Characterization and genome sequence of the genetically unique Escherichia bacteriophage vB_EcoM_IME392.

In this study, a novel Escherichia coli-specific bacteriophage, vB_EcoM_IME392, was isolated from chicken farm sewage in Qingdao, China. The genome of IME392 was found by next-generation sequencing to be 116,460 base pairs in length with a G+C content of 45.4% (GenBank accession number MH719082). BLASTn results revealed that only 2% of the genome sequence of IME392 shows sequence similarity to known phage sequences in the GenBank database, which indicates that IME392 is a novel bacteriophage. Transmission electron microscopy showed that IME392 belongs to the family Myoviridae. The host range, the multiplicity of infection, and a one-step growth curve were also determined.

Pathogenic Escherichia coli-Specific Bacteriophages and Polyvalent Bacteriophages in Piglet Guts with Increasing Coliphage Numbers after Weaning.

Postweaning diarrhea in pigs is mainly caused by pathogenic Escherichia coli and is a major source of revenue loss to the livestock industry. Bacteriophages dominate the gut virome and have the potential to regulate bacterial communities and thus influence the intestinal physiology. To determine the biological characterization of intestinal coliphages, we isolated and identified the fecal coliphages of healthy preweaned and postweaned piglets from the Nanjing and Chuzhou pig farms. First, ahead of coliphage isolation, 87 E. coli strains were isolated from healthy or diarrheal fecal samples from three pig farms, of which 8 were pathogenic strains, including enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC). Of the E. coli strains, 87.3% possessed drug resistance to three antibiotics. Using these 87 E. coli strains as indicator hosts, we isolated 45 coliphages and found a higher abundance in the postweaning stage than in the preweaning stage (24 versus 17 in the Nanjing and 13 versus 4 in the Chuzhou farm). Furthermore, each farm had a single most-prevalent coliphage strain. Pathogenic E. coli-specific bacteriophages were commonly detected (9/10 samples in the Nanjing farm and 7/10 in the Chuzhou farm) in guts of sampled piglets, and most had significant bacteriostatic effects (P < 0.05) on pathogenic E. coli strains. Three polyvalent bacteriophages (N24, N30, and C5) were identified. The N30 and C5 strains showed a genetic identity of 89.67%, with mild differences in infection characteristics. Our findings suggest that pathogenic E. coli-specific bacteriophages as well as polyvalent bacteriophages are commonly present in piglet guts and that weaning is an important event that affects coliphage numbers. IMPORTANCE Previous studies based on metagenomic sequencing reported that gut bacteriophages profoundly influence gut physiology but did not provide information regarding the host range and biological significance. Here, we screened coliphages from the guts of preweaned and postweaned piglets against indicator hosts, which allowed us to identify the pathogenic E. coli-specific bacteriophages and polyvalent bacteriophages in pig farms and quantify their abundance. Our approach complements sequencing methods and provides new insights into the biological characterizations of bacteriophage in the gut along with the ecological effects of intestinal bacteriophages.

Characteristics of Bacteriophage Isolates and Expression of Shiga Toxin Genes Transferred to Non Shiga Toxin-Producing E. coli by Transduction.

A risk analysis of Shiga toxin (Stx)-encoding bacteriophage was carried out by confirming the transduction phage to non-Stx-producing Escherichia coli (STEC) and subsequent expression of the Shiga toxin genes. The virulence factor stx1 was identified in five phages, and both stx1 and stx2 were found in four phages from a total of 19 phage isolates with seven non-O157 STEC strains. The four phages, designated as φNOEC41, φNOEC46, φNOEC47, and φNOEC49, belonged morphologically to the Myoviridae family. The stabilities of these phages to temperature, pH, ethanol, and NaClO were high with some variabilities among the phages. The infection of five non-STEC strains by nine Stx-encoding phages occurred at a rate of approximately 40%. Non-STEC strains were transduced by Stx-encoding phage to become lysogenic strains, and seven convertant strains had stx1 and/or stx2 genes. Only the stx1 gene was transferred to the receptor strains without any deletion. Gene expression of a convertant having both stx1 and stx2 genes was confirmed to be up to 32 times higher for Stx1 in 6% NaCl osmotic media and twice for Stx2 in 4% NaCl media, compared with expression in low-salt environments. Therefore, a new risk might arise from the transfer of pathogenic genes from Stx-encoding phages to otherwise harmless hosts. Without adequate sterilization of food exposed to various environments, there is a possibility that the toxicity of the phages might increase.

Lysis of cells of diverse bacteria by l,d-peptidases of Escherichia coli bacteriophages RB43, RB49 and T5.

AIMS: The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance. METHODS AND RESULTS: Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4ß) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts. CONCLUSIONS: Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.

Exploring the Remarkable Diversity of Culturable Escherichia coli Phages in the Danish Wastewater Environment.

