Genomic and Phenotypic Analysis of Salmonella enterica Bacteriophages Identifies Two Novel Phage Species

Abstract

Bacteriophages (phages) are potential alternatives to chemical antimicrobials against pathogens of public health significance. Understanding the diversity and host specificity of phages is important for developing effective phage biocontrol approaches. Here, we assessed the host range, morphology, and genetic diversity of eight Salmonella enterica phages isolated from a wastewater treatment plant. The host range analysis revealed that six out of eight phages lysed more than 81% of the 43 Salmonella enterica isolates tested. The genomic sequences of all phages were determined. Whole-genome sequencing (WGS) data revealed that phage genome sizes ranged from 41 to 114 kb, with GC contents between 39.9 and 50.0%. Two of the phages SB13 and SB28 represent new species, Epseptimavirus SB13 and genera Macdonaldcampvirus, respectively, as designated by the International Committee for the Taxonomy of Viruses (ICTV) using genome-based taxonomic classification. One phage (SB18) belonged to the Myoviridae morphotype while the remaining phages belonged to the Siphoviridae morphotype. The gene content analyses showed that none of the phages possessed virulence, toxin, antibiotic resistance, type I-VI toxin-antitoxin modules, or lysogeny genes. Three (SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins. Whole-genome-based phylogeny of the eight phages with their 113 homologs revealed three clusters A, B, and C and seven subclusters (A1, A2, A3, B1, B2, C1, and C2). While cluster C1 phages were predominantly isolated from animal sources, cluster B contained phages from both wastewater and animal sources. The broad host range of these phages highlights their potential use for controlling the presence of S. enterica in foods.

Keywords: Salmonella; bacteriophage; comparative phylogenomics; diversity; food safety; host specificity.

Figure 1

Maximum likelihood tree of 55 Salmonella strains representing 30 serovars used for phage propagation and host specificity in this study. The Salmonella strains used for propagating the phages in this study are indicated with a red star. Core-genome sequence alignment was generated from draft genomes using MAFFT v7.453 and the tree that was bootstrapped with 1000 replicates for node support was constructed using FastTree v2.1.11. The scale bar at the bottom represents nucleotide substitution per site.

Figure 2

Heat map of the lytic spectra of isolated phages with scoring of lysis, where 0 indicates no lysis and 1 to 3 indicate clearing (3 indicates complete clear lysis).

Figure 3

Maximum likelihood phylogenetic tree showing the relatedness of the phages under study. The phage genomes’ alignment was generated using MAFFT v7.453 and the tree that was bootstrapped with 1000 replicates for node support was constructed using IQtree v2.2. The scale bar at the bottom shows nucleotide substitution per site. Phages in the light green rectangle belonged to cluster A; those within the blue rectangle were designated as cluster B phages, and the others were singletons. The gray and white boxes depict the presence or absence of tailspike proteins in the phages.

Figure 4

Homology and gene synteny comparison of phages isolated in this study with previously sequenced phages. BlastN comparisons of the phages in this study with their closest references. (A) SB9 and SB10 with Salmonella phage fuchur; (B) SB13 with Escherichia phage Supergirl; (C) SB28 with the reference Salmonella phage E1; (D) SB3 and SB15 with Salmonella phage vB_SenS_AG11; (E) SB6 with Salmonella phage oselot, and (F) SB18 with Erwinia phage phiEa21-4. The direction of arrows indicates the DNA strand direction. Figures were generated using EasyFig.

Figure 5

Maximum likelihood phylogenetic tree showing the relatedness of phages under study, with previously identified Salmonella phages having a coverage of ≥80% and nucleotide homology of ≥95%. The phage genomes’ alignment was generated using MAFFT v7.453 and the maximum likelihood tree that was bootstrapped with 1000 replicates for node support was constructed using IQtree v2.2. The first three blocks show the source, host, and serovars (for the Salmonella host) of the phages, while the other blocks indicate the presence and absence of tailspike proteins. The scale bar at the bottom indicates the nucleotide differences amongst them. The phages under study are labeled in blue.

Figure 6

Transmission electron microscopy (TEM) images of representative phages from different clusters. (A) Cluster A phages: subcluster A1: SB28; subcluster A2: SB9, SB10, and SB13. (B) Cluster B phages: SB3 and SB15. (C) Cluster C phages: subcluster C1: SB6; subcluster C2: SB18.