Two Novel Bacteriophage Species Against Hybrid Intestinal Pathogenic Escherichia coli/Extraintestinal Pathogenic Escherichia coli Strains

Abstract

Background:

Colibacillosis caused by Escherichia coli is one of the main problems in the swine industry. In addition, the emergence of antimicrobial resistance and the combination of virulence genes among pathotypes have led to the emergence of more virulent pathogenic E. coli strains. Phage therapy has become a promising approach to address these issues.

Materials and Methods:

Virulence genes for intestinal pathogenic E. coli (IPEC) and extraintestinal pathogenic E. coli (ExPEC) were investigated in pathogenic E. coli isolated from pigs. In addition, two potential phages, vB_EcoM-RPN187 and vB_EcoM-RPN226, isolated in our previous study, were further characterized in this study.

Results:

Both phages were lytic and were highly effective at 20–37°C. Interestingly, they infected the hybrid IPEC/ExPEC strains. vB_EcoM-RPN187 and vB_EcoM-RPN226 possess 167 kbp of linear double-stranded DNA without virulence or antibiotic resistance genes and may be classified as new phage species in the genera Mosigvirus and Tequatrovirus, respectively.

Conclusion:

Both phages could be promising candidates for phage therapy against pathogenic E. coli.

Introduction

In the swine industry, production losses have a negative financial impact on both producers and consumers when expenses and supply–demand dynamics are considered. Colibacillosis caused by Escherichia coli is one of the main causes of production loss. This infectious disease is recognized as one of the most serious causes of illness and death in piglets. There are two main categories of E. coli known as intestinal pathogenic E. coli (IPEC) and extraintestinal pathogenic E. coli (ExPEC).

The former category, also called diarrheagenic E. coli (DEC), consists of six well-studied pathotypes, which are enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC). In contrast, ExPEC can be divided into uropathogenic E. coli (UPEC), neonatal meningitis-associated E. coli (NMEC), septicemic E. coli (SePEC), avian pathogenic E. coli (APEC), and endometrial pathogenic E. coli (EnPEC).

These pathotypes are identified based on the presence of virulence genes and their pathotypic traits.^1,2 The combination of virulence genes among pathotypes, and between IPEC and ExPEC, has led to the emergence of more virulent pathogenic E. coli strains. STEC O2:H6 harbored UPEC virulence-associated genes, leading to diarrhea and urinary tract infection. It was later called a STEC/UPEC hybrid pathogen, which caused both intestinal and extraintestinal diseases.³ It has been suggested that hybrid E. coli pathogens should be considered as a public health concern.⁴

Generally, antibiotics are the first option for treating infected pigs. Nevertheless, this treatment is not always successful because various pathogenic strains select for antibiotic resistance. A high number of ETEC (59%) and ETEC/STEC (53%) isolates were resistant to at least six drug categories. In addition, 14 of 15 hybrid ETEC/STEC isolates resisted colistin, a restricted antibiotic, raising serious concerns.⁵

Moreover, hybrid UPEC/EAEC isolates from ambulatory patients showed multidrug resistance profiles. Further clinical research was recommended because of the increased probability of developing pyelonephritis.⁶ Although different classes of antibiotics are available to treat bacterial infectious diseases, the emergence of multidrug-resistant bacteria has become a critical issue that needs to be considered and has led to more difficult treatment.

Phages are known as potential bioagents for bacterial control in a variety of applications such as phage therapy, disinfectants, and food safety.⁷ Strictly lytic phages have generally been preferred for therapeutic purposes as they only possess a lytic life cycle that destroys the infected bacterial cells, whereas temperate phages have been circumvented due to their capacity to facilitate transduction, which mediates gene transfer between bacteria and may increase bacterial virulence. Temperate phages may be an option in some circumstances.⁸ Despite its economic importance, phage-host specificity, and increasing interest in phage therapy, there have only been a few reports of phages associated with swine pathogens in Thailand.^9–11

