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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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Phylogentic placement of German EHEC O104:H4 outbreak strain.Minimum-spanning tree based on allelic profiles of E. coli core genome genes (n = 1,144) portraying the phylogenetic relationship of the EHEC O104:H4 outbreak strain (LB226692), the historical EHEC 01-09591 (HUSEC041), additional E. coli strains representing the most common EHEC serotypes, intestinal and extraintestinal E. coli pathovars and commensals, from the NCBI RefSeq database. In addition, an in silico generated hypothetical O104:H4 progenitor is included. Each dot represents an allelic profile, the number on connecting lines represent the number of alleles that differ between two profiles. The different pathovars (EHEC, EAEC, ExPEC, EPEC, ETEC, commensals) are defined by colors and the EHEC serotypes are indicated.
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pone-0022751-g003: Phylogentic placement of German EHEC O104:H4 outbreak strain.Minimum-spanning tree based on allelic profiles of E. coli core genome genes (n = 1,144) portraying the phylogenetic relationship of the EHEC O104:H4 outbreak strain (LB226692), the historical EHEC 01-09591 (HUSEC041), additional E. coli strains representing the most common EHEC serotypes, intestinal and extraintestinal E. coli pathovars and commensals, from the NCBI RefSeq database. In addition, an in silico generated hypothetical O104:H4 progenitor is included. Each dot represents an allelic profile, the number on connecting lines represent the number of alleles that differ between two profiles. The different pathovars (EHEC, EAEC, ExPEC, EPEC, ETEC, commensals) are defined by colors and the EHEC serotypes are indicated.

Mentions: Sequencing on the Ion Torrent PGM™ sequencer was completed within 62 hours, leading to the public release of the draft assembly of outbreak strain LB226692 on June 3 (Fig. 1, Table S1). Sequence data of the closely related historical isolate 01-09591 was also generated while the outbreak was still occurring. Genome assemblies based on the PGM™ reads showed that both of these HUS-causing strains (LB226692 and 01-09591) carry genes typically found in two different E. coli pathotypes, specifically EAEC and EHEC. Genome wide phylogenetic analysis based on core chromosomal ORFs (n = 1,144) demonstrated the close relationship of the LB226692 and 01-09591 strains to the previously sequenced EAEC strain 55989 (NCBI acc. no. NC_011478), and indicated that these strains are only distantly related to the commonly isolated EHEC serotypes (Fig. 3). However, unlike typical EAEC strains, both LB226692 and 01-09591 have an stx2-harboring prophage integrated in wrbA, which is also the integration site for stx2-phages in EHEC O157:H7 outbreak strains EDL933 [13] and Sakai (RIMD 0509952) [14]. The wrbA gene of EAEC 55989 is not occupied by a prophage. Furthermore, the IrgA homologue adhesin encoding gene (iha), which is responsible for adherence to epithelial cells and has been found in eae-negative STEC [15], is present in all three strains. In contrast to the two HUSEC041 complex strains, 55989 does not harbor the tellurite resistance encoding genes (ter). These characteristics led to the development of a rapid PCR-based test of stx2, O104 lipopolysaccharide (LPS) gene (rfbO104), H4 flagellin-encoding gene (fliCH4), and terD for the detection of the HUSEC041 complex [16].


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)

Phylogentic placement of German EHEC O104:H4 outbreak strain.Minimum-spanning tree based on allelic profiles of E. coli core genome genes (n = 1,144) portraying the phylogenetic relationship of the EHEC O104:H4 outbreak strain (LB226692), the historical EHEC 01-09591 (HUSEC041), additional E. coli strains representing the most common EHEC serotypes, intestinal and extraintestinal E. coli pathovars and commensals, from the NCBI RefSeq database. In addition, an in silico generated hypothetical O104:H4 progenitor is included. Each dot represents an allelic profile, the number on connecting lines represent the number of alleles that differ between two profiles. The different pathovars (EHEC, EAEC, ExPEC, EPEC, ETEC, commensals) are defined by colors and the EHEC serotypes are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022751-g003: Phylogentic placement of German EHEC O104:H4 outbreak strain.Minimum-spanning tree based on allelic profiles of E. coli core genome genes (n = 1,144) portraying the phylogenetic relationship of the EHEC O104:H4 outbreak strain (LB226692), the historical EHEC 01-09591 (HUSEC041), additional E. coli strains representing the most common EHEC serotypes, intestinal and extraintestinal E. coli pathovars and commensals, from the NCBI RefSeq database. In addition, an in silico generated hypothetical O104:H4 progenitor is included. Each dot represents an allelic profile, the number on connecting lines represent the number of alleles that differ between two profiles. The different pathovars (EHEC, EAEC, ExPEC, EPEC, ETEC, commensals) are defined by colors and the EHEC serotypes are indicated.
Mentions: Sequencing on the Ion Torrent PGM™ sequencer was completed within 62 hours, leading to the public release of the draft assembly of outbreak strain LB226692 on June 3 (Fig. 1, Table S1). Sequence data of the closely related historical isolate 01-09591 was also generated while the outbreak was still occurring. Genome assemblies based on the PGM™ reads showed that both of these HUS-causing strains (LB226692 and 01-09591) carry genes typically found in two different E. coli pathotypes, specifically EAEC and EHEC. Genome wide phylogenetic analysis based on core chromosomal ORFs (n = 1,144) demonstrated the close relationship of the LB226692 and 01-09591 strains to the previously sequenced EAEC strain 55989 (NCBI acc. no. NC_011478), and indicated that these strains are only distantly related to the commonly isolated EHEC serotypes (Fig. 3). However, unlike typical EAEC strains, both LB226692 and 01-09591 have an stx2-harboring prophage integrated in wrbA, which is also the integration site for stx2-phages in EHEC O157:H7 outbreak strains EDL933 [13] and Sakai (RIMD 0509952) [14]. The wrbA gene of EAEC 55989 is not occupied by a prophage. Furthermore, the IrgA homologue adhesin encoding gene (iha), which is responsible for adherence to epithelial cells and has been found in eae-negative STEC [15], is present in all three strains. In contrast to the two HUSEC041 complex strains, 55989 does not harbor the tellurite resistance encoding genes (ter). These characteristics led to the development of a rapid PCR-based test of stx2, O104 lipopolysaccharide (LPS) gene (rfbO104), H4 flagellin-encoding gene (fliCH4), and terD for the detection of the HUSEC041 complex [16].

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