Structural Analysis of Jumbo Coliphage phAPEC6.

The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of Escherichia coli virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid. A capsid inner body rod is also present, measuring about 91 nm by 18 nm and oriented along the portal axis. In the phAPEC6 contractile tail, 25 hexameric stacked rings can be distinguished, built of the identified tail sheath protein (Gp277). Cryo-EM reconstruction reveals the base of the unique hairy fibers observed during an initial transmission electron microscopy (TEM) analysis. These very unusual filaments are ordered at three annular positions along the contractile sheath, as well as around the capsid, and may be involved in host interaction.

Genome-wide screens reveal Escherichia coli genes required for growth of T1-like phage LL5 and V5-like phage LL12.

The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.

A Quest of Great Importance-Developing a Broad Spectrum Escherichiacoli Phage Collection.

Shigella ssp. and enterotoxigenic Escherichiacoli 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.

Still Something to Discover: Novel Insights intoEscherichia coli Phage Diversity and Taxonomy.

The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof 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-, and8% 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-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.

Escherichia coli bacteriophage Gostya9, representing a new species within the genus T5virus.

Escherichia coli bacteriophage Gostya9 (genus T5virus) was isolated from horse feces collected in Moscow, Russia, in 2013. This phage was associated in a single plaque with the previously reported phage 9g and was subsequently purified. Analysis of the complete genomic sequence of Gostya9 revealed that it is closely related to the T5-like bacteriophage DT57C, which had been isolated at the same location in 2007. These two viruses share 79.5% nucleotide sequence identity, which is below the 95% threshold applied currently to demarcate bacteriophage species. The most significant features distinguishing Gostya9 from DT57C include 1) the presence of one long tail fiber protein gene, 122c (ltf), instead of the two genes, ltfA and ltfB, that are present in DT57C; 2) the absence of the gene for the receptor-blocking lytic conversion lipoprotein precursor llp; and 3) the divergence of the receptor-recognition protein, pb5, which is only distantly related at the amino acid sequence level. The observed features of the Gostya9 adsorption apparatus are suggestive of a possible novel specificity for the final receptor and make this phage interesting for possible direct application in phage therapy of E. coli infections or as a source of receptor-recognition protein for engineering new phage specificities.

vB_EcoS_IME347 a novel T1-like Escherichia coli bacteriophage.

Advances in phage therapy and its application require more information on phage genome characteristics and host-phage interaction mechanisms. In this study, a so far unknown T1-like Escherichia coli phage was identified and named vB_EcoS_IME347 (IME347). The genome length of phage IME347 is 50,048 bp with a G + C content of 49.7%. BLASTn alignment showed that the phage has its highest homology (identity 78%, query cover 72%) with phage SRT8 (GenBank: MF996376). Electron microscopy showed that phage IME347 has an icosahedral head and a long non-contractiled tail, features of the family Siphoviridae. Phylogenetic analysis of the large subunit of the terminal enzyme and tail fiber protein revealed that phage IME347 is a novel member of the T1 virus. Furthermore, through comparative genomics, silencing mutation, phage spotting assay, and phage adsorption assay, an E. coli BL21 TonB-dependent receptor YncD was identified to be responsible for phage IME347 adsorption and entry. The identification of the phage receptor YncD enriches the phage receptor database and provides a theoretical basis for bacteriophage therapy.

Identification and characterization of new broad host-range rV5-like coliphages C203 and P206 directed against enterobacteria.

We isolated and characterized two novel rV5-like lytic bacteriophages from independently collected food samples. Nucleotide sequence analysis revealed that these phages have linear double-stranded DNA genomes comprising 138,073 bp with 213 CDS and 5 tRNA genes. The two genomes contain completely identical nucleotide sequence, albeit there is a 10,718 bp-long shift in the sequence. The GC content of the phage genomes was 43.7% and they showed high general homology to rV5-like phages. The new phages were termed C203 and P206. The genome of both phages contains a unique ORF that encodes for a putative phage homing endonuclease. The phage produced clear plaques with a burst size of approx. 1000 viral particles and a latent period of 60 min. Morphological investigation indicated that the new phages are members of the family Myoviridae with an approximate head length of 85 nm, tail length of 75 nm, and a head width of 96 nm. C203 and P206 exhibit a broad and uniform host range, which included enterohemorrhagic Escherichia coli strains of serogroup O157, multi drug resistant (MDR) E. coli strains of various sero- and pathotypes, and both Shigella sonnei and S. dysenteriae strains. C203 and P206 both effectively reduced the number of living EHEC O157:H7 Sakai in experimentally inoculated minced meat. The same broad host range, the lack of any virulence related genes, the stability and its short latent period suggest that these newly found phages could be suitable candidates as a bio-control agents against food-borne pathogenic Enterobacteria.