In our previous work, vB_EcoM-RPN187 and vB_EcoM-RPN226 phages were isolated using multidrug-resistant E. coli M187 and M226, isolated from diarrheal pig intestines, respectively, as hosts.¹² Both phages were combined with other potential phages to form a phage cocktail, which successfully reduced the number of fecal E. coli in artificially E. coli-infected nursery pigs.¹²

In this report, the virulence factors associated with IPEC and ExPEC in both E. coli host strains and other susceptible E. coli strains are presented. Furthermore, other characteristics and the complete genomes of both phages are reported to reveal their capability and safety for use in phage therapy against pathogenic E. coli. The taxonomy of both phages is also classified based on genome annotation.

Materials and Methods

Bacteria and bacteriophages used in this study

Bacteriophages vB_EcoM-RPN187 and vB_EcoM-RPN226 were previously isolated from drained water samples collected from pork stalls.¹² In this study, 29 other bacterial strains were used to determine the phage host range (Supplementary Table S1). All strains were cultured in tryptic soy broth (TSB) for 16–18 h before each experiment and phage enumeration.

Virulence factor investigation

The investigation was conducted using the colony polymerase chain reaction (PCR) technique with 23 primers. These primers are specific to E. coli uspA, faeG, fanA, fasA, fedA, fimF41a, elt, sta, stb,¹³ stx₁, stx₂, eaeA, hlyA,¹⁴ stx2e,¹⁵ csgA,¹⁶ aggR,¹⁷ fimH, kpsMTII, ibeA, fyuA, traT,¹⁸ cdtI-VB,¹⁹ and agn43 genes²⁰ (Supplementary Table S2). A fresh single colony of each E. coli strain (E. coli M158–M245 isolated from diarrheal pigs) was partly picked and suspended in sterilized deionized water.

The colony PCR mixture consisted of 2 μL of the cell suspension, 0.2 pmol of each primer, 12.5 μL of hot-start PCR master mix (Apsalagen, Thailand) and was adjusted to 25 μL using deionized water. The reactions were carried out for 35 cycles as follows: initial denaturation at 94°C for 10 min, denaturation at 94°C for 1 min, annealing at different temperatures (Supplementary Table S2) for 30 s, extension at 72°C for 30 s (60 s for the uspA and fyuA genes), and final extension at 72°C for 10 min. PCR products were visualized by agarose gel electrophoresis.

Host range determination

Phages were further explored for their efficiency against 29 other bacterial strains, including 22 E. coli strains, 1 Klebsiella pneumoniae strain, 5 Salmonella enterica strains, and 1 Staphylococcus aureus strain (Supplementary Table S1). The experiment was performed using the double layer agar (DLA) method. The efficiency of plating (EOP) value was determined to indicate phage capacity for lysing susceptible bacteria.²¹

EOP at different temperatures

The EOP was also investigated at a range of temperatures (10–45°C) following Seeley and Primrose²² with some modifications. The test was conducted using the DLA method and incubated at 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, 42°C, and 45°C for 18–48 h. The EOP value can be calculated by dividing the phage titer at the tested temperature by the highest phage titer at an optimal temperature. The phage efficiency was indicated as previously described.²¹

Thermal and pH stabilities

To determine temperature stability, phage suspensions in SM buffer were incubated at 4°C, 28°C, 37°C, 50°C, 60°C, and 70°C for 1 h. For pH stability, the phage suspension was 100-fold diluted in TSB at different pH levels (2–11) and incubated at 37°C for 1 h. After incubation, the samples were immediately subjected to DLA. The phage titer was calculated to determine phage stability compared with the initial phage titer.

One-step growth curve

Phage one-step growth was performed according to the method described by Imklin and Nasanit.²¹ In brief, phage suspension was mixed with host bacteria in 30 mL of TSB at a multiplicity of infection of 0.001 and incubated for 5 min. Centrifugation was performed to discard free phages. Cell pellets were resuspended in pre-warmed TSB at the same volume. The samples were taken every 5 min for phage enumeration.

