Genomic analysis of Salmonella bacteriophages revealed multiple endolysin ORFs and importance of ligand-binding site of receptor-binding protein

Abstract

Salmonella is a prevalent foodborne pathogen causing millions of global cases annually. Antimicrobial resistance is a growing public health concern, leading to search for alternatives like bacteriophages. A total of 97 bacteriophages, isolated from cattle farms (n = 48), poultry farms (n = 37), and wastewater (n = 5) samples in Türkiye, were subjected to host-range analysis using 36 Salmonella isolates with 18 different serotypes. The broadest host range belonged to an Infantis phage (MET P1-091), lysing 28 hosts. A total of 10 phages with the widest host range underwent further analysis, revealing seven unique genomes (32-243 kb), including a jumbophage (>200 kb). Except for one with lysogenic properties, none of them harbored virulence or antibiotic resistance genes, making them potential Salmonella reducers in different environments. Examining open reading frames (ORFs) of endolysin enzymes revealed surprising findings: five of seven unique genomes contained multiple endolysin ORFs. Despite sharing same endolysin sequences, phages exhibited significant differences in host range. Detailed analysis unveiled diverse receptor-binding protein sequences, with similar structures but distinct ligand-binding sites. These findings emphasize the importance of ligand-binding sites of receptor-binding proteins. Additionally, bacterial reduction curve and virulence index revealed that Enteritidis phages inhibit bacterial growth even at low concentrations, unlike Infantis and Kentucky phages.

Keywords: Salmonella; antibiotic resistance; bacteriophages; biopreservation; endolysin; ligand-binding site.

Figure 1.

Comparison of genome organization of MET_P1_100_107 and MET_P1_179_112. *Genes are shaded based on sequence identity (0% white, 100% black). The different colors represent clusters of genes that exhibit similarities between two phage genome.

Figure 2.

Virulence index of phages. Each experiment was conducted in triplicate, and the standard deviations were computed accordingly.