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As a foodborne pathogen capable of causing severe illnesses, early detection of Escherichia coli O157:H7 (E. coli O157:H7) is crucial for ensuring food safety. While Förster resonance energy transfer (FRET) is an efficient and precise detection technique, there remains a need for amplification strategies to detect low concentrations of E. coli O157:H7. In this study, we presented a phage (M13)-induced "one to many" FRET platform for sensitively detecting E. coli O157:H7. The aptamers, which specifically recognize E. coli O157:H7 were attached to magnetic beads as capture probes for separating E. coli O157:H7 from food samples. The peptide O157S, which specifically targets E. coli O157:H7, and streptavidin binding peptide (SBP), which binds to streptavidin (SA), were displayed on the P3 and P8 proteins of M13, respectively, to construct the O157S-M13K07-SBP phage as a detection probe for signal output. Due to the precise distance (≈3.2 nm) between two neighboring N-terminus of P8 protein, the SA-labeled FRET donor and acceptor can be fixed at the Förster distance on the surface of O157S-M13K07-SBP via the binding of SA and SBP, inducing FRET. Moreover, the P8 protein, with ≈2700 copies, enabled multiple FRET (≈605) occurrences, amplifying FRET in each E. coli O157:H7 recognition event. The O157S-M13K07-SBP-based FRET sensor can detect E. coli O157:H7 at concentration as low as 6 CFU/mL and demonstrates excellent performance in terms of selectivity, detection time (≈3 h), accuracy, precision, practical application, and storage stability. In summary, we have developed a powerful tool for detecting various targets in food safety, environmental monitoring, and medical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) and Enterotoxigenic E. coli (ETEC) have been found to readily develop biofilms on cucumber (Cucumis sativus L.), presenting a significant risk to the safety of ready-to-eat vegetables. This study aimed to assess the effectiveness of the lytic bacteriophage vB_EcoM_SQ17 (SQ17) against EHEC O157:H7 and ETEC biofilms on cucumber. Here, we evaluated the efficacy of phage SQ17 on the formation and reduction of biofilms formed by EHEC O157:H7 and ETEC strains on various surfaces, including polystyrene, poly-d-lysine precoated films, and fresh-cut cucumber, at different temperatures. Phage SQ17 significantly inhibited ETEC biofilm formation, reducing the number of adhered cells by 0.15 log CFU/mL at 37 °C. Treatment with phage SQ17 also significantly decreased the number of adhered cells in established biofilms via SEM observation. Moreover, phage SQ17 effectively reduced the biomass of EHEC O157:H7 and ETEC biofilms by over 54.8 % at 37 °C after 24 h of incubation. Following phage treatment, the viability of adhered EHEC O157:H7 cells decreased by 1.37 log CFU/piece and 0.46 log CFU/piece in biofilms on cucumber at 4 °C and 25 °C, respectively. Similarly, the viability of ETEC cells decreased by 1.07 log CFU/piece and 0.61 log CFU/piece in biofilms on cucumber at 4 °C and 25 °C, respectively. These findings suggest that phage SQ17 shows promise as a potential strategy for eradicating pathogenic E. coli biofilms on cucumber., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Listeria monocytogenes was the etiologic agent in nearly all recent outbreaks in North America attributed to pasteurized dairy products, whereas Escherichia coli O157 infections were responsible for most of the rare, yet serious complications from outbreaks involving unpasteurized dairy. This study determined the susceptibility of selected strains of L. monocytogenes and Shiga toxin-producing E. coli (STEC) to commercial phage preparations and their ability to control these pathogens in pasteurized and raw milk during 7-day storage at 7 °C. Both phage products demonstrated high lytic efficiency against 17 strains of L. monocytogenes whereas the efficiency of E. coli phages was more variable against 11 strains of O157 and non-O157 STEC. Broth microdilution assays identified effective endpoint multiplicities of infection (MOI) ranging from log 2.53 to 5.13, which differed between strains of L. monocytogenes and phage products. Mean log MOIs of E. coli phages against STEC also varied within and between products from 0.43 to 7.05. Despite these observations, the change in counts over time of three L. monocytogenes strains exposed to phage in pasteurized milk (log MOI 6) was similar with counts ∼ 4 log CFU/mL lower than control at day 7. Results for STEC O157 varied by strain but counts were lower than control in all cases by 72 h thorough day 7. Titers on isolates of both pathogens isolated from pasteurized milk indicated that the surviving populations were less susceptible to phage. The addition of a phage preparation to raw milk did not reduce populations of either pathogen or affect the change in counts of any strain over time when compared to control. Reduced efficacy in raw milk may be attributed to reduced phage binding as titers in raw milk decreased steadily (∼2 log PFU/mL) during storage. Commercial phage products may be a promising pathogen control intervention for pasteurized dairy products, warranting further investigation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dennis D'Amico reports financial support was provided by National Institute of Food and Agriculture and The George Walker Milk Research Fund. Products used in this research were provided free of charge by Intralytix and Micreos food Safety- DD If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bacteriophages (phages) have gained considerable attention as effective antimicrobial agents that infect and kill pathogenic bacteria. Based on this feature, phages have been increasingly used to achieve food safety. They are stored in a medium or buffer to ensure stability; however, they cannot be directly applied to food under these conditions due to reasons such as regulatory considerations and concerns about marketability. This study developed a stabilizing solution that allowed the maintenance of phage activity for extended periods at room temperature while being directly applicable to food. The stability of phages stored in distilled water was relatively low. However, adding a stabilizer composed of sugars and salts improved the survival rates of phages significantly, resulting in stability for up to 48 weeks at room temperature. When Escherichia coli O157:H7-contaminated vegetables were washed with tap water containing phages, the phages reduced the pathogenic E. coli count by over 90% compared with washing with tap water alone. Additionally, when pathogenic E. coli -contaminated vegetables were placed in a phage-coated container and exposed to water, the coating of the container dissolved, releasing phages and lysing the pathogenic E. coli . This led to a significant 90% reduction in pathogenic E. coli contamination compared to that after water rinsing. These results suggest an effective and economical method for maintaining phage activity and establishing the potential for commercialization through application in the food industry.

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC , the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance., Competing Interests: The authors declare no conflict of interest.

Engineered by nature, biological entities are exceptional building blocks for biomaterials. These entities can impart enhanced functionalities on the final material that are otherwise unattainable. However, preserving the bioactive functionalities of these building blocks during the material fabrication process remains a challenge. We describe a high-throughput protocol for the bottom-up self-assembly of highly concentrated phages into microgels while preserving and amplifying their inherent antimicrobial activity and biofunctionality. Each microgel is comprised of half a million cross-linked phages as the sole structural component, self-organized in aligned bundles. We discuss common pitfalls in the preparation procedure and describe optimization processes to ensure the preservation of the biofunctionality of the phage building blocks. This protocol enables the production of an antimicrobial spray containing the manufactured phage microgels, loaded with potent virulent phages that effectively reduced high loads of multidrug-resistant Escherichia coli O157:H7 on red meat and fresh produce. Compared with other microgel preparation methods, our protocol is particularly well suited to biological materials because it is free of organic solvents and heat. Bench-scale preparation of base materials, namely microporous films (the template for casting microgels) and pure concentrated phage suspension, requires 3.5 h and 5 d, respectively. A single production run, that yields over 1,750,000 microgels, ranges from 2 h to 2 d depending on the rate of cross-linking chemistry. We expect that this platform will address bottlenecks associated with shelf-stability, preservation and delivery of phage for antimicrobial applications, expanding the use of phage for prevention and control of bacterial infections and contaminants., (© 2024. Springer Nature Limited.)

