Phage Group Research Articles

MP05-12 IN VITRO EFFECT OF E.COLI PHAGES ON E.COLI BIOFILM MODEL BUILT ON URETERAL STENT

You have accessJournal of UrologyCME1 Apr 2023MP05-12 IN VITRO EFFECT OF E.COLI PHAGES ON E.COLI BIOFILM MODEL BUILT ON URETERAL STENT Leonid Boyarsky, Ortal Yerushalmi, Ronen Hazan, Amitay Lorber, Vladimir Yutkin, Ofer Gofrit, and Mordechai Duvdevani Leonid BoyarskyLeonid Boyarsky More articles by this author , Ortal YerushalmiOrtal Yerushalmi More articles by this author , Ronen HazanRonen Hazan More articles by this author , Amitay LorberAmitay Lorber More articles by this author , Vladimir YutkinVladimir Yutkin More articles by this author , Ofer GofritOfer Gofrit More articles by this author , and Mordechai DuvdevaniMordechai Duvdevani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To assess, in vitro, the ability of E.coli phages to eradicate biofilms built on ureteral stents with an intention to develop an additional treatment option for chronic urinary infection in patients with indwelling ureteral stent. METHODS: Phages were grown on isolated E.coli cultures. Phage host range of infection was determined with plaque assay method. The urine stability of phages was assessed. Biofilm model was built separately on 96 well plates and on ureteral stent. Following biofilm infection, the phage lytic with and without ampicillin was assessed by colony forming units (CFU) test and scanning electron microscopy (SEM). RESULTS: Ampicillin resistant, E.coli strain O5 was found as sensitive to a group of phages designated ECOY 1-8 and was therefore selected as a study strain. ECOY5 was found to be stable in urine and was selected as a study phage. In the study group, the CFU test showed 10- fold and 100- fold decrease in E.coli CFU growth for well plate and stent models respectively. The lytic effect of phage treatment was visualized on SEM images. CONCLUSIONS: Use of the ECYO5 phage alone or in combination with ampicillin appears to be very effective in breaking down the biofilm of E.coli. Further research is needed to develop phage-based products for stent associated infections including other uropathogens. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e48 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Leonid Boyarsky More articles by this author Ortal Yerushalmi More articles by this author Ronen Hazan More articles by this author Amitay Lorber More articles by this author Vladimir Yutkin More articles by this author Ofer Gofrit More articles by this author Mordechai Duvdevani More articles by this author Expand All Advertisement PDF downloadLoading ...

Isolation of phages infecting the abundant freshwater Actinobacteriota order ‘Ca. Nanopelagicales’

Low-GC Actinobacteriota of the order ‘Ca. Nanopelagicales’ (also known as acI or hgcI clade) are abundant in freshwaters around the globe. Extensive predation pressure by phages has been assumed to be the reason for their high levels of microdiversity. So far, however, only a few metagenome-assembled phages have been proposed to infect them and no phages have been isolated. Taking advantage of recent advances in the cultivation of ‘Ca. Nanopelagicales’ we isolated a novel species of its genus ‘Ca. Planktophila’. Using this isolate as bait, we cultivated the first two phages infecting this abundant bacterial order. Both genomes contained a whiB-like transcription factor and a RNA polymerase sigma-70 factor, which might aid in manipulating their host’s metabolism. Both phages encoded a glycosyltransferase and one an anti-restriction protein, potential means to evade degradation of their DNA by nucleases present in the host genome. The two phage genomes shared only 6% of their genome with their closest relatives, with whom they form a previously uncultured family of actinophages within the Caudoviricetes. Read recruitment analyses against globally distributed metagenomes revealed the endemic distribution of this group of phages infecting ‘Ca. Nanopelagicales’. The recruitment pattern against metagenomes from the isolation site and the modular distribution of shared genes between the two phages indicate high levels of horizontal gene transfer, likely mirroring the microdiversity of their host in the evolutionary arms race between host and phage.

