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The aim of this study was to gain further insight into the diversity of Escherichia coli phages followed by enhanced work on taxonomic issues in that field. Therefore, we present the genomic characterization and taxonomic classification of 50 bacteriophages against E. coli isolated from various sources, such as manure or sewage. All phages were examined for their host range on a set of different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms. Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and 8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb. Annotation and comparison with databases showed similarities in particular to T4- and T5-like phages, but also to less-known groups. Though various phages against E. coli are already described in literature and databases, we still isolated phages that showed no or only few similarities to other phages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny and classification of the newly isolated phages using VICTOR resulted in the proposal of new genera and led to an enhanced taxonomic classification of E. coli phages.

Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. We cultivated temperate phages produced by 900 E. coli strains isolated from 648 fecal samples of children 1-year-old. Next, we isolated coliphages directly from the viral fraction of the same fecal samples. We found that 63% of strains hosted phages, while 24% of the viromes contained phages targeting E. coli. 150 of these phages, half recovered from strain supernatants, half from virome (73% temperate and 27% virulent) were tested for their host range on 75 E. coli strains isolated from the same cohort. Temperate phages barely infected the gut strains, whereas virulent phages killed up to 68% of them. We conclude that in fecal samples from children, temperate coliphages dominate, while virulent ones have greater infectivity and broader host range, likely playing a role in gut microbiota dynamics.

Phages drive bacterial diversity, profoundly influencing microbial communities, from microbiomes to the drivers of global biogeochemical cycling. Aiming to broaden our understanding of Escherichia coli (MG1655, K-12) phages, we screened 188 Danish wastewater samples and isolated 136 phages. Ninety-two of these have genomic sequences with less than 95% similarity to known phages, while most map to existing genera several represent novel lineages. The isolated phages are highly diverse, estimated to represent roughly one-third of the true diversity of culturable virulent dsDNA Escherichia phages in Danish wastewater, yet almost half (40%) are not represented in metagenomic databases, emphasising the importance of isolating phages to uncover diversity. Seven viral families, Myoviridae, Siphoviridae, Podoviridae, Drexlerviridae, Chaseviridae, Autographviridae, and Microviridae, are represented in the dataset. Their genomes vary drastically in length from 5.3 kb to 170.8 kb, with a guanine and cytosine (GC) content ranging from 35.3% to 60.0%. Hence, even for a model host bacterium, substantial diversity remains to be uncovered. These results expand and underline the range of coliphage diversity and demonstrate how far we are from fully disclosing phage diversity and ecology.

Background Amidst rising antimicrobial resistance, bacteriophage (phage) therapy has re-emerged as a pivotal weapon against multidrug-resistant pathogens. Jumbo phages, distinguished by large genomes, show particular therapeutic promise. Yet current understanding of jumbo phages is still lacking. Methods Phage was isolated from domestic sewage. The biological properties of JP4 was characterized via transmission electron microscopy, stability tests, one-step growth curve. The genome of JP4 were elucidated by sequencing and bioinformatics tools. Structural proteins were identified via mass spectrometry. Bactericidal and biofilm eradication activities were evaluated using bacterial turbidity measurements and crystal violet assays, respectively. Statistical significance was determined by using one-way ANOVA in GraphPad Prism. Results Phage JP4 has an icosahedral head (approximately 110 nm in diameter) and a contractile tail (about 120 nm in length). JP4 possesses a linear dsDNA genome of 370,741 bp, encoding 738 proteins and 8 tRNAs. Phylogenetic analysis revealed that JP4 is a new member of the Asteriusvirus genus, and shares close evolutionary relationships with Escherichia phage UB. Additionally, mass spectrometry identified four novel structural protein encoding genes of JP4. Phage JP4 exhibited rapid infection cycle, high stability, potent in vitro bactericidal activity, and strong inhibitory effect on E. coli biofilms. Conclusions Phage JP4 is a new member of the Asteriusvirus genus. As a lytic jumbo phage with rapid bactericidal activity and strong biofilm degradation capacity, JP4 is a promising therapeutic candidate against E. coli O157:H7 infections. This study provides insights into the diversity and clinical potential of jumbo phages in combating pathogens.