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy., (© 2024. The Author(s).)

Four tailspike proteins (TSP1-4) of Escherichia coli O157:H7 bacteriophage CBA120 enable infection of multiple hosts. They form a branched complex that attaches to the tail baseplate. Each TSP recognizes a different lipopolysaccharide on the membrane of a different bacterial host. The 335 N-terminal residues of TSP4 promote the assembly of the TSP complex and anchor it to the tail baseplate. The crystal structure of TSP4-N 335 reveals a trimeric protein comprising four domains. The baseplate anchor domain (AD) contains an intertwined triple-stranded β-helix. The ensuing XD1, XD2 and XD3 β-sheet containing domains mediate the binding of TSP1-3 to TSP4. Each of the XD domains adopts the same fold as the respective XD domains of bacteriophage T4 gp10 baseplate protein, known to engage in protein-protein interactions via its XD2 and XD3 domains. The structural similarity suggests that XD2 and XD3 of TSP4 also function in protein-protein interactions. Analytical ultracentrifugation analyses of TSP4-N 335 and of domain deletion proteins showed how TSP4-N 335 promotes the formation of the TSP quaternary complex. TSP1 and TSP2 bind directly to TSP4 whereas TSP3 binding requires a pre-formed TSP4-N 335 :TSP2 complex. A 3-dimensional model of the bacteriophage CBA120 TSP complex has been developed based on the structural and ultracentrifuge information., (© 2022. The Author(s).)

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Type II toxin-antitoxin (TA) systems are classically composed of two genes that encode a toxic protein and a cognate antitoxin protein. Both genes are organized in an operon whose expression is autoregulated at the level of transcription by the antitoxin-toxin complex, which binds operator DNA through the antitoxin's DNA-binding domain. Here, we investigated the transcriptional regulation of a particular TA system located in the immunity region of a cryptic lambdoid prophage in the Escherichia coli O157:H7 EDL933 strain. This noncanonical paaA2-parE2 TA operon contains a third gene, paaR2 , that encodes a transcriptional regulator that was previously shown to control expression of the TA. We provide direct evidence that the PaaR2 is a transcriptional regulator which shares functional similarities to the lambda CI repressor. Expression of the paaA2-parE2 TA operon is regulated by two other transcriptional regulators, YdaS and YdaT, encoded within the same region. We argue that YdaS and YdaT are analogous to lambda Cro and CII and that they do not constitute a TA system, as previously debated. We show that PaaR2 primarily represses the expression of YdaS and YdaT, which in turn controls the expression of paaR2 - paaA2-parE2 operon. Overall, our results show that the paaA2-parE2 TA is embedded in an intricate lambdoid prophage-like regulation network. Using single-cell analysis, we observed that the entire locus exhibits bistability, which generates diversity of expression in the population. Moreover, we confirmed that paaA2-parE2 is addictive and propose that it could limit genomic rearrangements within the immunity region of the CP-933P cryptic prophage. IMPORTANCE Transcriptional regulation of bacterial toxin-antitoxin (TA) systems allows compensation of toxin and antitoxin proteins to maintain a neutral state and avoid cell intoxication unless TA genes are lost. Such models have been primarily studied in plasmids, but TAs are equally present in other mobile genetic elements, such as transposons and prophages. Here, we demonstrate that the expression of a TA system located in a lambdoid cryptic prophage is transcriptionally coupled to the prophage immunity region and relies on phage transcription factors. Moreover, competition between transcription factors results in bistable expression, which generates cell-to-cell heterogeneity in the population, but without, however, leading to any detectable phenotype, even in cells expressing the TA system. We show that despite the lack of protein sequence similarity, this locus retains major lambda prophage regulation features.

The increasing prevalence of foodborne diseases caused by Escherichia coli O157:H7 as well as its ability to form biofilms poses major threats to public health worldwide. With increasing concerns about the limitations of current disinfectant treatments, phage-derived depolymerases may be used as promising biocontrol agents. Therefore, in this study, the characterization, purification, and application of a novel phage depolymerase, Dpo10, specifically targeting the lipopolysaccharides of E. coli O157, was performed. Dpo10, with a molecular mass of 98 kDa, was predicted to possess pectate lyase activity via genome analysis and considered to act as a receptor-binding protein of the phage. We confirmed that the purified Dpo10 showed O-polysaccharide degrading activity only for the E. coli O157 strains by observing its opaque halo. Dpo10 maintained stable enzymatic activities across a wide range of temperature conditions under 55°C and mild basic pH. Notably, Dpo10 did not inhibit bacterial growth but significantly increased the complement-mediated serum lysis of E. coli O157 by degrading its O-polysaccharides. Moreover, Dpo10 inhibited the biofilm formation against E. coli O157 on abiotic polystyrene by 8-fold and stainless steel by 2.56 log CFU/coupon. This inhibition was visually confirmed via fieldemission scanning electron microscopy. Therefore, the novel depolymerase from E. coli siphophage exhibits specific binding and lytic activities on the lipopolysaccharide of E. coli O157 and may be used as a promising anti-biofilm agent against the E. coli O157:H7 strain., (© 2021. The Microbiological Society of Korea.)

Escherichia coli O157:H7 is a global concern that causes serious diseases, such as hemolytic uremic syndrome and bloody diarrhea. To control E. coli O157:H7 in food, a novel siphophage, BECP10, that targets the O157 serotype was isolated and characterized. Unlike other E. coli phages, BECP10 can only infect E. coli O157 strains, and thus, did not infect other strains. The 48 kbp genome of BECP10 contained 76 open reading frames (ORFs), including 33 putative functional ORFs. The phage did not contain lysogeny-related modules or toxin-associated genes, suggesting that the phage might be strictly lytic. The tail spike protein (TSP) sequence had very low homology with the reported T1-like phages, indicating that TSP might be related to this unique host spectrum. The specific O-antigen residue of E. coli O157:H7 may be a key factor for phage infection by adsorption and receptor identification. The phage exhibited strong antibacterial activity against E. coli O157:H7 over a broad pH range and showed little development of phage-insensitive mutants. The phage sustained viability on the burger patties and reduced E. coli O157:H7 to a non-detectable level without the emergence of resistant cells at low temperatures for five days. Therefore, phage BECP10 might be a good biocontrol agent for E. coli O157:H7-contaminated food matrices.

Escherichia coli O157:H7 strains often produce Shiga toxins encoded by genes on lambdoid bacteriophages that insert into multiple loci as prophages. O157 strains were classified into distinct clades that vary in virulence. Herein, we used PCR assays to examine Shiga toxin (Stx) prophage occupancy in yehV , argW , wrbA , and sbcB among 346 O157 strains representing nine clades. Overall, yehV was occupied in most strains (n = 334, 96.5%), followed by wrbA (n = 213, 61.6%), argW (n = 103, 29.8%), and sbcB (n = 93, 26.9%). Twelve occupancy profiles were identified that varied in frequency and differed across clades. Strains belonging to clade 8 were more likely to have occupied sbcB and argW sites compared to other clades ( p < 0.0001), while clade 2 strains were more likely to have occupied wrbA sites ( p < 0.0001). Clade 8 strains also had more than the expected number of occupied sites based on the presence of stx variants ( p < 0.0001). Deletion of a 20 kb non-Stx prophage occupying yehV in a clade 8 strain resulted in an ~18-fold decrease in stx2 expression. These data highlight the complexity of Stx prophage integration and demonstrate that clade 8 strains, which were previously linked to hemolytic uremic syndrome, have unique Stx prophage occupancy profiles that can impact stx2 expression.

