Isolation and Characterization of the New Lytic Bacteriophage KEC4 Against Escherichia coli MDR Strains

TL;DR

This paper describes a new bacteriophage, KEC4, that can kill drug-resistant Escherichia coli and reduce the minimum inhibitory concentrations of certain antibiotics.

Contribution

The paper introduces a novel lytic bacteriophage, KEC4, isolated from a Russian river, with potential for treating multidrug-resistant E. coli infections.

Findings

KEC4 can lyse 14 out of 31 E. coli clinical isolates with multiple resistance patterns.

The presence of KEC4 reduced the MICs of several antibiotics by up to 16-fold in some isolates.

KEC4 forms round plaques and has a genome of 145,125 bp with no lysogeny-associated proteins.

Abstract

Being first applied for the treatment of infectious diseases of the gut at the start of the 20th century, bacteriophages are again now considered as alternative antimicrobial tools for targeting antibiotic-resistant enterobacteria. Here, we report the new bacteriophage Escherichia phage KEC4 isolated from the Kukshum River (Chuvash Republic, Russia), lysing Escherichia coli and belonging to the Septuagintavirus genus. The genome consists of 145,125 bp with a GC content of 41.3% and contains 6 tRNA and 303 protein-coding sequences. Among them, only 72 encode proteins with known functions, while no proteins potentially associated with lysogeny can be identified. The bacteriophage forms round and pure plaques 0.3–1 mm in diameter and is capable of lysing 14 of 31 E. coli clinical isolates with multiple resistance patterns. Furthermore, in the presence of KEC4, the MICs of meropenem and kanamycin decreased 16-fold in the reference strain. In clinical multidrug-resistant isolates, a 16-fold decrease in the MIC was observed for aminoglycosides (amikacin and gentamicin) for the isolate NKC1, and an eight-fold drop in the MIC of ceftriaxone was observed for isolate 167, with no increase in the efficiency of aminoglycosides. Finally, a four-fold increase in the efficiency of both azithromycin and gentamicin was detected in isolate 5767. Taken together, these data characterize the new Escherichia phage KEC4 as a promising tool for the treatment of infections associated with Escherichia coli, while a preliminary assessment of both the isolate specificity of the phage and an antimicrobial susceptibility test would be required for successful elimination of the pathogen.

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1. Introduction

The family Enterobacteriaceae represents a large and heterogeneous group of Gram-negative, non-spore-forming, rod-shaped bacteria [1]. Members of this group inhabit diverse ecosystems, including the normal intestinal microbiota of humans and animals, soil, water, and food products [2]. The broad distribution of enterobacteria across ecological niches is associated with their exceptional adaptability: they can survive under a wide range of pH values (from 3.8 to 9) and temperatures (from 10 °C to 45 °C) [3]. Particularly dangerous among them are the causative agents of systemic diseases with chronic consequences, such as hemolytic–uremic syndrome (Escherichia coli O157:H7) and typhoid fever (Salmonella spp.) [4]. According to the World Health Organization (WHO), 550–600 million people worldwide annually develop infectious diarrhea after consuming contaminated food [5], where Salmonella enterica and E. coli are the major foodborne bacterial pathogens [6].

E. coli is the most extensively studied bacterium of the Enterobacteriaceae family [7]. According to the literature, nosocomial E. coli strains are constituents of the “healthy” intestinal microbiome in approximately 90% of the global population [8]. Moreover, E. coli strains are among the first microorganisms to colonize the gastrointestinal tract of newborns [9]. However, certain serotypes of E. coli can cause human infectious diseases accompanied by intoxication, fever, and gastrointestinal lesions (various forms of diarrhea, hemorrhagic colitis, hemolytic–uremic syndrome, etc.) and less frequently act as causative agents of urinary and biliary tract infections. Currently, E. coli is also considered as a potential contributor to inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis—immune-mediated, progressive disorders with a complex pathogenesis and unclear etiology [10]. The key role of E. coli in IBD development is supported by the elevated presence of virulent strains in clinical samples from affected patients [11]. Furthermore, E. coli is associated with up to 50% of urinary tract infections (UTIs), accounting for 404.6 million cases in both males and females each year and 236,786 fatalities [12]. Thus, a detailed investigation of the biological properties, pathogenicity mechanisms, and resistance profiles of enterobacteria such as E. coli is required.

