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Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.

Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y, Zhang W, McLaughlin SF, Henkhaus JK, Leopold B, Bielaszewska M, Prager R, Brzoska PM, Moore RL, Guenther S, Rothberg JM, Karch H - PLoS ONE (2011)

Bottom Line: Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past.The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC).In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hygiene, University Münster, Münster, Germany.

ABSTRACT
An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

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Whole chromosomal Optical Maps of the EHEC O104:H4 outbreak and related strains.Optical Maps were created from current outbreak isolates (LB isolates) and 01-09591 using NcoI as described. An in silico reference map was also created from the published EAEC 55989 sequence [11]. Optical and in silico maps were compared to reveal shared and unique elements. Shared restriction fragments are white/un-highlighted. Regions shared by the current outbreak isolates but unique relative to 01-09591 and EAEC 55989 are highlighted in red, regions unique to 01-09591 are highlighted in green, and regions unique to EAEC 55989 are highlighted in yellow. Perceived minor variations in banding patterns are due to fragment sizes less than 2 kb and therefore not included in subsequent analysis (see Methods).
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pone-0022751-g006: Whole chromosomal Optical Maps of the EHEC O104:H4 outbreak and related strains.Optical Maps were created from current outbreak isolates (LB isolates) and 01-09591 using NcoI as described. An in silico reference map was also created from the published EAEC 55989 sequence [11]. Optical and in silico maps were compared to reveal shared and unique elements. Shared restriction fragments are white/un-highlighted. Regions shared by the current outbreak isolates but unique relative to 01-09591 and EAEC 55989 are highlighted in red, regions unique to 01-09591 are highlighted in green, and regions unique to EAEC 55989 are highlighted in yellow. Perceived minor variations in banding patterns are due to fragment sizes less than 2 kb and therefore not included in subsequent analysis (see Methods).

Mentions: Optical Mapping data using four outbreak strains, 01-09591 and EAEC 55989 also corroborates the common ancestor model (Fig 6). If it is assumed that the hypothetical common O104:H4 ancestor contains the shared genomic regions of the outbreak strains, then LB226692 evolved by three insertion events, 01-09591 by one insertion event, and EAEC 55989 by six insertion events. All of these events would be unconstrained, meaning that the nature and location of the insertions leading to one strain would be independent of the insertions that lead to the other strains. According to the linear model, following the time of isolation, EAEC 55989 leads to 01-09591, which then leads to LB226692. In this model, EAEC 55989 loses six regions and gains one new genomic region to give rise to 01-09591. Then, 01-09591 loses this recently gained region and gains three additional new regions leading to the current outbreak strain. Although the total number of genomic changes is the same in both models, the linear model imposes an additional constraint of the gain and subsequent loss of the same genetic region.


Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.

Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y, Zhang W, McLaughlin SF, Henkhaus JK, Leopold B, Bielaszewska M, Prager R, Brzoska PM, Moore RL, Guenther S, Rothberg JM, Karch H - PLoS ONE (2011)

Whole chromosomal Optical Maps of the EHEC O104:H4 outbreak and related strains.Optical Maps were created from current outbreak isolates (LB isolates) and 01-09591 using NcoI as described. An in silico reference map was also created from the published EAEC 55989 sequence [11]. Optical and in silico maps were compared to reveal shared and unique elements. Shared restriction fragments are white/un-highlighted. Regions shared by the current outbreak isolates but unique relative to 01-09591 and EAEC 55989 are highlighted in red, regions unique to 01-09591 are highlighted in green, and regions unique to EAEC 55989 are highlighted in yellow. Perceived minor variations in banding patterns are due to fragment sizes less than 2 kb and therefore not included in subsequent analysis (see Methods).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140518&req=5

pone-0022751-g006: Whole chromosomal Optical Maps of the EHEC O104:H4 outbreak and related strains.Optical Maps were created from current outbreak isolates (LB isolates) and 01-09591 using NcoI as described. An in silico reference map was also created from the published EAEC 55989 sequence [11]. Optical and in silico maps were compared to reveal shared and unique elements. Shared restriction fragments are white/un-highlighted. Regions shared by the current outbreak isolates but unique relative to 01-09591 and EAEC 55989 are highlighted in red, regions unique to 01-09591 are highlighted in green, and regions unique to EAEC 55989 are highlighted in yellow. Perceived minor variations in banding patterns are due to fragment sizes less than 2 kb and therefore not included in subsequent analysis (see Methods).
Mentions: Optical Mapping data using four outbreak strains, 01-09591 and EAEC 55989 also corroborates the common ancestor model (Fig 6). If it is assumed that the hypothetical common O104:H4 ancestor contains the shared genomic regions of the outbreak strains, then LB226692 evolved by three insertion events, 01-09591 by one insertion event, and EAEC 55989 by six insertion events. All of these events would be unconstrained, meaning that the nature and location of the insertions leading to one strain would be independent of the insertions that lead to the other strains. According to the linear model, following the time of isolation, EAEC 55989 leads to 01-09591, which then leads to LB226692. In this model, EAEC 55989 loses six regions and gains one new genomic region to give rise to 01-09591. Then, 01-09591 loses this recently gained region and gains three additional new regions leading to the current outbreak strain. Although the total number of genomic changes is the same in both models, the linear model imposes an additional constraint of the gain and subsequent loss of the same genetic region.

Bottom Line: Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past.The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC).In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hygiene, University Münster, Münster, Germany.

ABSTRACT
An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

Show MeSH
Related in: MedlinePlus