A New Kayfunavirus-like Escherichia Phage vB_EcoP-Ro45lw with Antimicrobial Potential of Shiga Toxin-Producing Escherichia coli O45 Strain

Lytic bacteriophages are re-considered as a solution to resolve antibiotic-resistant rampage. Despite frequent foodborne outbreaks caused by the top six non-O157 Shiga-toxin-producing Escherichia coli (STEC), the current interventions are not sufficiently effective against each serogroup, particularly O45. Therefore, this study aimed to characterize a new short-tailed phage, vB_EcoP-Ro45lw (or Ro45lw), as an alternative antimicrobial agent for STEC O45 strains. Phage Ro45lw belongs to the Kayfunavirus genus within the Autographiviridae family and shares no close evolutionary relationship with any reference phages. Ro45lw contains a tail structure composed of a unique tail fiber and tail tubular proteins A and B, likely to produce enzymatic activity against the target bacterial cells besides structural function. Additionally, the phage genome does not contain virulent, antibiotic-resistant, or lysogenic genes. The phage has a latent period of 15 min with an estimated burst size of 55 PFU/CFU and is stable at a wide range of pH (pH4 to pH11) and temperatures (30 °C to 60 °C). Regardless of the MOIs (MOI = 0.1, 1, and 10) used, Ro45lw has a strong antimicrobial activity against both environmental (E. coli O45:H-) and clinical (E. coli O45:H2) strains at 25 °C. These findings indicate that phage Ro45lw has antimicrobial potential in mitigating pathogenic STEC O45 strains.


Introduction
Shiga-toxin-producing Escherichia coli (STEC) strains, including O157 and the top six non-O157 serogroups, are renowned for producing Shiga toxin, capable of causing severe illnesses, such as hemolytic-uremic syndrome (HUS), and are implicated in many foodborne outbreaks in the United States and around the world [1]. STEC, in general, has contributed to approximately 265,000 illnesses, 3600 hospitalizations, and 30 deaths yearly [2]. Although the O157 serogroup is the primary pathogen contributing to STECassociated outbreaks, the rate of non-O157 STEC infections has increased in recent years [3]. Among non-O157 STEC serogroups, STEC O45 strains have scarcely been implicated in any recent incidences but are still regulated as adulterants for certain types of food [4,5]. Despite the low prevalence of STEC O45 infection, precautions for this pathogen are still needed and can easily be overlooked. E. coli O45:H2 was associated with sporadic cases of bloody diarrhea from previous foodborne outbreaks, deriving from ill food workers and consuming contaminated meat products [4,6,7]. Our previous study also showed Microorganisms 2023, 11, 77 2 of 14 that E. coli O45:H2 of clinical or outbreak origin shared a close evolutionary relationship with the outbreak strain E. coli O103:H2, one of the most prevalent serogroups associated with recent foodborne outbreaks [8]. Most importantly, our genomic results showed that E. coli O45:H2 harbored similar pathogenicity factors as E. coli O103:H2 and could still cause severe illness if food producers or administrators overlook the precautionary interference to prevent human infection. A previous study found the genetic correlation of E. coli O104:H4 strain associated with a 2011 outbreak in Germany with the same strain, E. coli O104:H4 (HUSEC041), isolated 10 years ago in the same area where this particular serogroup of E. coli was not even within the radar [9]. The food industry has established standardized antimicrobial measurements, such as lactic acid and peroxyacetic acid, to prevent contamination of STEC O157 for years. However, these intervention technologies may not be sufficient to control each top six non-O157 STEC serogroups, and the efficacy varies depending on the contamination levels [10]. Therefore, an alternative option of antimicrobial interventions for these pathogens is needed.
Bacteriophages, also known as phages, are bacteria-killing viruses with high diversity and are ubiquitous in the ecosystem [11]. Phages can recognize their receptor proteins on the target bacterial cell walls to initiate phage infection [12]. For a lytic phage, phage DNA will then hijack the genomic machinery of the bacterial host to produce phage progenies, released via lysis of the bacterial cell [13]. Therefore, lytic phages are host-specific and are very helpful in treating bacterial infections, especially those with the development of antibiotic resistance, without affecting the biennial background flora [14]. Many studies have indicated the antimicrobial potential of lytic phages against various STEC strains, particularly O157 serogroup [15,16]. In contrast to chemical-based antimicrobial agents, the phage application has few adverse effects on the quality of food products, such as sprouts [17]. Consumer awareness of phage application in food is low; however, the Generally Recognized As Safe (GRAS) approved phage products shed new light on alternative antimicrobial agents for foodborne pathogens [18]. Thus, this study aimed to characterize Escherichia phage vB_EcoP-Ro45lw (or Ro45lw) isolated from non-fecal compost for the potential antimicrobial activity against STEC O45.