Detection and Level Evaluation of Antibodies Specific to Environmental Bacteriophage I11mO19 and Related Coliphages in Non-Immunized Human Sera

Bacteriophages (phages) are viruses infecting bacteria. They are widely present in the environment, food, and normal microflora. The human microbiome is a mutually interdependent network of bacteria, bacteriophages, and human cells. The stability of these tri-kingdom interactions may be essential for maintaining immunologic and metabolic health. Phages, as with each other's antigens, may evoke an immune response during a human's lifetime and induce specific antibody generation. In this manuscript, we labeled these antibodies as naturally generated. Naturally generated antibodies may be one of the most important factors limiting the efficacy of phage therapy. Herein, we attempted to determine the physiological level of these antibodies specific to a population bacteriophage named I11mO19 in human sera, using an ELISA-based assay. First, we purified the phage particles and assessed the immunoreactivity of phage proteins. Then, affinity chromatography was performed on columns with immobilized phage proteins to obtain a fraction of human polyclonal anti-phage antibodies. These antibodies were used as a reference to elaborate an immunoenzymatic test that was used to determine the level of natural anti-phage antibodies. We estimated the average level of anti-I11mO19 phage antibodies at 190 µg per one milliliter of human serum. However, immunoblotting revealed that cross-reactivity occurs between some proteins of I11mO19 and two other coliphages: T4 and ΦK1E. The antigens probably share common epitopes, suggesting that the determined level of anti-I11mO19 phage might be overestimated and reflects a group of antibodies reactive to a broad range of other E. coli phages. Anti-I11mO19 antibodies did not react with Pseudomonas bacteriophage F8, confirming specificity to the coliphage group. In this work, we wanted to show whether it is possible to determine the presence and level of anti-phage antibodies in nontargeted-immunized sera, using an immunoenzymatic assay. The conclusion is that it is possible, and specific antibodies can be determined. However, the specificity refers to a broader coliphage group of phages, not only the single phage strain.

Genomic Analysis of Two Novel Bacteriophages Infecting Acinetobacter beijerinckii and halotolerans Species.

Bacteriophages are the most diverse genetic entities on Earth. In this study, two novel bacteriophages, nACB1 (Podoviridae morphotype) and nACB2 (Myoviridae morphotype), which infect Acinetobacter beijerinckii and Acinetobacter halotolerans, respectively, were isolated from sewage samples. The genome sequences of nACB1 and nACB2 revealed that their genome sizes were 80,310 bp and 136,560 bp, respectively. Comparative analysis showed that both genomes are novel members of the Schitoviridae and the Ackermannviridae families, sharing ≤ 40% overall nucleotide identities with any other phages. Interestingly, among other genetic features, nACB1 encoded a very large RNA polymerase, while nACB2 displayed three putative depolymerases (two capsular depolymerases and one capsular esterase) encoded in tandem. This is the first report of phages infecting A. halotolerans and beijerinckii human pathogenic species. The findings regarding these two phages will allow us to further explore phage-Acinetobacter interactions and the genetic evolution for this group of phages.

PCR Assay for Rapid Taxonomic Differentiation of Virulent Staphylococcus aureus and Klebsiella pneumoniae Bacteriophages.

Phage therapy is now seen as a promising way to overcome the current global crisis in the spread of multidrug-resistant bacteria. However, phages are highly strain-specific, and in most cases one will have to isolate a new phage or search for a phage suitable for a therapeutic application in existing libraries. At an early stage of the isolation process, rapid screening techniques are needed to identify and type potential virulent phages. Here, we propose a simple PCR approach to differentiate between two families of virulent Staphylococcus phages (Herelleviridae and Rountreeviridae) and eleven genera of virulent Klebsiella phages (Przondovirus, Taipeivirus, Drulisvirus, Webervirus, Jiaodavirus, Sugarlandvirus, Slopekvirus, Jedunavirus, Marfavirus, Mydovirus and Yonseivirus). This assay includes a thorough search of a dataset comprising S. aureus (n = 269) and K. pneumoniae (n = 480) phage genomes available in the NCBI RefSeq/GenBank database for specific genes that are highly conserved at the taxonomic group level. The selected primers showed high sensitivity and specificity for both isolated DNA and crude phage lysates, which permits circumventing DNA purification protocols. Our approach can be extended and applied to any group of phages, given the large number of available genomes in the databases.

Diverse Marine T4-like Cyanophage Communities Are Primarily Comprised of Low-Abundance Species Including Species with Distinct Seasonal, Persistent, Occasional, or Sporadic Dynamics.