Exploring bacterial resistance to antibiotics remains a critical aspect in developing effective strategies for the prevention and treatment of various infectious diseases associated with enterobacteria. Bacteria possess diverse tools to resist antimicrobials [13], and understanding their molecular mechanisms may facilitate the development of alternative therapeutic approaches, such as modifying the structure of existing antibiotics or implementing combined antibiotic therapy. However, the number of antibiotic-resistant strains rises faster than new effective and safe antibiotics are developed, thus challenging the creation of alternative biopharmaceutical agents. Among them is the use of bacteriophages, bacterial viruses recognized as the most abundant biological entities on Earth [14]. Thus, phages comprise up to 97.9% of the total viral population of the gastrointestinal tract [15,16]. Upon infection, a bacteriophage attaches to the bacterial cell wall using specialized capsid proteins and injects its genetic material into the bacterial cytoplasm [17]. After entering the cell, the bacteriophage may initiate either a lysogenic or a lytic cycle [18]. In the lysogenic cycle, the phage integrates into the bacterial genome and replicates passively with the host as a prophage. In the lytic cycle, the bacteriophage produces lytic enzymes that degrade the bacterial cell wall, releasing newly formed viral particles [19].

Compared to conventional antibiotics, phage therapy has several important advantages [20,21,22]. Phages are generally highly specific to their host bacteria and do not lyse other microbial species, thus retaining the microbiota of the host [23]. Secondly, phages are capable of targeting multidrug-resistant strains in both humans and animals, as well as have been successfully used to treat biofilm-associated infections [24,25]. Moreover, a combination of two types of antibacterial agents, such as antibiotics and bacteriophages, demonstrates greater efficacy than the use of either agent individually [26]. The combined use of these drugs has its advantages: enhanced antimicrobial activity of the drugs, increased bacterial cell permeability to antibiotics, and prevention of the development of bacterial resistance to bacteriophages and/or antibiotics [27]. To achieve the best results in the treatment of bacterial infections, it is important to follow the correct sequence and take into account the timing of administration of the bacteriophage and antibiotic [28]. In vitro studies have shown that maximum efficiency in killing bacterial planktonic cells and cells in biofilms is observed when bacteriophages are added before antibiotics [29]. At the same time, the simultaneous use of these two agents leads to a minimal reduction in the number of viable bacterial cells in biofilms [30]. And the preliminary addition of a bacteriophage allows us to reduce the concentration of antibiotics required to suppress bacterial cells [31].

Nevertheless, a number of challenges limit the spread of phage application. Among them, the development of bacterial resistance to phages, frequent specificity to only part of the clinical isolate of the host species, phage-mediated transduction of bacterial DNA, interactions with the immune system, regulatory issues and some other issues limit the use of phage therapy [32]. On the other hand, the screening of new phages is required for a deeper understanding of the phage–host interactions and the development of phage cocktails lysing a wide range of bacterial strains and not provoking a fast development of bacterial tolerance to the viral infection.

In this study we characterize a novel bacteriophage, KEC4, isolated from water samples from the Kukshum River (Chuvash Republic, Russia). Its lytic potential, host specificity, and potential for combined use with antibiotics were discovered.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

Escherichia coli ATCC 25922 was used here as the host strain. Clinical isolates of E. coli, 5496, 5785, 3399, 5422, 5428, 3745, 5924, 2263, 3588, 5441, 5463, NKC1, 167, 397, 1691, 6066, 5767, 5853, 6132, 5402, 308, 2086, 343, 5447, 928, NKC17, 2084, 382, 100, 214, 165, and 345, provided by the Kazan Research Institute of Epidemiology and Microbiology, were used for the strain-specificity testing. Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae subsp. pneumoniae ATCC 13883 bacteria were maintained and grown on LB medium (Lysogeny Broth) (%: tryptone 1.0; yeast extract 0.5; NaCl 0.5; pH 8.5; the 3×LB contained all compounds in a three-fold-higher amount), and an antibiotic susceptibility test was performed on full Muller–Hinton broth. The solid media contained an additional 1.5% agar, while the semi-liquid media contained 0.8% agar.

