ORIGINAL RESEARCH
published: 13 February 2018
doi: 10.3389/fmicb.2018.00202
Identification and Characterization
of T5-Like Bacteriophages
Representing Two Novel Subgroups
from Food Products
Domonkos Sváb 1, Linda Falgenhauer 2, Manfred Rohde 3, Judit Szabó 4,
Trinad Chakraborty 2* and István Tóth 1*
1 Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest,
Hungary, 2 Institute of Medical Microbiology, Justus Liebig University Giessen and German Center for Infection Research
(DZIF), Partner Site Giessen-Marburg-Langen, Giessen, Germany, 3 Central Facility for Microscopy, Helmholtz Centre for
Infection Research, HZI, Braunschweig, Germany, 4 Department of Medical Microbiology, Faculty of Medicine, University of
Debrecen, Debrecen, Hungary
During recent years, interest in the use of bacteriophages as biocontrol agents
against foodborne pathogens has increased, particularly for members of the family
Enterobacteriaceae, with pathogenic Escherichia coli, Shigella, and Salmonella strains
Edited by:
among them. Here, we report the isolation and characterisation of 12 novel T5-like
Stephen Tobias Abedon,
The Ohio State University, bacteriophages from confiscated food samples. All bacterophages effectively lysed
United States E. coli K-12 strains and were able to infect pathogenic E. coli strains representing
Reviewed by: enterohaemorrhagic (EHEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), and
Bob Gordon Blasdel,
KU Leuven, Belgium enteroinvasive (EIEC) pathotypes, Shigella dysenteriae, S. sonnei strains, as well as
Victor Krylov, multidrug-resistant (MDR) E. coli and multiple strains representing different Salmonella
Research Institute of Vaccines and
enterica serovars. All the bacteriophages exhibited Siphoviridae morphology. Whole
Sera. Mechnikov, Russian Academy of
Medical Sciences (RAS), Russia genome sequencing of the novel T5-like bacteriophages showed that they represent two
*Correspondence: distinct groups, with the genome-based grouping correlating to the different host spectra.
István Tóth As these bacteriophages are of food origin, their stability and lack of any virulence genes,
toth.istvan@agrar.mta.hu
Trinad Chakraborty as well as their broad and mutually complementary host spectrum makes these new T5-
trinad.chakraborty@mikrobio. like bacteriophages valuable candidates for use as biocontrol agents against foodborne
med.uni-giessen.de
pathogenic enterobacteria.
Specialty section: Keywords: bacteriophages, T5-like phages, phage genomics, Enterobacteriaceae, ESBL E. coli, food safety,
This article was submitted to bio-control
Antimicrobials, Resistance and
Chemotherapy,
a section of the journal INTRODUCTION
Frontiers in Microbiology
Received: 27 September 2017 Several members of the Enterobacteriaceae family are considered significant foodborne pathogens,
Accepted: 29 January 2018 with enterohemorrhagic Escherichia coli (EHEC) and Shigella strains capable of causing
Published: 13 February 2018 life-threatening disease even at a very small infectious dose (reviewed in Croxen et al., 2013;
Citation: Anderson et al., 2016). Many of these infections are treated with antibiotic therapy, but this
Sváb D, Falgenhauer L, Rohde M, is threatened by the increasing occurrence of multidrug-resistant (MDR) strains (reviewed in
Szabó J, Chakraborty T and Tóth I Szmolka and Nagy, 2013). The development of novel antibiotics has significantly slowed down in
(2018) Identification and the past decades, as it is becoming increasingly hard to find agents with new effect mechanisms or
Characterization of T5-Like
targets, and also because of limited economic returns (Hunter, 2012). More recently, attention has
Bacteriophages Representing Two
Novel Subgroups from Food turned on the development of “evolution-proof” antibiotics (Bell and MacLean, 2017) and as an
Products. Front. Microbiol. 9:202. alternative answer to this challenge, there is also a renewed interest in the study of bacteriophages
doi: 10.3389/fmicb.2018.00202 capable of lysing pathogenic bacteria.
Frontiers in Microbiology | www.frontiersin.org 1 February 2018 | Volume 9 | Article 202
Sváb et al. Novel T5-Like Bacteriophages
As biocontrol agents, phages can be applied either as a to serve as biocontrol agents against fooborne pathogenic
treatment of ongoing infections (phage therapy) as in the case Enterobacteria.
of Staphylococcus aureus strains, or for foodborne pathogens, the Using E. coli K-12 strains for propagation, we isolated 12 new
treatment of alimentary food as a preventive measure (reviewed T5-like lytic bacteriophages from individual food samples, and
by Johnson et al., 2008 and Hagens and Loessner, 2010). There proceeded with their detailed characterisation. Whole genome
are already several examples of commercially available phage sequence analysis revealed that these bacteriophages do not
cocktails to be utilized against various foodborne pathogens. harbor undesirable virulence-related genes. They represent two
There is a phage cocktail aimed against E. coli O157 to be distinct new genotypes among T5-like bacteriophages and proved
applied to food surfaces (Carter et al., 2012), as well as cocktails to be capable of lysing a wide array of pathogenic E. coli, Shigella,
against Shigella (Soffer et al., 2017), Listeria monocytogenes and and Salmonella strains.
