The lambda - P22 problem.

Lambda and P22 are members of 2 families of tailed phages and have limited genomic relationships. Both form hybrids with many phages. P22 appears as a hybrid of mixed ancestry. Despite their similarities, lambda and P22 and their relatives form 2 distinct lineages and must be classified separately.

Feces of feedlot cattle contain a diversity of bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli.

This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.

Leuconostoc bacteriophages from blue cheese manufacture: long-term survival, resistance to thermal treatments, high pressure homogenization and chemical biocides of industrial application.

Nine Leuconostoc mesenteroides phages were isolated during blue cheese manufacture yielding faulty products with reduced eye formation. Their morphologies, restriction profiles, host ranges and long-term survival rates (25°C, 8°C, -20°C and -80°C) were analysed. Based on restriction analysis, six of them were further examined regarding resistance to physical (heat and high pressure homogenization, HPH) and chemical treatments (ethanol, sodium hypochlorite, peracetic acid, biocides A, C, E and F). According to their morphology, L. mesenteroides phages studied in the present work belonged to the Caudovirales order and Siphoviridae family. Six distinct restriction patterns were obtained with EcoRV, HindIII, ClaI and XhoI enzymes, revealing interesting phage diversity in the dairy environment. No significant reductions in phage counts were observed after ten months of storage at -20°C and -80°C, while slightly and moderate decrease in phage numbers were noticed at 8°C and 25°C, respectively. The phages subjected to heat treatments generally showed high resistance at 63°C and moderate resistance at 72°C. However, 80°C for 30 min and 90°C for 2 min led to complete inactivation of viral particles. In general, the best ethanol concentration tested was 75%, as complete inactivation for most Leuconostoc phages within 30 min of incubation was achieved. Peracetic acid, and biocides A, C, E and F were highly effective when used at the same or at a moderately lower concentration as recommended by the producer. Usually, moderate or high concentrations (600-1,600 ppm) of sodium hypochlorite were necessary to completely inactivate phage particles. Leuconostoc phages were partially inactivated by HPH treatments as remaining viral particles were found even after 8 passes at 100 MPa. This is the first report of L. mesenteroides phages isolated from an Argentinean dairy cheese plant. The results of this work could be useful for establishing the most effective physical and chemical treatments for inactivating phages in industrial plants and laboratory environments.

A giant Pseudomonas phage from Poland.

A novel giant phage of the family Myoviridae is described. Pseudomonas phage PA5oct was isolated from a sewage sample from an irrigated field near Wroclaw, Poland. The virion morphology indicates that PA5oct differs from known giant phages. The phage has a head of about 131 nm in diameter and a tail of 136 × 19 nm. Phage PA5oct contains a genome of approximately 375 kbp and differs in size from any tailed phages known. PA5oct was further characterized by determination of its latent period and burst size and its sensitivity to heating, chloroform, and pH.

Anna S. Tikhonenko: Electron microscopist extraordinary.

Anna Sergeyevna Tikhonenko (1925-2010) is to be remembered for the excellency of her electron microscopical work, particularly with bacteriophages. She published 113 articles and one book, Ultrastructure of Bacterial Viruses (Izdadelstvo Nauka, Moscow 1968; Plenum Press, New York, 1972). It included 134 micrographs and a complete overview of the 316 phages then examined by electron microscopy. Most micrographs were of exceptional quality. This book, a rarity in those days of strict separation of Soviet and Western research, was the first bacteriophage atlas in the literature and presented a morphological classification of phages into five categories of family level, similar to a scheme presented in 1965 by D.E. Bradley (J Royal Microsc Soc 84:257-316). Her book remains one of the fundamentals of phage research.

Isolation and characterization of a rare waterborne lytic phage of Salmonella enterica serovar Paratyphi B.