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.

Bacteriophages (phages) have been extensively utilized as antibacterial agents in the food industry because of their host-specificity. However, their application in polymer films has been limited because of the lack of a strong attachment method for phage to the surface. We developed an antibacterial film by covalently immobilizing Escherichia coli (E. coli)-specific phage T4 on a polycaprolactone (PCL) film. The chemical bond formation was confirmed by XPS analysis, and the covalent attachment of phage T4 effectively inhibited E. coli growth even after external stimulation of the film by sonication. When applied as a packaging film for raw beef inoculated with E. coli O157:H7, the chemically functionalized PCL film showed approximately 30-fold higher bacterial inhibitory effects than the film with physically adsorbed phage T4. These results indicate the promising application potential of chemically functionalized PCL film with phage T4 as an antibacterial food packaging material against the foodborne pathogen E. coli., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Due to the nascency of bacteriophage-based pathogen detection technologies, several practical hurdles stand in the way between providing promising proof-of-concept data and development of robust detection platforms. One such hurdle, and the focus of this work, is the development of methods for transitioning laboratory stocks of bacteriophage into functional, consistent, and shelf-stable delivery methods in commercial detection kits. Research described here was undertaken to evaluate two methods for their ability to store the bacteriophage ɸV10 at ambient temperature without aqueous storage solutions while limiting loss of viability. ɸV10 is a temperate bacteriophage which solely infects the zero-tolerance food adulterant Escherichia coli O157:H7 and has been genetically modified to generate a detectable phenotype in host cells. In order to integrate this reporter bacteriophage into food-borne pathogen detection methodologies, two methods of processing phage suspensions for long-term, ambient storage were evaluated: printing solutions onto pieces of dissolvable paper and lyophilizing suspensions with sucrose. Applying phage to dissolvable paper yielded key attributes to consider when addressing phage viability, however, optimized methodology still resulted in an approximate five-log reduction in titer of viable phage. Lyophilization of ɸV10 with various concentrations of the cryoprotectant molecule, sucrose, yielded losses of approximately 0.3-log after 120 days of storage at 23°C. Liquid storage buffer samples with and without sucrose saw a reduction of viable phage of at least 3.9-log in the same period. Additionally, the ability for ɸV10 to form lysogens in an E. coli O157:H7 host was not negatively affected by lyophilization. Drying ɸV10 at ambient temperature drastically reduces the viability of the phage. However, lyophilizing ɸV10 in the presence of sucrose is an effective method for dehydration and storage of the phage in ambient environmental conditions for an extended time lending to commercial application and integration into foodborne pathogen detection methodologies., Competing Interests: Bruce Applegate is the founder of Phicrobe an LLC located in West Lafayette, Indiana, which has licensed technology related to using ΦV10 and its derivatives for the detection of E. coli O157:H7. This does not alter our adherence to PLOS ONE policies on sharing data and materials

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a -encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.

Acquisition of new prophages that are able to increase the bacterial fitness by the lysogenic conversion is believed to be an important strategy of bacterial adaptation to the changing environment. However, in contrast to the factors determining the range of bacteriophage lytic activity, little is known about the factors that define the lysogenization host range. Bacteriophage phi24B is the paradigmal model of Stx-converting phages, encoding the toxins of the Shiga-toxigenic E. coli (STEC). This virus has been shown to lysogenize a wide range of E. coli strains that is much broader than the range of the strains supporting its lytic growth. Therefore, phages produced by the STEC population colonizing the small or large intestine are potentially able to lysogenize symbiotic E. coli in the hindgut, and these secondary lysogens may contribute to the overall patient toxic load and to lead to the emergence of new pathogenic STEC strains. We demonstrate, however, that O antigen effectively limit the lysogenization of the wild E. coli strains by phi24B phage. The lysogens are formed from the spontaneous rough mutants and therefore have increased sensitivity to other bacteriophages and to the bactericidal activity of the serum if compared to their respective parental strains.

The objective of this study was to identify and characterize five different lytic bacteriophages specific to Escherichia coli O157:H7. vB&#95;EcoM-P12, vB&#95;EcoM-P13, vB&#95;EcoM-P23, and vB&#95;EcoM-P34 phages belonged to the Myoviridae family and vB&#95;EcoS-P24 phage was in the Siphoviridae family. Their plaque sizes changed between 0.48 ± 0.03 and 0.90 ± 0.03 mm in diameter. stx1 and stx2 virulent gene regions were absent in the genome of five Eco-phages and their genome size was 33 kbp. The protein band profiles of the five phages were found to be different from each other. Their latent period, burst size, and burst time changed between 10-15 min, 72-144 PFU/cell and 20-35 min, respectively. Multiplicity of infection values and mutant frequency of the phages were among 0.1-0.001 and 1.14 × 10 -7 -3.69 × 10 -8 , respectively. The phages had strong lytic activity against their host bacteria ( E. coli NCTC 12900, ATCC 43888, and ATCC 35150) at 5-37 ℃ and adsorbed to their host cells by 92.7-97.5% in the first five minutes of incubation. These phages are thought to be good candidates as therapeutic and biocontrol agents against E. coli O157:H7 in the veterinary science and food industry due to short latent period, high burst size, rapid development in host cells, high lytic activity, high adsorption rate, stability over a wide pH range and high temperature, and absence of stx1 and stx2 genes.

Escherichia coli O157:H7 is an important foodborne pathogen that has become a major worldwide factor affecting the public safety of food. Bacteriophage has gradually attracted attention because of its ability to kill specific pathogens. In this study, a lytic phage of E. coli O157:H7, named FEC14, was isolated from hospital sewage. Transmission electron microscopy analysis showed that phage FEC14 had an isometric head 80 ± 5 nm in diameter and a contractile tail whose terminal spikes present an umbrella-like structure. Phage FEC14 revealed 158,639 bp double-stranded DNA, with the G+C content of 44.6%, 209 ORFs and four tRNAs. Genome DNA of FEC14 could not be digested by some endonucleases. Many of the features of phage FEC14 are very similar to those of the newly classified genus "Kuttervirus", including morphology, genome size and organization, etc. Phage FEC14 is proposed to be a new isolate of genus "Kuttervirus" within the family Ackermannviridae, moreover, the endonuclease resistance of phage FEC14, has priority over other genera of bacteriophages for its use in biocontrol of foodborne pathogens.