Cyanophages exert important top-down controls on their cyanobacteria hosts; however, concurrent analysis of both phage and host populations is needed to better assess phage-host interaction models. We analyzed picocyanobacteria Prochlorococcus and Synechococcus and T4-like cyanophage communities in Pacific Ocean surface waters using five years of monthly viral and cellular fraction metagenomes. Cyanophage communities contained thousands of mostly low-abundance (<2% relative abundance) species with varying temporal dynamics, categorized as seasonally recurring or non-seasonal and occurring persistently, occasionally, or sporadically (detected in ≥85%, 15-85%, or <15% of samples, respectively). Viromes contained mostly seasonal and persistent phages (~40% each), while cellular fraction metagenomes had mostly sporadic species (~50%), reflecting that these sample sets capture different steps of the infection cycle-virions from prior infections or within currently infected cells, respectively. Two groups of seasonal phages correlated to Synechococcus or Prochlorococcus were abundant in spring/summer or fall/winter, respectively. Cyanophages likely have a strong influence on the host community structure, as their communities explained up to 32% of host community variation. These results support how both seasonally recurrent and apparent stochastic processes, likely determined by host availability and different host-range strategies among phages, are critical to phage-host interactions and dynamics, consistent with both the Kill-the-Winner and the Bank models.

Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an Nλ-like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtainedonly when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1.

Characteristics of Environmental Klebsiella pneumoniae and Klebsiella oxytoca Bacteriophages and Their Therapeutic Applications.

In recent years, multidrug-resistant (MDR) strains of Klebsiella pneumoniae have spread globally, being responsible for the occurrence and severity of nosocomial infections. The NDM-1-kp, VIM-1 carbapenemase-producing isolates as well as extended-spectrum beta lactamase-producing (ESBL) isolates along with Klebsiella oxytoca strains have become emerging pathogens. Due to the growing problem of antibiotic resistance, bacteriophage therapy may be a potential alternative to combat such multidrug-resistant Klebsiella strains. Here, we present the results of a long-term study on the isolation and biology of bacteriophages active against K. pneumoniae, as well as K. oxytoca strains. We evaluated biological properties, morphology, host specificity, lytic spectrum and sensitivity of these phages to chemical agents along with their life cycle parameters such as adsorption, latent period, and burst size. Phages designated by us, vB_KpnM-52N (Kpn52N) and VB_KpnM-53N (Kpn53N), demonstrated relatively broad lytic spectra among tested Klebsiella strains, high burst size, adsorption rates and stability, which makes them promising candidates for therapeutic purposes. We also examined selected Klebsiella phages from our historical collection. Notably, one phage isolated nearly 60 years ago was successfully used in purulent cerebrospinal meningitis in a new-born and has maintained lytic activity to this day. Genomic sequences of selected phages were determined and analyzed. The phages of the sequenced genomes belong to the Slopekvirus and Jiaodavirus genus, a group of phages related to T4 at the family level. They share several features of T4 making them suitable for antibacterial therapies: the obligatorily lytic lifestyle, a lack of homologs of known virulence or antibiotic resistance genes, and a battery of enzymes degrading host DNA at infection.

The Evolutionary History of a DNA Methylase Reveals Frequent Horizontal Transfer and Within-Gene Recombination.

Inteins, often referred to as protein introns, are highly mobile genetic elements that invade conserved genes throughout the tree of life. Inteins have been found to invade a wide variety of key genes within actinophages. While in the process of conducting a survey of these inteins in actinophages, we discovered that one protein family of methylases contained a putative intein, and two other unique insertion elements. These methylases are known to occur commonly in phages as orphan methylases (possibly as a form of resistance to restriction-modification systems). We found that the methylase family is not conserved within phage clusters and has a disparate distribution across divergent phage groups. We determined that two of the three insertion elements have a patchy distribution within the methylase protein family. Additionally, we found that the third insertion element is likely a second homing endonuclease, and that all three elements (the intein, the homing endonuclease, and what we refer to as the ShiLan domain) have different insertion sites that are conserved in the methylase gene family. Furthermore, we find strong evidence that both the intein and ShiLan domain are partaking in long-distance horizontal gene transfer events between divergent methylases in disparate phage hosts within the already dispersed methylase distribution. The reticulate evolutionary history of methylases and their insertion elements reveals high rates of gene transfer and within-gene recombination in actinophages.

Phages for treatment of Escherichia coli infections.

