in human medicine and animal production. Phage cocktails, comprising multiple phages, are commonly employed to broaden the host range and prevent or delay the development of phage resistance. There are numerous techniques and protocols available to evaluate the lytic activity of bacteriophages, with the most commonly used methods being Spot Test Assays, Efficiency of Plating (EOP), and infection assays in liquid culture. However, there is currently no standardization for which analyses should be employed and the possible differences among them in order to precisely determine the host range of phages and the composition of a cocktail. A preliminary selection using the Spot Test Assay resulted in four phages for subsequent evaluation against a panel of 36 Salmonella isolates of numerous serovars. Comparing EOP and infection assays in liquid culture revealed that EOP could underestimate the lytic activity of phages, directly influencing phage cocktail development. Moreover, the phage cocktail containing the four selected phages was able to control or remove biofilms formed by 66% (23/35) of the isolates, including those exhibiting low susceptibility to phages, according to EOP. Phages were characterized genomically, revealing the absence of genes associated with antibiotic resistance, virulence factors, or integrases. According to confocal laser scanning microscopy analysis, the biofilm maturation of one Salmonella isolate, which exhibited high susceptibility to phages in liquid culture and 96-well plates biofilm viability assays but had low values for EOP, was found to be inhibited and controlled by the phage cocktail. These observations indicate that phages could control and remove Salmonella biofilms throughout their growth and maturation process, despite their low EOP values. Moreover, using infection assays in liquid culture enables a more precise study of phage interactions for cocktail design timelessly and effortlessly. Hence, integrating strategies and techniques to comprehensively assess the host range and lytic activity of bacteriophages under different conditions can demonstrate more accurately the antibacterial potential of phage cocktails.
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