Isolation, characterization, and genomic analysis of the novel T4-like bacteriophage ΦCJ20

Abstract

Pathogenic Escherichia coli infections have been consistently reported annually. The basic characteristics and genome of the newly isolated ΦCJ20 from swine feces was analyzed. To determine basic characteristics, dotting assays and double-layer agar assays were conducted. Bacteriophage particles were analyzed via transmission electron microscopy. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed to determine the sizes of major structural proteins. The complete genome of the phage was analyzed. Bacteriophage particles were identified as Myoviridae, with a head measuring 110.57 ± 1.89 nm and a contractile tail measuring 107.97 ± 3.20 nm and were found to infect E. coli. Major structural proteins of ΦCJ20 showed two well-pronounced bands of approximately 53.6 and 70.9 kDa. The genome size of ΦCJ20 was 169,884 bp, and 118 of 307 open reading frames were annotated. This study provides a baseline for the development of E. coli infection treatment strategies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-021-00906-y.

Introduction

Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly present in various animals (Jang et al., 2017). Most of these bacterial strains are nonpathogenic; however, some strains can cause opportunistic infections (De Souza et al., 2019). Diarrheagenic strains of E. coli can be classified as enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli, (EIEC) according to the serotype (Al-Gallas et al., 2007; Nataro and Kaper, 1998). Infection with these pathogenic E. coli causes mild symptoms such as diarrhea and vomiting, and these symptoms will generally resolve without intervention. However, in severe cases, death can be caused by complications. In 2020, the Centers for Disease Control and Prevention reported that of 167 people infected with E. coli O157:H7 across the United States of America, 85 required hospitalization, and 15 had hemolytic uremic syndrome (HUS) (CDC, 2020). In addition, outbreaks of E. coli infection can occur via food production. Although antibiotics are commonly used to treat pathogenic E. coli infection, some pathogenic E. coli cause complications, such as HUS, during antibiotic administration (Muhlen et al., 2020). Furthermore, antibiotic overuse can cause side effects, such as the emergence of multidrug-resistant bacteria (Sidjabat and Paterson, 2015). Thus, efforts have been made to reduce the use of antibiotics (Duclohier, 2010; Fischer et al., 2013).

Because of these problems, bacteriophages (also known as phages) are being considered as potential antibiotic substitutes (Romero-Calle et al., 2019). Phages are ubiquitous but commonly found in the same habitat as their bacterial host (Kutter and Sulakvelidze, 2004). Phages are safe for use in humans and animals because phages do not infect eukaryotic cells (Necel et al., 2020). Recently, some phages were authorized as generally recognized as safe by the US Food and Drug Administration (Kim et al., 2019). Moreover, phages do not influence food flavor or nutritional value (Yang et al., 2017). Some commercial phage preparations are already used in foodstuffs to control pathogenic bacteria (Perera et al., 2015).

Although phage therapy may have applications as an antibiotic substitute, this approach has limitations (Kakasis and Panitsa, 2019). For example, because of the host specificity of phages, collection of large amounts of different phages is required for the treatment of diverse bacterial infections (Necel et al., 2020). Thus, the narrow host specificity of phages may be a major problem. To overcome this issue, a phage cocktail may be used to target multiple organisms (Fischer et al., 2013). Alternatively, the host range can be expanded or altered through genetic engineering. T4-like phages, which show great virulence, may be safe for numerous phage therapy applications, and the genes associated with receptor specificity have been explored (Chen et al., 2017). Additionally, some enzymes from phages are effective for controlling multiple bacterial strains. For example, endolysin from E. coli can inhibit not only the original host but also other ones (Zhang et al., 2015). Therefore, isolation of new T4-like phages is important for complementing the narrow host range, easily altering the host range by manipulating receptor specificity-related genes, and targeting multiple strains of bacteria.

Accordingly, in this study, the basic and genetic characteristics of ΦCJ20, a T4-like phage newly isolated from swine feces, were investigated to determine the potential for controlling E. coli.