A lytic phage of Salmonella serovar Paratyphi B, named φSPB, was isolated from surface waters of the Pavana River in India. Phage φSPB is a member of the Podoviridae family and is morphologically similar to the 7-11 phages of the C3 morphotype of tailed phages, characterized by a very long, cigar-shaped head. The head measured approximately 153 × 57 nm, and the tail size was 12 × 7 nm. The phage was stable over a wide range of pH (4-9) and temperature (4-40 °C). The adsorption rate constant was 4.7 × 10(-10). Latent and eclipse periods were 10 and 15 min, respectively, and the burst size was 100 plaque-forming units/infected cell after 25 min at 37 °C. The phage DNA was 59 kb in size. Ten major proteins were observed on SDS-PAGE, although some of these proteins could be bacterial contaminants. This is the first report of Salmonella enterica subsp. enterica serovar Paratyphi B phage of C3 morphotype from India that has many unique features, such as high replication potential, short replication time, and stability over a wide range of pH and temperature, making it a promising biocontrol agent against the drug-resistant strains of Salmonella Paratyphi B.

Fake virus particles generated by fluorescence microscopy.

Many laboratories are actively studying the abundance and roles of viruses in natural ecosystems. In these studies, the presence and number of viral particles is usually determined using fluorescent dyes. However, DNA associated with membrane-derived vesicles (MVs), gene transfer agents (GTAs), or cell debris can produce fluorescent dots that can be confused with viral particles. We suspect that fluorescence counting can lead to overestimation of virus numbers and even suggest the presence of viruses when there are none. Future studies in environmental virology should acknowledge this point and consider how to bypass this problem. Besides trying to improve discrimination between virions and MVs, we suggest adopting less holistic approaches, focusing on the detection of known virus groups and the isolation of new viruses from a broader range of hosts.

Sad State of Phage Electron Microscopy. Please Shoot the Messenger.

Two hundred and sixty publications from 2007 to 2012 were classified according to the quality of electron micrographs; namely as good (71); mediocre (21); or poor (168). Publications were from 37 countries; appeared in 77 journals; and included micrographs produced with about 60 models of electron microscopes. The quality of the micrographs was not linked to any country; journal; or electron microscope. Main problems were poor contrast; positive staining; low magnification; and small image size. Unsharp images were frequent. Many phage descriptions were silent on virus purification; magnification control; even the type of electron microscope and stain used. The deterioration in phage electron microscopy can be attributed to the absence of working instructions and electron microscopy courses; incompetent authors and reviewers; and lenient journals. All these factors are able to cause a gradual lowering of standards.

Murphy's law-if anything can go wrong, it will: Problems in phage electron microscopy.

The quality of bacteriophage electron microscopy appears to be on a downward course since the 1980s. This coincides with the introduction of digital electron microscopes and a general lowering of standards, possibly due to the disappearance of several world-class electron microscopists The most important problem seems to be poor contrast. Positive staining is frequently not recognized as an undesirable artifact. Phage parts, bacterial debris, and aberrant or damaged phage particles may be misdiagnosed as bacterial viruses. Digital electron microscopes often seem to be operated without magnification control because this is difficult and inconvenient. In summary, most phage electron microscopy problems may be attributed to human failure. Journals are a last-ditch defense and have a heavy responsibility in selecting competent reviewers and rejecting, or not, unsatisfactory articles.

Who went into phage research?

A total of 30,000 phage papers, books, or book chapters, published between 1965 and 2010, were analyzed for the ethnic origins of 14,429 first authors. Their names represent 40 linguistic domains or geographic areas and at least 70 languages. British and German names predominate. Results broadly concur with statistics on the frequency of publications by country and show the growing role of Third-World countries in phage research. Irish and Jewish scientists are prominent. Historical and societal factors appear to be very important elements in the advancement of science.

Characterization of 4 T1-like lytic bacteriophages that lyse Shiga-toxin Escherichia coli O157:H7.

Bacteriophages are associated with reduced fecal shedding of Shiga-toxin-producing Escherichia coli O157:H7 (STEC O157:H7) in cattle. Four phages exhibiting activity against 12 of 14 STEC O157:H7 strains, representing 11 common phage types, were isolated. Phages did not lyse non-O157 E. coli, with 11 of the 12 STEC strains exhibiting extreme susceptibility (average multiplicity of infection (MOI) = 0.0003-0.0007). All phages had icosahedral heads with tapered, noncontractile tails, a morphology indicative of T1-like Siphoviridae. Genome size of all phages was ∼44 kb, but EcoRІ or HindIII digestion profiles differed among phages. Based on restriction enzyme digestion profiles, phages AHP24, AHS24, and AHP42 were more related (66.7%-82.4%) to each other than to AKS96, while AHP24 and AHS24, isolated from the same feedlot pen, exhibited the highest identity (88.9%-92.3%). Phages AHP24 and AHS24 exhibited the broadest host range and strongest lytic activity against STEC O157:H7, making them strong candidates for biocontrol of this bacterium in cattle.