2.2. Isolation of Bacteriophage

The isolation of bacteriophages was performed as described in [33]. Briefly, in the first stage, 100 mL of the water sample from the Kukshum River (Chuvash Republic, Russia, 56.105419, 47.439386) was passed through a paper filter to remove sediments and debris. The resulting filtrate was then additionally filtered through a nitrocellulose membrane with a pore size of 0.45 μm. In the last step, the sample was filtered through a syringe filter with a pore size of 0.22 μm. The final sterile filtrate was mixed with three-fold concentrated LB broth at a ratio of 2:1 and an equal volume of an overnight culture of E. coli ATCC 25922 in LB broth, thus obtaining a final ratio of components in the suspension of 2:1:1. The obtained suspension was incubated for 24 h at 30 °C with shaking. Then, bacterial cells were removed by centrifugation at 4000 rpm and 4 °C for 30 min. The supernatant was harvested and treated with chloroform (3% of the volume) for 24 h at 4 °C. Subsequently, the samples were centrifuged at 4000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm syringe filter and tested against the target bacterial strains.

2.3. Plaque Formation Assay

The bacterial lysis was investigated by using a double-layer approach. For that, 15 mL of LB agar was poured into Petri dishes to form the first layer. Next, 3 mL of 0.8% LB agar premixed with 30 μL of bacterial culture (10^7^ CFU/mL) was loaded onto the surface of the first layer. Then, 20 μL of the phage lysate was dropped onto the surface, and the Petri dish was tilted to form a line according to the “dripping drop” approach. After absorption of the liquid, the dish was incubated at 37 °C for 24 h.

To assess the morphology of the plaques, the phage suspension was diluted until a titer, at which individual plaques were visible and spread across the entire Petri dish using the Gratia assay, with modifications. The dish was incubated for 24 h at 37 °C, after which plaque morphology was evaluated.

2.4. PFU Count

PFUs were counted by preparing serial ten-fold dilutions of the bacteriophage suspensions from 10^−1^ to 10^−12^ in sterile 0.9% NaCl. For that, 10 μL of the phage lysate was added to 90 μL of 0.9% NaCl, followed by sequential ten-fold dilutions. An amount of 5 μL of each dilution was dropped onto the agar surface with absorbed bacterial culture according to the Otto method [30]. After drying, the Petri dishes were incubated at 37 °C for 24 h. PFUs were counted as follows, and median values were defined from all replicates:

$$[eqn]$$

where N is the phage titer, y is the number of negative colonies, v is the volume of the phage filtrate used, and x is the dilution factor of the phage suspension.

2.5. Assessment of Bacteriophage Specificity Using a Five-Point Scale (Cross-Rating System)

To assess the lytic activity and strain specificity of bacteriophage, its ability to lyse different clinical isolates was investigated by the Otto method [34]. The sensitivity of clinical isolates to the bacteriophage was scored according to the “cross” scale—four crosses (“++++”) or three crosses (“+++”) indicated high lytic activity, two crosses (“++”) moderate activity, and one cross (“+”) low activity.

2.6. Phage Stability Under Different Conditions

To determine thermal stability, a phage suspension was prepared in SM buffer (50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO_4_, pH 7.5) at a final concentration of 1.0 × 10^6^ PFUs/mL and incubated at −20, 4, 25, 37, 50, 60, and 70 °C for 1 h. To assess pH stability, the phage suspension was added to the SM buffer and adjusted with NaOH or HCl to pH values of 3, 4, 5, 7, 9, 10 and 11 until a final titer of 1.0 × 10^6^ PFUs/mL and incubated at 25 °C for 18 h. A phage titer was then determined in all resulting solutions using the double-layer agar (LB top agar) method. All experiments were performed in three biological replicates.