Phage Isolation
Escherichia phage Ro45lw was previously isolated with E. coli O45:H-from a non-fecal compost sample [19]. Phage propagation was performed by mixing 50 µL phage lysate (~10 6 PFU/mL) with 100 µL of overnight E. coli O45:H-culture per plate using a doublelayer plaque assay. After incubation at 37 • C for 20 h, 5 mL SM buffer was poured on top of the plate and then incubated at room temperature overnight to elute the phage. Later, the elution was centrifuged at 8000× g for 5 min, followed by filtration through a 0.22 µm filter membrane prior to downstream analysis.

Bacterial Culture
A bacterial panel consisting of the top 6 non-O157 STEC-the serogroups O26, O45, O103, O111, O121, O145-E. coli O157:H7, generic E. coli, and Salmonella enterica strains was obtained from the Produce Safety and Microbiology (PSM) Research Unit at the U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Western Regional Research Center (WRRC), Albany, CA, USA for the host range tests in this study. E. coli O45:H-was used for phage propagation, quantification, and the one-step growth curve. Fresh bacterial culture was prepared in 10 mL tryptic soy broth (TSB; Becton Dickinson, Sparks, MD, USA) inoculated with 1 µL loopful of individual strain and incubated overnight at 37 • C before use.

Genomic Analysis
Phage Ro45lw was purified through a CsCl gradient and subsequently subjected to DNA extraction, followed by DNA library preparation before sequencing as previously Microorganisms 2023, 11, 77 3 of 14 described [20]. Later, the samples were loaded into a MiSeq Reagent Kit v2 (500-cycle) and sequenced on MiSeq platform (Illumina, San Diego, CA, USA), generating approximately 14 million 2 × 250 bp paired-end reads. The updated assembly and annotation were generated using the previous methods [21] and deposited in National Center for Biotechnology Information (NCBI) database. Briefly, raw sequence reads were subjected to FASTQC and trimming via Trimmomatic with a setting of Q30. De novo assembly was conducted on the resulting quality reads using Unicycler Galaxy v0.4.6.0 (SPAdes v2.5.1), followed by annotation via the Prokka pipeline Galaxy 1.13 [22] with default settings. Subsequently, the annotation was manually curated with Universal Protein Resource (UniProt) database [23] using Geneious (v11.0.3, Biomatters, Auckland, New Zealand). tRNAscan-SE (v2.0) server was used to confirm the predicted tRNAs in the phage genome [24]. Genome map of Ro45lw was performed using the CGview server beta (https://proksee.ca/, accessed on 15 November 2022).
The evolutionary tree of Ro45lw with its close-related reference phages was analyzed using the Virus Classification and Tree Building Online Resource (VICTOR) based on the whole-genome sequence at the amino acid level [25]. Phylogenetic analyses of the amino acid sequences were conducted on the putative tail fiber protein (ORF_44), tail tubular protein B (ORF_37), lysozyme (ORF_17), and putative class II holin (ORF_45) using the methods as previously described [20]. In brief, the amino acid sequences were aligned using the ClustalW (version 1.2.3), and the phylogenetic tree was performed using the MEGA11 program, with the maximum composite likelihood method and 500 bootstrap replications.

Transmission Electron Microscopy (TEM)
Purified phage Ro45lw was CsCl-purified before being subjected to negative staining and examined in a transmitted electron microscope (FEI Tecnai G2), as previously described [20].