Materials and methods

Bacteriophage and culture conditions

Swine feces from an agricultural corporation in Chungcheong-do, Republic of Korea, was suspended in 50 mL SM buffer [50 mM Tris–HCl, 0.1 M NaCl, and 8 mM MgSO₄·7H₂O (pH 7.5)]. The mixture was centrifuged at 4,000 × g for 10 min, and the supernatant was filtered through a 0.45-μm membrane filter (Advantec, Tokyo, Japan) to prepare a phage suspension. Subsequently, the filtered sample (18 mL) was mixed with 2 mL ETEC FC02 culture (OD₅₄₀ = 0.2) in Luria–Bertani broth (LB; Becton, Dickinson and Company, Sparks, MD, USA) and incubated with vigorous shaking for 18 h at 30 °C. The presence or absence of the phage was tested using dotting assays (Kim et al., 2019). Briefly, 100 μL of a host bacterial culture (OD₅₄₀ = 0.2) was added to 5 mL LB soft agar (0.7% agar), which was poured onto LB agar plates (1.5% agar). Before solidification, 5 μL phage lysate was inoculated onto the plates, followed by incubation for 18 h at 30 °C.

Preparation of a high-titer solution

Production of the phage titer was performed as described by Bandara et al. (2012), with slight modifications. Briefly, the phage solution was inoculated into 100 mL of the host bacterial culture (OD₅₄₀ = 0.2) in LB broth and incubated for 18 h at 30 °C with vigorous shaking. After incubation, the culture was centrifuged at 11,000 × g for 10 min at 4 °C, and the supernatant was filtered through a 0.45-μm membrane filter. Next, 20% (w/v) polyethylene glycol (PEG 8000; Sigma, St. Louis, MO, USA) and 1 M NaCl were added to the filtered phage lysate and then cooled on ice for at least 2 h. The mixture was centrifuged at 110,000 × g for 10 min at 4 °C, and the supernatant was then discarded. Next, the phage pellet was resuspended in SM buffer, and an equal volume of chloroform was added. The mixture was centrifuged at 4,000 × g for 10 min, and the aqueous layer was filtered through a 0.45-μm membrane filter. To determine the phage concentration, a double-layer agar assay was performed. Briefly, 100 μL of serially diluted phage solution and a host bacterial culture (OD₅₄₀ = 0.2) were inoculated into 5 mL LB soft agar, which was poured onto LB agar plates and incubated for 18 h at 30 °C. After incubation, the plaques were counted, and the number of plaque-forming units (PFUs) was calculated.

Transmission electron microscopy (TEM)

The morphology of ΦCJ20 was examined using field-emission TEM (JEM-2100; JEOL, Tokyo, Japan) at Korea Basic Science Institute (Chuncheon, Republic of Korea). A high-titer phage solution (> 10⁸ PFU/mL) was negatively stained with 2% (w/v) uranyl acetate. Micrographs with a magnification between 100,000 × and 150,000 × were taken using a One view Gatan CMOS camera. Particle size was calculated using ImageJ, developed by the NIH (https://imagej.nih.gov/ij/), software.

Determination of the bacteriophage host range and lytic efficacy

To determine the host range of ΦCJ20, several E. coli strains, as well as other Gram-positive and Gram-negative strains, were evaluated using dotting assays. The plates were incubated for 18 h at 30 °C. In addition, assessment of productive infection against each host bacterium was carried out using the double-layer agar technique. The efficiency of plating (EOP) was calculated as the number of phage particles on the target bacterium divided by the number of phage particles on the most infectious bacterium. The EOP value was classified as high production efficiency (0.5 or more), medium production efficiency (0.1 or better but less than 0.5), or low production efficiency (less than 0.1).

pH and temperature stability test

To verify the stability of ΦCJ20 at various acidity levels, the pH of the phage solution was adjusted from 2 to 12, and the solution was incubated for 2 h at room temperature. To measure the stability of the phage at various temperatures, the phage solution was incubated at 4 °C, 20 °C, 37 °C, 50 °C, 65 °C, or 80 °C for 2 h. After incubation, the stability was assessed using a double-layer agar technique with the bacterial host E. coli BL21(DE3).

One-step growth experiment

To assess the one-step growth of ΦCJ20, the phage solution was inoculated into 5 mL E. coli BL21(DE3) culture (OD₅₄₀ = 0.15) in LB broth and incubated for 5 min at 30 °C. The mixture was centrifuged at 4000 × g for 10 min and then replaced with an equal volume of fresh LB broth. The phage concentration was measured every 10 min for 100 min using the double-layer agar technique. Burst size was calculated as the number of phage particles at the initial plateau phase divided by the number of phage particles at the initial maturation phase.