Salmonella enterica (reviewed by Abedon, 2017). There have also
been clinical trials of phage cocktails against diarrheagenic E. coli MATERIALS AND METHODS
on human subjects (Sarker et al., 2012, 2016), but these did not
yield results regarding the efficacious use of phage cocktails for Isolation of Bacteriophages
such infections. Bacteriophages were isolated from food samples confiscated on
Bacteriophage T5 was one of the first enterobacterial phages the Hungarian border by the customs agents. Samples underwent
to be characterized in-depth in the 1950s, and since then the first steps of the ISO 16654:2001 method for isolating E. coli
several similar bacteriophages have been reported. Their most O157. Briefly, 5 g of the food samples were homogenized in 10
unique feature is a two-step DNA transfer during infection volumes of tryptic soy broth (TSB) supplemented with bile salts,
(reviewed in Davison, 2015). Later, partly because of the renewed and incubated for 24 h at 42◦C. Bacterium-free samples of these
interest of utilizing bacteriophages as antibacterial agents, the precultures were spread or spotted onto layered 0.7% soft agar
bacteriophage T5 genomewas fully sequenced (Wang et al., 2005) plates containing the E. coli K-12 derivative C600 as propagating
and new members of this bacteriophage group have also been strain, as described by Strauch et al. (2001). After overnight
characterized (Kim and Ryu, 2011; Golomidova et al., 2015). All incubation at 37◦C, single plaques were picked and purified by
T5 bacteriophages seem to share a common genomic structure re-propagation on E. coli C600 as well as at E. coli MG1655 at
where pre-early, early and late regions can be distinguished least three times, until high titer ( 1011≥ PFU/ml) bacteriophage
depending on the time of transcription during the infectious stocks were produced. The list of the isolated bacteriophages and
cycle (reviewed by Davison, 2015). In recent studies, several T5- their origin is given in Table 1.
like bacteriophages were proposed as potential candidates for
use against various foodborne pathogens, including Salmonella Testing Host Specificity and Efficiency of
enterica serovar Typhimurium (Kim and Ryu, 2011; Piya et al., Plating
2015) as well as against E. coli O157:H7 strains (Niu et al., The host specificity of the isolated bacteriophages was
2012; Hong et al., 2014), with promising in vivo results investigated by spot assays on the strains listed in Table 2
produced in live sheep with the phage CEV2 (Raya et al., as described before. In addition to reference strains and strains
2011). from our strain collection, extended-spectrum β-lactamase
In a previous study, we assessed the risk posed by foodborne (ESBL)-producing multidrug-resistant (MDR) E. coli strains
pathogens present in illegally imported foodstuff in Europe, isolated from human clinical samples were investigated.
with a special emphasis on STEC (Nagy et al., 2015). We MDR strains were isolated at the University of Debrecen
hypothesized that bacteriophages capable of lysing foodborne
pathogenic bacteria should be also capable of surviving and
replicating in the same food. Therefore, a potent source for
bacteriophage candidates to be applied as biocontrol against TABLE 1 | Origin and subgrouping of new T5-like bacteriophages.
foodborne bacteria could be the very same food samples. As
pointed out by Abedon (2017), studies aiming toward phage Phage no. Foodstuff of origin Country of origin Subgroup
therapy tend to focus on successes on therapeutic application,
chee24 cattle cheese Bulgaria chee24
and neglect in-depth characterisation of phages. Studies
pork27 row pork meat Serbia chee24
on the potential therapeutic application of phages against
pork29 row pork meat Serbia chee24
enterobacterial infections (Sarker et al., 2016) suggest that a
saus47N pork sausage Serbia chee24
preventive approach, i.e., prior eradication of pathogens from
saus111K pork sausage Ukraine chee24
food with phages before consumption could be a more succesful
poul124 poultry meat Ukraine chee24
application.
In light of these considerations we aimed to isolate chee130_1 cattle cheese Ukraine chee130_1
bacteriophages from the food samples that are capable of saus132 pork sausage Ukraine chee130_1
lysing foodborne enteric pathogens to extensively characterize poul149 poultry meat Ukraine chee130_1
their host spectrum with other phenotypic features, and to chee158 cattle cheese Ukraine chee130_1
characterize their genomes. This in-depth characterisation would cott162 cattle cottage cheese Ukraine chee130_1
serve to determine whether the isolated phages can be considered saus176N pork sausage Hungary chee130_1
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Sváb et al. Novel T5-Like Bacteriophages
TABLE 2 | Host specificity and efficiency of plating of the two subgroups of new T5-like bacteriophages.
