A two-phage cocktail demonstrated potent antibacterial and antibiofilm efficacy against multidrug-resistant Salmonella enterica serovar Pullorum under in vitro conditions, according to research published in BMC Microbiology by Ghazale Faridfar, Taghi Zahraei Salehi, Ramak Yahyaraeyat, Iradj Ashrafi Tamai, and Ramin Mazaheri Nezhad Fard. Addressing the substantial challenge that multidrug-resistant Salmonella enterica serovar Pullorum presents to commercial poultry production, the investigators explored lytic bacteriophages as alternative antimicrobial strategies. Under the tested laboratory conditions, the combined phage formulation achieved greater antibacterial activity than either single phage alone, decreasing bacterial growth by approximately 94 percent based on optical density measurements at 570 nanometers. Furthermore, the combination sustained continuous bacterial growth suppression for at least 12 hours even when administered at an ultra-low multiplicity of infection of 0.0001.

The LP23 phage was recovered from industrial poultry wastewater collected in Ardabil, while the OP23 phage was sourced from mixed cattle and poultry farm wastewater gathered in Tehran. To isolate, propagate, and characterize both viral candidates, the research team utilized a previously isolated and characterized multidrug-resistant Salmonella Pullorum strain, designated Salehi114, as the bacterial host. Following isolation, both phages were subjected to a comprehensive experimental framework encompassing host range determination, plaque morphology examination, transmission electron microscopy, one-step growth kinetics, physicochemical stability testing, antibacterial assays, and evaluations of their impact on biofilm biomass.

Evaluation of bacterial host range demonstrated that the two phages possess markedly different infectivity spectra across bacterial taxa. The LP23 phage exhibited a broader host range across tested Salmonella serovars, whereas OP23 demonstrated the ability to cross bacterial genus boundaries, showing lytic activity against both Salmonella and Escherichia coli O2:K1. Plaque morphology assessments highlighted further phenotypic differences between the two viruses. The LP23 phage generated large, clear plaques measuring 4 to 5 millimeters in diameter on bacterial lawns, whereas OP23 formed noticeably smaller plaques measuring between 0.5 and 1 millimeter. High-resolution transmission electron microscopy confirmed structural distinctions between the two isolates, revealing that both LP23 and OP23 possess distinct tailed-phage morphologies.

Quantitative one-step growth kinetics established contrasting viral replication dynamics and infection parameters for the two isolated bacteriophages. The LP23 phage displayed an optimal multiplicity of infection of 0.01 and produced a viral burst size of 340 plaque-forming units per infected host cell. In contrast, the OP23 phage functioned with an optimal multiplicity of infection of 0.1, generating a larger burst size of 415 plaque-forming units per infected host cell.

When exposed to varying chemical environments, both LP23 and OP23 retained their infectivity across a broad pH range spanning from pH 5 to pH 11. In thermal tolerance testing, both bacteriophages exhibited marked resilience, with residual viral infectivity still detected after the phages were exposed to a temperature of 70 degrees Celsius for 60 minutes.

The combined LP23 and OP23 cocktail exhibited robust disruption of bacterial biomass across both nascent and established biofilm structures. In biofilm formation assays, introducing the dual-phage mixture reduced biofilm biomass by approximately 60 percent. When applied to pre-existing bacterial communities in preformed biofilm assays, the cocktail reduced biofilm biomass by up to approximately 54 percent.

In their assessment of the overall experimental findings, the researchers noted that LP23 and OP23 exhibit distinct and potentially complementary biological characteristics under the evaluated in vitro conditions. Their demonstrated antibacterial performance, broad pH stability, thermal resistance, and ability to diminish biofilm biomass support further investigation of both phages, individually and in combination, as potential bacteriophage-based biocontrol candidates against multidrug-resistant Salmonella Pullorum. However, the authors underscored that the current evidence is confined to laboratory models. Comprehensive genomic characterization alongside subsequent in vivo animal trials remains essential to rigorously establish the genomic safety, biological efficacy, and practical applicability of the phages prior to potential field implementation in poultry production.

Subject of Research: Lytic bacteriophage characterization and biocontrol potential against multidrug-resistant Salmonella Pullorum

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

Article References: Faridfar, G., Zahraei Salehi, T., Yahyaraeyat, R., Ashrafi Tamai, I., & Mazaheri Nezhad Fard, R. (2026). Isolation and comparative characterization of broad and narrow-spectrum lytic bacteriophages infecting Salmonella enterica serovar Pullorum. BMC Microbiology. https://doi.org/10.1186/s12866-026-05837-4

Image Credits: AI Generated

DOI: 10.1186/s12866-026-05837-4

Keywords: bacteriophages, Salmonella enterica serovar Pullorum, multidrug resistance, poultry biocontrol, phage cocktail, vB_TZahraeiS_LP23, vB_TZahraeiS_OP23, Salehi114, poultry wastewater, biofilm biomass, one-step growth kinetics, burst size, transmission electron microscopy, lytic phages, Escherichia coli O2:K1, thermal stability, host range