Enumeration of bacteriophages by double agar overlay plaque assay.
ABSTRACT The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described.
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ABSTRACT: Background: The rise in antibiotic-resistant Pseudomonas aeruginosa and the considerable difficulty in eradicating it from patients has re-motivated the study of bacteriophages as a therapeutic option. For this to be effective, host range and viability following nebulization need to be assessed. Host-range has not previously been assessed for the Liverpool Epidemic Strain (LES) isolates that are the most common cystic fibrosis-related clone of P. aeruginosa in the UK. Nebulization studies have not previously been linked to clinically relevant phages. Methods: 84 phenotypically variable isolates of the LES were tested for susceptibility to seven bacteriophages known to have activity against P. aeruginosa. Five of the phages were from the Eliava Institute (IBMV) and 2 were isolated in this study. The viability of the two bacteriophages with the largest host ranges was characterized further to determine their ability to be nebulized and delivered to the lower airways. Phages were nebulized into a cascade impactor and the phage concentration was measured. Results: The bacteriophages tested killed between 66%-98% of the 84 Liverpool Epidemic Strain isolates. Two isolates were multi phage resistant, but were sensitive to most first line anti-Pseudomonal antibiotics. The amount of viable bacteriophages contained in particles that are likely to reach the lower airways (<4.7 μm) was 1% for the Omron and 12% AeroEclipse nebulizer. Conclusions: Individual P. aeruginosa bacteriophages can lyse up to 98% of 84 phenotypically diverse LES strains. High titers of phages can be effectively nebulized.Journal of Aerosol Medicine and Pulmonary Drug Delivery 02/2015; DOI:10.1089/jamp.2014.1172 · 2.40 Impact Factor
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ABSTRACT: Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. Copyright © 2014 Elsevier Inc. All rights reserved.Virology 02/2015; 477. DOI:10.1016/j.virol.2014.12.035 · 3.28 Impact Factor
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ABSTRACT: In order to control Pectobacterium carotovorum subsp. carotovorum, a novel virulent bacteriophage PM2 was isolated. Bacteriophage PM2 can infect 48% of P. carotovorum subsp. carotovorum and 78% of P. carotovorum subsp. brasilliensis but none of atrosepticum, betavasculorum, odoriferum and wasabiae isolates had been infected with PM2. PM2 phage belongs to the family Myoviridae, and contains a large head and contractile tail. It has a 170,286 base pair genome that encodes 291 open reading frames (ORFs) and 12 tRNAs. Most ORFs in bacteriophage PM2 share a high level of homology with T4-like phages including IME08, RB69, and JS98. Phylogenetic analysis based on the amino acid sequence of terminase large subunits confirmed that PM2 is classified as a T4-like phage. It contains no integrase- or no repressor-coding genes related to the lysogenic cycle, and lifestyle prediction using PHACT software suggested that PM2 is a virulent bacteriophage.The plant pathology journal 03/2015; 31(1):83-9. DOI:10.5423/PPJ.NT.09.2014.0099 · 0.76 Impact Factor