Characterization of Shiga Toxin 2a Encoding Bacteriophages Isolated From High-Virulent O145:H25 Shiga Toxin-Producing Escherichia coli.

Silje N Ramstad,Arne M Taxt,Jørgen V Bjørnholt,Jon Bohlin,Yngvild Wasteson,Lin T Brandal,Bjørn-Arne Lindstedt

doi:10.3389/fmicb.2021.728116

Abstract

Shiga toxin-producing Escherichia coli (STEC) may cause severe disease mainly due to the ability to produce Shiga toxins (Stx) encoded on bacteriophages. In Norway, more than 30% of the reported cases with STEC O145:H25 develop hemolytic uremic syndrome (HUS), and most cases, with known travel history, acquired the infection domestically. To describe phage characteristics associated with high virulence, we extracted the Stx2a phage sequences from eight clinical Norwegian O145:H25 STEC to conduct in-depth molecular characterization using long and short read sequencing. The Stx2a phages were annotated, characterized, and compared with previously published Stx2a phages isolated from STEC of different serotypes. The Norwegian O145:H25 Stx2a phages showed high sequence identity (>99%) with 100% coverage. The Stx2a phages were located at the integration site yciD, were approximately 45 kbp long, and harbored several virulence-associated genes, in addition to stx2a, such as nanS and nleC. We observed high sequence identity (>98%) and coverage (≥94%) between Norwegian O145:H25 Stx2a phages and publicly available Stx2a phages from O145:H25 and O145:H28 STEC, isolated from HUS cases in the USA and a hemorrhagic diarrhea case from Japan, respectively. However, low similarity was seen when comparing the Norwegian O145:H25 Stx2a phage to Stx2a phages from STEC of other serotypes. In all the Norwegian O145:H25 STEC, we identified a second phage or remnants of a phage (a shadow phage, 61 kbp) inserted at the same integration site as the Stx2a phage. The shadow phage shared similarity with the Stx2a phage, but lacked stx2a and harbored effector genes not present in the Stx2a phage. We identified a conserved Stx2a phage among the Norwegian O145:H25 STEC that shared integration site with a shadow phage in all isolates. Both phage and shadow phage harbored several virulence-associated genes that may contribute to the increased pathogenicity of O145:H25 STEC.

Highlights

Infection with Shiga toxin-producing Escherichia coli (STEC) may lead to severe symptoms, such as hemorrhagic diarrhea and hemolytic uremic syndrome (HUS) (Karch et al, 2005)
STEC NIPH1–NIPH8 were the only STEC using yciD as integration site for the Stx2a phages (Table 1). These findings suggest that the Stx2a phages of the Norwegian high-virulent O145:H25 STEC are most likely of the same phage type and they were present in O145:H25 STEC from both core genome multilocus sequence typing (cgMLST) clusters (Figure 1), indicating that this Stx2a phage variant likely is dominating in Norwegian human clinical O145:H25 STEC isolates
This study was based on the hypothesis that characteristics of the Stx2a phages might explain the high occurrence of HUS in Norwegian patients with STEC O145:H25 infections

Summary

Introduction

Infection with Shiga toxin-producing Escherichia coli (STEC) may lead to severe symptoms, such as hemorrhagic diarrhea and hemolytic uremic syndrome (HUS) (Karch et al, 2005). 5% of STEC infections in European Union (EU) countries and Norway lead to HUS (Brandal et al, 2015; Naseer et al, 2017; European Food Safety Authority European Centre for Disease Prevention Control, 2019; Jenssen et al, 2019). In the EU, STEC of serogroups O26, O157, O145, O80, and O111 are most commonly associated with HUS cases (European Food Safety Authority European Centre for Disease Prevention Control, 2019). In Norway, STEC with Stx subtypes Stx2a, Stx2c, and Stx2d are defined as high-virulent, and their identification triggers strict infection control measures including close followup of cases (Norwegian Institute of Public Health, 2021). The process leading to transcription of the late phage genes, including stx, is induced by the onset of the bacterial SOS response. The bacterial SOS response can be activated by, for instance, exposure to Mitomycin C, UV irradiation, and antimicrobials (Kruger and Lucchesi, 2015)

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