Bacteriophages with the ability to degrade uropathogenic Escherichia coli biofilms.

Escherichia coli-associated urinary tract infections (UTIs) are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC) have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages) as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12) which were able to lyse 80.5% of a subset (42) of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages' genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2-12 h of incubation.

Bacteriophage electron microscopy.

Since the advent of the electron microscope approximately 70 years ago, bacterial viruses and electron microscopy are inextricably linked. Electron microscopy proved that bacteriophages are particulate and viral in nature, are complex in size and shape, and have intracellular development cycles and assembly pathways. The principal contribution of electron microscopy to bacteriophage research is the technique of negative staining. Over 5500 bacterial viruses have so far been characterized by electron microscopy, making bacteriophages, at least on paper, the largest viral group in existence. Other notable contributions are cryoelectron microcopy and three-dimensional image reconstruction, particle counting, and immunoelectron microscopy. Scanning electron microscopy has had relatively little impact. Transmission electron microscopy has provided the basis for the recognition and establishment of bacteriophage families and is one of the essential criteria to classify novel viruses into families. It allows for instant diagnosis and is thus the fastest diagnostic technique in virology. The most recent major contribution of electron microscopy is the demonstration that the capsid of tailed phages is monophyletic in origin and that structural links exist between some bacteriophages and viruses of vertebrates and archaea. DNA sequencing cannot replace electron microscopy and vice versa.

Morphology and genome sequence of phage ϕ1402: A dwarf myovirus of the predatory bacterium Bdellovibrio bacteriovorus.

Phages are among the simplest biological entities known and simultaneously the most numerous and ubiquitous members of the biosphere. Among the three families of tailed dsDNA phages, the Myoviridae have the most structurally sophisticated tails which are capable of contraction, unlike the simpler tails of the Podoviridae and Siphoviridae. Such "nanomachines" tails are involved in both efficient phage adsorption and genome injection. Their structural complexity probably necessitates multistep morphogenetic pathways, involving non-structural components, to correctly assemble the structural constituents. For reasons probably related, at least in part, to such morphological intricacy, myoviruses tend to have larger genomes than simpler phages. As a consequence, there are no well-characterized myoviruses with a size of less than 40 kb. Here we report on the characterization and sequencing of the 23,931 bp genome of the dwarf myovirus ϕ1402 of Bdellovibrio bacteriovorus. Our genomic analysis shows that ϕ1402 differs substantially from all other known phages and appears to be the smallest known autonomous myovirus.

Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions.

Poultry intestinal material, sewage and poultry processing drainage water were screened for virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm non-contractile tails characteristic of members of the family Siphoviridae in the order Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses resulted in the identification of the putative pre-neck/appendage protein and a minor structural protein encoded by large open reading frames. Variants of the portal protein were identified, and several mycobacteriophage gp6-like protein variants were detected in large amounts relative to other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had major differences in the central portion of the protein between the two phage gene products. Based on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type, using a head-full DNA packaging strategy.

The first phage electron micrographs.

The first phage electron micrographs were published in 1940 in Germany and proved the particulate nature of bacteriophages. Phages and infected bacteria were first examined raw and unstained. US American scientists introduced shadowing and freeze-drying. Phages appeared to be tailed and morphologically heterogeneous. Phage types identified by early electron microscopy include enterobacteriophages T4, T1, T7, T5, 7-11, ViI and Pseudomonas phage PB1. This paper retraces the development of early virus electron microscopy till the introduction of negative staining.

Morphology of Salmonella Typhimurium typing phages of the Lilleengen set.

The Lilleengen scheme for typing Salmonella enterica serovar Typhimurium consists of 12 tailed phages. Ten phages are podoviruses and morphologically identical to Salmonella phage P22. Two phages are siphoviruses and identical to flagella-specific phage chi.