Diseases due to infections by pathogenic Escherichia coli strains are on the rise and with the growing antimicrobial resistance among bacterial pathogens, including this group. Thus, alternative therapeutic options are actively investigated. Among these alternatives is phage therapy. In the case of E. coli, the combination of the well understood biology of this species and its bacteriophages represents a good guiding example for the establishment of phage therapy principles against this and other pathogenic bacteria. In this chapter, the procedures toward the development of phage therapy against pathogenic E. coli with the use of T-even group of phages are discussed. These steps involve the isolation, purification, characterisation and large-scale production of these phages, with formulation of phage cocktails for in vitro and in vivo studies. The main emphasis is made on phage therapy of enteropathogenic E. coli O157:H, which is one of the prominent human pathogens but persists as a commensal bacterium in many food animals. The implementation of phage therapy against E. coli O157:H within the One Health framework in carrier animals and for treatment of meat, vegetables, fruits and other agricultural produce thus would allow controlling and interrupting the transmission routes of this pathogen to the human food chain and preventing human disease. Examples of successful control and elimination of E. coli O157:H are given, while the problems encountered in phage treatment of this pathogen are also discussed.

Comparison of bacterial suppression by phage cocktails, dual-receptor generalists, and coevolutionarily trained phages.

The evolution and spread of antibiotic-resistant bacteria have renewed interest in phage therapy, the use of bacterial viruses (phages) to combat bacterial infections. The delivery of phages in cocktails where constituent phages target different modalities (e.g., receptors) may improve treatment outcomes by making it more difficult for bacteria to evolve resistance. However, the multipartite nature of cocktails may lead to unintended evolutionary and ecological outcomes. Here, we compare a 2-phage cocktail with a largely unconsidered group of phages: generalists that can infect through multiple, independent receptors. We find that λ phage generalists and cocktails that target the same receptors (LamB and OmpF) suppress Escherichia coli similarly for ~2 days. Yet, a "trained" generalist phage, which previously adapted to its host via 28 days of coevolution, demonstrated superior suppression. To understand why the trained generalist was more effective, we measured the resistance of bacteria against each of our phages. We find that, when bacteria were assailed by two phages in the cocktail, they evolved mutations in manXYZ, a host inner-membrane transporter that λ uses to move its DNA across the periplasmic space and into the cell for infection. This provided cross-resistance against the cocktail and untrained generalist. However, these mutations were ineffective at blocking the trained generalist because, through coevolutionary training, it evolved to bypass manXYZ resistance. The trained generalist's past experiences in training make it exceedingly difficult for bacteria to evolve resistance, further demonstrating the utility of coevolutionary phage training for improving the therapeutic properties of phages.

Broad host range may be a key to long-term persistence of bacteriophages infecting intestinal Bacteroidaceae species

The longitudinal studies have found that the human gut microbiota is stable over time with some major bacterial lineages or even strains persisting for years. This was recently extended to gut bacteriophages using the metagenomic data. Here, we focused on cultivation of the major Bacteroidetes of human gut, the Bacteroides and Phocaeicola strains, and their bacteriophages from two healthy donors. The persistence of Bacteroides and Phocaeicola species and strains was confirmed. We isolated 28 genetically different phages grouped into seven distinct clusters, two of these were new. Moreover, the bacteriophages from several groups, although being genetically quite homogeneous, had the ability to infect the strains belonging to different species isolated from several sampling time-points and different donors. We propose that the ability to infect several host species, which differ in their nutritional niches, may promote long-term persistence of dominant gut bacteriophage groups.

Recombination Events in Putative Tail Fibre Gene in Litunavirus Phages Infecting Pseudomonas aeruginosa and Their Phylogenetic Consequences.

Recombination is the main driver of bacteriophage evolution. It may serve as a tool for extending the phage host spectrum, which is significant not only for phages' ecology but also for their utilisation as therapeutic agents of bacterial infections. The aim of this study was to detect the recombination events in the genomes of Litunavirus phages infecting Pseudomonas aeruginosa, and present their impact on phylogenetic relations within this phage group. The phylogenetic analyses involved: the whole-genome, core-genome (Schitoviridae conserved genes), variable genome region, and the whole-genome minus variable region. Interestingly, the recombination events taking place in the putative host recognition region (tail fibre protein gene and the adjacent downstream gene) significantly influenced tree topology, suggesting a strong phylogenetic signal. Our results indicate the recombination between phages from two genera Litunavirus and Luzeptimavirus and demonstrate its influence on phage phylogeny.

