Characterization and genomic analysis of DSF2: a novel lytic phage infecting multidrug-resistant Shigella

Multidrug-resistant Shigella flexneri (MDR S. flexneri) serotype 2a is the predominant cause of shigellosis in China, presenting a major public health challenge amid escalating antibiotic resistance and limited treatment options. Bacteriophages are gradually emerging as a highly promising alternative to antibiotics because of its highly specific bactericidal ability. However, only 113 Shigella phage genomes are available in NCBI as of August 2025, highlighting the need for novel lytic phages targeting prevalent MDR strains.

A novel lytic phage, vB_SflP_DSF2 (DSF2), was isolated from untreated sewage at the 305 Hospital of PLA using MDR S. flexneri 2a strain 301 as host. Morphology was examined by transmission electron microscopy. Host range and efficiency of plating were determined against 41 bacterial strains (33 Shigella, 6 Escherichia coli, and others) using double-layer agar spot assays. One-step growth curves, pH and thermal stability, and biological properties were assessed using standard plaque assays. The complete genome was sequenced via Illumina NovaSeq, with comparative genomic and phylogenetic analyses performed using VIRIDIC, TerL phylogeny, AlphaFold structural predictions, and Swiss-Model for protein structure comparisons.

The DSF2 is a Schitoviridae phage with an elongated prolate head, short non-contractile tail. It produces haloed 1-2 mm plaques indicating depolymerase activity, with a 60-min latent period and 115 PFU/cell burst size. The DSF2 remains stable from 4 °C to 50 °C and active at pH 4-10, selectively lysing all S. flexneri serotype 2/X strains. Genomic analysis revealed that DSF2 possesses a 72,532 bp dsDNA genome with a G+C content of 44.89%, containing 89 predicted open reading frames. The DSF2 harbors no virulence or antibiotic resistance genes. Closest relative Shigella virus Moo19 shares 94.1% identity, defining the DSF2 as a new species. The prolate head of DSF2 closely resembles that of Escherichia coli phage PH444, driven by divergent Hoc-like head decoration, despite the conservation of capsid and portal proteins when compared to Shigella virus Moo19.

The DSF2 represents a novel Schitoviridae species that expands the limited Shigella phage repertoire, offering precision biocontrol against MDR S. flexneri serotype 2/X with minimal microbiome disruption. Hoc-like head decoration likely drives DSF2's unique prolate morpholog through intercapsomer angular constraints.