Diversity and Host Range of Shiga Toxin-Encoding Phage

The foodborne pathogen Escherichia coli O157:H7 is responsible for a reported 73,000 cases of illness per year in the United States (16). Disease can range from watery and bloody diarrhea to the life-threatening hemolytic uremic syndrome. A major virulence factor responsible for disease is the production of Shiga toxin (Stx). In Stx-producing E. coli (STEC) such as E. coli O157:H7, Stx can be present in two forms: Stx1, which is essentially identical to Stx from Shigella dysenteriae, or Stx2, which is about 55% homologous to Stx1 at the amino acid level (reviewed in reference 20). A STEC strain can produce either Stx1 or Stx2 or both; however, severe disease has been epidemiologically linked to the presence of Stx2 (3, 24). The biology of Stx production is unusual. The genes for Stx are encoded in the late gene region of lysogenic phage (21, 28). Late phage genes are expressed only when the phage are engaged in the replicative or lytic cycle (reviewed in reference 11). For E. coli O157:H7, the toxin genes are silent during lysogeny; however, if the phage are induced to enter the lytic cycle, phage and toxin production will occur (37). Induction to the lytic cycle can occur after exposure of the bacteria to DNA damaging agents, such as UV light or mitomycin C (18), or to antibiotics, such as the quinolones (41), which stall DNA synthesis. In addition, neutrophil activation products such as hydrogen peroxide have been shown to induce Stx production (35). Previous studies indicated that Stx2-encoding phages are highly variable (10, 17, 19, 34, 36). Wagner et al. (36) demonstrated that Stx2 phages from STEC clinical isolates displayed different host ranges and differed in toxin production, suggesting that phage heterogeneity could play a role in disease outcome. Indeed, Muniesa et al. (19) have shown that E. coli O157:H7 isolates from a single outbreak were lysogenized with different toxin-encoding phages. Furthermore, the severity of disease symptoms observed in different patients correlated with in vitro toxin production by their O157:H7 isolate. These studies suggest that phage variability can influence pathogenic fitness. Shiga toxin-encoding phage can infect non-O157 E. coli. Phage infection can have two outcomes. In some instances, infection of non-toxin-producing E. coli will result in lysogeny, where the phage genome becomes incorporated into the bacterial chromosome (11). Lysogeny has been demonstrated to occur in vivo in a mouse model of disease (1, 7). Lysogeny with toxin-encoding phage has important implications for the evolution of new pathogenic strains. It has been shown that Shigella sonnei can become lysogenized with Stx-encoding phage, which has the potential to convert them to a more pathogenic form (32). The other outcome of phage infection is lytic infection. Lytic infection of non-O157 E. coli can lead to production of phage and Stx. Our investigators have shown that lytic infection of non-toxin-producing E. coli with Stx-encoding phage in vitro can increase Stx production by more than 1,000-fold (7). Furthermore, in a mouse model of disease, intestinal Stx2 production was dramatically increased in some cases when the toxin-encoding phage were able to infect non-toxin-producing E. coli (7). Epidemiological studies of E. coli O157:H7 suggest that following exposure, many individuals develop intestinal symptoms; however, only a small percentage of patients go on to develop severe, life-threatening systemic sequelae, such as hemorrhagic colitis or hemolytic uremic syndrome (16). Certain combinations of toxin-encoding phage and host intestinal flora could result in highly elevated levels of intestinal Stx production, and these individuals may be at increased risk for developing severe systemic disease. Human intestinal flora is highly variable and, furthermore, the host range of Stx-encoding phages is highly variable (2, 9, 22). It is currently unknown how often individuals possess intestinal flora that is susceptible to the toxin-encoding phages. In this study, we examined phage characteristics and immunity profiles of six Stx2-encoding phages from highly related clinical E. coli O157:H7 isolates from the Cincinnati area, with an emphasis on assessing their ability to undergo lysogenic integration and/or lytic infection in a variety of E. coli strains. The Stx2-encoding phages were found to be highly variable, even when the E. coli O157:H7 isolates were identical by pulsed-field gel electrophoresis (PFGE) analysis, consistent with the hypothesis that variations in phage host range may play a role in pathogenic fitness.