The burst size was calculated by dividing the average phage titer at each time point by the average initial phage titer, and growth curves were constructed. It should be noted that the starting point of the graph in this study corresponds to a particular time when the cell pellet was resuspended. In addition, the latent period started from the beginning of the infection, when phages and bacteria were encountered, to the beginning of the burst period.

Complete genome analysis

Phage genomic DNA was extracted using the phenol-chloroform method.²¹ Illumina sequencing by synthesis technology (Macrogen, Korea) was utilized to perform short-read sequencing of 101 bases using a Nextera XT DNA Library Preparation Kit and a TruSeq Nano DNA Kit for vB_EcoM-RPN187 and vB_EcoM-RPN226, respectively.

The raw reads were trimmed and filtered using Trimmomatic (v0.36)²³ to obtain good-quality bases (∼98%) at a phred quality score of 30 for both phage genomes. FastQC (v0.11.5) was used to check the overall quality of the data. The obtained reads were subjected to de novo assembly using SPAdes (v3.13.0)²⁴ to create a single contig representing the phage genome. Filtering, trimming, and assembly were performed by Macrogen (the Republic of Korea).

The nucleotide sequences of both ends of the received contig were verified by the PCR technique before genome annotation. Each PCR product of contig ends was amplified using the designed primers RPN187-F 5′-GGCACAAAGAAGTCCTCGAA-3′ and RPN187-R 5′-GCCAATGATGCCACCAACAAC-3′ for vB_EcoM-RPN187, and RPN226-F 5′-GGAGCGAATAACGGAACAACC-3′ and RPN226-R 5′-CCTCTACGAGTTTCTGTGGCA-3′ for vB_EcoM-RPN226. Open reading frames (ORF) were predicted using Glimmer 3²⁵ through Geneious Prime (v2023.2.1) and GeneMarkS (v3.26)²⁶ with default settings.

Amino acid sequences were translated and extracted from Geneious Prime in the FASTA format. Protein functions were predicted by comparing amino acid sequences using BLASTp against the nonredundant protein sequences (nr) database with an E-value cutoff of 1E-10. NCBI-conserved domains, HHpred, and Uniprot were additionally used in conjunction with the BLASTp results to identify putative protein functions. Only those proteins that met at least 90% identity from the BLASTp results and contained conserved domains were assigned as functional proteins, whereas other proteins were hypothetical proteins.

Aragorn (v1.2.41) was used to find tRNA gene with the default setting.²⁷ The genome sequence was annotated and submitted to the GenBank database through Geneious Prime. Amino acid sequences of the terminase large subunit (TerL) and major capsid protein (MCP) were aligned before constructing phylogenetic trees using MEGA 7 with 1000 bootstrap replicates under the maximum likelihood method with the Jones–Taylor–Thornton model.²⁸ Nearest-Neighbor-Interchange and default NJ/BioNJ methods were combined for tree topology analyses.

To determine the similarity between both vB_EcoM-RPN187 and vB_EcoM-RPN226 phages and their relatives, whole genome sequences were subjected to BLASTn and PAirwise Sequence Comparison (PASC) analysis with default settings.²⁹ Consequently, each closest relative according to the PASC result was genetically compared with each phage DNA sequence using a linear comparison application, Easyfig (v2.2.2).³⁰ The complete genome sequences of Escherichia phages vB_EcoM-RPN187 and vB_EcoM-RPN226 are available from GenBank under the accession numbers OL770074 and OL770073, respectively.

Raw reads of the vB_EcoM-RPN187 and vB_EcoM-RPN226 genomes were submitted to the sequence read archive (SRA) repository following this information: BioProject: PRJNA1055028 and PRJNA1055045, BioSample: SAMN38976064 and SAMN38976473, SRA accession: SRR27315206 and SRR27297327, respectively.

Statistical analysis

The statistical significance of the data in the thermal and pH stability tests was determined by a paired-samples t-test using IBM SPSS Statistics version 23. A value of p < 0.05 was considered to be statistically significant.