Rapid turnover of mobile elements drives the plasticity of bacterial genomes. Integrated bacteriophages (prophages) encode host-adaptive traits and represent a sizable fraction of bacterial chromosomes. We hypothesized that natural selection shapes prophage integration patterns relative to the host genome organization. We tested this idea by detecting and studying 500 prophages of 69 strains of Escherichia and Salmonella. Phage integrases often target not only conserved genes but also intergenic positions, suggesting purifying selection for integration sites. Furthermore, most integration hotspots are conserved between the two host genera. Integration sites seem also selected at the large chromosomal scale, as they are nonrandomly organized in terms of the origin–terminus axis and the macrodomain structure. The genes of lambdoid prophages are systematically co-oriented with the bacterial replication fork and display the host high frequency of polarized FtsK-orienting polar sequences motifs required for chromosome segregation. matS motifs are strongly avoided by prophages suggesting counter selection of motifs disrupting macrodomains. These results show how natural selection for seamless integration of prophages in the chromosome shapes the evolution of the bacterium and the phage. First, integration sites are highly conserved for many millions of years favoring lysogeny over the lytic cycle for temperate phages. Second, the global distribution of prophages is intimately associated with the chromosome structure and the patterns of gene expression. Third, the phage endures selection for DNA motifs that pertain exclusively to the biology of the prophage in the bacterial chromosome. Understanding prophage genetic adaptation sheds new lights on the coexistence of horizontal transfer and organized bacterial genomes.

Background Urinary tract infections (UTIs) caused by antibiotic-resistant bacteria have become a significant public health concern. The increasing ineffectiveness of antibiotics has led to a renewed focus on investigating other strategies, such as bacteriophages, to target specific pathogenic bacteria and prevent future resistance. Results This study reports the isolation and characterization of bacteriophage vB_Eco_ZCEC08 targeting uropathogenic Escherichia coli (UPEC). Phage vB_Eco_ZCEC08 is morphologically a non-contractile tailed phage that exhibits strong lytic activity against UPEC with a short latent period of less than 15 min and a lysis time of 20 min to produce a high burst of around 900 phage particles per host cell. vB_Eco_ZCEC08 phage activity demonstrated exceptional stability against temperature [-80–60 ̊C], pH [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 – 11 ], UV exposure and incubation in artificial human urine. The phage effectively reduced UPEC counts over a range of infection rates, with MOI 1 the most effective, and which resulted in the limited emergence of phage-insensitive bacteria. A whole-genome study of the 47.926 bp vB_Eco_ZCEC08 phage identified one tRNA gene and 84 predicted genes. Comparative genomics and phylogenetic analysis suggest that the vB_Eco_ZCEC08 phage belongs to the same genus as the Salmonella phage vB_SenS_ST1 but represents a new species. Phage vB_Eco_ZCEC08 showed minimal cytotoxicity against human urinary bladder cancer and skin fibroblast cell lines. Conclusion vB_Eco_ZCEC08 phage demonstrates strong selective lytic activity against UPEC in the absence of any lysogenic behavior. These properties coupled with inherent physiochemical stability and low cytotoxicity support the development of vB_Eco_ZCEC08 as an alternative treatment for multidrug-resistant UPEC.

Background Escherichia coli -associated urinary tract infections (UTIs) are among the most prevalent bacterial infections in humans. Typically, antibiotic medication is used to treat UTIs, but over the time, growth of multidrug resistance among these bacteria has created a global public health issue that necessitates other treatment modalities, such as phage therapy. Methods The UPEC strain PSU-5266 (UE-17) was isolated from human urine samples, while phages were obtained from wastewater. These phages were characterized through host range analysis, stability studies, adsorption assays, and electron microscopy. Additionally, genomic, phylogenetic, and proteomic analyses were conducted to provide further insights. Results The current study describes the isolation and characterization of four Escherichia coli phages designated as UE-S5a, UE-S5b, UE-M3 and UE-M6. Bactericidal assays depicted that all bacteriophages exhibited a strong lytic ability against uropathogenic E. coli (UPEC) strain PSU-5266 (UE-17). The phages displayed a broad host range (31–41%) among 104 tested isolates and adsorption rate of 15–20 min. They were stable within pH range of 5–11 and temperature range of 4 to 55 °C. Electron microscopy showed that all phages have icosahedral heads (70–74 nm) and short non-contractile tails, thus exhibiting a podovirus morphology. Sequencing results showed that they have linear double stranded DNA, genome of 73 to 76 kb in length, with GC content of 42% and short direct terminal repeats. Their genomes contain 84–88 predicted genes with putative functions predicted to 42–48% of gene products. The phylogenetic and comparative genomic analysis results depicted that these phages, sharing > 98% sequence similarity, are new members of genus Gamaleyavirus of subfamily Enquatrovirinae , in the Schitoviridae family. Mass spectrometric analysis of purified phage particles identified 44–56 phage particle-associated proteins (PPAPs) belonging to various functional groups such as lysis proteins, structural proteins, DNA packaging related proteins, and proteins involved in replication, metabolism and regulation. In addition, no genes encoding virulence factors, antibiotic resistance or lysogeny factors were identified. Conclusion The overall findings suggest that these bacteriophages are potential candidates for phage therapy in treating UTIs caused by UPEC strains.