Strain Pathotype/serovar /species Serogroup or serotype Phage chee24 Phage chee130_1 Strain reference
II95-36 EIEC O121 − − this study
20 EIEC O124 − − this study
Bra2 26 EIEC O152 +++ +++ this study
Saigon EIEC O164 − − this study
E2348/69 EPEC O127:H6 − + Iguchi et al., 2009
536 UPEC O6:K15:H31 − − Hochhut et al., 2006
IHE3034 ExPEC O18:K1:H7 − − Moriel et al., 2010
E250 APEC O1:K1:H7 − − Tóth et al., 2003
T22 atypical O157:H43 − − Tóth et al., 2009
E22 EHEC O103:H2 ++ − Marchès et al., 2003
Sakai EHEC O157:H7 − − Hayashi et al., 2001
EDL933 EHEC O157:H7 − − Perna et al., 2001
ICC169 Citrobacter rodentium N/A − − Wiles et al., 2005
20080 Shigella dysenteriae 1A N/A +++ − this study
M90T Shigella flexneri N/A − − this study
20038 Shigella boydii N/A − − this study
866-F Shigella sonnei N/A +++ +++ Allué-Guardia et al., 2011
20045 Shigella sonnei N/A +++ − this study
1201 Salmonella Typhimurium 1 N/A +++ − this study
1202 Salmonella Infantis N/A + − this study
1203 Salmonella Panama N/A +++ − this study
1199 Salmonella Typhi N/A − − this study
1198 Salmonella Gallinarum N/A − − this study
1200 Salmonella Enteritidis N/A − − this study
5871* ESBL E. coli O15 − − this study
18531** ESBL E. coli O73 ++ + this study
29095** ESBL E. coli O90 − − this study
H10407 ETEC O78:H11:K80 + + Evans et al., 1973
MG1655 E. coli K-12 O16:H48 +++ +++ Blattner et al., 1997
The range of EOP values relative to the titer of the bacteriophages on Escherichia coli MG1655 is symbolized as follows: +++: between 1 and 10-3; ++: between 10-4-10-6; +: 10-7
and below; −: no lysis.
*Resitance to ESBL, Ciprofloxacin, Gentamicin, Tetracycline.
**Resistance to ESBL, Ciprofloxacin, Gentamicin, Sulphamethoxazole/Trimethoprim.
and Salmonella strains were kindly provided by László One Step Growth Experiments
Makrai (University of Veterinary Medicine, Budapest). One step growth experiment to determine burst size was
Shigella flexneri M90T was kindly contributed by Zoltán conducted on the representative bacteriophages chee24 and
Tigyi (University of Pécs). Enteroinvasive E. coli (EIEC) and chee130_1 on E. coli MG1655, according to the description of
Shigella strains (except for S. sonnei 866-F) originated from the E. Bassiri1 with somemodifications. Briefly, 5 108× bacteria were
Hungarian National Collection of Medical Bacteria (HNCMB), mixed with 5 × 106 bacteriophages in Luria broth (LB), setting
Budapest. the multiplicity of infection (MOI) to 0.01. The mixture was
The ESBL resistant E. coli strains were isolated from human incubated for 10min at room temperature for initial adsorption,
extraintestinal infections, the Shigella flexneri strain was isolated then diluted 104-fold, and in a total volume of 10ml incubated
from human feces and the Salmonella strains were isolated at 37
◦C for 1 h. Hundred microliter aliquots were taken every
from food or from animals. These isolates were identified 5min and plated on layered soft agar for counting. Experiments
biochemically and were serotyped using O specific immunsera were run at three independent times in two parallels each. Burst
(data not shown). size was determined as a ratio of average bacteriophage count
Efficiency of plating (EOP) was determined by applying serial of the baseline and the average bacteriophage count after the
dilutions of bacteriophage suspensions in spot assays. The ratio of burst.
bacteriophage titer on each strain to the titer measured on E. coli
MG1655 was considered the EOP of the bacteriophage on the 1http://www.sas.upenn.edu/LabManuals/biol275/Table_of_Contents_files/13-
given strain. PhageGrowth.pdf Accessed on 20th September, 2017.