Results

Virulence genes in E. coli strains

Of the 23 tested genes, 3–6 virulence genes were detected in the tested E. coli. These included E. coli uspA, stb, stx2, csgA, fimH, kpsMTII, fyuA, traT, and agn43 genes (Supplementary Table S1). Two types of fimbriae-associated genes were found in E. coli M170, but no toxin or other tested virulence genes were identified. In contrast, at least 4 virulence-associated genes were detected in 13 other E. coli strains. These results indicated that most E. coli strains possessed both IPEC and ExPEC virulence genes.

Host range determination

The host range test (Supplementary Table S2) revealed that vB_EcoM-RPN187 was specific to four E. coli strains. vB_EcoM-RPN226 effectively infected nine E. coli strains. Several susceptible E. coli strains carried a combination of both IPEC and ExPEC virulence genes. Notably, the phages could infect pathogenic E. coli, causing diarrhea in pigs and mastitis and metritis in cows.

EOP at different temperatures

The results showed that vB_EcoM-RPN187 and vB_EcoM-RPN226 formed clear plaques at all tested temperatures (Supplementary Fig. S1). vB_EcoM-RPN187 was highly capable of producing plaques against its host at 20–37°C, whereas medium effectiveness was observed at 15°C. However, the phage had low EOP at 10°C, 42°C, and 45°C. The optimal temperature was in the range of 20–37°C. vB_EcoM-RPN226 was highly capable of forming plaques against its host at 30°C, whereas it was moderately effective at 25°C and 37°C. This phage had poor plating efficiency at 10°C, 15°C, 20°C, 42°C, and 45°C.

Thermal and pH stabilities

Both phages survived at a range of temperatures (10–45°C) for 1 h. However, a significant reduction of ∼1–2 log PFU/mL was observed at 60°C and 70°C (supplementary Fig. S2). In addition, they were stable in the pH range 3–10 (Supplementary Fig. S3). A decrease in vB_EcoM-RPN187 titer was observed below pH 4 and above pH 10. vB_EcoM-RPN226 was more stable than vB_EcoM-RPN187 at pH 4 and 10. No plaque-forming units were recovered after exposure to pH 2 and 11 for either phage.

One-step growth curve

The latent and burst periods were ∼20 and 40 min, respectively, for vB_EcoM-RPN187. They were ∼25 and 55 min, respectively, for vB_EcoM-RPN226. The burst sizes of these phages were 19 ± 2 PFU/infected cell (Supplementary Fig. S4).

Complete genome analysis

The genome of vB_EcoM-RPN187 is a linear double-stranded DNA with 167,158 bp and 37.8% GC content. It possesses 265 ORFs and 10 tRNA genes. ORFs were categorized into six groups based on their functions (Fig. 1a). Of the total 265 ORFs, 103 ORFs were predicted to encode functional proteins. Importantly, no virulence, antimicrobial resistance, or toxin genes were detected in the phage genome. The BLAST search information is provided in Supplementary Table S3. Phylogenetic trees demonstrated the genetic relationship between vB_EcoM-RPN187 and virus members in the genus Mosigvirus (Fig. 2).

FIG. 1.

The annotated genome map of vB_EcoM-RPN187 (a) and vB_EcoM-RPN226 (b). The arrows and numbers indicate the transcriptional direction and the nucleotide positions, respectively. All ORFs are highlighted based on predicted protein functions. ORFs, open reading frames.

FIG. 2.

Phylogenetic analysis based on the amino acid sequence alignment of terminase large subunit (a) and major capsid protein (b) between vB_EcoM-RPN187 (black circle) and its closest relative, S143_2 (open circle) and vB_EcoM-RPN226 (black square) and its closest relative, vB_EcoM_SYGD1 (open square), and other phages. Sequences were gathered from the GenBank database. The phylogenetic trees were constructed by MEGA7 using the maximum likelihood method with 1000 bootstrap replications.

