Bacteriophages were isolated from food samples confiscated on the Hungarian border by the customs agents. Samples underwent the first steps of the ISO 16654:2001 method for isolating E. coli O157. Briefly, 5 g of the food samples were homogenized in 10 volumes of tryptic soy broth (TSB) supplemented with bile salts, and incubated for 24 h at 42°C. Bacterium-free samples of these precultures were spread or spotted onto layered 0.7% soft agar plates containing the E. coli K-12 derivative C600 as propagating strain, as described by Strauch et al. (2001). After overnight incubation at 37°C, single plaques were picked and purified by re-propagation on E. coli C600 as well as at E. coli MG1655 at least three times, until high titer (≥10¹¹ PFU/ml) bacteriophage stocks were produced. The list of the isolated bacteriophages and their origin is given in Table 1.

The host specificity of the isolated bacteriophages was investigated by spot assays on the strains listed in Table 2 as described before. In addition to reference strains and strains from our strain collection, extended-spectrum β-lactamase (ESBL)-producing multidrug-resistant (MDR) E. coli strains isolated from human clinical samples were investigated. MDR strains were isolated at the University of Debrecen and Salmonella strains were kindly provided by László Makrai (University of Veterinary Medicine, Budapest). Shigella flexneri M90T was kindly contributed by Zoltán Tigyi (University of Pécs). Enteroinvasive E. coli (EIEC) and Shigella strains (except for S. sonnei 866-F) originated from the Hungarian National Collection of Medical Bacteria (HNCMB), Budapest.

The ESBL resistant E. coli strains were isolated from human extraintestinal infections, the Shigella flexneri strain was isolated from human feces and the Salmonella strains were isolated from food or from animals. These isolates were identified biochemically and were serotyped using O specific immunsera (data not shown).

Efficiency of plating (EOP) was determined by applying serial dilutions of bacteriophage suspensions in spot assays. The ratio of bacteriophage titer on each strain to the titer measured on E. coli MG1655 was considered the EOP of the bacteriophage on the given strain.

One step growth experiment to determine burst size was conducted on the representative bacteriophages chee24 and chee130_1 on E. coli MG1655, according to the description of E. Bassiri¹ with some modifications. Briefly, 5 × 10⁸ bacteria were mixed with 5 × 10⁶ bacteriophages in Luria broth (LB), setting the multiplicity of infection (MOI) to 0.01. The mixture was incubated for 10 min at room temperature for initial adsorption, then diluted 10⁴-fold, and in a total volume of 10 ml incubated at 37°C for 1 h. Hundred microliter aliquots were taken every 5 min and plated on layered soft agar for counting. Experiments were run at three independent times in two parallels each. Burst size was determined as a ratio of average bacteriophage count of the baseline and the average bacteriophage count after the burst.

Heat stability of the bacteriophages was tested as follows: 1 ml stocks containing a 100-fold dilution of a bacteriophage stock in LB were incubated for 1 h at 25°C, 37°C, 42°C, and 80°C. After incubation, the titer of the treated bacteriophage stocks was determined with spot assay on E. coli MG1655. Experiments were performed in two parallels on each temperature.

Stability of the bacteriophages under different pH values was tested by incubating 100-fold dilutions of the bacteriophages for 1 h at 37°C in 1 ml LB pH-adjusted to 3, 5, 7, and 9 with HCl and NaOH solutions. After incubation, the titer of the treated bacteriophage stocks was determined with spot assay on E. coli MG1655. Experiments were performed in two parallels on each pH value.

Bacteriophages were investigated by transmission electron microscopy (TEM). Briefly, drops of a high titer bacteriophage suspensions were placed on parafilm, absorbed onto carbon film, washed in TE buffer (10 mM TRIS, 1 mM EDTA, pH 6.9) and negatively-stained with 2% aqueous uranyl acetate, pH 5.0. Carbon film was collected with 300 mesh copper grids and access negative-stain was removed with filter paper and subsequentely air-dried. Samples were examined in a TEM 910 transmission electron microscope (Carl Zeiss, Oberkochen) at an acceleration voltage of 80 kV. Images were recorded digitally at calibrated magnifications with a Slow-Scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Münster, Germany). Contrast and brightness were adjusted with Adobe Photoshop CS3.