Phages in urban wastewater have the potential to disseminate antibiotic resistance.

A total of 29 Escherichia coli phages were isolated from wastewater samples collected from an urban wastewater treatment plant and were characterised by host range determination, transmission electron microscopy, antibiotic resistance gene identification and phage transduction. ß-Lactam resistance genes (blaCMY, blaTEM, blaSHV, blaCTX-M and blaOXA) were amplified on phage DNA by PCR. Of nine host range patterns observed, six were able to multiply in three or more indicator strains, including Shiga toxin-producing E. coli (STEC). Twelve E. coli phages were able to grow in all four E. coli O157 strains tested. The blaTEM gene was detected in 15 phages, of which 6 were able to transduce blaTEM into E. coli ATCC 13706. These data suggest that phages with broad host range are prevalent in the urban environment and can serve as a natural reservoir of antibiotic resistance genes. These phages can also transfer antibiotic resistance genes via phage transduction and may contribute to the dissemination of antibiotic resistance in the environment.

The complete genome sequence of PE3-1, a novel E. coli O153 phage.

A novel virulent phage PE3-1 against E. coli O153 was isolated from an aeration tank in a wastewater treatment plant. Transmission electron microscopy images showed that phage PE3-1 had an icosahedral head and a short tail, which revealed that it was a member of the family Podoviridae of the order Caudovirales. The complete PE3-1 genome consisted of 39,093 bp and was a linear double-stranded DNA with an average GC content of 49.93 %. Phage PE3-1 showed homology to the T7-like phages in 48 open reading frames (ORFs), but it differed from previously reported E .coli phages in morphology and bioinformatics analysis. This indicated that phage PE3-1 is a new member of the genus T7 virus.

Total coliphages removal by activated sludge process and their morphological diversity by transmission electron microscopy.

This study was conducted to isolate phages in treated sewage collected from wastewater treatment plant, and explore their morphological diversity by transmission electron microscopy (TEM). Fates of total bacteriophages and their reduction by biological treatment were also assayed. Phages were isolated using the plaque assay then negatively stained and observed by electron microscope. Electron micrographs showed different types of phages with different shapes and sizes. The majority of viruses found in treated sewage ranged from 30 to 100 nm in capsid diameter. Many of them were tailed, belonging to Siphoviridae, Myoviridae and Podoviridae families. Non-tailed phage particles were also found at a low rate, presumably belonging to Leviviridae or Microviridae families. This study shows the diversity and the abundance of bacteriophages in wastewater after biological treatment. Their persistence in wastewater reused in agriculture should raise concerns about their potential role in controlling bacterial populations in the environment. They should be also included in water treatment quality controlling guidelines as fecal and viral indicators.

Isolation, genome sequencing and functional analysis of two T7-like coliphages of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, which results in significant economic losses to the poultry industry worldwide. Due to the drug residues and increased antibiotic resistance caused by antibiotic use, bacteriophages and other alternative therapeutic agents are expected to control APEC infection in poultry. Two APEC phages, named P483 and P694, were isolated from the feces from the farmers market in China. We then studied their biological properties, and carried out high-throughput genome sequencing and homology analyses of these phages. Assembly results of high-throughput sequencing showed that the structures of both P483 and P694 genomes consist of linear and double-stranded DNA. Results of the electron microscopy and homology analysis revealed that both P483 and P694 belong to T7-like virus which is a member of the Podoviridae family of the Caudovirales order. Comparative genomic analysis showed that most of the predicted proteins of these two phages showed strongest sequence similarity to the Enterobacteria phages BA14 and 285P, Erwinia phage FE44, and Kluyvera phage Kvp1; however, some proteins such as gp0.6a, gp1.7 and gp17 showed lower similarity (<85%) with the homologs of other phages in the T7 subgroup. We also found some unique characteristics of P483 and P694, such as the two types of the genes of P694 and no lytic activity of P694 against its host bacteria in liquid medium. Our results serve to further our understanding of phage evolution of T7-like coliphages and provide the potential application of the phages as therapeutic agents for the treatment of diseases.

