Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction

Abstract

Enterohemorrhagic E. coli (EHEC) is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS) and nervous system complications. Shiga toxin 2 (Stx2) is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage ϕ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424). Among 38 commensal E. coli strains from healthy children below 5 years, 15 were lysogenized by the ϕ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli ϕ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H2O2 treatment, 8 out of 13 commensal lysogens produced more ϕ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced) produced higher levels of phage than the H2O2 induced NIPH-11060424. An especially high frequency of HUS (60%) was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak.

The effect of antimicrobials on verocytotoxin bacteriophage transduction under bovine rumen fluid and broth conditions

“…A range of peak concentrations were 6.7 6.6 6.6 7.3 6.0 5.9 5.7 6.3 7.0 6.6 6.8 6.2 6.5 6.9 6.0 6.7 5.9 6.6 5.8 4 6.9 6.9 6.9 6.8 6.7 6.7 6.5 5.8 5.9 6.1 6.9 6.9 6.5 6.2 6.7 6.7 5.6 5.7 4.9 5.9 6 6.8 6.2 6.6 6.5 5.9 6.5 5.7 5.6 6.6 5.9 6.8 6.1 6.5 6.5 6.6 6.5 5.5 5.5 5.9 5.6 27 7.2 6.5 6.5 6.6 6.2 6.7 6.6 6.5 6.5 6.7 7.2 6.1 6.2 6.2 6.5 6.7 6.5 5.8 6.5 6.6 51 6.8 6.6 6.2 6.2 6.6 6.4 6.3 6.2 6.1 6.0 6.8 5. phages are constitutively replicating at any given time via the lytic cycle. This finding has been previously reported (Livny and Friedman, 2004;Iversen et al, 2015). Such a mechanism would ensure phage persistence even if bacterial inactivation processes were rapid and the VTEC hosts did not survive for sufficient time to allow phage release before being killed (Martinez-Castillo and Muniesa, 2014).…”

“…However, these temperate phages may also replicate lytically. At any given time, a small percentage of the bacteriophage population will be in the lytic phase (Livny and Friedman, 2004;Iversen et al, 2015). However, in unfavourable conditions (e.g.…”

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

“…Classical techniques for the evaluation prophage induction employ plaque assays (21,24), protein assays (6,10), and quantification of gene expression (25); however, these methods inform only on prophage induction at a population level. Using a fluorescent reporter gene to replace stx 2 was done in previous studies to investigate the induction of Stx2 prophages (6,26).…”

“…The outbreak strain is a novel pathotype with the serotype O104:H4, combining the virulence factors of STEC and enteroaggregative E. coli (EAEC) (7). Stx2-phages can also establish lysogeny in non-STEC, including enteropathogenic, enteroinvasive, and enterotoxigenic E. coli and commensal E. coli strains in vitro (9)(10)(11). This demonstrates that Stx2-phages have a broad host range among strains of E. coli.…”

“…Human neutrophils in the intestine release antibacterial molecules, including H 2 O 2 , which stimulate Stx and phage production (19). The Stx-phage released in the intestine may transduce to commensal strains and thus amplify toxin production and exacerbate disease symptoms (10). Hydrostatic pressure, a physical method of food preservation, also induces Stx prophages (18,21).…”