Bacteriophage DNA was isolated from bacteriophage stocks with a concentration of at least 10¹¹ PFU/ml. The phenol-chloroform method described by Sambrook et al. (1987) was used for isolation with the modifications outlined by Tóth et al. (2016). Briefly, to remove any non-bacteriophage related DNA and RNA from the 700 μl bacteriophage suspension, the sample was treated with amplification grade DNase I and RNase A (Sigma Aldrich) in 10 μg/ml final concentration for 30 min at 37°C. For bacteriophage lysis, proteinase K (Sigma Aldrich) was added in 6.6 μg/ml final concentration to the lysis buffer (50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 0.5% SDS) and incubated at 65°C for 30 min. The suspension was cooled to room temperature and mixed with 750 μl of a 1:1 mixture of equilibrated phenol and chloroform. After 5 min incubation at room temperature, proteins were removed by centrifugation at 10,000 × g for 5 min. The aqueous phase was transferred to a clean Eppendorf tube and bacteriophage DNA was precipitated after adding 0.1 volume of 3 M potassium acetate, pH 5.5 with 0.7 volume of isopropanol at ice for 20 min. DNA was collected by centrifugation at 13,000 × g at 10 min, washed with 600 μ1 70% ethanol and dissolved in 30 μl 10 mM Tris–HCl, 1 mM EDTA, pH 8.0 buffer.

Genomic DNA sequencing libraries were prepared using the Nextera XT kit (Illumina, Eindhoven, NL). Sequencing was performed using Nextseq Mid-output reagent kit v2 (2 × 150 bp) on an Illumina NextSeq 500. Assembly was performed with SPAdes (Bankevich et al., 2012). The genome was annotated using the RAST server (Overbeek et al., 2014). The genes orf5c (T5.035) and the bacteriophage DNA polymerase (T5.122) were used for phylogenetic analysis. The DNA sequences of these two genes from all bacteriophages determined in this study and of T5 reference genomes (Supplementary Table 1) were aligned using Clustal Omega (Sievers et al., 2011). The phylogenetic tree was visualized using MEGA5.2 (Hall, 2013). For whole-bacteriophage sequence phylogeny, the program VICTOR (Meier-Kolthoff and Goeker, 2017) was used with default settings and all T5 reference genomes (Supplementary Table 1). Determination of sequence homologies and the identification of SNPs were conducted by using the BLAST tools available at the NCBI website. Genome alignments were visualized with Easyfig (Sullivan et al., 2011) and modified using Inkscape. The left and right repeats were defined using a pile-up analysis of raw sequencing reads after mapping with CLC workbench 9.0 (Qiagen, Hilden, Germany). The repeats were defined as >130% of average coverage at the particular site (30% more coverage than average coverage).

The whole genome sequences of all bacteriophages determined in the current study were deposited in GenBank database under accession nos. MF431730- MF431741.

By using E. coli K-12 derivative strains MG1655 and C600 as indicator and propagating strain, we isolated 12 new bacteriophages from different food samples. The origin, as well as the subgrouping of the bacteriophages are summarized in Table 1. The bacteriophages were isolated from either meat, meat products (e.g., sausages) or cheese.

All the new isolated bacteriophages showed Siphoviridae morphology, with an average head length of ~85 nm, head width of ~75 nm and a flexible, non-contractile tail with a length of ~200 nm. As a representative, the morphology of T5-like bacteriophage chee24 is shown (Figure 1).

To assess the host specificity of the bacteriophages, we tested their lytic capacity on a large variety of enterobacterial pathogenic and ESBL multidrug-resistant clinical strains listed in Table 2. We found that the novel T5-like bacteriophages showed two distinct lysis patterns, which we denote by a representative bacteriophage of each: bacteriophage chee24 (group chee24) and bacteriophage chee130_1 (group chee130_1). The grouping of the bacteriophages is indicated in Table 1. EOP values were determined by spot assay for these two bacteriophages. The lysis spectrum and EOP values are given in Table 2.

Apart from K-12 derivative strains, several E. coli, Shigella, and Salmonella strains representing different pathotypes and serovars proved to be susceptible to the bacteriophages. While bacteriophage chee130_1 lysed enteropathogenic E. coli (EPEC), EIEC, and Shigella sonnei strains, bacteriophage 24 induced lysis on Salmonella serovars, EHEC O103:H2, and Shigella dysenteriae strains. Both bacteriophages showed lysis on enterotoxigenic E. coli (ETEC) type strain H10407. It has to be noted however, that in each case EOP values were orders of magnitude lower when compared to K-12 strains, and on some wild type strains the plaque morphology was opaque, unlike the clear plaques of ~2 mm in diameter, observed with the strain MG1655.

Burst size was determined with one step growth experiments on E. coli MG1655 (Figure 2). The burst sizes of bacteriophage chee24 and chee130_1 proved to be different, in the case of the former, it was around 1,000 PFU/cell, while in the case of chee130_1, the burst size was around 44 PFU/cell. In both cases the burst occurred ~45 min after the end of the initial absorption.

Heat stability experiments showed that incubation of 10¹⁰ PFU bacteriophage stocks at 25°C, 37°C and 42°C for 1 h did not significantly affect the EOP of the bacteriophages (data not shown). Incubation at 80°C, however, caused 10⁴-fold average decrease in the bacteriophage titer.

A pH value of 3 completely deactivated the bacteriophages, as no plaques were observable even in the spots of the concentrated suspensions. On the other hand, no significant decrease in titer could be observed at pH values 5, 7, and 9 after 1 h incubation (data not shown).