2.7. Determination of the Minimum Inhibitory Concentration

To assess the minimum inhibitory concentration of the antimicrobials, either a disk diffusion test or two-fold serial dilutions in Muller–Hinton broth in 96-well plates (SPL Life Sciences, Pocheon-si, Republic of Korea) was performed according to the EUCAST rules for antimicrobial susceptibility testing [35]. The concentrations of antibiotics were as follows: amikacin 0.03125–32 µg/mL, gentamicin 0.00097–1 µg/mL, meropenem 0.00048–0.5 µg/mL, and kanamycin 0.0156–16 µg/mL. After dilution of the antibiotics, all wells were inoculated with bacterial culture to a final density of 10^7^ CFU/mL. Plates were incubated for 24 h at 37 °C. Experiments were performed in biological triplicates.

For bacterial growth visualization, resazurin sodium salt was added to the wells to a final concentration of 120 μM, followed by visual assessment (Alamar blue test). Those wells where no reduction of blue, non-fluorescent resazurin to pink, fluorescent resorufin could be observed were considered as containing no bacterial growth. The minimum inhibitory concentration of the antimicrobial was defined as its lowest concentration in a well with no detected bacterial growth after 24 h of incubation, and the median value of the MIC obtained in all replicates was calculated.

The susceptibility of clinical isolates to antimicrobials was assessed by using the disk diffusion test, as recommended by EUCAST [31]. Briefly, disks of antimicrobials (azlocillin (75 µg/disc), aztreonam (30 µg/disc), amikacin (30 µg/disc), gentamicin (10 µg/disc), levofloxacin (5 µg/disc), piperacillin (100 µg/disk), tobramycin (10 µg/disk), ceftazidime (10 µg/disc), and ciprofloxacin (5 µg/disc)) (NICP—Research Center for Pharmacotherapy, St. Petersburg, Russia) were placed onto LB agar inoculated with an E. coli suspension (with a density of 10^7^ CFUs/mL), and the plates were incubated for 24 h. The diameter of the growth inhibition zone was measured across the area in which growth was prominently reduced, and the results were interpreted according to available breakpoints as sensitive (S), resistant (R), or sensitive at increased exposure (I).

2.8. Assessing Synergy Between the KEC4 Phage and Antibiotics

The synergy between the KEC4 phage and antibiotics (meropenem, amikacin, gentamicin, kanamycin, ciprofloxacin, azithromycin and ceftriaxone) was assessed in a method similar to MIC testing in 96-well plates with modifications. A series of two-fold microdilutions of the antibiotics (selected for each isolate depending on the susceptibility pattern of the latter) at concentrations from 0.03125 to 64 μg/mL were prepared in a plate with a final volume of 100 μL. Then, the E. coli cell suspension (10^7^ CFUs/mL) was premixed in a ratio of 1:1 in LB broth with the phage (final titer of 10^7^ PFUs/mL). Finally, 100 μL of the obtained suspension was added to the antibiotic solution, thus decreasing the antibiotic’s concentration two-fold and obtaining a range of 0.015625–32 μg/mL. Each test was performed three times and contained growth control of bacteria without the addition of a phage or any antimicrobial agents. The plates were incubated for 24 h at 37 °C without shaking, and the bacterial viability was assessed with the Alamar blue test. The ratio of the antimicrobial’s MIC in the absence or presence of KEC4 was calculated as the MIC fold change. The synergy was considered significant if the MIC fold change was four or higher.

2.9. Isolation of Viral DNA

The DNA of the bacteriophage was isolated using phenol–chloroform extraction with preliminary enzymatic treatment of phage lysates with RNase A, DNase I, and proteinase K (Biolabmix, Novosibirsk, Russia) in accordance with the manufacturer’s instructions. To 1 mL of the phage lysate, 500 μL of a phenol–chloroform mixture was added at a ratio of 1:1. The samples were vigorously shaken for 3 min and then incubated at 70 °C for 30 min, periodically inverting the tubes every 5 min. The tubes were centrifuged at 12,000 rpm for 10 min, and the upper phase was transferred to a new tube. An equal volume of isopropanol was added, and the mixture was vigorously shaken for 10–15 min and centrifuged at 12,000 rpm for 10 min. The supernatant was discarded, and the resulting pellet was washed with 200 μL of 70% ethanol followed by centrifugation at 10,000 rpm for 2 min. This step was repeated three times. After washing, the pellet was dissolved in 60 μL of sterile water.

