Bacteriophages, also known as phages, are viruses that infect bacteria. Until recently they have been ignored by most of the scientific community, but their impact upon our world is enormous. They are the most abundant lifeform on the globe and drive the diversity and abundance of bacteria around us, including, in many instances, the pathogenic profiles of many of mankind's most feared bacterial pathogens. This article focuses on how a group of bacteriophages, Stx-phages, which carry the genes encoding Shiga toxin, have driven and are driving the emergence of Shiga toxin-producing pathogens such as the infamous Escherichia coli O157:H7. Since the emergence of this foodborne pathogen as a cause of significant human disease in 1982, more than 500 different serogroups of E. coli have been reported to produce Shiga toxin, as well as a few other organisms. These events and many more are all controlled by the biology of Stx-phages.

A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt 2 ), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.Verocytotoxigenic Escherichia coli (VTEC) is a serious pathogen of considerable public health concern worldwide. Infection is usually characterized by bloody diarrhea and can be life threatening due to the subsequent development of hemolytic-uremic syndrome mediated by verocytotoxins (VTs), of which there are two forms, VT1 and VT2. In almost all cases, the VT genes are carried on temperate bacteriophages (VT phages). Although E. coli O157 is the most commonly isolated VTEC serogroup in the United Kingdom, North America, and Japan, more than 30 disease-causing non-0157 VTECs have been described (1) and over 100 serotypes are capable of producing VT (6). VT production has been observed in other members of the Enterobacteriaceae, including Enterobacter cloacae (8) and Citrobacter freundii (12), but was first described in Shigella dysenteriae as Shiga toxin (3). The localization of vt genes on a bacteriophage was first described by Smith et al. (13), but their acquisition by pathogenic E. coli strains remained anomalous because only nonpathogenic (rough) E. coli strains could apparently be infected with VT phage. Previously, the vt 2 gene of a bacteriophage (24 B ), isolated from an E. coli O157 strain, had been inactivated by insertion of a selectable marker (kanamycin resistance) (10). This provided an ideal opportunity to investigate the host range of a lysogenic VT bacteriophage and thus its potential to transfer the ability to produce VT between E. coli and related gram-negative bacteria.The host range of this recombinant VT2 phage (24 B ::Kan) was determined by infection of pathogenic and commensal strains of E. coli and other Enterobacteriaceae strains from human and animal sources. Lysogens were detected by spreading phage-infected cultures of the host bacteria (100 l) onto Luria-Bertani Miller (LB) agar (Difco) plates containing kanamycin (50 g ml Ϫ1 ). As it is clear that some phage infections create lysogens and do not result in a lytic infection, plaque assays may not necessarily detect all infectious phage particles. Induction of the VT phage lytic cycle is RecA dependent (7). RecA plays a central role in the SOS response of E. coli, during which phage-mediated lysis is induced. The recA441 mutant E. coli K-12 strain...

The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx 1 and/or stx 2 ) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx 1 or stx 2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage , and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

BackgroundStx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages.ResultsThe genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system.ConclusionsThe data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.

Enteric viruses cause gastrointestinal illness and are commonly transmitted through the fecal-oral route. When wastewater is released into river systems, these viruses can contaminate the environment. Our results show that we can use viromics to find the range of potentially pathogenic viruses that are present in the environment and identify prevalent genotypes. The ultimate goal is to trace the fate of these pathogenic viruses from origin to the point where they are a threat to human health, informing reference-based detection methods and water quality management.