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Sváb et al. Novel T5-Like Bacteriophages
Heat Stability Tests Bacteriophage Genome Sequencing and
Heat stability of the bacteriophages was tested as follows: 1ml Analysis
stocks containing a 100-fold dilution of a bacteriophage stock Genomic DNA sequencing libraries were prepared using the
in LB were incubated for 1 h at 25◦C, 37◦C, 42◦C, and 80◦C. Nextera XT kit (Illumina, Eindhoven, NL). Sequencing was
After incubation, the titer of the treated bacteriophage stocks was performed using Nextseq Mid-output reagent kit v2 (2 × 150
determined with spot assay on E. coliMG1655. Experiments were bp) on an Illumina NextSeq 500. Assembly was performed with
performed in two parallels on each temperature. SPAdes (Bankevich et al., 2012). The genome was annotated
using the RAST server (Overbeek et al., 2014). The genes orf5c
pH Tolerance Tests (T5.035) and the bacteriophage DNA polymerase (T5.122) were
Stability of the bacteriophages under different pH values was used for phylogenetic analysis. The DNA sequences of these two
tested by incubating 100-fold dilutions of the bacteriophages for genes from all bacteriophages determined in this study and of T5
1 h at 37◦C in 1ml LB pH-adjusted to 3, 5, 7, and 9 with HCl reference genomes (Supplementary Table 1) were aligned using
and NaOH solutions. After incubation, the titer of the treated Clustal Omega (Sievers et al., 2011). The phylogenetic tree was
bacteriophage stocks was determined with spot assay on E. coli visualized usingMEGA5.2 (Hall, 2013). For whole-bacteriophage
MG1655. Experiments were performed in two parallels on each sequence phylogeny, the program VICTOR (Meier-Kolthoff and
pH value. Goeker, 2017) was used with default settings and all T5 reference
genomes (Supplementary Table 1). Determination of sequence
Determination of Bacteriophage homologies and the identification of SNPs were conducted by
Morphology using the BLAST tools available at the NCBI website. Genome
alignments were visualized with Easyfig (Sullivan et al., 2011)
Bacteriophages were investigated by transmission electron
and modified using Inkscape. The left and right repeats were
microscopy (TEM). Briefly, drops of a high titer bacteriophage
defined using a pile-up analysis of raw sequencing reads after
suspensions were placed on parafilm, absorbed onto carbon
mapping with CLC workbench 9.0 (Qiagen, Hilden, Germany).
film, washed in TE buffer (10mM TRIS, 1mM EDTA, pH 6.9)
The repeats were defined as >130% of average coverage at the
and negatively-stained with 2% aqueous uranyl acetate, pH 5.0.
particular site (30% more coverage than average coverage).
Carbon film was collected with 300 mesh copper grids and access
negative-stain was removed with filter paper and subsequentely Sequence Accession Numbers
air-dried. Samples were examined in a TEM 910 transmission The whole genome sequences of all bacteriophages determined
electron microscope (Carl Zeiss, Oberkochen) at an acceleration in the current study were deposited in GenBank database under
voltage of 80 kV. Images were recorded digitally at calibrated accession nos. MF431730- MF431741.
magnifications with a Slow-Scan CCD-Camera (ProScan, 1024×
1024, Scheuring, Germany) with ITEM-Software (Olympus Soft
Imaging Solutions, Münster, Germany). Contrast and brightness RESULTS
were adjusted with Adobe Photoshop CS3. Isolation of Bacteriophages
By using E. coli K-12 derivative strains MG1655 and C600
Bacteriophage DNA Isolation as indicator and propagating strain, we isolated 12 new
Bacteriophage DNA was isolated from bacteriophage stocks with bacteriophages from different food samples. The origin, as well
a concentration of at least 1011 PFU/ml. The phenol-chloroform as the subgrouping of the bacteriophages are summarized in
method described by Sambrook et al. (1987) was used for Table 1. The bacteriophages were isolated from either meat, meat
isolation with the modifications outlined by Tóth et al. (2016). products (e.g., sausages) or cheese.
Briefly, to remove any non-bacteriophage related DNA and
RNA from the 700 µl bacteriophage suspension, the sample was Morphology
treated with amplification grade DNase I and RNase A (Sigma All the new isolated bacteriophages showed Siphoviridae
Aldrich) in 10µg/ml final concentration for 30min at 37◦C. For morphology, with an average head length of∼85 nm, head width
bacteriophage lysis, proteinase K (Sigma Aldrich) was added in of ∼75 nm and a flexible, non-contractile tail with a length
6.6µg/ml final concentration to the lysis buffer (50mM Tris– of ∼200 nm. As a representative, the morphology of T5-like
HCl (pH 8.0), 10mM EDTA, 0.5% SDS) and incubated at 65◦C bacteriophage chee24 is shown (Figure 1).
for 30min. The suspension was cooled to room temperature
and mixed with 750 µl of a 1:1 mixture of equilibrated phenol Host Spectrum, Efficiency of Plating, Burst
and chloroform. After 5min incubation at room temperature, Size, and Stability
proteins were removed by centrifugation at 10,000× g for 5min. To assess the host specificity of the bacteriophages, we tested
The aqueous phase was transferred to a clean Eppendorf tube and their lytic capacity on a large variety of enterobacterial pathogenic
bacteriophage DNA was precipitated after adding 0.1 volume of and ESBL multidrug-resistant clinical strains listed in Table 2.
