Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7

Abstract

Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10⁻⁸ ml/min and 1.91 × 10⁻⁸ ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10⁻² CFU/ml for S. Typhimurium and 4.58 × 10⁻⁵ CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.

Fig 1

Electron microscopic image of phage SFP10 negatively stained with 0.2% uranyl acetate. Scale bar, 50 nm.

Fig 2

One-step growth curve analysis of S. Typhimurium SL1344 (A) and E. coli O157:H7 ATCC 43890 (B) infected by SFP10 phage. E, eclipse period; L, latent period; B, burst size. Closed circles, non-chloroform-treated sample; open squares, chloroform-treated sample. The error bars indicate standard deviations.

Fig 3

Confirmation of a phage SFP10 receptor by deletion and complementation of rfaL (A) and Tn5 mutation and complementation of rfbG (B) involved in LPS biosynthesis in S. Typhimurium SL1344. The phage sensitivities of wild-type and mutant strains were tested using an adsorption assay with SFP10 phage. Diamonds, wild-type strain (SFP10 sensitive); squares, E. coli MG1655 (SFP10 resistant). (A) Triangles, ΔrfaL deletion mutant; circles, ΔrfaL deletion mutant complemented with the pUHE21-lacI^q::rfaL expression vector. (B) Triangles, ΔrfbG/Tn5 mutant; circles, ΔrfbG/Tn5 mutant complemented with the pUHE21-lacI^q::rfbG expression vector. The error bars indicate the standard deviations in triplicate experiments.

Fig 4

Bacterial challenge test of phage SFP10 with S. Typhimurium SL1344 (A) and E. coli O157:H7 ATCC 43890 (B). The graphs show viable-cell counts of samples collected every hour. Each strain was infected with phage SFP10 when the OD at 600 nm was 1.0. Circles, non-SFP10-infected sample; squares, SFP10-infected sample. The error bars indicate standard deviations.

Fig 5

Genome map of phage SFP10. The outer circle indicates the gene coding regions by strand. The color of each gene refers to the functional category: phage structure (blue), replication/recombination/repair (yellow), nucleotide metabolism (pink), transcription (orange), translation (green), or additional function (purple). The arrowheads in the first inner circle indicate the locations of tRNAs. The inner circle with a red line indicates the GC content. The legends for phage structural proteins are blue. The scale units are base pairs.

Fig 6

Phylogenetic analysis of MCPs from various bacteriophages. The MCPs were compared by ClustalW multiple alignments, and the phylogenetic tree was generated by the MEGA4 program using the neighbor-joining method with P distance values.

Fig 7

Comparative analysis of three phage genomes (A) and gene clusters involved in host specificity for infection from three phage genomes (B). (A) Phage SFP10 (middle), Shigella phiSboM-AG3 phage (top), and Salmonella phage Vi01 (bottom). The variable regions in the three phage genomes involved in host specificity for infection are boxed. (B) The white arrows indicate host specificity genes, and the gray arrows indicate hypothetical proteins. The identities of amino acids between homologous genes are indicated as percentages.