Multiplicity of infection (MOI)

The MOI was defined as the number of phage particles divided by the number of host cells, as described by Abbasifar (2013), with slight modifications. Briefly, E. coli BL21 (DE3) was inoculated into fresh LB broth at a concentration of 1.0 × 10⁸ CFU/mL. Then, the culture was infected with ΦCJ20 at different ratios (MOI: 0.01, 0.1, 1.0, and 10) and incubated at 30 °C. The control group was treated with SM buffer. Every 3 h, the mixture was sampled and optical density (OD) was measured at 540 nm on a microplate reader (Multiskan FC; Thermo, Waltham, MA, USA).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

To display major structural proteins of ΦCJ20, SDS-PAGE was performed. A phage titer was mixed with 5 × sample buffer (Elpis Biotech, Daejeon, Republic of Korea) and heated for 5 min at 95 °C. The separation was carried out from 20 to 40 mV on 8% acrylamide gels. Subsequently, gels were stained with Coomassie brilliant blue G250 (Bio-Rad Laboratories Inc., Hercules, CA, USA) for visualization and destained with deionized water. Band size was predicted using ImageJ software.

Whole-genome sequencing and analysis of bacteriophage nucleic acids

Genomic DNA from ΦCJ20 was extracted with a Phage Isolation Kit (Norgen Bioteck, ON, Canada). Whole-genome sequencing of the phage was performed using next-generation sequencing on a HiSeq 4000 sequencer (Illumina, San Diego, CA, USA). A DNA fragment library was prepared using a TruSeq DNA PCR Free Kit (Illumina). The BLAST program at the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/) was employed for the comparison of sequences. Open reading frames (ORFs) were annotated through SnapGene (http://www.snapgene.com/) and compared using NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) with ATG, GTG, and TTG as the start codon.

A tRNA search was carried out using tRNAscan-SE 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/index.html) (Lowe and Chan, 2016). Some sequences were aligned and analyzed in MEGA X (https://www.megasoftware.net/). The maximum likelihood method was used to construct a phylogenetic tree.

Sequence accession number

The genomic sequence of ΦCJ20 (accession no. MT533174) was assigned from NCBI GenBank.

Statistical analysis

All assays were performed in triplicate. The data were analyzed using IBM SPSS Statistics 25 software (IBM Corp., Armonk, NY, USA). One-way analysis of variance and Duncan’s multiple-range tests were carried out with the characterization data. Results with p values less than 0.01 were considered statistically significant.

Results and discussion

Morphological analysis by TEM

To verify the morphological characteristics of ΦCJ20, micrographs were acquired by TEM (Fig. 1). The phage morphology included an icosahedral head and a contractile tail. Therefore, ΦCJ20 was classified as Myoviridae. The head and tail lengths of the phage were 110.57 ± 1.89 and 107.97 ± 3.20 nm, respectively.

Fig. 1.

Transmission electron micrographs of ΦCJ20 virions negatively stained with 2% (w/v) uranyl acetate. (A) An expanded sheath. (B) A shrunken sheath. Scale bars: 200 nm

Almost all prokaryote viruses composed of double-stranded DNA belong to the families Myoviridae, Siphoviridae, and Podoviridae, which are classified based on their shape (Ackermann, 2007). The ratio of Myoviridae is approximately 25.1% in the families mentioned above (Ackermann, 2001). The shape of ΦCJ20 illustrates Myoviridae, with a contractile tail covered by a contractile sheath. Tailed phages are typically reported to have a head of 30–160 nm and tail length of 10–800 nm (Ackermann, 2001), and the length of ΦCJ20 was within this range (Fig. 1).