3M potassium acetate, pH 5.5 with 0.7 volume of isopropanol at We found that the novel T5-like bacteriophages showed two
ice for 20min. DNA was collected by centrifugation at 13,000 × distinct lysis patterns, which we denote by a representative
g at 10min, washed with 600 µ1 70% ethanol and dissolved in 30 bacteriophage of each: bacteriophage chee24 (group chee24) and
µl 10mM Tris–HCl, 1mM EDTA, pH 8.0 buffer. bacteriophage chee130_1 (group chee130_1). The grouping of
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Sváb et al. Novel T5-Like Bacteriophages
Whole Genome Sequence Analysis
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
FIGURE 1 | Transmission electron micrograph of bacteriophage 24 showing (GenBank AY543070.1) are shown in Supplementary Tables 2, 3.
Siphoviridae morphology.
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
the bacteriophages is indicated in Table 1. EOP values were after the time of their transcription, as well as a terminal repeat
determined by spot assay for these two bacteriophages. The lysis region on the 5′ and 3′ end, which is 9,968 bp long in chee24-
spectrum and EOP values are given in Table 2. like phages, and 8,717 bp in group chee130_1 phages. For group
Apart from K-12 derivative strains, several E. coli, Shigella, chee24 bacteriophages, the bacteriophage pork27 differed by 1
and Salmonella strains representing different pathotypes and SNP in the terminal repeat, while for group chee130_1 phages,
serovars proved to be susceptible to the bacteriophages. While all terminal repeats of other than chee130_1 differed from those
bacteriophage chee130_1 lysed enteropathogenic E. coli (EPEC), present in the prototype, with saus132, chee158, cott162 and
EIEC, and Shigella sonnei strains, bacteriophage 24 induced lysis saus176N having 3 SNPs, and poul149 harboring 4 SNPs.
on Salmonella serovars, EHEC O103:H2, and Shigella dysenteriae At the nucleotide level, the newly identified T5-like
strains. Both bacteriophages showed lysis on enterotoxigenic bacteriophages not only have the same genome size within
E. coli (ETEC) type strain H10407. It has to be noted however, the two subgroups—group chee24 and group chee130_1-, but
that in each case EOP values were orders of magnitude lower when compared to the “type bacteriophage” of their groups
when compared to K-12 strains, and on some wild type strains as reference bacteriophage chee24 or chee130_1, respectively,
the plaque morphology was opaque, unlike the clear plaques of they differ in only 2–5 nucleotide positions. It has to be noted
∼2mm in diameter, observed with the strain MG1655. that in the case of bacteriophage pork27 and saus47N, one SNP
Burst size was determined with one step growth experiments causes a premature stop codon in bacteriophage protein genes
on E. coli MG1655 (Figure 2). The burst sizes of bacteriophage of unknown function. In the case of bacteriophages saus132,
chee24 and chee130_1 proved to be different, in the case of poul149 and chee158 there are premature stop codons in a
the former, it was around 1,000 PFU/cell, while in the case of bacteriophage tail fiber gene. A summary of these genomic
chee130_1, the burst size was around 44 PFU/cell. In both cases locations is given in Table 3.
the burst occurred∼45min after the end of the initial absorption. None of the new characterized bacteriophage genomes
Heat stability experiments showed that incubation of 1010 contained any known virulence, toxin, antibiotic resistance-
PFU bacteriophage stocks at 25◦C, 37◦C and 42◦C for 1 h did encoding and virulence regulator genes.
not significantly affect the EOP of the bacteriophages (data not
shown). Incubation at 80◦C, however, caused 104-fold average Phylogenetic Analysis
decrease in the bacteriophage titer. To reveal the phylogenetic position of bacteriophages chee24
A pH value of 3 completely deactivated the bacteriophages, as and chee130_1 among T5-like phages, several comparisons
no plaques were observable even in the spots of the concentrated were conducted. Comparison based on the genes orf5c
suspensions. On the other hand, no significant decrease in titer (T5.035) and the bacteriophage DNA polymerase (T5.122) of
could be observed at pH values 5, 7, and 9 after 1 h incubation bacteriophage T5 (AY692264.1) showed that while bacteriophage
(data not shown). 24 proved to be the closest relative of E. coli bacteriophages
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Sváb et al. Novel T5-Like Bacteriophages
FIGURE 2 | One step growth curves of new T5-like bacteriophages on E. coli MG1655 strain. PFU values of bacteriophage chee24 are marked with triangles, those
of bacteriophage chee130_1 are marked with squares. The error bars indicate standard deviations from the results of the three independent triplicate experiments.