According to the United States Food and Drug Administration (FDA) agency, bacteriophage solutions targeting the serotype O157:H7 are Generally Recognized as Safe (GRAS) to control STEC during beef processing. However, outbreaks involving the "Big Six" STEC increased the industry concern about those serotypes. The objective of this study was to test the efficacy of MS bacteriophages to reduce the "Big Six" non-O157 STEC in beef. The lysing efficacy of phages isolated for each specific serotype varied from 96.2% to 99.9% in vitro. When applied to contaminated trim, reductions ranging from 0.7 to 1.3 Log of all STEC were observed in ground beef. Bacteriophages may provide an additional barrier against the "Big Six" STEC in ground beef. Results of this research provide support documentation to the FDA to extend GRAS status for bacteriophages as processing aids against all adulterant STEC., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

With an increase in the consumption of non-heated fresh food, foodborne shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most problematic pathogens worldwide. Endolysin, a bacteriophage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been garnering interest as a powerful antimicrobial agent. In this study, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4°C- 55°C). The optimum temperature and pH ranges of LysECP26 were identified at 37°C-42°C and pH 7- 8, respectively. NaCl supplementation did not affect the lytic activity. Although LysECP26 was limited in that it could not pass the outer membrane, E. coli O157: H7 could be effectively controlled by adding ethylenediaminetetraacetic acid (EDTA) and citric acid (1.44 and 1.14 log CFU/ml) within 30 min. Therefore, LysECP26 may serv as an effective biocontrol agent for gram-negative pathogens, including E. coli O157:H7.

Salmonella Enteritidis, Salmonella Typhimurium, and Escherichia coli O157:H7 are the most important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages are increasingly considered as novel antibacterial agents to control foodborne pathogens. In this study, 8 Salmonella phages and 10 E. coli O157:H7 phages were isolated from chicken products. A polyvalent phage PS5 capable of infecting S. Enteritidis, S. Typhimurium, and E. coli O157:H7 was further characterized and its efficacy in reducing these foodborne pathogens was evaluated in in vitro and in foods. Morphology, one-step growth, and stability assay showed that phage PS5 was a myovirus, with relatively short latent periods, large burst sizes, and high stability. Genome sequencing analysis revealed that the genome of PS5 does not contain any genes associated to antibiotic resistance, toxins, lysogeny, and virulence factors. In broth, phage PS5 significantly decreased the viable counts of all the three bacterial hosts by more than 1.3 log CFU/mL compared to controls after 2 h of incubation at 4 °C and 24 °C. In foods, treatment with PS5 also resulted in significant reductions of viable counts of all the three bacterial hosts compared to controls at temperatures tested. This is the first report on single phage capable of simultaneously controlling S. Enteritidis, S. Typhimurium and E. coli O157:H7 in both in vitro and in foods., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

After High Event Periods, beef subprimals are usually removed from vacuum and treated with antimicrobials. After re-packaging, subprimals are tested to verify the presence of STEC. In this study, bacteriophage and organic acids were applied on beef contaminated with STEC O157:H7 to evaluate the efficiency of industry practices. Beef samples inoculated with STEC were treated with bacteriophage, lactic acid, and peroxyacetic acid and kept under vacuum or aerobic conditions. STEC loads were evaluated 30 min and 6 h after antimicrobial applications. Under aerobic conditions for 30 min and 6 h, phage reduced STEC in beef by approximately 1.4 log whereas organic acids led to a 0.5 log reduction. Under vacuum for 30 min, bacteriophage significantly reduced STEC by 1 log. No effects were observed when samples were treated with organic acids. Under vacuum after 6 h, bacteriophage reduced STEC loads by 1.4 log, lactic acid reduced by 0.6 log, and no effects were observed when peroxyacetic acid was applied., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest associated with this research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The characterization of a recently isolated bacteriophage, vB&#95;Eco4M-7, which effectively infects many, though not all, Escherichia coli O157 strains, is presented. The genome of this phage comprises double-stranded DNA, 68,084 bp in length, with a GC content of 46.2%. It contains 96 putative open reading frames (ORFs). Among them, the putative functions of only 35 ORFs were predicted (36.5%), whereas 61 ORFs (63.5%) were classified as hypothetical proteins. The genome of phage vB&#95;Eco4M-7 does not contain genes coding for integrase, recombinase, repressors or excisionase, which are the main markers of temperate viruses. Therefore, we conclude that phage vB&#95;Eco4M-7 should be considered a lytic virus. This was confirmed by monitoring phage lytic development by a one-step growth experiment. Moreover, the phage forms relatively small uniform plaques (1 mm diameter) with no properties of lysogenization. Electron microscopic analyses indicated that vB&#95;Eco4M-7 belongs to the Myoviridae family. Based on mass spectrometric analyses, including the fragmentation pattern of unique peptides, 33 phage vB&#95;Eco4M-7 proteins were assigned to annotated open reading frames. Importantly, genome analysis suggested that this E. coli phage is free of toxins and other virulence factors. In addition, a similar, previously reported but uncharacterized bacteriophage, ECML-117, was also investigated, and this phage exhibited properties similar to vB&#95;Eco4M-7. Our results indicate that both studied phages are potential candidates for phage therapy and/or food protection against Shiga toxin-producing E. coli, as the majority of these strains belong to the O157 serotype.

Escherichia coli O157:H7 and Salmonella enterica subsp. enterica are the pathogens that frequently cause foodborne illness. Bacteriophage applications have been proposed as effective for preventing food contamination caused by these pathogenic bacteria. Escherichia phage KIT03 was isolated from the soil of a poultry farm in Kyoto, Japan. KIT03 can infect Escherichia coli O157:H7 and Salmonella enterica serotypes Choleraesuis and Enteritidis. One-step growth analysis revealed that KIT03 can propagate within its initial host (E. coli NBRC 3972), E. coli O157:H7 and S. Choleraesuis with an approximate burst size of 39, 51 and 37 phage particles per infected cell, respectively. The morphological type and genome annotation suggested that KIT03 belongs to the family Myoviridae, subfamily Tevenvirinae, genus Tequatrovirus. In vitro challenge tests demonstrated that KIT03 can lyse the tested bacteria and suppress their growth. Based on the susceptibility test and adsorption assay of KIT03 with E. coli K-12 BW25113 mutants, it was proposed that KIT03 may recognise and infect bacteria with a deficient outer core of lipopolysaccharides.

Shigella ssp. and enterotoxigenic Escherichia coli are the most common etiological agents of diarrheal diseases in malnourished children under five years of age in developing countries. The ever-growing issue of antibiotic resistance and the potential negative impact of antibiotic use on infant commensal microbiota are significant challenges to current therapeutic approaches. Bacteriophages (or phages) represent an alternative treatment that can be used to treat specific bacterial infections. In the present study, we screened water samples from both environmental and industrial sources for phages capable of infecting E. coli laboratory strains within our collection. Nineteen phages were isolatedand tested for their ability to infect strains within the ECOR collection and E. coli O157:H7 Δstx. Furthermore, since coliphages have been reported to cross-infect certain Shigella spp., we also evaluated the ability of the nineteen phages to infect a representative Shigella sonnei strain from our collection. Based on having distinct (although overlapping in some cases) host ranges, ten phage isolates were selected for genome sequence and morphological characterization. Together, these ten selected phages were shown to infect most of the ECOR library, with 61 of the 72 strains infected by at least one phage from our collection. Genome analysis of the ten phages allowed classification into five previously described genetic subgroups plus one previously underrepresented subgroup.