One-Step Growth Curve
A one-step growth curve of phage Ro45lw was conducted on E. coli O45:H1-, based on the previous method with subtle modification [20]. In brief, fresh bacterial culture was prepared in 10 mL of TSB at 37 • C for 20 h; the next day, 0.2 mL of the overnight culture was sub-cultured in 19.8 mL of TSB and incubated for 2 h at 37 • C to reach log phase of the bacterial growth. Thereafter, phage lysate of Ro45lw was added to the log-phase bacterial solution (MOI of 0.01) supplemented with CaCl 2 at 10 mM and incubated at 37 • C for 5 min to allow phage adsorption onto the membranes of the bacterial cells. The phage-bacterial mixture was centrifuged at 10,000× g for 5 min at 4 • C to discard supernatant. Later, the bacterial pellet was washed with 2 mL of TSB and then resuspended into 20 mL of fresh TSB. Next, 0.3 mL of resuspended culture was added to 29.7 mL of TSB and incubated at 37 • C throughout the duration of the experiment (35 min). Meanwhile, quantification of phage-infected bacteria was determined at incubation time 0 by mixing 50 µL of the 30 mL phage-bacterial mixture (no filtration) with 100 µL of fresh overnight bacterial culture and 5 mL of molten 50% tryptic soy agar (TSA) and pouring into a Petri dish with 10 mL bottom TSA (also known as double-layer plaque assay). Additionally, 1 mL of the sample was obtained from the 30 mL phage-bacterial mixture every five min and subjected to 0.22-µm membrane filtration. The titers of Ro45lw were determined using the double-layer plaque assay, where plates were incubated at 37 • C overnight. The experiment was conducted in three replications to estimate the latent period and burst size of phage Ro45lw.

pH and Temperature Stability
A range of pH values from pH3 to pH12 was used to test the stability of Ro45lw at 37 • C for 20 h using the method as previously described with minor changes [20]. In brief, 100 µL of phage Ro45lw was added to 5 mL of SM buffer with final pH values of 3, 4, 5, 7, 10, and 12 and incubated at 37 • C for 20 h. Viable phage particles of Ro45lw were determined against E. coli O45:Hvia the double-layer plaque assay.
For the temperature stability test, bulk phage lysate of Ro45lw was prepared by mixing the original phage lysate with SM buffer (pH = 7) at 1:9 (v/v) ratio. Later, an aliquot of 1 mL Ro45lw solution was dispensed in several sterile microcentrifuge tubes and subjected to heat treatment, including 30, 40, 50, 60, and 70 • C, for 20 min, 40 min, and 60 min. The temperatures covered were inclusive of the general conditions encountered in food-related environments. Phage titers were determined using the double-layer plaque assay.

Host Range and Efficiency of Plating (EOP)
The host range test of Ro45lw against non-pathogenic E. coli, E. coli O157:H7, top six non-O157 STEC, and three Salmonella strains was conducted using a spot test assay with three different dilutions of phage lysate (10 −1 to 10 −3 ) as previously described [26]. The bacterial strains showing lysis were further subjected to the efficiency of plating (EOP) assay to determine productive infection of phage Ro45lw from the tested bacterial strains versus the primary host strain for producing the phage progenies [27]. Briefly, fresh bacterial cultures were prepared in TSB at 37 • C overnight and used for quantification of Ro45lw by the double-layer plaque assay with diluted phage lysate using four successive dilutions (10 −3 to 10 −7 ). The plates were incubated at 37 • C for 18 h. The experiment was conducted in three replications. Generally, a high phage-producing efficiency had an EOP of 0.5 or more; a medium-producing efficiency had an EOP above 0.1 but below 0.5; a low-producing efficiency had an EOP between 0.001 and 0.1; inefficient phage production was any value lower than 0.001.

Bacterial Challenge Assay
The bacterial challenge assay was performed to measure the effects of phage Ro45lw with different MOIs on bacterial growth based on bacterial optical density at a wavelength of 600 nm (OD 600 ) as previously described with minor changes [21]. In brief, fresh bacterial culture of E. coli O45:H-(RM10729), E. coli O45:H16 (RM13752), and E. coli O45:H2 (SJ7) was prepared in TSB at 37 • C overnight and further diluted in TSB to 1 × 10 4 CFU/mL. An aliquot of 190 µL of diluted bacterial solution per well was added to a 96-well plate. Later, 10 µL of phage Ro45lw with different titers was added to each sample well to reach MOIs of 0.05, 0.5, and 5; the control group contained only bacterial solution without phage. At 25 • C, readings programmed to OD 600 were measured using a spectrophotometer (Promega, Madison, WI, USA) every 30 min for 17 h. The experiment was conducted in 3 replications.