Host range and lytic efficacy

Next, we performed host range experiments to determine the adsorption spectrum of ΦCJ20 for various bacterial strains. 11 E. coli strains and nine other Gram-positive and Gram-negative bacterial strains were examined using dotting assays. The lytic ability of the phage differed across the tested bacterial strains. The phage formed clear plaques against six E. coli strains, namely, FC01, FC02, FC04, DH5α, BL21(DE3) and ATCC 25922. Interestingly, ΦCJ20 formed clear plaques against Shigella flexneri ATCC 12022. In contrast, other Gram-negative species, including Cronobacter sakazakii, and Gram-positive species, including Lactobacillus spp., Bacillus spp., and Staphylococcus aureus were not infected by the phage (no lysis; Table 1). The EOP analysis showed another result. Although high production efficiency was observed for E. coli DH5α and BL21(DE3), low production efficiency was detected for the other three ETEC strains, i.e., FC01, FC02, and FC04. Remarkably, high production efficiency was observed for S. flexneri.

Table 1.

The host range of ΦCJ20 and EOPs against all the bacterial strains used in this study

Bacterial species	Culture collection number	Lysis(ΦCJ20)a	EOPsb	Sourcec
Escherichia coli	FC01	+	< 0.001	1
	FC02	+	< 0.001	1
	FC03	−		1
	FC04	+	< 0.001	1
	FC05	−		1
	FC06	−		1
	FC07	−		1
	DH5α	+	0.81	2
	BL21(DE3)	+	1.00	2
	ATCC 35150	−		3
	ATCC 25922	+	< 0.001	3
Cronobacter sakazakii	ATCC 29544	−		3
Shigella flexneri	ATCC 12022	+	0.98	3
Staphylococcus aureus	ATCC 13565	−		3
Bacillus cereus	ATCC 14579	−		3
Bacillus atrophaeus	ATCC 9372	−		3
Lactobacillus pentosus	ATCC 8041	−		3
Lactobacillus plantarum	ATCC 14917	−		3
Lactobacillus fermentum	ATCC 9338	−		3
Lactobacillus brevis	ATCC 13648	−		3

^aRelative lytic activity was determined by measuring the number of plaques. + , lysis; −, no lysis

^bEOP values are shown as the mean ± standard deviations from three independent experiments

^c1: this study; 2: strains purchased from the Enzynomics; 3: strains purchased from American Type Culture Collection

The host range of newly isolated ΦCJ20 infected a few E. coli strains and S. flexneri ATCC 12022. S. flexneri causes shigellosis, symptomatic of severe diarrhea, and closely related E. coli (Goodridge, 2013). Therefore, phages of these two species would be similar in physical, chemical, and genetic properties. In fact, phages that infect both E. coli and Shigella spp. were reported (Chang and Kim, 2011; Goodridge et al., 2003).

According to Mirzaei and Nilsson (2015), dotting assays are frequently employed to identify phages appropriate for bacterial infection. Nevertheless, dotting assays can exaggerate the virulence and host range (Mirzaei and Nilsson, 2015). For this reason, the host range was comparatively analyzed using dotting assays and EOP assays. Consequently, ΦCJ20 infected E. coli FC01, FC02, FC04, DH5α, BL21 (DE3), ATCC 25922, and S. flexneri ATCC 12022 as determined using EOP assays. High production efficiency was observed for DH5α, BL21 (DE3), and S. flexneri. Interestingly, compared to BL21 (DE3), which has the highest infectivity, the phage infected the second highest for S. flexneri (EOP = 0.98). By contrast, ΦCJ20 was classified as showing low production efficiency (< 0.1) against FC01, FC02, FC04, and ATCC 25922. Although differences between the two methods may be related to bacteriocins in the phage lysate and the presence of prophage harboring genes coding for the phage resistance system in the bacterial genome, further studies are needed to assess these mechanisms.

Stability and kinetics of ΦCJ20

To determine the stability of ΦCJ20, the phage was exposed to various pH and temperature conditions (Fig. 2A, B). The number of phages was maintained between pH 5 to 10, and a slight decrease was observed at pH 3–4. Moreover, no plaques were observed at pH 2, 10, or 11. As shown in Fig. 2B, the number of plaques decreased at 37 °C and 50 °C in comparison with that at 4 °C and 20 °C, and the phage was not observed at 65 °C or 80 °C.

Fig. 2.

