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Comparative Study
. 2012;7(5):e37362.
doi: 10.1371/journal.pone.0037362. Epub 2012 May 23.

A comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the German E. coli O104:H4 outbreak strain

Chad R Laing  1 , Yongxiang Zhang, Matthew W Gilmour, Vanessa Allen, Roger Johnson, James E Thomas, Victor P J Gannon
Affiliations
Comparative Study

A comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the German E. coli O104:H4 outbreak strain

Chad R Laing et al. PLoS One. 2012.

Abstract

Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The neighbor-net visualization of the Stx2 “pan-genome” among E. coli Stx2-bacteriophage and a bacteriophage from Shigella dysenteriae ( Table 1 ).
The “pan-genome” consists of 500 bp sequence fragments present in at least 3 bacteriophage sequences at an 85% sequence identity threshold. Labels represent the host-source of the bacteriophage as serotype followed by strain name. Red shading: Stx2-phage from O104:H4 outbreak isolates (light); O111:H- strain JB1-95 (dark). Purple shading: Stx2-phage from O157:H7 lineage I strains; related Stx2-bacteriophge; O145:H2 strain 4.0967. Green shading: Stx2-phage from O157:H7 lineage I/II strains; the sub-group of lineage I/II Stx2-phage most closely related to the O104:H4 isolate phage. Yellow shading: Stx2c-phage from O157:H7 lineage I/II strains and bacteriophage 2851 (light); O157:H7 lineage II strains (dark).
Figure 2
Figure 2. Maximum likelihood phylogram with aLRT branch-support values of the Stx2 “pan-genome” data among E. coli Stx2-bacteriophage and a bacteriophage from Shigella dysenteriae ( Table 1 ).
Labels represent the host-source of the bacteriophage as serotype followed by strain name. Red shading: Stx2-phage from O104:H4 outbreak isolates (light); O111:H- strain JB1-95 (dark). Purple shading: Stx2-phage from O157:H7 lineage I strains; related Stx2-bacteriophge; O145:H2 strain 4.0967. Green shading: Stx2-phage from O157:H7 lineage I/II strains; the sub-group of lineage I/II Stx2-phage most closely related to the O104:H4 isolate phage. Yellow shading: Stx2c-phage from O157:H7 lineage I/II strains and bacteriophage 2851 (light); O157:H7 lineage II strains (dark).
Figure 3
Figure 3. Maximum likelihood phylogram with aLRT branch-support values based on the complete pan-genome of the Stx2-containing genomic sequences in Table 1 .
Red shading: O104:H4 outbreak isolates (light); O111:H- strain JB1-95 (dark). Purple shading: O157:H7 lineage I strains; O145:H2 strain 4.0967. Green shading: O157:H7 lineage I/II strains; the sub-group of lineage I/II strains with Stx2-phage most closely related to the O104:H4 isolate phage. Yellow shading: O157:H7 lineage II strains.
Figure 4
Figure 4. The progressiveMauve alignment of the Stx2-bacteriophage closely related to the O104:H4 outbreak isolates .
Bounded boxes indicate similar sequence composition among sequences. The black box indicates the location of the stx2ab gene cluster in each sequence.
Figure 5
Figure 5. Maximum likelihood phylogram with aLRT branch-support values of the MAFFT alignment of the Stx2-bacteriophage closely related to the O104:H4 outbreak isolates.
Figure 6
Figure 6. Stx2-production by E. coli O104:H4 outbreak-related strain ON-2011 and E. coli O157:H7 strains EDL933 and Sakai in un-induced, and mitomycin C-induced states as measured by a Stx2-specific ELISA.
Error bars represent standard deviations from three independent replicates.
Figure 7
Figure 7. Stx2-mRNA copy number differences between E. coli O104:H4 outbreak-related strain ON-2011 and E. coli O157:H7 strain EDL933 under ciprofloxacin, norfloxacin and mitomycin C treatments.
NT =  no treatment. Error bars represent standard deviations from three independent replicates.
See this image and copyright information in PMC

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