2.10. Whole-Genome Sequencing and Bioinformatic Analysis

Shotgun sequencing of the phage genome was performed with paired-end 150 bp reads on the Illumina HiSeq platform (San Diego, CA, USA). Read quality after sequencing was assessed using FastQC [36], and genome assembly was performed using SPAdes 3.15.5. Genome annotation was performed using Pharokka v1.3.0 [37]. A homology search was conducted using BLAST [38]. The annotated genomic sequence of Escherichia phage KEC4 was deposited into NCBI, Bethesda, MA, USA GenBank, and is available under accession number PX673861.

For phylogenetics, a search for related sequences was first performed using the NCBI core database, followed by multiple sequence alignment (MSA) in MAFFT v7.463 (L-INS-I algorithm) [39], and the IQ-TREE v2.0.4 [40] maximum likelihood method with an automatic search of the best substitution model was used to construct a phylogenetic tree. Intergenomic similarity comparisons were conducted using VIRIDIC v1.1 [41]. A BLAST search for virulence factors was conducted using the VFDB database [42,43]. The genomic map was plotted using Clinker [44]. The Abricate v1.2.4 toolbox (https://github.com/tseemann/abricate, accessed on 13 December 2025) and VFDB database [45] were used for the screening of virulence factors and toxin-encoding and antibiotic resistance genes in the genome.

2.11. Data Analysis

All experiments were performed in three independent biological replicates, with three technical repeats in each run, if another is not mentioned. For MIC values and PFU count, the median values were calculated. The significance of differences between medians was assessed by using the non-parametric Kruskal–Wallis ANOVA test with Dunn’s post hoc test for multiple comparisons (p < 0.05).

3. Results

3.1. Isolation of the Bacteriophage KEC4

The bacteriophage KEC4 was isolated from water samples of the Kukshum River, Chuvash Republic, by filtration followed by co-cultivation of the phage with E. coli ATCC 25922 as a host strain. The phage suspension had a titer of 3 × 10^9^ PFU/mL. On the lawn, the KEC4 phage provides full lysis of bacterial cells, forming zones with clean edges (Figure 1a), while remaining cell debris is visible. As well, it forms round and pure plaques 0.3–1 mm in diameter (Figure 1b). KEC4 did not lyse another Enterobacteria or P. aeruginosa; the strain-specificity tests revealed that KEC4 was able to lyse 14 of 31 E. coli clinical isolates (45%), including nine MDR isolates (Table 1).

Phage KEC4 exhibited stability profiles under various conditions. Thermostability assays revealed no significant titer reduction after 1 h of incubation at temperatures ranging from 4 to 50 °C. However, the titer decreased 10-fold at 60 °C, and complete inactivation was observed at 70 °C (Figure 2a). In the pH tolerance tests (Figure 2b), the phage remained active within a pH range of 5.0 to 9.0, with a 10-fold reduction at pH 4.0 and pH 10.0. A complete inactivation occurred at pH 3.0 and pH 11.0.

3.2. Genome Sequencing, Annotation and Genomic Analysis

Sequencing of the Escherichia phage KEC4 genome was performed using 150 bp paired-end reads on the Illumina HiSeq platform. De novo assembly with SPAdes v3.15.5 produced a single contig of 145,125 bp with an average coverage of 119x. The genome has a GC content of 41.3%, which is substantially lower than that of typical Escherichia coli chromosomes (50.8% GC for the reference K-12 genome). A total of 303 protein-coding genes and six tRNA genes were predicted. Putative functions were assigned to 72 proteins, whereas the remaining 231 ORFs were annotated as hypothetical. The genome map (Figure 3) indicates a broadly modular architecture typical for tailed dsDNA phages, with recognizable blocks corresponding to DNA packaging and virion morphogenesis, replication and nucleotide metabolism, and lysis-related functions. However, the modularity is not sharply compartmentalized, because several functionally related genes are dispersed rather than tightly co-localized (Figure 3).

