Complete Genome Sequences for 36 Canadian Salmonella enterica Serovar Typhimurium and I 1,4,[5],12:i:- Isolates from Clinical and Animal Sources.

Here, we report the complete genome sequences for 36 Canadian isolates of Salmonella enterica subsp. enterica serovar Typhimurium and its monophasic variant I 1,4,[5]:12:i:- from both clinical and animal sources. These genome sequences will provide useful references for understanding the genetic variation within this prominent serotype.

The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5.

BACKGROUND: Bacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs. METHODS: We examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set. RESULTS: Phage rV5 is a member of the Myoviridae family possessing an icosahedral head of 91 nm between opposite apices. The extended tail measures 121 x 17 nm and has a sheath of 44 x 20 nm and a 7 nm-wide core in the contracted state. It possesses a 137,947 bp genome (43.6 mol%GC) which encodes 233 ORFs and six tRNAs. Until recently this virus appeared to be phylogenetically isolated with almost 70% of its gene products ORFans. rV5 is closely related to coliphages Delta and vB-EcoM-FY3, and more distantly related to Salmonella phages PVP-SE1 and SSE-121, Cronobacter sakazakii phage vB_CsaM_GAP31, and coliphages phAPEC8 and phi92. A complete shotgun proteomic analysis was carried out on rV5, extending what had been gleaned from the genomic analyses. Host range studies revealed that rV5 is active against several other E. coli.

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.

Escherichia coli O157:H7 typing phage V7 is a T4-like virus.

The complete genome sequence of the Escherichia coli O157:H7 typing phage V7 was determined. Its double-stranded DNA genome is 166,452 bp long, encoding 273 proteins and including 11 tRNAs. This virus belongs to the genus T4-like viruses within the subfamily Tevenvirinae, family Myoviridae.

The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1.

Based upon whole genome and proteome analysis, Escherichia coli O157:H7-specific bacteriophage (phage) wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage phiEa21-4. The genome characteristics of phage wV8 (size 88.49 kb, mol%G+C 38.9, 138 ORFs, 23 tRNAs) are very similar to those of phage Felix O1 (86.16 kb, 39.0 mol%G+C, 131 ORFs and 22 tRNAs) and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.

Enumeration of bacteriophages by double agar overlay plaque assay.

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.

Enumeration of bacteriophages by the direct plating plaque assay.

A method is described for determination of the concentration of infectious phage particles by the direct plating plaque assay, which is simpler and faster than the double agar overlay plaque procedure outlined in the previous chapter.

Enumeration of bacteriophages using the small drop plaque assay system.

The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. Described here is a drop plaque assay, which, being simpler, faster and more efficient than either the classical overlay or direct plating methods, enhances efficiency in processing large numbers of samples.

Preparation and characterization of anti-phage serum.

This chapter describes a method for the generation of polyclonal antibodies against bacteriophages and how these may be assayed immunochemically and biologically.

Generalized transduction by lytic bacteriophages.

As interest in lytic phages as antimicrobial therapies or as treatments to reduce environmental contamination with pathogenic bacteria has increased, so has the need to determine if the use of lytic phages may lead to dissemination of virulence factors through generalized transduction, as occurs with temperate phages. Here we describe simple methods we have developed to determine if a lytic phage, rV5, can mediate generalized transduction in Escherichia coli O157:H7. These sensitive methods can be easily adapted to study generalized transduction between virulent and avirulent strains of bacteria.