Novel Salmonella Phage, vB_Sen_STGO-35-1, Characterization and Evaluation in Chicken Meat

Abstract

Salmonellosis is one of the most frequently reported zoonotic foodborne diseases worldwide, and poultry is the most important reservoir of Salmonella enterica serovar Enteritidis. The use of lytic bacteriophages (phages) to reduce foodborne pathogens has emerged as a promising biocontrol intervention for Salmonella spp. Here, we describe and evaluate the newly isolated Salmonella phage STGO-35-1, including: (i) genomic and phenotypic characterization, (ii) an analysis of the reduction of Salmonella in chicken meat, and (iii) genome plasticity testing. Phage STGO-35-1 represents an unclassified siphovirus, with a length of 47,483 bp, a G + C content of 46.5%, a headful strategy of packaging, and a virulent lifestyle. Phage STGO-35-1 reduced S. Enteritidis counts in chicken meat by 2.5 orders of magnitude at 4 °C. We identified two receptor-binding proteins with affinity to LPS, and their encoding genes showed plasticity during an exposure assay. Phenotypic, proteomic, and genomic characteristics of STGO-35-1, as well as the Salmonella reduction in chicken meat, support the potential use of STGO-35-1 as a targeted biocontrol agent against S. Enteritidis in chicken meat. Additionally, computational analysis and a short exposure time assay allowed us to predict the plasticity of genes encoding putative receptor-binding proteins.

Keywords: Salmonella Enteritidis; Salmonella–phage in food; Siphoviridae; receptor-binding proteins; siphoviral morphotype.

Figure 1

One-step growth curve of STGO-35-1. The phage concentration (PFU/mL) was followed over time after infection of Salmonella with phage STGO-35-1. A latency period (in blue) of 30 min (±10 min) and a burst size (in red) of 122 (±10 PFU/mL) can be observed.

Figure 2

Transmission electron microscopy shows a morphology showing that this virus has a capsid, with tail and without contractile neck with siphoviral morphology. The magnification was at 100,000× in 85 kV, visualized using Fiji3 microscopy [42].

Figure 3

(a) Genome map of STGO-35-1, made with Artemis and DNAPlotter [52]. The color coding of genes indicates the functional categories of putative proteins: capsid proteins (green); lysozyme (black); CDSs and hypothetical proteins (gray); and tail proteins (red). The GC skew is represented in the inner circle and with purple indicating below average and yellow above average. (b) Genomic and phenotypic characterization of phage STGO 35-1. (c) The packaging strategies predicted in PhageTerm [25].

Figure 4

Phylogenetic relatedness of STGO-35-1 to other siphoviral morphotypes of phages. (I). Phylogenetic tree based on COBALT [56], using neighbor joining [55]. Phage most closely related to STGO-35-1 in red squares were marked. (II). Comparison of Salmonella phage STGO 35-1 (1) with the three most closely related phages: Salmonella phage KFS-SE2 (4), Salmonella phage Akira (3), and Salmonella 6795_sal3 (2). The color coding of genes indicates putative functional categories: capsid proteins (green); lysozyme (black); CDSs and hypothetical proteins (gray); tail proteins (red).

Figure 5

Chicken meat assay of S. Enteritidis and phage. This analysis represented the four experimental groups considered in this study in chicken meat pieces at a size of 2 cm² approx. Experimental groups were inoculated with: (1) phage lysate at 4 × 10⁶ PFU/mL (black circle/broken lines); and (2) Salmonella (SE^nal) at 4 × 10⁶ CFU/mL (red triangles/broken lines) (MOI = 1). (3) Salmonella controls (red triangles/unbroken lines) were prepared with only Salmonella. (4) Phage control with only phage (black circle/ unbroken lines). The standard deviation was calculated between the three replicates per day, and statistically significant differences (estimated with ANOVA at p < 0.05) are indicated by different letters.