The virion morphogenesis region includes the expected head and tail genes (major capsid protein, head decoration, tail sheath, baseplate components, and tail assembly chaperone). A prominent feature is a dense adsorption-related segment: five predicted tail fiber protein genes are scattered within an approximately 27 kbp region, consistent with a complex and potentially flexible receptor-binding apparatus. The genome encodes a rich set of genes associated with nucleic acid metabolism and nucleotides, including a DNA polymerase, primase/helicase, additional helicase and exonuclease functions, DNA ligase, multiple endonucleases (including HNH endonucleases), Holliday junction resolvase (RuvC-like), and enzymes supporting nucleotide metabolism such as ribonucleotide reductase subunits and thymidylate synthase. Collectively, this repertoire is consistent with a substantial degree of replication autonomy relative to smaller, more host-dependent phage genomes. In the DNA packaging module, the terminase large subunit (TLS) is encoded in a split form, with a shorter 5′ part and a longer 3′ part separated by an intervening HNH endonuclease gene. A similar HNH-associated splitting of TLS genes is observed for other Vequintavirinae phages and is not restricted to a single genus of this subfamily. In addition, the annotation includes a gene labeled as an anti-restriction nuclease (Figure 3), suggesting the presence of a dedicated countermeasure against bacterial restriction systems as part of the KEC4 defense–counter-defense apparatus.

Although lysis-associated functions are readily identifiable, they are distributed across the genome rather than forming a single compact lysis module (Figure 3). Specifically, the genome appears to encode two candidates for cell wall degradation enzymes that can function as endolysins: (i) an endolysin predicted to act as a murein transglycosylase and (ii) a peptidase annotated as an L-alanyl-D-glutamate endopeptidase. These genes are separated by long stretches of unrelated ORFs, and additional lysis-associated components (for example, the Rz-like spanin pair) occur elsewhere in the genome. This dispersed organization contrasts with the tight clustering of lysis genes observed in some other phage groups [46].

A BLAST search using the NCBI Vore database found the highest degree of identity to be 96.35% for the genomes of Escherichia phage A5-4, classified by the International Committee on Taxonomy of Viruses (ICTV) as Septuagintavirus sv54 (GenBank accession NC_130866.1, query sequence coverage 95%), unclassified Escherichia phage W70 (GenBank accession OP778610.1, query sequence coverage 90%), and Escherichia phage A73 (Septuagintavirus A73, GenBank accession NC_112204.1, query sequence coverage 94%). According to the ICTV standards, viruses are classified as belonging to the same species when their whole-genome BLASTN identity exceeds 95%; if their identity exceeds 70%, they belong to the same genus. Based on the results of our alignments, KEC4 can be considered as a new species of E. coli bacteriophages. Comparisons of intergenomic similarity using VIRIDIC (Figure 3) place KEC4 within the Septuagintavirus cluster inside the subfamily Vequintavirinae, which is assigned directly to the class Caudoviricetes [47]. The closest genomes in this cluster include Escherichia phages A73 (Septuagintavirus A73), A5-4, A7_1, W70, and vB_EcoM-LTH01. Pairwise intergenomic similarity values for KEC4, accounting for coverage, are 92.4% to A73, 92.1% to A5-4, 91.5% to A7_1, 89.5% to W70, and 89.9% to vB_EcoM-LTH01 (Figure 4). These values support the assignment of KEC4 to the genus Septuagintavirus of the Vequintavirinae subfamily.

