Complete genome sequence of the salmonella enterica serovar enteritidis bacteriophages fSE1C and fSE4C isolated from food matrices

Salmonella enterica serovar Enteritidis is one of the most common causes of Salmonellosis worldwide. Utilization of bacteriophages as prophylactic agents is a practical solution to prevent Salmonellosis in ready-to-eat products. Shelf stability is one of the desirable properties for prophylactic bacteriophages. Here, we describe the phenotype, genome, and phylogeny of fSE1C and fSE4S Salmonella bacteriophages. fSE1C and fSE4S were previously isolated from pickle sauce and ground beef respectively and selected for their significant shelf stability. fSE1C and fSE4S showed a broad S. enterica serovar range, infecting several Salmonella serovars. The viral particles showed an icosahedral head structure and flexible tail, a typical morphology of the Siphoviridae family. fSE1C and fSE4C genomes consists of dsDNA of 41,720 bp and 41,768 bp with 49.73% and 49.78% G + C, respectively. Comparative genomic analysis reveals a mosaic relationship between S. enterica serovar Enteritidis phages isolated from Valparaiso, Chile.


Introduction
The current methodologies to inactivate bacterial pathogens in ready-to-eat products are not infallible. Foodborne diseases caused by non-typhoid Salmonella still have an enormous impact on public health [1,2]. Salmonella enterica serotype Enteritidis is one of the most common causes of non-typhoid Salmonellosis with contaminated food [3][4][5]. The increasing cases of Salmonellosis together with the emergence of antibiotic resistant strains have led to efforts searching for new methods to control Salmonella colonization in ready-to-eat products. Traditional methods to reduce bacterial contamination (U.V., steam, and dry heat) face the problems of food organoleptic properties deterioration and lack of prophylactic protection once the product is contaminated. Also, some of these approaches used in the food industry to reduce contamination by food borne pathogens cannot be directly applied to fresh fruits, vegetables, and raw meat [6]. Despite technical advances to avoid transmission of bacterial pathogens throughout the food chain, novel strategies are still required to fulfill consumer demands to minimize chemical preservatives in fresh food products. Bacteriophage-based biocontrol has a great potential to enhance microbiological safety based on their long history of safe use, relatively easy handling, high and specific antimicrobial activity and public acceptance [7].
Shelf stability is one of the desirable characteristics that a bacteriophage must have for its effective utilization in fresh food [6]. Previously, we isolated the bacteriophages fSE1C and fSE4S from pickle sauce and ground beef respectively [8]. These bacteriophages have a significant stability in shelf conditions and in food matrices with respect to other Salmonella bacteriophages [8], making fSE1C and fSE4S excellent candidates to be used in readyto-eat products. Here, we report the phenotypic characteristics, genome sequence, and phylogeny of fSE1C and fSE4S bacteriophages isolated from food matrices in Valparaiso, Chile.