Tyroviruses are a new group of temperate phages that infect Bacillus species in soil environments worldwide

BackgroundBacteriophages are widely considered to be highly abundant and genetically diverse, with their role in the evolution and virulence of many pathogens becoming increasingly clear. Less attention has been paid on phages preying on Bacillus, despite the potential for some of its members, such as Bacillus anthracis, to cause serious human disease.ResultsWe have isolated five phages infecting the causative agent of anthrax, Bacillus anthracis. Using modern phylogenetic approaches we place these five new Bacillus phages, as well as 21 similar phage genomes retrieved from publicly available databases and metagenomic datasets into the Tyrovirus group, a newly proposed group named so due to the conservation of three distinct tyrosine recombinases. Genomic analysis of these large phages (~ 160–170 kb) reveals their DNA packaging mechanism and genomic features contributing to virion morphogenesis, host cell lysis and phage DNA replication processes. Analysis of the three tyrosine recombinases suggest Tyroviruses undergo a prophage lifecycle that may involve both host integration and plasmid stages. Further we show that Tyroviruses rely on divergent invasion mechanisms, with a subset requiring host S-layer for infection.ConclusionsUltimately, we expand upon our understanding on the classification, phylogeny, and genomic organisation of a new and substantial phage group that prey on critically relevant Bacillus species. In an era characterised by a rapidly evolving landscape of phage genomics the deposition of future Tyroviruses will allow the further unravelling of the global spread and evolutionary history of these Bacillus phages.

Co-transfer of functionally interdependent genes contributes to genome mosaicism in lambdoid phages.

Lambdoid (or Lambda-like) phages are a group of related temperate phages that can infect Escherichia coli and other gut bacteria. A key characteristic of these phages is their mosaic genome structure, which served as the basis for the 'modular genome hypothesis'. Accordingly, lambdoid phages evolve by transferring genomic regions, each of which constitutes a functional unit. Nevertheless, it is unknown which genes are preferentially transferred together and what drives such co-transfer events. Here we aim to characterize genome modularity by studying co-transfer of genes among 95 distantly related lambdoid (pro-)phages. Based on gene content, we observed that the genomes cluster into 12 groups, which are characterized by a highly similar gene content within the groups and highly divergent gene content across groups. Highly similar proteins can occur in genomes of different groups, indicating that they have been transferred. About 26 % of homologous protein clusters in the four known operons (i.e. the early left, early right, immunity and late operon) engage in gene transfer, which affects all operons to a similar extent. We identified pairs of genes that are frequently co-transferred and observed that these pairs tend to be near one another on the genome. We find that frequently co-transferred genes are involved in related functions and highlight interesting examples involving structural proteins, the cI repressor and Cro regulator, proteins interacting with DNA, and membrane-interacting proteins. We conclude that epistatic effects, where the functioning of one protein depends on the presence of another, play an important role in the evolution of the modular structure of these genomes.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Biological and genomic characteristics of two bacteriophages isolated from sewage, using one multidrug-resistant and one non-multidrug-resistant strain of Klebsiella pneumoniae.

Bovine mastitis caused by multi-drug resistant (MDR) Klebsiella pneumoniae is difficult to treat with antibiotics, whereas bacteriophages may be a viable alternative. Our objective was to use 2 K. pneumoniae strains, 1 MDR and the other non-MDR, to isolate phages from sewage samples and compare their biological and genomic characteristics. Additionally, phage infected mouse mammary gland was also analyzed by H&E staining and ELISA kits to compare morphology and inflammatory factors, respectively. Based on assessments with double agar plates and transmission electron microscopy, phage CM_Kpn_HB132952 had clear plaques surrounded by translucent halos on the bacterial lawn of K. pneumoniae KPHB132952 and belonged to Siphoviridae, whereas phage CM_Kpn_HB143742 formed a clear plaque on the bacterial lawn of K. pneumoniae KPHB143742 and belonged to Podoviridae. In 1-step growth curves, CM_Kpn_HB132952 and CM_Kpn_HB143742 had burst sizes of 0.34 and 0.73 log10 PFU/mL, respectively. The former had a latent period of 50 min and an optimal multiplicity of infection (MOI) of 0.01, whereas for the latter, the latent period was 30 min (MOI = 1). Phage CM_Kpn_HB132952 had better thermal and acid–base stability than phage CM_Kpn_HB143742. Additionally, both phages had the same host range rate but different host ranges. Based on Illumina NovaSeq, phages CM_Kpn_HB132952 and CM_Kpn_HB143742 had 140 and 145 predicted genes, respectively. Genomic sequencing and phylogenetic tree analysis indicated that both phages were novel phages belonging to the Klebsiella family. Additionally, the histopathological structure and inflammatory factors TNF-α and IL-1β were not significantly different among phage groups and the control group. In conclusion, using 1 MDR and 1 non-MDR strain of K. pneumoniae, we successfully isolated two phages from the same sewage sample, and demonstrated that they had distinct biological and genomic characteristics.