The BLASTn results revealed the similarity between vB_EcoM-RPN187 and the most familiar Escherichia phage S143_2 (MZ189261), with 98.2% identity and 96.0% query cover. The PASC results indicated that ∼94% of the entire nucleotide sequence of vB_EcoM-RPN187 vastly matched that of Escherichia phage S143_2 (Table 1). Genome comparison using the BLASTn algorithm illustrated the reciprocal evolutionary relationship between vB_EcoM-RPN187 and S143_2, with nucleotide similarity ranging from 69% to 100% (Fig. 3a).

Table 1.

The Nucleotide Alignment of Whole Genome Sequences of vB_EcoM-RPN187 and Its Closest Relatives Using Pairwise Sequence Comparison

The overall nucleotide sequence identity (%)	Accession number	Bacteriophage	Genome size (kb)	% GC content	No. of protein encoding gene	No. of tRNA
—	OL770074	Escherichia phage vB_EcoM-RPN187	167.16	37.8	275	10
94.07	MZ189261	Escherichia phage S143_2	168.77	37.7	270	10
93.48	MN850579	Escherichia phage mogra	168.72	37.7	266	2
93.01	NC_029091	Escherichia phage APCEc01	168.77	37.7	274	2
92.83	MK047718	Escherichia phage p000y	169.87	37.7	276	10
92.81	NC_024124	Escherichia phage vB_EcoM_JS09	169.15	37.6	273	2
92.51	NC_041863	Escherichia coli O157 typing phage 3	168.73	37.6	272	2
92.34	KT184310	Enterobacteria phage ATK48	169.73	37.6	218	2
92.26	NC_055708	Enterobacteria phage ATK47	170.02	37.6	215	2
92.26	NC_054940	Shigella phage JK45	170.74	37.6	275	2
91.98	NC_055781	Escherichia phage p000v	167.80	37.6	264	1

FIG. 3.

The linear comparison of full-length genomes between vB_EcoM-RPN187 and Escherichia phage S143_2 (MZ189261) (a) and between vB_EcoM-RPN226 and Escherichia phage vB_EcoM_SYGD1 (MW883059) (b). The dark-light gray gradients present nucleotide similarity.

The genome of vB_EcoM-RPN226 is a linear double-stranded DNA of 167,390 bp and 35.6% GC content. It consists of 265 ORFs and 8 tRNA genes (Fig. 1b). Among these, 115 ORFs were predicted to encode functional proteins (Supplementary Table S4). Importantly, no virulence, antimicrobial resistance, or toxin genes were detected in the phage genome. The phylogenetic trees of TerL (Fig. 2a) and MCP (Fig. 2b) amino acid sequences revealed the closest genetic relationship between vB_EcoM-RPN226 and Escherichia phages in the genus Tequatrovirus.

According to the BLASTn results of the whole genome sequence, vB_EcoM-RPN226 was most closely related to Escherichia phage vB_EcoM_SYGD1 (MW883059), with 96.4% identity and 93.0% query coverage. Furthermore, the PASC result proved that the whole nucleotide sequence of vB_EcoM-RPN226 closely matched that of the Escherichia phage vB_EcoM_SYGD1 by about 89.5% (Table 2). With nucleotide similarity ranging from 67% to 100%, genome comparison using the BLASTn algorithm demonstrated the reciprocal evolutionary relationship between vB_EcoM-RPN226 and vB_EcoM_SYGD1 (Fig. 3b).

Table 2.

The Nucleotide Comparison of Complete Genome Sequences of the vB_EcoM-RPN226 Phage and Its Closest Relatives Conducted by Pairwise Sequence Comparison