Background Bacteriophages are the most genetically diverse biological entities in nature. Our current understanding of phage biology primarily stems from studies on a limited number of model bacteriophages. Jumbo phages, characterized by their exceptionally large genomes, are less frequently isolated and studied. Some jumbo phages exhibit remarkable genetic diversity, unique infection mechanisms, and therapeutic potential. Methods In this study, we describe the isolation of Sharanji, a novel Escherichia coli jumbo phage, isolated from chicken feces. The phage genome was sequenced and analyzed extensively through gene annotation and phylogenetic analysis. The jumbo phage was phenotypically characterized through electron microscopy, host range analysis, and survival at different pH and temperatures, and one-step growth curve assay. Finally, Sharanji mediated infection of E. coli is studied through fluorescence microscopy, to analyze its mechanism of infection compared to well-studied nucleus-forming jumbo phages. Results Whole genome sequencing reveals that Sharanji has a genome size of 350,079 bp and is a phage encompassing 593 ORFs. Genomic analysis indicates that the phage belongs to the Asteriusvirus genus and is related to E. coli jumbo phages PBECO4 and 121Q. Phenotypic analysis of isolated phage Sharanji, indicates that the phage size is 245.3 nm, and it is a narrow-spectrum phage infecting E. coli K12 strains, but not other bacteria including avian pathogenic E. coli . Infection analysis using microscopy shows that Sharanji infection causes cell filamentation. Furthermore, intracellular phage nucleus-like structures were not observed in Sharanji-infected cells, in contrast to infection by ΦKZ-like jumbo phages. Conclusions Our study reports the isolation and characterization of Sharanji, one of the large E. coli jumbo phages. Both genotypic and phenotypic analyses suggest that Sharanji serves as a unique model system for studying phage-bacteria interactions, particularly within the context of non-nucleus-forming jumbo phages. Further exploration of jumbo phages holds promise for uncovering new paradigms in the study of microbial viruses.

Five lytic bacteriophages specific for Salmonella enterica and Escherichia coli were isolated from wastewater in Minnesota. These phages, designated vB_Sal_EH1, vB_Sal_EH2, vB_Sal_EH3, vB_Sal_EH4, and vB_Sal_EH7, were characterized, and their genomes were sequenced. Phylogenetic analysis showed that they grouped within the genus Epseptimavirus, with genome sizes ranging from 108,554 to 115,218 bp. All five phages exhibited lytic activity against both S. enterica and Shiga-toxin-producing E. coli O157:H7. Transposon mutagenesis of the host genome identified the outer membrane protein BtuB as essential for phage infection, suggesting that it is a putative receptor. Genome sequence comparisons revealed genetic loci that are variable among the isolated phages and potentially influence their host specificity and virulence.

The widespread misuse of antibiotics has accelerated the emergence of multidrug-resistant bacterial strains, presenting a major threat to global public health. Bacteriophages (phages), owing to their host-specific lytic activity and self-replicating nature, have emerged as promising alternatives or adjuncts to conventional antibiotic therapies. In this study, a lytic phage targeting avian pathogenic Escherichia coli (APEC) was isolated from farm wastewater. The phage's morphological characteristics, host range, optimal multiplicity of infection (MOI), one-step growth curve, pH stability, thermal stability, chloroform sensitivity, and in vitro antibacterial activity were determined. Subsequently, the therapeutic efficacy of the phage was evaluated in a pigeon model. In this study, we isolated and characterized a lytic phage, designated vB_EcoM_P3322, from farm wastewater targeting APEC. Transmission electron microscopy classified vB_EcoM_P3322 within the Myoviridae family. The phage exhibited broad lytic activity against five Escherichia coliserotypes (O8:H10, O15:H18, O51:H20, O149:H20, and O166:H6). Optimal biological parameters included a multiplicity of infection (MOI) of 1, a latent period of 10 minutes, an 80-minute burst period, and a burst size of 252 PFUs/cell. vB_EcoM_P3322 maintained stable lytic activity across a pH range of 5-9 and temperatures from 4°C to 50°C, although it was sensitive to chloroform. In vitro, the phage effectively suppressed bacterial growth within 6 hours at MOIs of 0.1, 1, and 10. Whole-genome sequencing revealed a 151,674 bp double-stranded DNA genome encoding 279 predicted open reading frames. No virulence factors, toxin genes, antibiotic resistance genes, or lysogeny-related elements were identified, affirming its safety for therapeutic application. Phylogenetic analysis indicated 98.44% nucleotide identity (97% coverage) with phage vB_EcoM_Ro121c4YLVW (GenBank: NC_052654), suggesting a close evolutionary relationship. In a pigeon infection model, vB_EcoM_P3322 treatment significantly improved survival and reduced histopathological damage in the liver and spleen. Metagenomic analysis of duodenal contents revealed a marked reduction (P < 0.01) in E. coli abundance in the treatment group, indicating selective pathogen clearance and modulation of gut microbiota. In summary, vB_EcoM_P3322 displays broad-spectrum lytic activity, robust environmental stability, potent antibacterial efficacy both in vitro and in vivo, and a safe genomic profile. These attributes support its potential as a novel biocontrol agent for managing APEC infections in poultry farming.