Hybrid Vigor: Importance of Hybrid λ Phages in Early Insights in Molecular Biology.

Laboratory-generated hybrids between phage λ and related phages played a seminal role in establishment of the λ model system, which, in turn, served to develop many of the foundational concepts of molecular biology, including gene structure and control. Important λ hybrids with phages 21 and 434 were the earliest of such phages. To understand the biology of these hybrids in full detail, we determined the complete genome sequences of phages 21 and 434. Although both genomes are canonical members of the λ-like phage family, they both carry unsuspected bacterial virulence gene types not previously described in this group of phages. In addition, we determined the sequences of the hybrid phages λ imm21, λ imm434, and λ h434 imm21. These sequences show that the replacements of λ DNA by nonhomologous segments of 21 or 434 DNA occurred through homologous recombination in adjacent sequences that are nearly identical in the parental phages. These five genome sequences correct a number of errors in published sequence fragments of the 21 and 434 genomes, and they point out nine nucleotide differences from Sanger's original λ sequence that are likely present in most extant λ strains in laboratory use today. We discuss the historical importance of these hybrid phages in the development of fundamental tenets of molecular biology and in some of the earliest gene cloning vectors. The 434 and 21 genomes reinforce the conclusion that the genomes of essentially all natural λ-like phages are mosaics of sequence modules from a pool of exchangeable segments.

Two Newly Isolated Enterobacter-Specific Bacteriophages: Biological Properties and Stability Studies.

In an era of antibiotic therapy crisis caused by spreading antimicrobial resistance, and when recurrent urinary tract infections constitute a serious social and medical problem, the isolation and complex characterization of phages with a potential therapeutic application represents a promising solution. It is an inevitable, and even a necessary direction in the development of current phage research. In this paper, we present two newly isolated myoviruses that show lytic activity against multidrug-resistant clinical isolates of Enterobacter spp. (E. cloacae, E. hormaechei, and E. kobei), the genomes of which belong to a poorly represented phage group. Both phages were classified as part of the Tevenvirinae subfamily (Entb_43 was recognized as Karamvirus and Entb_45 as Kanagawavirus). Phage lytic spectra ranging from 40 to 60% were obtained. The most effective phage-to-bacteria ratios (MOI = 0.01 and MOI = 0.001) for both the phage amplification and their lytic activity against planktonic bacteria were also estimated. Complete adsorption to host cells were obtained after about 20 min for Entb_43 and 10 min for Entb_45. The phage lysates retained their initial titers even during six months of storage at both −70 °C and 4 °C, whereas storage at 37 °C caused a complete loss in their activity. We showed that phages retained their activity after incubation with solutions of silver and copper nanoparticles, which may indicate possible synergistic antibacterial activity. Moreover, a significant reduction in phage titers was observed after incubation with a disinfectant containing octenidinum dihydrochloridum and phenoxyethanol, as well as with 70% ethanol. The observed maintenance of phage activity during incubation in a urine sample, along with other described properties, may suggest a therapeutic potential of phages at the infection site after intravesical administration.

Metagenomic analysis of wastewater phageome from a University Hospital in Turkey.

Phage DNA analysis gives opportunity to understand living ecosystem of the environment where the samples are taken. In the present study, we analyzed phage DNA obtained from wastewater sample of university hospital sewage. After filtration, long high-speed centrifugation was done to collect phages. DNA was extracted from pellet by phenol chloroform extraction and used for NGS sequencing. The host profile, taxonomic and functional analyses were performed using MG-RAST, and ResFinder program was used for resistance gene detection. High amounts of reads belong to bacteriophage groups (~ 95%) from our DNA sample were obtained and all bacteriophage reads were found belonging to Caudovirales order and Myoviridae (56%), Siphoviridae (43%), and Podoviridae (0.02%) families. The most common host genera were Escherichia (88.20%), Salmonella (5.49%) and Staphylococcus (5.19%). SEED subsystems hits were mostly structural parts and KEGG Orthology hits were nucleotide- and carbohydrate metabolism-related genes. No anti-microbial resistance genes were detected. Our bacteriophage DNA purification method is favorable for phage metagenomic studies. Dominance of coliphages may explain infrequent Podoviridae. Dominancy of structural genes and auxiliary genes is probably due to abundance of lytic phages in our sample. Absence of antibiotic resistance genes even in hospital environment phages indicates that phages are not important carrier of resistance genes.