Classification and features
The bacteriophages fSE1C and fSE4S were isolated from pickle sauce and ground beef respectively, from samples obtained at the Central Market of Valparaiso, Chile, during 2013. Routine enrichment techniques [9] and the host, S. enterica serovar Enteritidis PT4 [8] were utilized for the isolation process. The two phages isolated formed clear plaques on the host bacterial lawn after 18 h of incubation at 37°C. The diameters of plaques were 1 mm for both phages ( Fig. 1a and b). fSE1C and fSE4S showed a productive lytic infection in different S. enterica serovars including S. enterica serovar Enteritidis (control), S. enterica serovar Infantis, S. enterica serovar Heidelberg, S. enterica serovar Typhi, S. enterica serovar Typhimurium, S. enterica serovar Paratyphi B and S. enterica serovar Pullorum. The bacteriophages have a different host range. fSE4S can have a productive lytic infection in S. enterica serovar Derby and S. enterica serovar Hadar in contrast to fSE1C [10]. The transmission electron microscopy showed that these bacteriophages have a typical morphology of the Siphoviridae family consisting of an icosahedral head (~50 nm), flexible long non-contractile tail (~150 nm) and base ( Fig. 1b and d).
The extracted nucleic acids from phage particles were treated with EcoRI, HindIII and HaeIII restriction enzymes. The genomic material of both phages was digested by these enzymes, revealing that their genomic material is dsDNA (Fig. 1e). The restriction enzyme patterns were similar for both phages (Fig. 1e). Taken together, these results indicated these phages belong to the Siphoviridae family [11]. Phylogenetic analysis, using the The evolutionary history was inferred using the Neighbor-Joining method [23]. The optimal tree with the sum of branch length = 2.55835582 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method [25] and are in the units of the number of base differences per site. The analysis involved 25 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 104441 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [26]. g. fSE1C bacteriophage genome map; the unique gene to fSE1C is indicated in red and the putative cas4 gene in blue; h. fSE4S bacteriophage genome map; the putative cas4 gene is indicated in blue. The internal circle show the G + C % in red and the A + T % in black. DNAPlotter was utilized for genome map visualization [33] complete bacteriophage genomes, showed that these phages are close related to f18SE [12], SSe and wksl3 Salmonella phages (Fig. 1f ). The bacteriophage SSe, wksl3 and f18SE are members of the proposed subfamily Jersyvirinae [12], genera Jersylikekvirus [13]. However our phylogenetic analysis, which includes the most recently sequenced Salmonella Siphoviridae bacteriophages, revealed that fSE1C, fSE4S, f18SE, SSe and wksl3 are distant members from the Jersylikekvirus genera (Fig. 1f ).
Genes encoding DNA polymerase, helicase, the major tail protein, portal protein, the terminase large subunit and the major capsidase, were predicted from the genomes of both phages and used for phylogenetic analysis ( Fig. 1g and h). DNA polymerase, helicase and the major tail protein are closely related to the bacteriophage f18SE [12] (Fig. 2). On the other hand, the portal protein and the terminase large subunit are closely related between both phages, but not related to the f18SE bacteriophage (Fig. 2). The major capsid subunit of the Fig. 2 Phylogenetic analysis of conserved genes of Siphoviridae bacteriophages. Phylogenetic tree of conserved gene on bacteriophages of Siphoviridae family, and fSE1C and fSE4S. The evolutionary history was inferred using the Neighbor-Joining method [23]. DNA Polymerase, helicase, major tail, portal protein, terminase, and major capside gene sequences were selected. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [24]. The evolutionary distances were computed using the p-distance method [25] and are in the units of the number of base differences per site. Evolutionary analyses were conducted in MEGA6 [26] phage fSE1C is closely related to f18SE, in contrast to fSE4S, which is closely related to the SETP3 phage (Fig. 2). Mosaicism is known to be prevalent in the family Siphoviridae, which is reflected in our results. However, the DNA polymerase, and helicase proteins presented similar phylogenic relationships, analogous to the complete bacteriophage genome phylogenic relationships (Fig. 1f ). Information on the isolation, classification, and general features of the phages fSE1C and fSE4S are presented in Table 1.

Genome sequencing information
Genome project history Genome sequencing of the bacteriophages fSE1C and fSE4S was performed as a part of a research project that aimed to sequence effective bacteriophages fore use in anti-Salmonella prophylactic cocktails for ready-to-eat products. Previously, we reported the genome sequence of the Salmonella bacteriophage f18SE isolated from the poultry industry in Valparaiso, Chile, during 2001, which has been tested successfully in vivo and in processed foods [14][15][16] as part of this project.
Genome sequencing of fSE1C and fSE4S was performed using the NGS Illumina MiSeq at Universidad Mayor, Center for Genomics and Bioinformatics (Huechuraba, Chile). The sequences were assembled using CLC Genomics Workbench 8.5.1 (Qiagen), resulting in single contigs. The assembled sequences were annotated by the PHASTER server [17,18] and the NCBI-PGAAP. The complete genome sequences and annotation information of both bacteriophages were submitted to GenBank under the accession numbers KT962832 (fSE1C) and KT881477 (fSE4S) ( Table 2).

Growth conditions and genomic DNA preparation
The bacteriophages fSE1C and fSE4S were isolated from pickle sauce and ground beef respectively using S. enterica serovar Enteritidis PT4 as host [8]. Isolation and propagation methods were those used routinely [9,19]. Briefly, the bacteriophages were enriched using a S.  [20].