The overall nucleotide sequence identity (%)	Accession number	Bacteriophage	Genome size (kb)	% GC content	No. of protein encoding gene	No. of tRNA
—	OL770073	Escherichia phage vB_EcoM-RPN226	167.39	35.6	265	8
89.53	MW883059	Escherichia phage vB_EcoM_SYGD1	171.26	35.3	271	8
87.75	NC_054904	Enterobacteria phage vB_EcoM_IME340	165.55	35.5	260	10
87.60	MT682709	Escherichia phage vB_EcoM_SP1	165.42	35.6	269	10
87.33	NC_054937	Salmonella phage pSe_SNUABM_01	172.36	35.4	276	6
87.30	MK327928	Escherichia phage vB_EcoM_G2133	168.96	35.3	273	8
87.26	NC_054925	Escherichia phage vB_EcoM_NBG2	166.08	35.4	261	10
87.25	MW822007	Escherichia phage BF15	167.8	35.4	267	8
87.01	KR269718	Escherichia phage HY03	170.77	35.3	269	7
86.92	NC_054930	Escherichia phage PP01	167.81	35.5	280	8
86.70	NC_054916	Escherichia phage vB_EcoM-G28	170.12	35.3	267	8

Discussion

Our previous study revealed that all E. coli strains resisted 2–6 antibiotics.¹² In addition, the fact that E. coli strains were isolated from lung specimens and intestines of diarrheal pigs suggests that they may have been involved in swine illnesses. In this study, the IPEC and ExPEC virulence genes were detected in the tested E. coli strains. For example, the stb gene encodes heat-stable enterotoxin b, which responds promptly to an infected host and causes moderate porcine diarrhea.³¹ Another toxin gene found in some E. coli isolates was the stx₂ gene, which encodes Shiga toxin 2 (Stx2).

This toxin is more cytotoxic than Stx1, indicating STEC pathogenicity, which causes hemorrhagic colitis.³² The product of the csgA gene is curli fimbriae, a type of adhesin that can be found in E. coli from the ExPEC category, especially UPEC, and is associated with biofilm formation and enhanced pathogenicity.³³ However, STEC, an IPEC pathotype, has been shown to carry this gene.³⁴ The traT gene encodes a complement resistance protein called protectine. ExPEC isolates that produce this protein are serum-resistant, promoting bacterial virulence as they can survive in the bloodstream and cause bacteremia.^35,36

It was suggested that those who possessed virulence genes of at least two DEC pathotypes should be referred to hetero-pathogenic strains. For hybrid pathogens, it was adopted for those that carried both DEC and ExPEC virulence genes or for DEC strains isolated from extraintestinal systems.³⁷ Notably, new threats to public health, both humans and animals, could arise from the emergence of drug-resistant E. coli hybrid strains. Interestingly, regarding the host range results, the phages vB_EcoM-RPN187 and vB_EcoM-RPN226 could effectively infect these pathogenic bacteria with at least three strains each.

vB_EcoM-RPN187 and vB_EcoM-RPN226 demonstrated outstanding ability to kill their hosts at temperatures between 20°C and 37°C. Therefore, they could be classified as members of mid-temperature phages.²² According to the stability results, it is expected that both phages would effectively traverse the gastrointestinal tract of piglets and maintain their efficacy under in vivo conditions through oral administration. Notably, the gastric and intestinal pH of piglets varied in a range of 3.0–4.1 and 5.1–6.5, respectively.³⁸

In addition, piglet body temperatures typically fall between 36.8°C and 39.2°C.³⁹ These inherent characteristics of both phages hold significant value, as they demonstrate a remarkable capacity to combat pathogens under diverse piglet physiological conditions. Furthermore, both phages exhibited rapid host cell lysis, as observed in the one-step growth experiment, which further enhances their potential as effective bactericidal agents.

Several genes in the vB_EcoM-RPN187 and vB_EcoM-RPN226 genomes are associated with phage–host interactions, for example, the discriminator of mRNA degradation protein, which is required to protect phage mRNAs from host RNase LS⁴⁰; RNA ligase A, which is responsible for repairing damaged phage tRNA caused by a host defense system⁴¹; and polynucleotide kinase and RNA ligase 2, both of which involve repairing nicked tRNA caused by a host response mechanism.⁴²

These genes demonstrate that the phages possess the ability to circumvent the defense mechanisms of their hosts. Moreover, the absence of virulence, antimicrobial resistance, and toxin genes was observed in both the phage genomes. These results suggested the suitability of phages for therapeutic purposes.