Statistical Analysis
Experiments subjected to statistical analysis were conducted in three individual repeats. The quantification of phages was calculated as PFU/mL, with logarithmical conversion for statistical analysis. The stress effect of pH (pH4 to pH10) was determined using a one-way analysis of variance (ANOVA) with statistical significance at a 5% level [28].

Genomic Features of Ro45lw
Phage Ro45lw had double-stranded DNA with 39,793 bp genome size and an average GC content of 52.2%. The phage was predicted to have the packaging mechanism belonging to direct terminal repeats, also known as DTR [29], with 190 bp terminal repeat region at both sides of the genome end [19]. Ro45lw also contained 50 open reading frames (ORFs), 6 promoters, and 1 terminator without tRNA ( Figure 1). Ro45lw had been classified in the genus of Kayfunavirus under the Autographiviridae family by the International Committee on Taxonomy of Virus (ICTV). Based on the ICTV taxonomy guideline, a new phage classified in the same genus shares a minimum 70% nucleotide identity of the full genome length of the phages in the particular genera via analysis of genomic tools, such as BLASTn or VIRIDIC [30]. The evolutionary tree of the VICTOR analysis showed that Ro45lw had a unique amino acid sequence in comparison to 65 reference phages belonging to the Kayfunavirus genus ( Figure 2). Among 50 ORFs, 26 were annotated with known functions, including structural proteins, DNA replication, DNA packaging, and cell lysis ( Table 1). The phage also contained regulatory genes, such as phi1.6, phi2.5, phi6.5, and phi17, which were also found in Enterobacteria phage T7.
at both sides of the genome end [19]. Ro45lw also contained 50 open reading frames (ORFs), 6 promoters, and 1 terminator without tRNA ( Figure 1). Ro45lw had been classified in the genus of Kayfunavirus under the Autographiviridae family by the International Committee on Taxonomy of Virus (ICTV). Based on the ICTV taxonomy guideline, a new phage classified in the same genus shares a minimum 70% nucleotide identity of the full genome length of the phages in the particular genera via analysis of genomic tools, such as BLASTn or VIRIDIC [30]. The evolutionary tree of the VICTOR analysis showed that Ro45lw had a unique amino acid sequence in comparison to 65 reference phages belonging to the Kayfunavirus genus ( Figure 2). Among 50 ORFs, 26 were annotated with known functions, including structural proteins, DNA replication, DNA packaging, and cell lysis ( Table 1). The phage also contained regulatory genes, such as phi1.6, phi2.5, phi6.5, and phi17, which were also found in Enterobacteria phage T7.   Table 1). The center of the genome map provides % GC content (black).

Phylogenetic Analysis
Phylogenetic analyses were conducted on the ORFs encoding the proteins associated with host range and bacterial cell lysis. Ro45lw contained a unique putative tail fiber protein sharing no close evolutionary relationship at the amino acid level with the counterfeit of any reference phage ( Figure 3A). Moreover, Ro45lw contained ORF_37 encoding tail tubular protein B, which shared a close evolutionary relationship with Cronobacter phage GW1 ( Figure 3B). The phage also harbored the gene encoding tail tubular protein A, with high amino acid similarity to the counterpart of Cronobacter phage GW1 (data not shown). Tail fiber, tail tubular proteins A and B are parts of the tail structure of a podophage [31]. For lysozyme (cell lysis), Ro45lw shared a high amino acid identity with the counterpart of Escherichia phage IMM-002 ( Figure 3C). On the contrary, the phage contained putative class II holin protein that did not share a close evolutionary relationship with that of the reference phages ( Figure 3D). counterpart of Escherichia phage IMM-002 ( Figure 3C). On the contrary, the phage contained putative class II holin protein that did not share a close evolutionary relationship with that of the reference phages ( Figure 3D).