Stability and kinetics of ΦCJ20. To counting of phage particles, double-layer agar assay was performed at 30 °C after treatment in each condition. (A) pH stability tests were performed under various conditions (pH 2–12) in LB broth at 30 °C. (B) Temperature tests were performed under various conditions (4 °C, 20 °C, 37 °C, 50 °C, 65 °C, and 80 °C) in LB broth at 30 °C. (C) One-step growth was measured every 10 min for 100 min. L, latent phase; M, maturation phase; P, plateau phase; B, burst size. (D) Inhibition of E. coli BL21 in LB broth at 30 °C for 24 h depending on the MOI of ΦCJ20. (○) Control, (●) treatment with ΦCJ20 at an MOI of 0.01, (□) treatment with ΦCJ20 at an MOI of 0.1, (■) treatment with ΦCJ20 at an MOI of 1.0, (△) treatment with ΦCJ20 at an MOI of 10

One-step growth experiments were carried out for 100 min (Fig. 2C). The curve indicated that the latent phase was 40 min after host infection and the maturation phase was between 40 and 80 min. The burst size of the phage was 7.5 particles/infected cell.

During the 24-h incubation time, there were no differences in the growth of E. coli BL21 treated with ΦCJ20 at MOIs of 0.01 and 0.1 (PFU/CFU) compared with that in the control group (Fig. 2D). Although host growth slowed at an MOI of 1.0, there were no differences compared with the control group after 12 h. At an MOI of 10, complete growth inhibition by the phage was observed.

Figure 2 shows the basic characteristics of ΦCJ20. As reported previously, the stability of the T4-like phage Bp7 (GenBank accession no. HQ829472) was determined at various pH values and temperatures (Zhang et al., 2013). ΦBp7 was stable between pH 6 and 9 and at temperatures less than 50 °C. Thus, our results indicated that ΦCJ20 was stable at a wider range of pH values and at a similar range of temperatures (Fig. 2A, B). The latent phase of T4-like phage vB_EcoM_VR20 was reported at 42 min, and the burst size was approximately 37 PFU/infected cell at 24 °C (Kaliniene et al., 2010). The one-step growth curve of ΦCJ20 resembled ΦvB_EcoM_VR20, except for burst size (Fig. 2C). Because ΦvB_EcoM_VR20 was reported as a low-temperature T4-like phage, for which the optimum temperature for plating was around 24 °C, the burst size of ΦCJ20 was calculated to be lower.

Proteomic analysis of phage structural proteins

The sizes of the major structural proteins of ΦCJ20 were determined by SDS-PAGE. More than five protein bands were found to range in size from 48 to 180 kDa (Fig. 3). In particular, the well-pronounced protein bands of ΦCJ20 were identified at approximately 53.6 and 70.9 kDa. A dominant protein band around 53.6 kDa was observed in the polyacrylamide gel (Fig. 3). Notably, the proteomic patterns of ΦCJ20 and ΦvB_EcoM_VR20 showed similarities on the gel (Kaliniene et al., 2010). The band corresponded to CJ20_155 of ΦCJ20 and VR20_186 of ΦvB_EcoM_VR20, and these two ORFs were identified as the T4 major capsid protein (gp23), with an approximate molecular weight of 55.6 kDa.

Fig. 3.

Structural proteins of ΦCJ20 separated by SDS-PAGE. Lane M, molecular mass marker; 1, ΦCJ20 structural proteins. Proteins were visualized using 8% (w/v) gels. Arrows indicate abundant proteins. All experiments were performed in triplicate

Genomic analysis

The genome of ΦCJ20 was 169,884 bp long and contained 307 protein-coding genes and two tRNA genes, with a G + C content of 40.4%. The functions of 118 genes were identified, including 10 transcription-related genes; six translation-related genes; 15 nucleotide metabolism-related genes; 24 DNA replication-, recombination-, repair-, packaging-, and processing-related genes; nine lysis-related genes, one host or phage interaction-related gene, 45 virion proteins, four chaperonins/assembly catalysts, and four host alteration/shutoff-related genes, as predicted using the NCBI SmartBLAST database (Fig. 4 and Table S1). A phylogenetic tree was constructed using the endolysin murine hydrolase gene sequences of the 17 species (Fig. 5). The 14 species included in the tree were phages of E. coli, and Lactobacillus phage Lb, Bacteroides phage crAss001, and Cronobacter phage vB_Csam_GAP31 served as the outgroups. The endolysin gene sequence of ΦCJ20 shared 78% similarity with that of ΦDK-13.