Background: Enterohemorrhagic Escherichia coli (E. coli) are intestinal pathogenic bacteria that cause life-threatening disease in humans. Their cardinal virulence factor is Shiga toxin (Stx), which is encoded on lambdoid phages integrated in the chromosome. Stx phages can infect and lysogenize susceptible bacteria, thus either increasing the virulence of already pathogenic bacterial hosts or transforming commensal strains into potential pathogens. There is increasing evidence that Stx phage-encoded factors adaptively regulate bacterial host gene expression. Here, we investigated the effects of Stx phage carriage in E. coli K-12 strain MG1655. We compared the transcriptome and phenotype of naive MG1655 and two lysogens carrying closely related Stx2a phages: ϕO104 from the exceptionally pathogenic 2011 E. coli O104:H4 outbreak strain and ϕPA8 from an E. coli O157:H7 isolate., Results: Analysis of quantitative RNA sequencing results showed that, in comparison to naive MG1655, genes involved in mixed acid fermentation were upregulated, while genes encoding NADH dehydrogenase I, TCA cycle enzymes and proteins involved in the transport and assimilation of carbon sources were downregulated in MG1655::ϕO104 and MG1655::ϕPA8. The majority of the changes in gene expression were found associated with the corresponding phenotypes. Notably, the Stx2a phage lysogens displayed moderate to severe growth defects in minimal medium supplemented with single carbon sources, e.g. galactose, ribose, L-lactate. In addition, in phenotype microarray assays, the Stx2a phage lysogens were characterized by a significant decrease in the cell respiration with gluconeogenic substrates such as amino acids, nucleosides, carboxylic and dicarboxylic acids. In contrast, MG1655::ϕO104 and MG1655::ϕPA8 displayed enhanced respiration with several sugar components of the intestinal mucus, e.g. arabinose, fucose, N-acetyl-D-glucosamine. We also found that prophage-encoded factors distinct from CI and Cro were responsible for the carbon utilization phenotypes of the Stx2a phage lysogens., Conclusions: Our study reveals a profound impact of the Stx phage carriage on E. coli carbon source utilization. The Stx2a prophage appears to reprogram the carbon metabolism of its bacterial host by turning down aerobic metabolism in favour of mixed acid fermentation.

Bacteriophage tailspike proteins mediate virion absorption through reversible primary receptor binding, followed by lipopolysaccharide or exopolysaccharide degradation. The Escherichia coli O157:H7 bacteriophage CBA120 genome encodes four distinct tailspike proteins, annotated as ORFs 210 through 213. Previously, we reported the crystal structure of ORF210 (TSP1). Here we describe the crystal structure of ORF212 (TSP3) determined at 1.85 Å resolution. As observed with other tailspike proteins, TSP3 assembles into a trimer. Each subunit of TSP3 has an N-terminal head domain that is structurally similar to that of TSP1, consistent with their high amino acid sequence identity. In contrast, despite sharing a β-helix fold, the overall structure of the C-terminal catalytic domain of TSP3 is quite different when compared to TSP1. The TSP3 structure suggests that the glycosidase active site resides in a cleft at the interface between two adjacent subunits where three acidic residues, Glu362 and Asp383 on one subunit, and Asp426 on a second subunit, are located in close proximity. Comparing the glycosidase activity of wild-type TSP3 to various point mutants revealed that catalysis requires the carboxyl groups of Glu362 and Asp426, and not of Asp383, confirming the enzyme employs two carboxyl groups to degrade lippopolysaccharide using an acid/base mechanism.

Background: Escherichia coli O157:H7 (O157) has been linked to numerous foodborne disease outbreaks. The ability to rapidly sequence and analyze genomes is important for understanding epidemiology, virulence, survival, and evolution of outbreak strains. In the current study, we performed comparative genomics to determine structural and functional features of the genome of a foodborne O157 isolate NADC 6564 and infer its evolutionary relationship to other O157 strains., Results: The chromosome of NADC 6564 contained 5466 kb compared to reference strains Sakai (5498 kb) and EDL933 (5547 kb) and shared 41 of its 43 Linear Conserved Blocks (LCB) with the reference strains. However, 18 of 41 LCB had inverse orientation in NADC 6564 compared to the reference strains. NADC 6564 shared 18 of 19 bacteriophages with reference strains except that the chromosomal positioning of some of the phages differed among these strains. The additional phage (P19) of NADC 6564 was located on a 39-kb insertion element (IE) encoding several hypothetical proteins, an integrase, transposases, transcriptional regulators, an adhesin, and a phosphoethanolamine transferase (PEA). The complete homologs of the 39-kb IE were found in E. coli PCN061 of porcine origin. The IE-encoded PEA showed low homology (32-33%) to four other PEA in NADC 6564 and PEA linked to mobilizable colistin resistance in E. coli but was highly homologous (95%) to a PEA of uropathogenic, avian pathogenic, and enteroaggregative E. coli. NADC 6564 showed slightly higher minimum inhibitory concentration of colistin compared to the reference strains. The 39-kb IE also contained dndBCDE and dptFGH operons encoding DNA S-modification and a restriction pathway, linked to oxidative stress tolerance and self-defense against foreign DNA, respectively. Evolutionary tree analysis grouped NADC 6564 with lineage I O157 strains., Conclusions: These results indicated that differential phage counts and different chromosomal positioning of many bacteriophages and genomic islands might have resulted in recombination events causing altered chromosomal organization in NADC 6564. Evolutionary analysis grouped NADC 6564 with lineage I strains and suggested its earlier divergence from these strains. The ability to perform S-DNA modification might affect tolerance of NADC 6564 to various stressors.

Phage therapy is getting considerable attention as a method for prophylaxis of food poisoning caused by Escherichia coli O157:H7, an important pathogen causing life-threatening bloody diarrhea. Despite previous studies have shown the feasibility of phage therapy to E. coli O157:H7, promising results have not been obtained in vivo yet. A major drawback of phage therapy is that bacteriophages have high specificity and cannot infect all the sub-strains of a particular pathogenic strain. To overcome this hurdle, we thought to establish a way to artificially expand the host-range of E. coli O157:H7-specific phages. To develop a proof-of-concept for this method, we focused on T2 phage, which cannot infect E. coli O157:H7 strains, and PP01 phage, which displays broad infectivity to them, and attempted to make T2 phage able to infect E. coli O157:H7 as efficiently as PP01. We report the trials of T2 genome editing using the CRISPR/Cas9 system and the modification of both long and short tail fibers of this phage based on comparison with PP01. The resultant recombinant showed the adsorption rate comparable to PP01. Thus, we provided the evidence that the short tail fiber of PP01 plays an important role in adsorption to E. coli O157:H7., (© FEMS 2019.)

Bacteriophage V32, a representative of bacterial viruses of the Myoviridae family Ounavirinae subfamily, is proposed for search and identification of E. coli O 157 serogroup, including Shiga-toxin producing E. coli O 157 :H 7 (STEC O 157 :H 7 ), among cultures of enterobacteria from the primary seeding of the material studied. Phage genome containes a linear double-stranded DNA of 87875 base pairs with G/C-content of 38.9% and includes 132 open reading frames (ORF). In the genome, there are no determinants of antibiotic resistance, virulence genes of STEC and other well-known pathogroups of E. coli. It has been established that phage V32 has lytic activity against all studied cultures of E. coli O 157 serogroup (n=183) isolated from people and farm animals in various regions of the Russian Federation, as well as in Japan and Italy. At the same time, the phage lyses only 6 of 182 strains (3.3%) of E. coli not belonging to the O 157 serogroup and is not active against strains of other enterobacteria. That is, the phage has a high specificity. The use of bacteriophage V32 as a diagnostic tool is a highly efficient, fast, cheap and simple method for identifying E. coli serogroup O 157 , including the serotype E. coli O 157 : H 7 , in any bacteriological laboratory without special equipment and special training of performers., Competing Interests: The authors declare no conflict of interest.