TABLE 3 | Single nucleotide polymorphisms shown by T5-like bacteriophages of the chee24-like subgroup (A) and the chee130_1-like subgroup (B) when compared to
the type bacteriophage of each subgroup.
A
Position in phage chee24 ORF Predicted function chee24 pork27 pork29 saus47N saus111K* poul124** Amino acid change
24634 42 Phage protein C A A C A A V65L
57794 101 Phage protein G G G A G G W22*
84550 129 Phage tail fibers C C C T T T G700D
84602 129 Phage tail fibers T G G G G G T683P
86896 130 Phage protein C A C C C C E59*
B
Position in phage chee130_1 ORF Predicted function chee130_1 saus132 poul149 chee158 cott162 saus176N Amino acid change
595 2 Phage protein G A A G G G synonymous
1774 4 Probable A1 protein C C C G C C G493A
1919 4 Probable A1 protein G G G G A G L445F
2501 4 Probable A1 protein A A G A A A synonymous
2707 4 Probable A1 protein G G G G G A R182W
3301 intergenic region A C C C C C N/A
3303 intergenic region T A A A A A N/A
50473 intergenic region T C C T T T N/A
86944 133 Phage tail fibers G G G A G G Q210*
86950 133 Phage tail fibers C C C C T C A212T
87643 133 Phage tail fibers G A A G G G Q443*
*In position 101429-101430, AA is deleted.
**In position 101429-101430, AA is deleted. At the beginning, ACT is inserted, and at the end, CGTG is inserted.
Letters in bold refer to nucleotides different from those found in phages chee24 and chee130_1 in the same positions, respectively.
DT57C and DT571/2, bacteriophage chee130_1 are closely on the single genes, putting the two representative phages
related to the Salmonella bacteriophages Stitch and SPC35 next to the phage genomes that also proved closest homologs
(Figure 3). with the BLAST analysis. An alignment of T5-like genomes
Whole-genome based phylogeny of all T5-like bacteriophages with mutual homologies including representatives of the two
clearly indicated that the phages isolated in the current study subgroups is presented in Figure 5, showing the 5′ end of the
are members of the T5-like bacteriophages (Figure 4). The late region as the most variable, which contains mainly tail fiber
topology of this tree is entirely different from the one based genes.
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Sváb et al. Novel T5-Like Bacteriophages
FIGURE 3 | Phylogenetic tree of T5-like bacteriophages based on the Clustal Omega alignment of the genes orf5c (T5.035, A) and the phage DNA polymerase
(T5.122, B) of bacteriophage T5 (AY692264.1). The scale represents homology %. The list of included T5-like phage genomes with their GenBank accession numbers
is given in Supplementary Table 1.
subunits of a HNH endonuclease, a receptor binding protein
(CDSADW80123.1) 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.
DISCUSSION
Using E. coli K-12 C600 strain as the host bacterium, we
isolated and characterized 12 novel T5-like bacteriophages
from independent food samples of various animal origin from
around the world These novel bacteriophages represented two
subgroups based on their host specificity. While the most
effective host of the bacteriophages were K-12 derivative
FIGURE 4 | Phylogenetic tree of T5-like bacteriophages made with the whole
E. coli strains, several strains representing important intestinal
genome-based VICTOR analysis. The scale represents homology %. The list
of included T5-like phage genomes with their GenBank accession numbers is pathotypes of E. coli, Shigella, and Salmonella also proved
given in Supplementary Table 1. susceptible to the bacteriophages. The Siphoviridae morphology
and the genomic characteristics including the genome size,
the genome architecture, the low GC content and the
Comparing the genomes of bacteriophage chee24 and sequence homologies showed that the bacteriophages are T5-like
chee130_1at the nucleotide-level, regions showing low homology bacteriophages.
to each other were the tail fiber region and an open reading frame An important feature of these newly found phages is that
encoding a bacteriophage-associated receptor binding protein they were isolated from food samples, unlike the previously
gene. All these features seem to contribute to the differences isolated T5-like bacteriophages, which originated from sewage
observed in the host specificity of the two bacteriophage groups. and fecal sources (Kim and Ryu, 2011; Golomidova et al., 2015).
At nucleotide level, the closest homolog to bacteriophage chee24 There is also one case where a T5-like phage was isolated
is bacteriophage SPC35 (GenBank no. HQ406778.). In addition, and characterized from a commercially available bacteriophage
two hypothetical proteins, a thymidylate synthase gene, and two cocktail designed against enteric pathogens (Piya et al., 2015).