Phages drive bacterial diversity, profoundly influencing microbial communities, from microbiomes to the drivers of global biogeochemical cycling. Aiming to broaden our understanding of Escherichiacoli (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.

New insights into the biodiversity of coliphages in the intestine of poultry.

Despite phages' ubiquitous presence and great importance in shaping microbial communities, little is known about the diversity of specific phages in different ecological niches. Here, we isolated, sequenced, and characterized 38 Escherichia coli-infecting phages (coliphages) from poultry faeces to gain a better understanding of the coliphage diversity in the poultry intestine. All phages belonged to either the Siphoviridae or Myoviridae family and their genomes ranged between 44,324 and 173,384 bp, with a G+C content between 35.5 and 46.4%. Phylogenetic analysis was performed based on single "marker" genes; the terminase large subunit, portal protein, and exonucleases, as well as the full draft genomes. Single gene analysis resulted in six distinct clusters. Only minor differences were observed between the different phylogenetic analyses, including branch lengths and additional duplicate or triplicate subclustering. Cluster formation was according to genome size, G+C content and phage subfamily. Phylogenetic analysis based on the full genomes supported these clusters. Moreover, several of our Siphoviridae phages might represent a novel unclassified phage genus. This study allowed for identification of several novel coliphages and provides new insights to the coliphage diversity in the intestine of poultry. Great diversity was observed amongst the phages, while they were isolated from an otherwise similar ecosystem.

Male-Specific and Somatic Coliphage Profiles from Major Aquaculture Areas in Republic of Korea.

Human and animal feces are important sources of various types of microbial contamination in water. Especially, enteric viruses, the major agents of waterborne infection, can attain long-term survival in water environments due to their strong resistance to various environmental factors including pH, salinity, and temperature. Coliphages are promising viral indicators for fecal contamination in water environments. Here, we investigated the seasonal and spatial distribution of male-specific and somatic coliphages in surface water and seawater at three major aquaculture areas, including Goseong Bay, Aphae Island, and Gomso Bay, in Republic of Korea over a period of 1 year. We selected 6 surface water and 14 seawater sampling sites for each study area and collected a total of 480 water samples from March 2014 to February 2015. Overall, surface water samples contained higher occurrences of coliphages than seawater samples. The high coliphage concentrations were detected in spring (March to May 2014). The differences in geographical features and patterns in land usage of the three aquaculture areas may have affected the coliphage concentration and occurrence. Moreover, environmental factors such as cumulative precipitation were strongly correlated with coliphage concentrations. Therefore, we suggest that further longitudinal studies on coliphage concentrations and distributions should be performed to support the application of coliphages in tracking fecal contamination in water.

LamB, OmpC, and the Core Lipopolysaccharide of Escherichia coli K-12 Function as Receptors of Bacteriophage Bp7.

Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions.IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38.

Virulent coliphages in 1-year-old children fecal samples are fewer, but more infectious than temperate coliphages.

Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. Here, we cultivate temperate phages produced by 900 E. coli strains isolated from 648 fecal samples from 1-year-old children and obtain coliphages directly from the viral fraction of the same fecal samples. We find that 63% of strains hosted phages, while 24% of the viromes contain 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.

Aptness of Escherichia coli host strain CB390 to detect total coliphages in Colombia.

Fecal bacteria have been used for more than a century as indicators of fecal contamination in water. In recent years, the monitoring of somatic and F-specific coliphages has been gradually included in guidelines and regulations as an additional parameter to reinforce water safety. The Escherichia coli host strain CB390 was tailored to detect both somatic and F-specific coliphages in a single test. The efficacy of this strain for bacteriophage detection, previously evaluated in Western Europe and North America, was assessed here for the first time in South America. The detection of somatic and F-specific coliphages by the strain CB390, as well as by standardized methods, was performed in drinking and river water and municipal and abattoir wastewaters. No statistical difference was found in the numbers of total coliphages detected by strain CB390 and the sum of somatic and F-specific coliphages determined separately by the standardized ISO methods. The data presented here provide further validation of the effectiveness of the host strain E. coli CB390 for the detection of total coliphages in waters in a single test and demonstrate its suitability for application in upper-middle income countries of the Americas (World Bank category).

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.

Riding the wave of genomics to investigate aquatic coliphage diversity and activity.

Bacteriophages infecting Escherichia coli (coliphages) have been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that is present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterized coliphages from three coastal locations in the United Kingdom and Poland. Comparative genomics and phylogenetic analysis of phage isolates facilitated the identification of putative new species within the genera Rb69virus and T5virus and a putative new genus within the subfamily Tunavirinae. Furthermore, genomic and proteomic analysis combined with host range analysis allowed the identification of a putative tail fibre that is likely responsible for the observed differences in host range of phages vB_Eco_mar003J3 and vB_Eco_mar004NP2.

Evaluation of a Male-Specific DNA Coliphage Persistence Within Eastern Oysters (Crassostrea virginica).