It was discovered that vB_EcoM-RPN187 and vB_EcoM-RPN226 were genetically closely related to phages in the genera Mosigvirus and Tequatrovirus, respectively. vB_EcoM-RPN187 has a prolate icosahedral head, a long tail with a contractile sheath, and long-tail fibers (supplementary Fig. S5a). These structural characteristics resemble those of Escherichia phage APCEc01, which is classified as a viral species within the genus Mosigvirus.⁴³

Genes encoding the long-tail fiber protein and long-tail fiber proximal subunit were also detected in the vB_EcoM-RPN187 genome. However, the APCEc01 phage has a larger genome than the vB_EcoM-RPN187 phage. This suggests that vB_EcoM-RPN187 may be considered a novel member of the Mosigvirus genus. When compared with its closest genetically related phage, Escherichia phage S143_2, a high percentage of whole genome similarity (94%) was observed.

Nevertheless, notable differences existed between the two phages. The whole genome comparison (Fig. 3a) illustrated a low percentage (light gray) similarity in the last detected structural protein, the tail protein, between vB_EcoM-RPN187 (ULA51974) and S143_2 (QWV60330). The BLASTp summary displayed the alignment of these amino acid sequences with 100% query coverage and 59.8% identity, indicating a distinct tail morphology. However, there is currently no publicly available report regarding the characteristics of Escherichia phage S143_2.

It was found that the vB_EcoM-RPN226 genomic DNA sequence was most closely related to that of vB_EcoM_SYGD1.⁴⁴ The vB_EcoM_SYGD1 genome comprises 171,255 bp with 35.3% GC content and a total of 271 ORFs and 8 tRNAs. Notably, vB_EcoM-RPN226 has a shorter nucleotide sequence and fewer ORFs than vB_EcoM_SYGD1, whereas the GC content percentage does not significantly differ. Linear DNA comparison, as shown in Figure 3b, highlights the genetic distinctions between the two phage genomes.

However, notable differences were observed in the stability characteristics of the two phages. Regarding its physical traits, vB_EcoM-RPN226 has a prolate icosahedral head, a long contractile tail, and long-tail fibers with baseplates (Supplementary Fig. S5b), similar to that of vB_EcoM_SYGD1. Although both phages exhibited substantial similarities in both genetic and morphological information, the presence of characteristic differences suggests that vB_EcoM-RPN226 may be considered a new member of the genus Tequatrovirus.

To classify phage taxonomy, phages whose entire genomes share >95% nucleotide identity are assigned as to the same species. Furthermore, the threshold for assigning phages to different genera is set at 70% nucleotide similarity of the complete genome.⁴⁵ According to the guidelines, phylogenetic trees, BLASTn, and PASC results, vB_EcoM-RPN187 and vB_EcoM-RPN226 may potentially represent new phage species in the genus Mosigvirus and Tequatrovirus, respectively.

Conclusion

In summary, vB_EcoM-RPN187 and vB_EcoM-RPN226 presented their capability of infecting multidrug-resistant hybrid pathogenic E. coli strains at a wide range of temperatures. These phages have shown resilience within the piglet body temperature range and varying pH levels of the digestive tract, thereby increasing their potential to reach bacterial targets in the gut. Furthermore, no undesirable genes were detected in their genomes. These characteristics establish vB_EcoM-RPN187 and vB_EcoM-RPN226 as novel Escherichia phages with promising potential as bactericidal agents for effectively managing the causative bacteria in swine production.

Authors' Contributions

The study conception, design, and supervision were contributed by R.N. Methodology and investigation were performed by N.I., P.S., and N.T. Formal analysis was performed by N.I. and P.S. Writing—original draft and visualization were done by N.I. Funding acquisition and project administration were contributed by P.L. and R.N. All authors read, commented, and approved the final article.

Author Disclosure Statement

No completing financial interests exist.

Funding Information

This study was supported by the Kasetsart University Research and Development Institute, KURDI (Grant No. FF(KU)17.64).