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Sváb et al. Novel T5-Like Bacteriophages
FIGURE 5 | Alignment of T5-like bacteriophage whole genomes. GenBank accession numbers of the phages are given in Supplementary Table 1.Arrows with different
colors represent ORFs associated with the genomic regions indicated at the top. The gray bar in the lower right corner shows the identity percentage associated with
the color of the bars connecting homologous regions.
Our results indicate that food can be a rich source of The closest homolog of the chee130_1 subgroup, EPS7 is able
bacteriophages effective against pathogenic bacteria potentially to lyse the same Salmonella serovars and seven unnamed E. coli
residing in the same environment. These phages maintain their strains (Hong et al., 2008), however, phage chee130_1 did not lyse
infective capacity in this environment suggesting that foodborne Salmonella strains. On the other hand, phage chee130_1 was able
phages are promising biocontrol candidates against foodborne to propagate on Shigella sonnei, EIEC and EPEC strains. All of
bacteria. these differences in host range underline the potential role that
Our results also show the huge diversity of bacteriophages, genes showing low homology to closely related bacteriophages
even from well-characterized groups such as T5-like (Table 4) might have importance in host specificity. In the case
bacteriophages, as both the chee24 and chee130_1 subgroups of chee130_1, ORF134 that is annotated as “phage tail fibers”
are different from the closest related bacteriophages. Our data has very low homology to the corresponding gene in EPS7 and
indicate that the most crucial differences are within genes seems to support this notion. It is interesting to note however,
encoding receptor-binding proteins (Table 4, Figure 5). The that in ORF133, another gene annotated as phage tail fiber,
closest colinear relative of bacteriophage chee24 is SPC35, there are premature stop codons in the case of bacteriophages
which was reported to lyse Salmonella Typhimurium and saus132, poul149 and chee158. These mutations nevertheless did
Enteritidis strains, as well as a large set of K-12 derivative and not seem to have any effect on the bacterial host specificity of
commensal E. coli (Kim and Ryu, 2011). However, the host these bacteriophages when compared to other members of their
spectrum of group chee24 phages is particularly broad, and subgroup (data not shown).
includes a strain representing one of the so-called “big six” Lee et al. (2016) suggested that besides bacteriophage
serotypes of EHEC (O103:H2), as well as an ESBL E. coli strain cocktails, a solution for simultaneous biocontrol of several
of serogroup O73 isolated from an extraintestinal infection. foodborne pathogens could be the cloning of several receptor
The differences in ORFs 145 and 149, annotated as “phage tail binding genes into a single bacteriophage. For either purpose,
fibers” and “receptor binding protein” could account for the obtaining knowledge about new bacteriophages capable of
discrepancy. infecting these pathogens and assessing their host spectrum is
We wanted to test the efficiency of the new T5-like phages on essential.
target strains, and the one step growth on E. coliMG1655 yielded Besides the above-cited studies of promising candidate phages
burst sizes comparable to those of the T5-like bacteriophage to be used as biocontrol agents against pathogenic bacteria, there
phiLLS (Amarillas et al., 2017). are already several commercially available phage cocktails aimed
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Sváb et al. Novel T5-Like Bacteriophages
TABLE 4 | List of genes and regions showing low homology or missing in chee24-like (A) and chee130_1-like (B) bacteriophages when compared to their closest
homolog bacteriophages.