Isolation and characterization of a T4-like phage with a relatively wide host range within Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, resulting in severe economic losses worldwide. Coliphages represent alternative antibacterial substitutes based on high lytic efficiency and few side-effects. However, the complete genome sequences information of APEC phages are limited, and knowledge of undesired genes and the narrow host range restrict their applications. In this study, we isolated a virulent phage QL01, with a relatively broad lytic spectrum (41 of 78 APEC strains). Transmission electron micrography showed it belonged to the family Myoviridae with an elongated head and a contractile tail. Whole genome sequencing revealed a linear double-stranded DNA (170,527 kb; GC content, 39.6%) with 275 possible ORFs. Comparative genome analysis revealed high homology between QL01 and other T4-like phages. However, it also showed some unique features, for example, ORF142 and ORF143, which encode IP9 and IP8, respectively, and may counteract host resistance only exist in a few T4-like phages such as IME08 and vB_EcoM_VR5. Furthermore, phage therapy in artificially infected ducks showed a 26.67% decrease in mortality compared with the untreated group. Our study indicates the potential antibacterial function of phage QL01 against APEC infections and highlights unique molecular features underlying the relatively broad host range.

Isolation and Characterization of Lytic Phage vB_EcoM_JS09 against Clinically Isolated Antibiotic-Resistant Avian Pathogenic Escherichia coli and Enterotoxigenic Escherichia coli.

OBJECTIVES: To characterize the lytic coliphage vB_EcoM_JS09 (phage JS09) isolated from sewage samples of a swine farm in Jiangsu Province, China, which infects antibiotic-resistant avian pathogenic Escherichia coli (APEC) and enterotoxigenic E. coli (ETEC). METHODS AND RESULTS: Transmission electron microscopy revealed that phage JS09 has an isometric icosahedral head (76 nm in diameter) and a long contractile tail (140 nm in length) and features a T-even morphology. Its latent period was 30 min and the average burst size was 79 phage particles per infected cell. It attached to the host cells within 9 min. JS09 could infect 16 clinically isolated APEC and ETEC strains and the laboratory-engineered E. coli K and B strains. Ten of the clinical isolates of E. coli were resistant to antibiotics. At a multiplicity of infection of 10, 3, 1, or 0.3, the phage caused rapid cell lysis within 2 h, resulting in 5- to 10-fold reductions in cell concentration. Sequencing of the JS09 genome revealed a 169.148-kb linear but circularly permuted and terminally redundant dsDNA with 37.98% G+C content. Two hundred seventy-three open reading frames were predicted to be coding sequences, 135 of which were functionally defined and organized in a modular format which includes modules for DNA replication, DNA packaging, structural proteins, and host cell lysis proteins. Phage JS09 is assigned to the Caudovirales order (Myoviridae phage family), and it is considered a T4-like phage based on its morphological, genomic, and growth characteristics. JS09 gp37, a receptor-binding protein (RBP) important for host cell infection, shares little homology with other RBP in the NCBI database, which suggests that the variable regions in gp37 determine the unique host range of phage JS09. Protein sequence comparisons cluster the putative 'RBP' of JS09 much more closely with those of Yersinia phage phiD1, phage TuIa, and phage TuIb. CONCLUSIONS: A novel lytic coliphage named JS09 was isolated from sewage samples of a swine farm in Jiangsu Province, China. It could infect antibiotic-resistant APEC and ETEC. The morphological, genomic, and growth characteristics of JS09 were studied, and this will be helpful for phage therapy in controlling diseases caused by APEC and ETEC.

Characterization of the morphology and genome of an Escherichia coli podovirus.