To examine the genomic structures and the potential diversity in the genomes, all the 12 newly isolated bacteriophage genome sequences were determined and detailed genomic comparison was conducted. Genome sequencing of the bacteriophages showed that all the bacteriophages have a linear double-stranded DNA genome, and belong to T5-like bacteriophages. The two subgroups with different host specificity also have minor genetic differences. The genome of the “group chee24” bacteriophages is 120,618 (bacteriophage pork27), 120,620 (bacteriophage saus111K), 120,622 (bacteriophage chee24, pork29 and saus47N) or 120,629 bp (bacteriophage poul124) long, and contains 165 (saus47N, saus111K, poul124), 166 (chee24, pork29) or 168 (pork27) protein-encoding CDS, as well as 18 tRNA genes, respectively. Its GC content is 39.3%. On the other hand, the genome size of the ‘group chee130_1’ bacteriophages is 121,986 bp, with 164 (saus132, poul149), 165 (cott162) or 167 (chee130_1, chee158, saus176N) protein-encoding CDS, 20 tRNA genes, and a GC content of 39.7%. These groups were congruent with the different host spectrum observable within the bacteriophages, indicating that the chee24-like and chee130_1-like bacteriophages form distinct genetic groups. The list of ORFs compared to each other and to the original T5 bacteriophage (GenBank AY543070.1) are shown in Supplementary Tables 2, 3. Members of both subgroups showed the characteristic functional regions in their genomes, which is a common feature of T5-like phages, having pre-early, early and late genomic regions named after the time of their transcription, as well as a terminal repeat region on the 5′ and 3′ end, which is 9,968 bp long in chee24-like phages, and 8,717 bp in group chee130_1 phages. For group chee24 bacteriophages, the bacteriophage pork27 differed by 1 SNP in the terminal repeat, while for group chee130_1 phages, all terminal repeats of other than chee130_1 differed from those present in the prototype, with saus132, chee158, cott162 and saus176N having 3 SNPs, and poul149 harboring 4 SNPs.

At the nucleotide level, the newly identified T5-like bacteriophages not only have the same genome size within the two subgroups—group chee24 and group chee130_1-, but when compared to the “type bacteriophage” of their groups as reference bacteriophage chee24 or chee130_1, respectively, they differ in only 2–5 nucleotide positions. It has to be noted that in the case of bacteriophage pork27 and saus47N, one SNP causes a premature stop codon in bacteriophage protein genes of unknown function. In the case of bacteriophages saus132, poul149 and chee158 there are premature stop codons in a bacteriophage tail fiber gene. A summary of these genomic locations is given in Table 3.

None of the new characterized bacteriophage genomes contained any known virulence, toxin, antibiotic resistance-encoding and virulence regulator genes.

To reveal the phylogenetic position of bacteriophages chee24 and chee130_1 among T5-like phages, several comparisons were conducted. Comparison based on the genes orf5c (T5.035) and the bacteriophage DNA polymerase (T5.122) of bacteriophage T5 (AY692264.1) showed that while bacteriophage 24 proved to be the closest relative of E. coli bacteriophages DT57C and DT571/2, bacteriophage chee130_1 are closely related to the Salmonella bacteriophages Stitch and SPC35 (Figure 3).

Whole-genome based phylogeny of all T5-like bacteriophages clearly indicated that the phages isolated in the current study are members of the T5-like bacteriophages (Figure 4). The topology of this tree is entirely different from the one based on the single genes, putting the two representative phages next to the phage genomes that also proved closest homologs with the BLAST analysis. An alignment of T5-like genomes with mutual homologies including representatives of the two subgroups is presented in Figure 5, showing the 5′ end of the late region as the most variable, which contains mainly tail fiber genes.

Comparing the genomes of bacteriophage chee24 and chee130_1at the nucleotide-level, regions showing low homology to each other were the tail fiber region and an open reading frame encoding a bacteriophage-associated receptor binding protein gene. All these features seem to contribute to the differences observed in the host specificity of the two bacteriophage groups. At nucleotide level, the closest homolog to bacteriophage chee24 is bacteriophage SPC35 (GenBank no. HQ406778.). In addition, two hypothetical proteins, a thymidylate synthase gene, and two subunits of a HNH endonuclease, a receptor binding protein (CDS ADW80123.1) of SPC35 is also absent in chee24, suggesting different host specificity. Bacteriophage EPS7 (GenBank no. CP000917) is the closest homolog to bacteriophage chee130_1 of the previously sequenced T5-like bacteriophages, and carries homologs to all receptor-associated proteins, albeit the similarity is not 100% in each case. Furthermore, chee130_1 lacks a few hypothetical proteins and AGC_0146 putative tail protein as well. Genes showing low homology and those that are absent from bacteriophages of either the chee24 or chee130_1subgroup are listed in Table 4.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.