Phylogenetic analysis based on the TLS amino acid sequences was performed using a dataset of 60 phages total (including KEC4), which combined phages recovered by BLAST searches and representative phages covering the major taxa that are phylogenetically close to Vequintavirinae (Figure 5). In the resulting tree, KEC4 clusters within the Vequintavirinae clade and groups with the Septuagintavirus-associated branch containing Escherichia phages A73 and A5-4 (Septuagintavirus A73 and Septuagintavirus sv54), together with closely related Escherichia phages W70, vB_EcoM-LTH01, and A7_1 (Figure 4). Notably, the closest lineage to the Vequintavirinae block among the sampled non-Vequintavirinae references is Rheinheimera phage vB_RspM_Barba18A (genus Barbavirus), which forms the nearest external branch adjacent to the Vequintavirinae cluster in this analysis (Figure 5). Thus, the results of the phylogenetic analysis support the suggested classification of phage KEC4 as a member of the genus Septuagintavirus of the Vequintavirinae subfamily.

The annotation-based screen did not identify canonical lysogeny markers (such as integrase or related site-specific recombination modules) and did not detect virulence factor candidates. Furthermore, the analysis performed by using the abricate v1.2.4 toolbox and VFDB database revealed no virulence factors, toxin-encoding genes, antibiotic resistance genes, or mobile genetic elements in the KEC4 genome, suggesting putative safety of the phage for therapeutic use. Together with the absence of recognizable lysogeny determinants, these results are consistent with a lytic lifestyle and support the potential suitability of KEC4 for antimicrobial applications, pending experimental validation.

3.3. Synergistic Effects of KEC4 with Antimicrobials

Since the successful treatment of infectious diseases solely by phage therapy occurs relatively rarely, the effect of the combined use of KEC4 with various antimicrobials was investigated by evaluating the MIC of drugs in the presence or absence of Escherichia phage KEC4 against bacteria with different susceptibilities to either the phage or antimicrobials.

In the first stage, the combined effect of antimicrobials with the bacteriophage was evaluated on the E. coli ATCC 25922 reference strain. For this purpose, the MIC of various antimicrobial was assessed in the absence or presence of KEC4, and MIC fold changes were evaluated. Thus, in the case of meropenem and kanamycin, the effectiveness of the drugs increased 16-fold, while no significant changes in the case of amikacin and gentamicin were observed (Table 2).

In clinical isolates, less optimistic results were obtained. Thus, while the combined use of bacteriophage KEC4 and aminoglycosides allowed a decrease in the MIC of aminoglycosides (amikacin and gentamicin) by 16-fold on E. coli NKC1 with resistance to various classes of antibiotics, no improvement in efficiency of either ceftriaxone or ciprofloxacin was reached. By contrast, an eight-fold drop in the MIC of ceftriaxone was observed for isolate 167, with no increase in the efficiency of aminoglycosides. Finally, a four-fold increase in efficiency of both azithromycin and gentamicin was detected in isolate 5767 despite no visible lysis in the Gratia test (Table 1). In all other cases no significant changes in the MIC of the antimicrobials were observed, indicating individual, isolate-specific recruitment of both the phage and the antimicrobials, although a positive result can be reported on the reference strains.

4. Discussion

Currently, infectious diseases caused by opportunistic enterobacteria with a wide profile of resistance are becoming a challenge worldwide, and a large number of antibacterial drugs are being developed: plant-based compounds, nanoparticle- or liposome-based drugs, vaccines, antimicrobial peptides, hydrolytic enzymes, probiotics and prebiotics, quorum-sensing inhibitor molecules, and bacteriophages [48,49,50]. Among the limitations of phage therapy, such problems as the strain specificity of phages [51] and bacterial resistance to viral infection can be overcome by the use of a mixture of various phages [52], which in turn requires the discovery of many novel bacteriophages with a wide specificity to different strains [53].

In this study, we report the isolation and investigation of the novel bacteriophage Escherichia phage KEC4. It demonstrated considerable specificity toward various E. coli isolates, showing high levels of lytic activity while not affecting another enterobacteria and P. aeruginosa, which reflects its potential effectiveness for therapeutic applications. Although some variability in the degree of lysis was observed, the overall pattern indicates sufficiently broad coverage of clinical isolates. This aspect is important for selecting bacteriophages in situations requiring specialized tools for eliminating particular strains [54]. The question regarding the targeting of commensal E. coli strains is more complicated, since the risk of potential damage of part of the residential microflora could be expected. Nevertheless, since 45% of the E. coli isolates present in Table 1 were targeted, one could expect that the same fraction of residential E. coli strains will be sensitive to the phage, thus underlying putative limitations for the treatment of infections of the gastrointestinal tract.