A
Phage chee24 Phage SPC35
ORF Predicted function CDS Predicted function
none intergenic region ADW79993.1 hypothetical protein
none intergenic region ADW79995.1 hypothetical protein
7 phage protein ADW79996.1 hypothetical protein
7 phage protein ADW79997.1 hypothetical protein
none intergenic region ADW80009.1 hypothetical protein
31 serine-threonine proteine phosphatase ADW80013.1 serine-threonine proteine phosphatase
41 phage protein ADW80023.1 hypothetical protein
41 phage protein ADW80025.1 hypothetical protein
none intergenic region ADW80038.1 hypothetical protein
none intergenic region ADW80040.1 hypothetical protein
91 thymidilate synthase ADW80064.1 thymidilate synthase
none intergenic region ADW80067.1 putative H-N-H-endonuclease P-TflVIII
99 phage protein ADW80071.1 hypothetical protein
none intergenic region ADW80078.1 hypothetical protein
134 phage tail-length tape measure protein ADW80108.1 pore-forming tail tip protein
145 phage tail fibers ADW80118.1 putative tail protein
149 phage-associated receptor-binding protein ADW80118.1 receptor-binding protein
B
Phage chee130_1 Phage EPS7
ORF Predicted function CDS Predicted function
9 phage protein ACB97450.1 hypothetical protein
none intergenic region ACB97451.1 hypothetical protein
none intergenic region ACB97452.1 hypothetical protein
none intergenic region ACB97458.1 hypothetical protein
none intergenic region ACB97467.1 hypothetical protein
26 phage protein ACB97475.1 hypothetical protein
28 phosphoesterase ACB97478.1 putative serine/threonine phosphatase
29 phage protein ACB97479.1 hypothetical protein
30 serine/threonine phosphatase ACB97480.1 hypothetical protein
31 phage associated homing endonuclease ACB97482.1 hypothetical protein
none intergenic region ACB97510.1 hypothetical protein
none intergenic region ACB97513.1 rho-associated, coiled-coil containing protein kinase 2
60 phage protein ACB97515.1 hypothetical protein
61 hypothetical protein ACB97516.1 hypothetical protein
none intergenic region ACB97518.1 hypothetical protein
65 phage protein ACB97519.1 hypothetical protein
101 unknown ACB97556.1 hypothetical protein
105 NAD-dependent protein deacetylase of SIR2 family ACB97560.1 putative Sir2-like protein
109 hypothetical protein ACB97569.1 hypothetical protein
134 phage tail fibers ACB97589.1 putative phage tail protein
139 phage tail-length tape measure protein ACB97594.1 pore-forming tail tip protein
139 phage tail-length tape measure protein ACB97595.1 pore-forming tail tip protein pb2
none intergenic region ACB97613.1 hypothetical protein
In the case of chee24-like subgroup the reference bacteriophage is SPC35 (GenBank HQ406778), in the case of chee130_1-like bacteriophages it is EPS7 (GenBank CP000917).
Corresponding, albeit in some cases non-homologous ORFs or intergenic regions are displayed in the same row.
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Sváb et al. Novel T5-Like Bacteriophages
at various foodborne enterobacterial pathogens (reviewed by shown to be stable during storage and application temperatures,
Abedon, 2017). However, according to our knowledge except for as well as a range of pH values usual in most of the food.
ShigaShield, which is aimed against Shigellae (Soffer et al., 2017), All the above-mentioned properties, coupled with their food
and the Microgen ColiProteus aimed against E. coli and Proteus origin, as well as their broad and partially overlapping,
(McCallin et al., 2013), none of the component phages have been supplementary host spectrum makes the new T5-like
sequenced. In order to assess the applicability of a phage either bacteriophages valuable candidates as effective bio-control
for biocontrol of for therapy, extensive knowledge of its genome agents against foodborne pathogenic enterobacteria.
is essential, to determine whether the phage carries “undesirable”
genes. In-depth knowledge of genes determining host specificity AUTHOR CONTRIBUTIONS
or those contributing to phage stability in different environments
are also valuable information when selecting bacteriophages for a IT and TC conceived and elaborated the study. DS, LF, MR, JS,
given therapeutic or biocontrol purpose. and IT conducted the phenotypic experiments. LF performed
As it has been noted by Brüssow (2012), the development of sequencing and provided bioinformatical tools. DS, LF, TC, and
an actual phage therapy treatment can take years from phage IT analyzed the results and composed themanuscript. All authors
isolation to performing clinical trials. This process includes for reviewed the manuscript.
exapmle the purification (Bourdin et al., 2014b), as well as
characterisation of host specificity (Bourdin et al., 2014a) and ACKNOWLEDGMENTS
testing the biological safety of the phages either in animals
(Raya et al., 2011) or human subjects (Sarker et al., 2012, 2016). This work was supported by the 7th EU Framework Programme
An extensive genetic database of phages isolated from different PROMISE project (project n. 265877), the Hungarian National
sources, representing different genuses and host spectra could Research, Development and Innovative Office (NKFIH, grant
speed up the process of selecting phages for clinical trials. 124335) and grants from the Bundesministerium fuer Bildung
In summary, we identified 12 new T5-like bacteriophages und Forschung (BMBF, Germany) within the German Center for
from food samples, which represent two new species within this Infection research (DZIF/grant number 8032808811 to TC).
bacteriophage group. Our study so far shows that they fulfill
important criteria listed by Hagens and Loessner (2010) for SUPPLEMENTARY MATERIAL
bacteriophages to be used as biocontrol: their genome sequence
was determined, they propagate well on a non-pathogenic host, The Supplementary Material for this article can be found
they lack any virulence, virulence regulator, toxin, and antibiotic online at: https://www.frontiersin.org/articles/10.3389/fmicb.
resistance genes. They also seem to be strictly lytic and were 2018.00202/full#supplementary-material
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(2001). Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. is permitted, provided the original author(s) and the copyright owner are credited
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(2015). Complete genome sequence of Salmonella enterica serovar comply with these terms.
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