Isolation and Characterization of Chi-like Salmonella Bacteriophages Infecting Two Salmonella enterica Serovars, Typhimurium and Enteritidis

Abstract

Salmonella enterica Serovar Typhimurium and Salmonella enterica Serovar Enteritidis are well-known pathogens that cause foodborne diseases in humans. The emergence of antibiotic-resistant Salmonella serovars has caused serious public health problems worldwide. In this study, two lysogenic phages, STP11 and SEP13, were isolated from a wastewater treatment plant in Jeddah, KSA. Transmission electron microscopic images revealed that both phages are new members of the genus “Chivirus” within the family Siphoviridae. Both STP11 and SEP13 had a lysis time of 90 min with burst sizes of 176 and 170 PFU/cell, respectively. The two phages were thermostable (0 °C ≤ temperature < 70 °C) and pH tolerant at 3 ≤ pH < 11. STP11 showed lytic activity for approximately 42.8% (n = 6), while SEP13 showed against 35.7% (n = 5) of the tested bacterial strains. STP11 and STP13 have linear dsDNA genomes consisting of 58,890 bp and 58,893 bp nucleotide sequences with G + C contents of 57% and 56.5%, respectively. Bioinformatics analysis revealed that the genomes of phages STP11 and SEP13 contained 70 and 71 ORFs, respectively. No gene encoding tRNA was detected in their genome. Of the 70 putative ORFs of phage STP11, 27 (38.6%) were assigned to functional genes and 43 (61.4%) were annotated as hypothetical proteins. Similarly, 29 (40.8%) of the 71 putative ORFs of phage SEP13 were annotated as functional genes, whereas the remaining 42 (59.2%) were assigned as nonfunctional proteins. Phylogenetic analysis of the whole genome sequence demonstrated that the isolated phages are closely related to Chi-like Salmonella viruses.

Keywords: Chi-like phages; S. Enteritidis; S. Typhimurium; bacteriophage; molecular characterization.

Figure 1

Plaque morphology and TEM of phage STP11 (A,C), and phage SEP13 (B,D). The scale bar corresponds to 100 nm.

Figure 2

The line graph shows the stability of the isolated phage for (A) Thermal treatment; (B) pH; (C) Organic solvent and detergent treatment for both phages (same results obtained). Mean values ± SD for each point are displayed as log10 (PFU/mL).

Figure 3

One-step growth kinetics of phages STP11 and SEP13 on their corresponding hosts. Results are displayed as means of three replicates ± SD and presented as log₁₀ (PFU/mL).

Figure 4

Lytic activities of phages STP11 (A) and Phage SEP13 (B) against their corresponding hosts at MOIs of 100 and 10,000. The point represents the average + SD of three replicate experiments.

Figure 5

The circular genome map of phage (A) STP11 and (B) SEP13. The map was constructed and visualized using the CGView server database. The outer circle designates the ORFs of the isolated phage predicted, coupled with their putative functions. The negative sign in the parenthesis indicated the position of the strand ((main strand (not labeled), complementary strand (−)). The most inner circle marked with a red landscape designates the GC content, while the 2nd inner ring with the green and purple landscape shows the GC skew −/+ (GC-skew ((G-C)/(G+C))). The CDSs whose functions have been determined are labeled (blue color) along with their positions; however, other CDSs without labels (black color) represent hypothetical proteins. The physical map is scaled in kbp.

Figure 6

Phylogenetic tree was made using the whole genome sequence (A) and amino acid sequence of the major capsid protein (B) of phage STP11 and SEP13 and phages sharing homology sequence identity retrieved from GenBank (NCBI). The sequences were aligned using ClustalW, and the tree was built using MEGA 7 software. The evolutionary history of 33 major capsid protein amino acid sequences and 37 core genes of the whole genome sequence were aligned and inferred using the Neighbor-Joining method and 1000 bootstrap replicates. The red dot highlights the isolated phages. The scale bar represents 20% nucleotide substitution and 10% amino acid substitution percentage for the whole genomic and capsid protein map, respectively.

Figure 6

Phylogenetic tree was made using the whole genome sequence (A) and amino acid sequence of the major capsid protein (B) of phage STP11 and SEP13 and phages sharing homology sequence identity retrieved from GenBank (NCBI). The sequences were aligned using ClustalW, and the tree was built using MEGA 7 software. The evolutionary history of 33 major capsid protein amino acid sequences and 37 core genes of the whole genome sequence were aligned and inferred using the Neighbor-Joining method and 1000 bootstrap replicates. The red dot highlights the isolated phages. The scale bar represents 20% nucleotide substitution and 10% amino acid substitution percentage for the whole genomic and capsid protein map, respectively.