We used whole-genome sequencing to investigate the evolutionary context of an emerging highly pathogenic strain of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in England and Wales. A timed phylogeny of sublineage IIb revealed that the emerging clone evolved from a STEC O157:H7 stx-negative ancestor ≈10 years ago after acquisition of a bacteriophage encoding Shiga toxin (stx) 2a, which in turn had evolved from a stx2c progenitor ≈20 years ago. Infection with the stx2a clone was a significant risk factor for bloody diarrhea (OR 4.61, 95% CI 2.24-9.48; p<0.001), compared with infection with other strains within sublineage IIb. Clinical symptoms of cases infected with sublineage IIb stx2c and stx-negative clones were comparable, despite the loss of stx2c. Our analysis highlighted the highly dynamic nature of STEC O157:H7 Stx-encoding bacteriophages and revealed the evolutionary history of a highly pathogenic clone emerging within sublineage IIb, a sublineage not previously associated with severe clinical symptoms.

Background: In many Asian countries including Bangladesh E. coli O157 are prevalent in animal reservoirs and in the food chain, but the incidence of human infection due to E. coli O157 is rare. One of the reasons could be inability of the organism from animal origin to produce sufficient amount of Shiga toxin (Stx), which is the main virulence factor associated with the severe sequelae of infection. This study aimed to fill out this knowledge gap by investigating the toxigenic properties and characteristics of stx phage of E. coli O157 isolated from animal sources in Bangladesh., Results: We analysed 47 stx 2 positive E. coli O157 of food/animal origin for stx 2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q 933 and Q 21 ) and stx 2 upstream region. Of the 47 isolates, 46 were positive for both stx 2a and stx 2d while the remaining isolate was positive for stx 2d only. Reverse Passive Latex Agglutination assay (RPLA) showed that 42/47 isolates produced little or no toxin, while 5 isolates produced a high titre of toxin (64 to 128). 39/47 isolates were positive for the Toxin Non-Producing (TNP) specific regions in the stx 2 promoter. Additionally, all isolates were negative for antiterminator Q 933 while a majority of isolates were positive for Q 21 gene suggesting the presence of defective stx phage. Of the yehV and wrbA phage insertion sites, yehV was found occupied in 11 isolates while wrbA site was intact in all the isolates. None of the isolates was positive for the virulence gene, cdt but all were positive for hlyA, katP, etpD and eae genes. Isolates that produced high titre Stx (n = 5) produced complete phage particles capable of infecting multiple bacterial hosts. One of these phages was shown to produce stable lysogens in host strains rendering the Stx2 producing ability., Conclusion: Despite low frequency in the tested isolates, E. coli O157 isolates in Bangladesh carry inducible stx phages and have the capacity to produce Stx2, indicating a potential risk of E. coli O157 infection in humans.

Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene ( sspA ) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens. IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication., (Copyright © 2018 Cowley et al.)

Escherichia coli O157:H7 has become a global public health and a food safety problem. Despite the implementation of control strategies that guarantee the safety in various products, outbreaks persist and new alternatives are necessary to reduce this pathogen along the food chain. Recently, our group isolated and characterised lytic bacteriophages against E. coli O157:H7 with potential to be used as biocontrol agents in food. To this end, phages need certain requirements to allow their manufacture and application. The aim of this study was to determine the physical stability and allergenic potential of free and microencapsulated (ME) bacteriophage cocktails against E. coli O157:H7. In vitro and in vivo studies were performed to determine phage survival under different pH, gastrointestinal conditions, temperature and UV light intensities. Results showed that the stability of ME phages was significantly (P<0.05) higher than free phages after ultraviolet irradiation, pH conditions between 3 to 7, and exposure to temperatures between at -80°C and 70°C. Both formulations were highly sensitive to very low pH in simulated gastric fluid, but stable in bile salts. In vivo studies in mice confirmed these phages passed through the gastrointestinal tract and were excreted in faeces. In silico, full-length alignment analysis showed that all phage proteins were negative for allergenic potential, but different predicting criteria classified seven phage proteins with a very low probability to be an allergen. In conclusion, these data demonstrated that microencapsulation provided a greater stability to phage formulation under stress conditions and assure a more suitable commercial formulation for the biological control of E. coli O157:H7.

Background: The formation of biofilms and subsequent encasement of bacterial cells in a complex matrix can enhance resistance to antimicrobials and sterilizing agents making these organisms difficult to eradicate and control. The aim of this study was to evaluate and compare the capacity of 40 E. coli O26 isolates of enterohemorrhagic E. coli (EHEC, n = 27), potential EHEC (pEHEC, n = 3), atypical enteropathogenic E. coli (aEPEC, n = 8) and non-toxigenic E. coli (NTEC, n = 2) from human and cattle sources to form biofilms on different surfaces, and determine whether extracellular matrix (ECM) components (cellulose, curli), motility, prophage insertion in mlrA and cell surface hydrophobicity could influence biofilm formation. Finally, the influence of biofilm formation on the sensitivity of isolates to quaternary ammonium compounds (QACs; Profoam, Kwiksan 22) and peracetic acid-based sanitizer (Topactive Des.) for 2 min on polystyrene plate were also evaluated., Results: Biofilm production on one surface may not indicate biofilm formation on a different surface. Biofilm was formed by different pathotypes on polystyrene (70%), stainless steel (87.5%) and glass slides (95%), however only 50% demonstrated pellicle formation. EHEC isolates were significantly more likely to form a pellicle at the air-liquid interface and biofilms on polystyrene surface at 48 h than aEPEC. Strains that don't produce ECM (curli or cellulose), harbor a prophage insertion in mlrA, and are non-motile have lower biofilm forming capacities than those isolates possessing combinations of these attributes. Hydrophobicity had no impact on biofilm formation. After 2 min exposure, none of the disinfectants tested were able to completely inactivate all cells within a biofilm regardless of pathotypes and the amount of biofilm formed., Conclusion: Pathotypes of E. coli O26 showed varying capacities to form biofilms, however, most EHEC strains had the capacity to form biofilm on all surfaces and at the air-liquid interface under the conditions used in this study. Biofilms provided a protective effect to E. coli O26 strains against the three sanitizers, previously shown to successfully control the growth of their planktonic counterparts. Whether the characteristics of biofilm forming and non-biofilm forming strains observed in this study reflect their attributes within the food and meat-processing environments is unknown. Further studies that represent the food and meat-processing environments are required.

Bacteriophages are genetic elements that play key roles in the evolution and diversification of bacterial genomes. The Shiga toxin (Stx)-encoding phage plays an important role in the horizontal transfer of the stx gene. However, the influence of the Stx phage integration on the physiological properties and gene expression pattern of the host have not been clearly resolved. In this study, we constructed the Sp5 lysogen through lysogenisation of E. coli K-12 by Sp5, an Stx2 phage in enterohaemorrhagic E. coli (EHEC) O157:H7 Sakai, and examined the effect of the resulting lysogen on cell motility under various growth conditions. Sp5 lysogenisation decreased cell motility and the expression of fliC, which encodes flagellin, under anaerobic conditions at 37°C. Sp5 also lowered the expression of fliA, which encodes the FliA-sigma factor responsible for the transcription of fliC, and flhD, which facilitates the expression of fliA. Sp5 lysogenisation reduced the amount of FlhD and FlhC expressed from the araBAD promoter, suggesting that one or more genes present in Sp5 represses flhDC at the post-transcriptional level. Flagellin is highly antigenic and triggers an immune response in the host. Thus, Sp5 might enhance its viability by repressing the expression of the flagellar regulon to circumvent the immune response of host cells.