Genome sequencing and assembly
The purified bacteriophage DNA was used to prepare the libraries (one library for each phage) with the Nextera kit (Illumina, San Diego, CA). High-throughput sequencing of the libraries was performed using a MiSeq (Illumina) with a 2x300bp paired-end run, with the reagent kit version 3 (600 cycles) at the Center for Genomics and Bioinformatics, Universidad Mayor, Chile. In total, about 127 and 317 million pairs of reads were obtained for fSE1C and fSE4S, respectively. Raw reads were assembled by using CLC Genomics Workbench 8.5.1. Coverage was calculated from the sequencing statistics, and final contig sizes were 2874× and 7590× for fSE1C and fSE4S, respectively ( Table 2).

Genome annotation
Contigs were annotated using a combination of automatic annotations by the PHASTER server [17,18], and the NCBI PGAAP. Functional annotation of protein coding genes was improved by RPS-BLAST searches against the CDD [21]. Signal sequence peptides and transmembrane helices were predicted by the Phobius software [22]. BLASTp searches against the NCBI nr database were also performed. The CRISPRs were predicted base on structure using the web base software Structure RNA finder. The evolutionary history was inferred using the Neighbor-Joining method [23]. The trees were drawn to scale. The percentage of replicate trees for the conserved proteins in the bootstrap test (1000 replicates) are shown next to the branches [24] (Fig. 2). The evolutionary distances were computed using the p-distance method [25] and are in the units of the number of base differences per site. The ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA6 [26].

Genome properties
The complete genomes of both phages were assembled into single circular contigs. Bacteriophage fSE1C contains 41,720 bp and has a G + C content of 49.73%. The bacteriophage fSE4S contains 41,768 bp and has a G + C content of 49.78%. The genome of fSE1C contains 53 predicted genes and fSE4S contains 52 predicted genes, with a total gene length between 186-3099 bp. We found in fSE1C genome 17 genes with rightward orientation, while 36 were leftward oriented, and in fSE4S genome 35 genes with rightward orientation and 17 were leftward ( Fig. 1g and h) (Table 3). Both phage genomes contain genes for replication, structure, and lysis. Open reading frames (ORFs) were found for putative homing endonuclease, helicase, and DNA polymerase. The ORFs for terminase (large and small subunit), head morphogenesis protein, major capside protein, putative tail  The total is based on the size of the genome in base pairs The total is based on the total number of protein coding genes in the genome protein, and tail fiber protein and a portal protein were found. Also, a lysozyme, holing-like classes I and putative endolysins were also found. Lysogeny related genes, like C2 of P22 [27], CI and Cro of λ [28], and others are absent from both phage genomes. The phage genomes closely related to fSE1C and fSE4S were Salmonella phages f18SE (GenBank accession no. KR270151), SSe3 (GenBank accession no. AY730274), and wsk13 (GenBank accession no. JX202565). Comparative analysis between both phages showed that their genomes are 43.09% similar and all 52 genes of fSE4S have orthologous in the fSE1C genome. These orthologous proteins have a similarity between 73.58 and 100%. The only gene different in the fSE1C genome encodes for a hypothetical protein (GI:952094085) of 108 aa with no ortholog in fSE4S, but present in f18SE and other lytic Salmonella bacteriophages.
Non-coding RNA prediction was similar in both bacteriophages, presenting the CRISPR-DR41 and CRISPR-DR23 single direct repeat. This prediction was coincident with the COGs analyses (Table 4), which detected the Cas4 protein family (cl00641) in both bacteriophages. Functional CRISPRs have been described in V. cholerae bacteriophages [29], however, the CRISPRs predicted for fSE1C and fSE4S seem not a completed CRISPR system.

Conclusions
The ORFs involved in structure, replication, host specificity (i.e., tail fibers and tailspikes) and DNA metabolism were found to be conserved in these two phages compared to other Salmonella enterica bacteriophages. However, the major capsid protein showed some diversity (Fig. 2) that might be related to the high shelf stability presented by fSE1C and fSE4S phages [8].
The Jersyvirine subfamily consists of three genera, "Jerseyvirus", "Sp3unavirus" and "K1gvirus" [13]. The Jersyvirine subfamily include a distinct morphotype, genomes of 40-44 kb (49.6-51.4 mol % G + C), a syntenic genome organization, high degree of nucleotide sequence identity, and strictly lytic cycle [30]. As mentioned previously, the Siphoviriade family presents considerable mosaicism [31,32] and although we distinguished a possible new genus for the subfamily Jersyvirinae (Fig. 1f), we considered that a high number of sequenced Jersyvirinae phages are required to propose a new genus.