Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2

Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
PLoS ONE (Impact Factor: 3.23). 11/2012; 7(11):e48228. DOI: 10.1371/journal.pone.0048228
Source: PubMed


In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

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    • " strains might have diverged recently from a common ancestor . Our results are in accordance with those of the study performed by Ahmad et al . , which showed that the Georgian isolates are the closest genomically to the outbreak strain so far ; however , several differences in their stx2 pro - phages , genomic islands , and plasmids are evident ( Ahmed et al . , 2012 ) . Furthermore , our study showed that the tran - scription levels of the stx2 gene differed between the Georgian and the outbreak strains . This might be due to differences in phage genotype in the outbreak and pre - outbreak strains ."
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    ABSTRACT: Background: A novel pathotype, Shiga toxin-producing Escherichia coli O104:H4, was the cause of a severe outbreak that affected European countries, mainly Germany, in 2011. The effect of different regimens of rifampicin and gentamicin were evaluated to determine possible treatment modes for the novel strain, and to evaluate the SOS response and its effect on toxin release. Materials and methods: Pulsed-field gel electrophoresis (PFGE) was performed on the novel E. coli O104:H4 pathotype and two pre-outbreak E. coli O104:H4 CDC strains. Transcript levels of the stx2 and recA gene (SOS response inducer) were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) in the novel E. coli O104:H4 samples subjected to different regimens of rifampicin and gentamicin. Consequently, reverse passive latex agglutination (RPLA) was used to determine the Stx2 titers in these samples. Western blot was performed to determine the LexA levels (SOS response repressor) in E. coli O104:H4. The efficacy of treatment with antimicrobial agents was assessed in BALB/c mice. Results: The outbreak and pre-outbreak strains are closely related as shown by PFGE, which demonstrated slight genomic differences between the three strains. The transcription level of the stx2 gene in the new pathotype was 1.41- and 1.75-fold that of the 2009 EL-2050 and 2009 EL-2071 pre-outbreak strains, respectively. Moreover, the transcription level of the stx2 gene in the new pathotype was substantially decreased as a result of treatment with the different concentrations of the antimicrobial agents, but was enhanced when the antibiotics were administered at two subinhibitory levels. RPLA data were in accordance with the qRT-PCR results. E. coli O104:H4 exposed to gentamicin at both sub-minimum inhibitory concentration (MIC) levels led to high transcription levels of the recA gene and lack of expression of the LexA protein, implying that the SOS response was activated. Rifampicin at both sub-MIC levels resulted in low transcript levels of the recA gene, indicating that the SOS response was not induced. In vivo, the highest survival rate in BALB/c mice was observed in the group that was treated with the minimum bactericidal concentration (MBC) of gentamicin. Conclusion: The use of antimicrobial agents in E. coli O104:H4 infection seems to be effective at the MIC and MBC levels. This provides a promising ground for treatment of E. coli O104:H4.
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    • "EAHEC O104:H4 is an example of a newly emerged pathogen that possesses novel combinations of acquired antibiotic resistance genes and virulence factors. In the case of EAHEC O104:H4 an enteroaggregative E. coli had acquired a stx2 shiga toxin gene, a characteristic feature of enterohaemorrhagic E. coli genomes, via a bacteriophage (Grad et al., 2013); a plasmid encoded CTX-M-15 gene (encoding the antibiotic resistance factor, extended-spectrum beta-lactamase) (Kunne et al., 2012); and a chromosomally-located complex antibiotic resistance gene cluster, identified recently as the region of divergence 1 (Frank et al., 2011; Januszkiewicz et al., 2011; Ahmed et al., 2012; Radosavljevic et al., 2014). The extensively antibiotic resistant E. coli clonal group ST131 is similarly a globally emergent pathogen with novel combinations of acquired antibiotic resistance genes and virulence factors (Johnson et al., 2010; Andersen et al., 2013; Banerjee and Johnson, 2013; Price et al., 2013). "
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    • "The initial assembly was performed as previously described with modifications [62]. Briefly, 454 WGS and PE reads were assembled using Newbler (v2.3), and contigs broken into 2-kb overlapping fragments. "
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