Furthermore, the KEC4 genome assembly and annotation revealed the absence of genes associated with lysogenic behavior (integrase, transposase and recombinase). This indicates that KEC4 functions exclusively in the lytic cycle, eliminating bacteria without the risk of viral integration into the bacterial genome and without long-term consequences [55]. Genome-based comparisons (VIRIDIC and TLS phylogenies) consistently place KEC4 within the genus Septuagintavirus in the subfamily Vequintavirinae, a lineage formally recognized in ICTV taxonomy [47]. Importantly, for interpretation and potential application, Vequintavirinae and rV5-like myophages are widely represented by virulent (obligately lytic) isolates; for example, the rV5-like group was originally defined to include lytic phages such as Escherichia coli phage rV5 and Salmonella phage PVP-SE1, and rV5 relatives constitute one of the major groups repeatedly recovered in systematic surveys of lytic coliphages [55,56]. At the same time, the KEC4 genome illustrates that although a broadly modular organization is recognizable, module boundaries are not always sharply expressed: several functionally linked genes are dispersed rather than tightly co-localized, including lysis-associated functions. Notably, the genome likely encodes two distinct endolysin candidates separated in the genome, one predicted to act as a murein transglycosylase and the other as an L-alanyl-D-glutamate endopeptidase. Another interesting feature is the split architecture of the large terminase subunit (a small 5′ ORF and a larger 3′ ORF). Furthermore, KEC4 also carries putative counter-defense functions against host restriction–modification.

The synergistic effects of phages with antimicrobials have been demonstrated in many works {Citation}. An up to 256-fold reduction in MIC was shown for meropenem and colistin []. The effect of the combined use of KEC4 with various antimicrobials demonstrated promising results only against the reference strain and in the case of aminoglycosides (Table 2), while for some clinical isolates the increase in ceftriaxone efficiency has also been detected. The molecular background of this phenomenon is discussable. It can be linked with the mechanism of action of aminoglycosides, which involves the disruption of protein synthesis in bacterial cells. Meanwhile, bacteriophages cause the lysis of the bacterial cell wall and may allow these antimicrobials to penetrate bacterial cells more easily compared to intact cells [57,58]. As well, in the work of [59] the authors suggested that exposure to a phage could resensitize XDR K. pneumoniae to amikacin, allowing it to produce potent phage–antibiotic synergy at sub-inhibitory doses. Beta-lactams, whose mechanism of action involves the inhibition of bacterial cell wall synthesis, may benefit from phage-induced cell wall damage, facilitating their binding to proteins involved in cell wall formation. As well, some phages were reported to interfere with efflux pumps, thus increasing the efficiency of carbapenems and cephalosporines []. Unfortunately, in the case of clinical isolates, the combined use of the bacteriophage KEC4 and antibiotics seemed similar or only slightly better compared to monotherapy with solely antimicrobial agents (Table 2). This fact can be related to either high-efficiency tools or antibiotic resistance in isolates, possibly carrying substantial inter-strain differences in antibiotic resistance mechanisms. Apparently, future studies should include an in-depth analysis of individual characteristics of each isolate, i.e., its own mechanisms of resistance and their possible interference with a viral infection to optimize strategies for the combined application of a phage and conventional antibiotics. Or, most likely, the isolates used in this study contacted various phages and also have active defense systems to survive in the presence of the phage.

Taken together, the results of this study indicate that the phage KEC4, a new member of the genus Septuagintavirus, exhibits significant lytic potential against various Escherichia coli clinical strains, including MDR ones with a wide profile of the resistance pattern, and shows no genetic markers of neither lysogenic behavior nor non-safety for therapeutic use like genes associated with virulence, toxins and antibiotic resistance. The phage also enhances the antimicrobial activity of conventional antibiotics and thus can be considered as a candidate for use in phage cocktails for the treatment of infections caused by resistant bacteria. Further in vivo testing in animal models is required for evaluation of its potency for implementation of KEC4 in practice.