Male-specific coliphages (MSCs) are currently used to assess the virologic quality of shellfish-growing waters and to assess the impact of sewage release or adverse weather events on bivalve shellfish. Since MSC can have either DNA or RNA genomes, and most research has been performed exclusively on RNA MSCs, persistence of M13, a DNA MSC, was evaluated for its persistence as a function of time and temperature within Eastern oysters (Crassostrea virginica). Oysters were individually exposed to seawater containing a total of 1010 to 1012 pfu of M13 for 24 h at 15 °C followed by maintenance in tanks with as many as 21 oysters in continuously UV-sterilized water for up to 6 weeks at either 7, 15, or 22 °C. Two trials for each temperature were performed combining three shucked oysters per time point which were assayed by tenfold serial dilution in triplicate. Initial contamination levels averaged 106.9 and ranged from 106.0 to 107.0 of M13. For oysters held for 3 weeks, log10 reductions were 1.7, 3.8, and 4.2 log10 at 7, 15, and 22 °C, respectively. Oysters held at 7 and 15 °C for 6 weeks showed average reductions of 3.6 and 5.1 log10, respectively, but still retained infectious M13. In total, this work shows that DNA MSC may decline within shellfish in a manner analogous to RNA MSCs.

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.

Use of coliphages to investigate norovirus contamination in a shellfish growing area in Republic of Korea.

A number of severe norovirus outbreaks due to the consumption of contaminated shellfish have been reported recently. In this study, we evaluated the distribution of coliphage densities to determine their efficacy as fecal indicators of enteric viruses, including noroviruses, in water samples collected from a shellfish growing area in Republic of Korea over a period of approximately 1 year. Male-specific and somatic coliphages in water samples were analyzed using the single agar layer method, and norovirus genogroups I and II, which infect mainly humans, were analyzed using duplex reverse transcription quantitative PCR. Male-specific and somatic coliphages were detected widely throughout the study area. Several environmental parameters, including salinity, precipitation, temperature, and wind speed were significantly correlated with coliphage concentrations (P < 0.05). Moreover, the concentrations of male-specific coliphages were positively correlated with the presence of human noroviruses (r = 0.443; P < 0.01). The geospatial analysis with coliphage concentrations using a geographic information system revealed that densely populated residential areas were the major source of fecal contamination. Our results indicate that coliphage monitoring in water could be a useful approach to prevent norovirus contamination in shellfish.

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.

Complete genome sequence of SRT8, a novel T1-like Escherichia coli bacteriophage.

In this study, an Escherichia coli virulent phage, SRT8, was isolated from sewage sludge samples collected from Jinan, Shandong Province, China. The genome of phage SRT8 consists of 49,579 bp with 47.83% G+C content. The phage genome contains 84 putative protein-coding genes, and no rRNA or tRNA genes. Comparative genomics analysis showed that the E. coli phage SRT8 is a member of a new species and belongs to the subfamily Tunavirinae, which includes T1-like phages.

Complete genome sequence of a novel virulent phage ST31 infecting Escherichia coli H21.

More and more virulent phages that are fundamental materials for phage therapy have been isolated, characterized and categorized on GenBank. Phage ST31 infecting Escherichia coli H21 was isolated from wastewater and sequenced using an Illumina Hiseq system. Opening reading frames were identified using PHASTER and predicted using BLASTp analysis. Genomic analyses revealed that this was a virulent phage containing a circular double-stranded DNA and that the complete genome consisted of 39,693 nucleotides with an average GC content of 49.98 %. This study may provide possible alternative materials for phage therapy.

Preliminary Source Tracking of Male-Specific (F+) RNA Coliphage on Lettuce as a Surrogate of Enteric Viruses Using Reverse Transcription-PCR.

The aim of this research was to preliminary track fecal source male-specific F+RNA coliphages including human and animals in lettuce. At first, two published virus extraction procedures of ultracentrifugation and PEG precipitation were compared using DAL assay for determining the recovery efficiency in lettuce spiked artificially with three concentrations (102, 104, 106 pfu/100 ml) of MS2 coliphage. The results showed that PEG precipitation had the highest recovery in which the recovery efficiency at the spiked level of 106 pfu/100 ml was 16.63 %. Aqueous phase obtained from the final step of PEG method was applied for enumeration of coliphage and viral RNA extraction in naturally contaminated lettuce samples (N = 30) collected from two sources (market and farm). The samples were then analyzed based on (I, II, III, and IV primer sets) using RT-PCR method. Coliphages were detected in 9 (60 %) and 12 (80 %) out of 15 market and farm samples, respectively, using DAL assay, whereas male-specific F+RNA coliphages were detected using the RT-PCR method in 9 (60 %) and 13 (86.6 %) out of 15 samples of market and farm, respectively. Based on the results, only genotype I of male-specific F+RNA coliphages was detected in lettuce samples and no sample tested was positive for other genotypes (II, III, and IV).