Phage Morphology
Phage Ro45lw had the morphology containing an icosahedral head of approximately 95 ± 5 nm in diameter and a short tail, showing the Podoviridae morphology ( Figure 4A). The phage also produced plaques with a distinct halo on the plaque assay plate ( Figure 4B).

Phage Morphology
Phage Ro45lw had the morphology containing an icosahedral head of approximately 95 ± 5 nm in diameter and a short tail, showing the Podoviridae morphology ( Figure 4A). The phage also produced plaques with a distinct halo on the plaque assay plate ( Figure  4B).

One-Step Growth Curve and Stability Test
Phage Ro45lw had a 15 min latent period against E. coli O45:H-(RM10729) with an average burst size of 55 progeny virions per infected cell after the incubation at 37 °C for 35 min ( Figure 5).

One-Step Growth Curve and Stability Test
Phage Ro45lw had a 15 min latent period against E. coli O45:H-(RM10729) wi average burst size of 55 progeny virions per infected cell after the incubation at 37 ° 35 min ( Figure 5).  For pH tolerance, the titers of phage Ro45lw dropped approximately 2 log PFU/mL (p < 0.05) at pH4 and 1 log PFU/mL (p < 0.05) at pH5, pH10, and pH11 after 20 h incubation at 37 • C ( Figure 6A). No phage was detected at either pH3 or pH12. For temperature stability, Ro45lw was stable at temperatures ranging from 30 • C to 50 • C for 1 h treatment but completely inactivated at 60 • C and 70 • C after 40 min and 20 min, respectively ( Figure 6B). For pH tolerance, the titers of phage Ro45lw dropped approximately 2 log PFU/mL (p < 0.05) at pH4 and 1 log PFU/mL (p < 0.05) at pH5, pH10, and pH11 after 20 h incubation at 37 °C ( Figure 6A). No phage was detected at either pH3 or pH12. For temperature stability, Ro45lw was stable at temperatures ranging from 30 °C to 50 °C for 1 h treatment but completely inactivated at 60 °C and 70 °C after 40 min and 20 min, respectively ( Figure  6B).

Host Range and Productive Infection
Phage Ro45lw was able to infect various STEC O45 strains of environmental and clinical origins and generic E. coli (ATCC 13706 & DH5α) strains; no Salmonella strains were subjected to the phage infection (Table 2). Except for the generic E. coli with inefficiency, both environmental E. coli O45:H16 strains had a high phage-producing efficiency, and one clinical E. coli O45:H2 possessed a medium phage-producing efficiency ( Table 2). α EOP was conducted for the spot test-positive strains, with a value calculated by the ratio of phage titer on the test bacterium versus the phage titer on the primary host bacterium. High production efficiency is EOP ≥ 0.5, medium production efficiency is 0.5 > EOP ≥ 0.1, low production efficiency is 0.1 > EOP > 0.001, and inefficiency of phage production is EOP ≤ 0.001. * R denotes no lysis in the spot test assay.ˆH was the primary host bacterial strain used for isolation.

Bacterial Challenge Assay of Ro45lw
The in vitro antimicrobial activities of Ro45lw at MOIs of 0.1, 1, and 10 were determined against environmental E. coli O45:H16 (RM13752) and clinical E. coli O45:H2 (SJ7) strains at 25 • C using a spectrophotometer (Figure 7). The results showed that the bacterial growth of both E. coli O45:H16 and E. coli O45:H6 strains were completely inhibited by Ro45lw regardless of MOIs used during the 17.5 h incubation (Figure 7).

Discussion
Phage Ro45lw is a new member of podophages with a short tail, showing genomic classification in the genus of Kayfunavirus within the Autographiviridae family. Ro45lw does not share a close evolutionary relationship at the amino acid level with any of the 65 reference phages used in this study which also aligns with the VICTOR classification analysis, showing no similarity to Ro45lw at the species level. The Ro45lw does not contain tRNA in the phage genome, which could be associated with a narrow host range of the phage [32]. This is related to high codon usage by the phages containing more tRNAs than those with few or none [32].
For podophages, T7-like phages in particular, the tail structure is composed of at least four proteins, including connector protein, tail tubular proteins, and fiber proteins [31]. Previous studies found that tail tubular protein A was associated with phage adherence

