Isolation and comparative characterization of broad and narrow-spectrum lytic bacteriophages infecting Salmonella enterica serovar Pullorum

Abstract Background Multidrug-resistant (MDR) Salmonella enterica serovar Pullorum poses a significant challenge to poultry production, highlighting the need for alternative antimicrobial strategies. Methods Two lytic-phenotype phages, designated vB_TZahraeiS_LP23 (LP23) and vB_TZahraeiS_OP23 (OP23), were isolated from industrial poultry wastewater (Ardabil) and mixed cattle–poultry farm wastewater (Tehran), respectively, using the previously isolated and characterized MDR S. Pullorum strain Salehi114 as the host. The phages were characterized based on host range, plaque morphology, transmission electron microscopy, one-step growth kinetics, physicochemical stability, antibacterial activity, and effects on biofilm biomass. The activity of the LP23–OP23 cocktail was also evaluated under the tested in vitro conditions. Results LP23 showed a broader host range in Salmonella serovars, whereas OP23 exhibited activity against both Salmonella and Escherichia coli O2:K1, indicating activity across the two tested bacterial genera. LP23 produced large clear plaques (4–5 mm), whereas OP23 produced smaller plaques (0.5–1 mm). Transmission electron microscopy revealed distinct tailed-phage morphologies for LP23 and OP23. One-step growth analysis showed burst sizes of 340 PFU per cell for LP23 and 415 PFU per cell for OP23, with optimal multiplicities of infection (MOIs) of 0.01 and 0.1, respectively. Both phages retained infectivity across a broad pH range (pH 5–11), and residual infectivity was detected after exposure to 70 °C for 60 min. The LP23–OP23 cocktail showed greater antibacterial activity than the individual phages under the tested conditions, reducing bacterial growth by approximately 94% based on OD 570 measurements and reducing biofilm biomass by approximately 60% in the biofilm formation assay and by up to approximately 54% in the preformed biofilm assay. The cocktail also maintained bacterial growth suppression for at least 12 h at an ultra-low MOI of 0.0001. Conclusions LP23 and OP23 exhibited distinct and potentially complementary biological characteristics under the tested in vitro conditions. Their antibacterial activity and effects on biofilm biomass support further investigation of these phages, individually and in combination, as potential bacteriophage-based biocontrol candidates against MDR S. Pullorum. Further genomic characterization and in vivo studies are required to assess their genomic safety, efficacy, and practical applicability.