Escherichia coli O157:H7 (E. coli O157:H7) is one of the most common pathogens in fresh vegetables and fruits, and most of the diseases produced by E. coli O157:H7 are associated with biofilms. Cold nitrogen plasma (CNP) is a cold sterilization technique which has no residue. However to completely eliminate the biofilm on the surface of vegetables the processing power and time of CNP have to be enhanced, which will impact on the quality of fruits and vegetables. Thus the sequential treatment of CNP and phage techniques was engineered in this study. Compared to treatment performed separately, sequential treatment not only had more mild treatment conditions as 400W CNP treatment for 2min and 5% phage treatment for 30min, but also exhibited more remarkable effect on eradicating E. coli O157:H7 biofilms in vitro and on vegetables. The population of E. coli O157:H7 was approximately reduced by 2logCFU/cm 2 after individual treatment of 5% phages for 30min or 500W CNP for 3min. While the sequential treatment of CNP (400W, 2min) and phages (5%, 30min) reduced the E. coli O157:H7 viable count in biofilm by 5.71logCFU/cm 2 . Therefore, the sequential treatment holds a great promise to improve the current treatment systems of bacterial contamination on different vegetable surfaces., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Escherichia coli serotype O157: H7 and E. coli O104: H4 are well known foodborne pathogens causing sever enteric illness. Using bacteriophages as biocontrol agents of some foodborne pathogens and multidrug-resistant (MDR) bacteria has a great attention nowadays. This study aims to test the effect of cocktail phages on the growth of some foodborne pathogens and MDR E. coli. Routine conventional PCR was used to confirm the identification of E. coli isolates. Double-layered culture technique was used to isolate phages from sewage water. Morphology of bacteriophage was described using transmission electron microscopy, and spot test was performed to determine host range of the phage cocktail. Phage cocktail of Siphoviridae and Podoviridae family infecting E. coli O157: H7, E. coli O104: H4 and untypeable E. coli (neither O157 nor O104) has been isolated from sewage water. Phage cocktail showed both lytic and lysogenic activity. Lytic activity was observed against E. coli O157: H7, E. coli O104: H4 isolates, Staphylococcus. aureus ATCC6538 and Pseudomonas aeruginosa ATCC 10145, while the lysogenic activity was observed against the untypeable strain. The tested phage cocktail showed a promising inhibitory action on E. coli O157: H7 and O104: H4, S. aureus ATCC6538 and P. aeruginosa ATCC 10145, suggesting the possibility of its use as a biocontrol tool or as natural food preservatives for many food products., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In recent years, phages used for the reduction of pathogenic bacteria have fostered many attentions, but they are liable to lost bioactivity in food due to the presence of acidic compounds, enzymes and evaporite materials. To improve the stability of phages, a chitosan edible film containing liposome-encapsulated phage was engineered in the present study. The characteristics of liposome-encapsulated phage and the chitosan film containing liposome-encapsulated phage were investigated. The encapsulation efficiency of phages in liposome reached 57.66±0.12%. Besides, the desirable physical properties of chitosan film were obtained. The chitosan film embedded with liposome-encapsulated phage exhibited high antibacterial activity against Escherichia coli O157:H7, without the impact on the sensory properties of beef. Hence, chitosan film containing liposome-encapsulated phage could be a promising antibacterial packaging for beef preservation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Escherichia coli O157:H7 is an important foodborne pathogen that causes severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Ruminant manure is a primary source of E. coli O157:H7 contaminating the environment and food sources. Therefore, effective interventions targeted at reducing the prevalence of fecal excretion of E. coli O157:H7 by cattle and sheep and the elimination of E. coli O157:H7 contamination of meat products as well as fruits and vegetables are required. Bacteriophages offer the prospect of sustainable alternative approaches against bacterial pathogens with the flexibility of being applied therapeutically or for biological control purposes. This article reviews the use of phages administered orally or rectally to ruminants and by spraying or immersion of fruits and vegetables as an antimicrobial strategy for controlling E. coli O157:H7. The few reports available demonstrate the potential of phage therapy to reduce E. coli O157:H7 carriage in cattle and sheep, and preparation of commercial phage products was recently launched into commercial markets. However, a better ecological understanding of the phage E. coli O157:H7 will improve antimicrobial effectiveness of phages for elimination of E. coli O157:H7 in vivo.

Background: Enterohemorrhagic Escherichia coli (EHEC) O145 are among the top non-O157 serogroups associated with severe human disease worldwide. Two serotypes, O145:H25 and O145:H28 have been isolated from human patients but little information is available regarding the virulence repertoire, origin and evolutionary relatedness of O145:H25. Hence, we sequenced the complete genome of two O145:H25 strains associated with hemolytic uremic syndrome (HUS) and compared the genomes with those of previously sequenced O145:H28 and other EHEC strains., Results: The genomes of the two O145:H25 strains were 5.3 Mbp in size; slightly smaller than those of O145:H28 and other EHEC strains. Both strains contained three nearly identical plasmids and several prophages and integrative elements, many of which differed significantly in size, gene content and organization as compared to those present in O145:H28 and other EHECs. Furthermore, notable variations were observed in several fimbrial gene cluster and intimin types possessed by O145:H25 and O145:H28 indicating potential adaptation to distinct areas of host colonization. Comparative genomics further revealed that O145:H25 are genetically more similar to other non-O157 EHEC strains than to O145:H28., Conclusion: Phylogenetic analysis accompanied by comparative genomics revealed that O145:H25 and O145:H28 evolved from two separate clonal lineages and that horizontal gene transfer and gene loss played a major role in the divergence of these EHEC serotypes. The data provide further evidence that ruminants might be a possible reservoir for O145:H25 but that they might be impaired in their ability to establish a persistent colonization as compared to other EHEC strains.