Discussion
Phage Ro45lw is a new member of podophages with a short tail, showing genomic classification in the genus of Kayfunavirus within the Autographiviridae family. Ro45lw does not share a close evolutionary relationship at the amino acid level with any of the 65 reference phages used in this study which also aligns with the VICTOR classification analysis, showing no similarity to Ro45lw at the species level. The Ro45lw does not contain tRNA in the phage genome, which could be associated with a narrow host range of the phage [32]. This is related to high codon usage by the phages containing more tRNAs than those with few or none [32].
For podophages, T7-like phages in particular, the tail structure is composed of at least four proteins, including connector protein, tail tubular proteins, and fiber proteins [31]. Previous studies found that tail tubular protein A was associated with phage adherence to its bacterial hosts, with hydrolytic activity targeting polysaccharides on the bacterial capsule [33,34]. However, the enzymatic function of tail tubular protein A, also known as depolymerase enzyme [35], likely contributes to the halo surrounding a plaque caused by phage Ro45lw. The same research team also found the additional enzymatic activity of tail tubular protein B, extracted from Yersinia phage, to inhibit bacterial growth and biofilm formation of Yersinia enterocolitica, besides structural function [36]. Due to the close evolutionary relationship to Cronobacter phage GW1 on both tail tabular protein A and B, phage Ro45lw might be able to infect Cronobacter commonly found in dry food, such as powdered milk [37]. The current results show that Ro45lw has a unique tail fiber protein sequence. The phage also encodes a lysozyme sharing high amino acid similarity to Escherichia phage IMM-002, which infects multiple STEC O6 strains [38]. This protein likely contributes to a strong affinity toward STEC strains because its C-terminal domain is related to recognizing and adsorbing bacterial lipopolysaccharide on the cell membrane [39]. The special tail structure and cell lysis enzyme likely render the antimicrobial activity of Ro45lw against clinical E. coli O45:H2 strains, which were resistant to the infection of another STEC O45-infecting phage, Sa45lw, previously isolated in our lab [20]. Furthermore, Ro45lw can suppress both environmental and clinical STEC O45 with various MOIs in this study. The genome of Ro45lw does not have virulent genes, any lysogenic genes, or any antibiotic-resistance genes that can jeopardize the phage application as an alternative antimicrobial agent.
Regarding growth parameters, Ro45lw has a short latent period of 15 min and a small burst size (55 PFU/CFU) against E. coli O45:H-. Ro45lw encodes a unique class II holin protein, one of the factors affecting the latent period of the phage [40]. Ro45lw contains transcriptional terminators and regulated genes found in T7 phage and may be associated with the phage's lysis time [41]. Despite Ro45lw having a small burst size and short latent period, it may rapidly establish multiple parallel infections on the target bacteria if the bacterial concentration is high [42]. Therefore, the initial titers of the phage may be more critical upon application than the subsequent infection by the phage progenies produced from the first infection to mitigate the target bacteria. The current results showed that Ro45lw is stable at a wide pH range but is more sensitive to thermal treatment than phage Sa45lw, which could sustain up to 65 • C for 1 h [20]. The overall phage stability of Ro45lw is adequate to support further various applications in the food-processing environment.
For antimicrobial activity, although Ro45lw has a narrow host range and a small burst size compared with the phage Sa45lw [20], the phage characterized in this study can infect STEC O45 of both environmental and clinical sources. The finding indicates that Ro45lw can target different receptor proteins on these STEC O45 strains from phage Sa45lw, and both phages may be used for developing a phage cocktail to enhance antimicrobial activity. Further study regarding in vitro bacterial reduction and phage application in a food model is necessary.
To conclude, the genetic evidence indicates that phage Ro45lw is a novel STEC O45infecting phage in the Kayfunavirus genus. The current findings show that phage Ro45lw has the antimicrobial potential to control pathogenic STEC O45 strains. Additionally, the phage-derived depolymerases with enzymatic activity encoded by Ro45lw may also be promising phage-derived antimicrobial agents to prevent bacterial pathogens. Funding: This research was funded by the USDA-ARS CRIS projects 2030-42000-0055-000-D and Henan major public welfare project 222102520041.

Data Availability Statement:
The phage Ro45lw sequence used in this study can be found in Gen-Bank, with the accession number MK301532.