Escherichia coli is an important opportunistic pathogen. It can cause sepsis and severe infection. The application of lytic bacteriophages to treat infectious diseases is an alternative to antibiotics. A lytic Escherichia coli phage, designated IME-EC2, was isolated from hospital sewage. Transmission electron microscopy revealed that IME-EC2 to be a member of the family Podoviridae. It had a 60-nm head and a 15-nm tail. Here, we present the complete genome sequence of this phage, which consists of 41,510 bp with an overall G+C content of 59.2 %. A total of 60 coding sequences (CDS) were identified, and the phage genome does not contain any tRNA genes. Forty percent of the unknown CDSs are unique to IME-EC2. This phage does not show significant similarity to other phages at the DNA level, which suggests that IME-EC2 could be a novel phage. One of the unique features identified in the IME-EC2 genome was a gene coding for a putative colanic-acid-degrading protein, which could allow the phage to degrade bacterial capsule and biofilms. Another unique feature is that IME-EC2 does not contain a terminase small subunit, which suggests that this phage may have a unique packaging mechanism. The present work provides novel information on phages and shows that this lytic phage or its products could be exploited to destroy bacterial biofilms and pathogenic E. coli.

Differing populations of endemic bacteriophages in cattle shedding high and low numbers of Escherichia coli O157:H7 bacteria in feces.

The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to Escherichia coli O157:H7 from cattle shedding high and low numbers of naturally occurring E. coli O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ≥ 10(4) CFU · g(-1) of E. coli O157:H7 in feces at a single time point were identified as supershedders (SS; n = 6), and cattle excreting <10(4) CFU · g(-1) of feces were identified as low shedders (LS; n = 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks. E. coli O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of E. coli O157:H7 isolated from feces was 14.4% and did not differ between LS and SS (P = 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%; P = 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting E. coli O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle.

Isolation, characterization and complete genome sequence of PhaxI: a phage of Escherichia coli O157 : H7.

Bacteriophages are considered as promising biological agents for the control of infectious diseases. Sequencing of their genomes can ascertain the absence of antibiotic resistance, toxin or virulence genes. The anti-O157 : H7 coliphage, PhaxI, was isolated from a sewage sample in Iran. Morphological studies by transmission electron microscopy showed that it has an icosahedral capsid of 85-86 nm and a contractile tail of 115×15 nm. PhaxI contains dsDNA composed of 156 628 nt with a G+C content of 44.5 mol% that encodes 209 putative proteins. In MS analysis of phage particles, 92 structural proteins were identified. PhaxI lyses Escherichia coli O157 : H7 in Luria-Bertani medium and milk, has an eclipse period of 20 min and a latent period of 40 min, and has a burst size of about 420 particles per cell. PhaxI is a member of the genus 'Viunalikevirus' of the family Myoviridae and is specific for E. coli O157 : H7.

Endemic bacteriophages: a cautionary tale for evaluation of bacteriophage therapy and other interventions for infection control in animals.

BACKGROUND: One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. METHODS: During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar "rogue" phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. RESULTS: The "rogue" phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152x8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the "T1-like viruses" isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of "rogue" phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.

Biosafety evaluation of bacteriophages for treatment of diarrhea due to intestinal pathogen Escherichia coli 3-2 infection of chickens.

Intestinal Escherichia coli caused diarrhea in chicken makes serious damage directly to the chicken culture industry. Bacteriophage therapy is able to control the diarrhea in chickens effectively. In this study, the biosafety of bacteriophages was evaluated for treating intestinal pathogenic E. coli, which induced diarrhea in chickens. Ten bacteriophages were isolated from feces of chickens with diarrhea using the ill-chicken intestinal pathogenic E. coli 3-2 as target organism. Three bacteriophages propagated on E. coli 3-2 with relative big and clear plaques were selected and used together for toxicity experiment and evaluating the effect of therapy on chicken weight gain. In 3 weeks of trial, no mice given with or without mixed bacteriophages died, and the weight of mice of the experimental group did not show significant difference to the control group after 3 weeks infection. Besides remarkable decreasing the death rate of chickens with diarrhea, treatment of mixed bacteriophages also promoted the weight gain and saved the diet consumption as the utilize rate of diet increased 11% compared with the control group. These observations indicated that a mixture of three bacteriophages would be biosafe for rapid and effective preventing pathogenic E. coli infections.

A suggested new bacteriophage genus: "Viunalikevirus".

We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.