Escherichia coli O157:H7, a major foodborne pathogen, is a major public health concern associated with life-threatening diseases such as hemolytic uremic syndrome. To alleviate this burden, a sensitive and rapid system is required to detect this pathogen in various kinds of foods. Herein, we propose a phage-based pathogen detection method to replace laborious and time-consuming conventional methods. We engineered an E. coli O157:H7-specific phage phiV10 to rapidly and sensitively detect this notorious pathogen. The luxCDABE operon was introduced into the phiV10 genome and allowed the engineered phage phiV10lux to generate bioluminescence proportional to the number of viable E. coli O157:H7 cells without any substrate addition. The phage phiV10lux was able to detect at least 1CFU/ml of E. coli O157:H7 in a pure culture within 40min after 5h of pre-incubation. In artificially contaminated romaine lettuce, apple juice (pH3.51), and ground beef, the reporter phage could detect approximately 10CFU/cm 2 , 13CFU/ml, and 17CFU/g of E. coli O157:H7, respectively. Taken together, the constructed reporter phage phiV10lux could be applied as a powerful tool for rapid and sensitive detection of live E. coli O157:H7 in foods., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Biofilm formation is a growing concern in the food industry. Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persists in food and food-related environments and subsequently produce biofilms. The efficacy of bacteriophage BPECO 19 was evaluated against three E. coli O157:H7 strains in biofilms. Biofilms of the three E. coli O157:H7 strains were grown on abiotic (stainless steel, rubber, and minimum biofilm eradication concentration [MBEC TM ] device) and biotic (lettuce) surfaces at different temperatures. The effectiveness of bacteriophage BPECO 19 in reducing preformed biofilms on these surfaces was further evaluated by treating the surfaces with a phage suspension (10 8 PFU/mL) for 2 h. The results indicated that the phage treatment significantly reduced (P  < 0.05) the number of adhered cells in all the surfaces. Following phage treatment, the viability of adhered cells was reduced by ≥3 log CFU/cm 2 , 2.4 log CFU/cm 2 , and 3.1 log CFU/peg in biofilms grown on stainless steel, rubber, and the MBEC TM device, respectively. Likewise, the phage treatment reduced cell viability by ≥2 log CFU/cm 2 in biofilms grown on lettuce. Overall, these results suggested that bacteriophages such as BPECO 19 could be effective in reducing the viability of biofilm-adhered cells., (© 2017 Institute of Food Technologists®.)

Phage therapy is an old method of combating bacterial pathogens that has recently been taken into consideration due to the alarming spread of antibiotic resistance. Escherichia coli O157:H7 is a foodborne pathogen that causes hemorrhagic colitis and life-threatening Hemolytic Uremic Syndrome (HUS). There are several studies on isolation of specific phages against E. coli O157:H7 and more than 60 specific phages have been published so far. Although in vitro experiments have been successful in elimination or reduction of E. coli O157:H7numbers, in vivo experiments have not been as promising. This may be due to escape of bacteria to locations where phages have difficulties to enter or due to the adverse conditions in the gastrointestinal tract that affect phage viability and proliferation. To get around the latter obstacle, an alternative phage delivery method such as polymer microencapsulation should be tried. While the present time results are not very encouraging the work should be continued as more efficient phage treatment regimens might be found in future., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Escherichia coli O157:H7 is a major food safety concern for the beef industry. Several studies have provided evidence that cattle hides are the main source of beef carcass contamination during processing and that reductions in the E. coli O157:H7 load on the hides of cattle entering processing facilities will lead to reductions in carcass contamination. Bacteriophages have been proposed as a novel preharvest antimicrobial intervention to reduce the levels of E. coli O157:H7 on cattle hides. The objective of this study was to evaluate a commercialized phage application administered in the lairage area of commercial beef processing plants for the ability to reduce E. coli O157:H7 contamination of cattle hides and carcasses. Cattle lots either received phage spray treatment (n = 289) or did not (n = 301), as they entered the lairage environments in two separate experiments at two different commercial beef processing plants. Hide and carcass samples were collected and analyzed for E. coli O157:H7 prevalence and concentration. Cattle hides receiving phage treatment had an E. coli O157:H7 prevalence of 51.8%, whereas untreated hides had a prevalence of 57.6%. For carcass samples, the E. coli O157 prevalence in treated and untreated samples was 17.1% and 17.6%, respectively. The results obtained from these experiments demonstrated that the treatment of cattle hides with bacteriophages before processing did not produce a significant reduction of E. coli O157:H7 on cattle hides or beef carcasses during processing.

Background: Escherichia coli O157:H7 and Shigella flexneri are well-known food-borne pathogens causing severe food poisoning at low infectious doses. Bacteriophages have been approved for food applications by the US Food and Drug Administration (FDA) and have been suggested as natural food preservatives to control specific food-borne pathogens. To develop a novel natural food preservative against E. coli O157:H7 and S. flexneri, a new bacteriophage needs to be isolated and characterized., Methodology/principal Findings: Bacteriophage HY01 infecting both E. coli O157:H7 and S. flexneri was isolated from a swine fecal sample. HY01 belongs to the family Myoviridae and is stable under various temperature and pH conditions. One-step growth curve analysis showed relatively short eclipse and latent periods as well as large burst size. The 167-kb genome sequence of HY01 was sequenced, and a comparative genome analysis with T4 for non-O157:H7 E. coli suggests that the receptor recognition protein of HY01 plays an important role in determination of host recognition and specificity. In addition, food applications using edible cabbage were conducted with two E. coli O157:H7 strains (ATCC 43890 and ATCC 43895), showing that treatment with HY01 inhibits these clinical and food isolates with >2 log reductions in bacterial load during the first 2 h of incubation., Conclusions/significance: HY01 can inhibit both E. coli O157:H7 and S. flexneri with large burst size and stability under stress conditions. The ability of HY01 to infect both E. coli O157:H7 and S. flexneri may be derived from the presence of two different host specificity-associated tail genes in the genome. Food applications revealed the specific ability of HY01 to inhibit both pathogens in food, suggesting its potential as a novel biocontrol agent or novel natural food preservative against E. coli O157:H7 and potentially S. flexneri., Competing Interests: The authors have declared that no competing interests exist.

Characteristics of E. coli O157:H7-specific infection bacteriophages (O157 coliphages) and broad-host-range bacteriophages for other E. coli serotypes (broad-host coliphages) were compared. The burst sizes of the two groups ranged from 40 to 176 PFU/infected cell. Distributions of the virulence factors stx1 , stx2 , ehxA , and saa between the two groups were not differentiated. Broad-host-range coliphages showed lower stability at 70°C, in relation to O157 coliphages. However, O157 coliphages showed high acid and ethanol tolerance by reduction of only 22% and 11% phages, respectively, under pH 3 and 70% ethanol for 1 h exposure. Therefore, these results revealed that the O157 coliphages might be more stable under harsh environments, which might explain their effective infection of the acid-tolerant E. coli O157:H7.

Sensitive and rapid detection of Escherichia coli O157:H7, one of the most notorious bacterial pathogens, is urgently needed for public health protection. Yet, the existing methods are either lack of speed or limited in discriminating viable and dead cells. Using a recombinant bacteriophage, here we report the development of a rapid and sensitive method for live E. coli O157:H7 detection. First, the wild-type PP01 phage was engineered with a tetracysteine (TC)-tag fused with the small outer capsid (SOC) protein. Then, this PP01-TC phage was used to inoculate bacterial sample for 30min. Specific infection and rapid replication of PP01-TC phage in viable E. coli O157:H7 host cell yields a large number of progeny phages with capsids displaying TC tags that can be fluorescently labeled by a membrane permeable biarsenical dye (FlAsH). The bright green fluorescence of single E. coli O157:H7 cells can be readily detected by flow cytometry (FCM) and fluorescence microscopy. High specificity of the assay was verified with seven other bacterial strains. Practical application in E. coli O157:H7 detection in drinks was successfully demonstrated with artificially contaminated 100% apple juice. In less than three hours (including sample preconcentration) and with 40mL of sample volume, as low as 1cfu/mL E. coli O157:H7 can be detected in the presence of large excess of other nontarget bacteria via fluorescence microscopic measurement. The as-developed TC-PP01-FlAsH approach shows a great potential in the safeguard of liquid food products by providing rapid, sensitive, and specific detection of live E. coli O157:H7., (Copyright © 2016 Elsevier B.V. All rights reserved.)