Fig. 4.

Map of protein-coding genes of ΦCJ20. Each arrow indicates a coding sequence (CDS), with different colors representing different functions: red, transcription; brown, translation; orange, nucleotide metabolism; yellow, DNA replication, recombination, repair, packaging, and processing; green, lysis; purple, host or phage interactions; dark blue, virion proteins (head); medium blue, virion proteins (neck); light blue, virion proteins (tail); pale blue, virion proteins (tail fiber); aqua, chaperonins/assembly catalysts; pink, host alteration/shutoff

Fig. 5.

Phylogenetic tree of lysozyme murine hydrolase gene sequences, as analyzed by the maximum likelihood method with 1000 bootstrap replicates. The arrow points to the position of ΦCJ20 in the tree

The whole-genome sequence of ΦCJ20 was similar to that of T4-like ΦvB_EcoM_VR20 (GenBank accession no. KP007360), with coverage of 94% and identity of 96.21% according to NCBI BLASTn. Except for predicted hypothetical proteins, 109 of all 118 functional predicted ORFs matched the following Enterobacteria phages: ΦvB_EcoM_VR20, ΦSP18 (GenBank accession no. GQ981382), ΦvB_EcoM_VR7 (GenBank accession no. HM563683), ΦvB_EcoM_VR25 (GenBank accession no. KP007361), and ΦvB_EcoM_VR26 (GenBank accession no. KP007362). Among these phages, ΦvB_EcoM_VR20 matched the largest number of 41 ORFs (Table S1). Interestingly, unlike the other four phages, ΦSP18 was found to infect Shigella sonnei (Kim et al., 2010). In addition, Lee et al. (2016) showed that ΦHY01 (GenBank accession no. KF925357) infected both E. coli O157:H7 and S. flexneri, and this ability may have been derived from the two different host specificity-associated tail genes in the genome (Lee et al., 2016). Indeed, tail- and tail fiber-associated genes in ΦCJ20 matched those of both ΦvB_EcoM_VR20 and ΦSP18 (Table S1). Furthermore, the predicted tail virion-associated proteins CJ20_136 and CJ20_164 were found to be related to the ORFs of ΦHY01 (data not shown). CJ20_136 corresponded to the gp27 baseplate hub subunit, whereas CJ20_164 was identified as a tail tube protein; these ORFs showed coverages of 98% and 100%, respectively, and identities of 62.76% and 71.17%, respectively. These findings suggested that ΦCJ20 may infect Shigella spp., as well as E. coli. As shown in Fig. 5, the endolysin gene sequence of ΦCJ20 (CJ20_230) showed 78% similarity with ΦDK-13 (GenBank accession no. MT611523). Endolysins can be applied in food processing systems as antibacterial agents for food preservation and enhanced food safety (Chang, 2020). The enzyme could be classified into five different classes, i.e., N-acetylmuramidases (lysozymes), N-acetylglucosaminidases, transglycosylases, endopeptidases, and N-acetylmuramoyl-l-alanine amidases (Oliveira et al., 2013). Among endolysins, muramidase is the rarest type (Chang, 2020; Miroshnikov et al., 2006). In a previous study, the endolysin from the T4-like phage Bp7 was shown to have antibacterial activity against not only E. coli but also Micrococcus lysodeikticus (Zhang et al., 2015). The endolysin of ΦCJ20 was shown to be similar to the lysozyme murein hydrolase of ΦBp7, with a coverage of 100% and an identity of 77.91% (data not shown). Hence, the endolysin of ΦCJ20 could have potential to antibacterial activity against the two bacterial strains mentioned above.

In summary, in this study, we investigated the basic characteristics and genome sequence of a newly isolated phage specific to E. coli and S. flexneri. For efficient and practical use in the food industry, further studies are needed to ascertain the antibacterial activity of the endolysin through genetic engineering and to determine the lytic ability of the phage against various pathogenic E. coli and Shigella species. Such follow-up studies on ΦCJ20 may facilitate the development of alternative therapies for antibiotic-resistant pathogens in the food industry.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.