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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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Plasmid profile of German EHEC O104:H4 outbreak strain and strain 01-09591.Comparison of the plasmid content of German EHEC O104:H4 outbreak strains and the 01-09591 (O104:H4; HUSEC041). Lane 1: molecular mass markers (plasmids R27 [169 kb]; R100 [90 kb]; V517 [54 kb]); lane 2: strain 01-09591 EHEC O104:H4; lane 3: German EHEC O104:H4 2011 outbreak strain LB226692; lane 4: German EHEC O104:H4 2011 outbreak strain 11-002097; lane 5: EHEC O157:H7 strain EDL 933; lane 6: E. coli 39R861 molecular size marker.
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pone-0022751-g004: Plasmid profile of German EHEC O104:H4 outbreak strain and strain 01-09591.Comparison of the plasmid content of German EHEC O104:H4 outbreak strains and the 01-09591 (O104:H4; HUSEC041). Lane 1: molecular mass markers (plasmids R27 [169 kb]; R100 [90 kb]; V517 [54 kb]); lane 2: strain 01-09591 EHEC O104:H4; lane 3: German EHEC O104:H4 2011 outbreak strain LB226692; lane 4: German EHEC O104:H4 2011 outbreak strain 11-002097; lane 5: EHEC O157:H7 strain EDL 933; lane 6: E. coli 39R861 molecular size marker.

Mentions: Plasmid profiling demonstrated that LB226692 and 01-09591 each harbor two large plasmids (Fig. 4; 83 and 90 kb and 75 and 95 kb, respectively). Sequence analysis shows that the smaller plasmid of LB226692 contains aggregative adherence fimbriae type I (AAF/I) but lacks the EAEC heat-stable enterotoxin encoding gene, astA. The larger plasmid is an incompatibility group I1 (IncI1) plasmid with high similarity to pEC_Bactec (NCBI acc. no. GU371927) that harbors TEM-1 and CTX-M-15 beta-lactamase genes [17]. The large plasmid of strain 01-09591 appears to be closely related to the IncI1 family plasmid pSERB1 (NCBI acc. no. AY686591) from EAEC strain C1096 [18]. This strain (01-09591) also has a TEM-1 beta-lactamase that is located on a sequence contig carrying a number of genes encoding plasmid functions; we propose that this locus is also carried on the larger plasmid. The smaller plasmid of 01-09591 is an EAEC plasmid containing AAF/III, which is astA-positive, and closely related to the plasmid from EAEC 55989. Vitek® 2 and E-test® resistance testing indicates that both strains (LB226692 and 01-09591) have a TEM-1 phenotype. The ESBL genotype (CTX-M-15) of strain LB226692 was also phenotypically confirmed.


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)

Plasmid profile of German EHEC O104:H4 outbreak strain and strain 01-09591.Comparison of the plasmid content of German EHEC O104:H4 outbreak strains and the 01-09591 (O104:H4; HUSEC041). Lane 1: molecular mass markers (plasmids R27 [169 kb]; R100 [90 kb]; V517 [54 kb]); lane 2: strain 01-09591 EHEC O104:H4; lane 3: German EHEC O104:H4 2011 outbreak strain LB226692; lane 4: German EHEC O104:H4 2011 outbreak strain 11-002097; lane 5: EHEC O157:H7 strain EDL 933; lane 6: E. coli 39R861 molecular size marker.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022751-g004: Plasmid profile of German EHEC O104:H4 outbreak strain and strain 01-09591.Comparison of the plasmid content of German EHEC O104:H4 outbreak strains and the 01-09591 (O104:H4; HUSEC041). Lane 1: molecular mass markers (plasmids R27 [169 kb]; R100 [90 kb]; V517 [54 kb]); lane 2: strain 01-09591 EHEC O104:H4; lane 3: German EHEC O104:H4 2011 outbreak strain LB226692; lane 4: German EHEC O104:H4 2011 outbreak strain 11-002097; lane 5: EHEC O157:H7 strain EDL 933; lane 6: E. coli 39R861 molecular size marker.
Mentions: Plasmid profiling demonstrated that LB226692 and 01-09591 each harbor two large plasmids (Fig. 4; 83 and 90 kb and 75 and 95 kb, respectively). Sequence analysis shows that the smaller plasmid of LB226692 contains aggregative adherence fimbriae type I (AAF/I) but lacks the EAEC heat-stable enterotoxin encoding gene, astA. The larger plasmid is an incompatibility group I1 (IncI1) plasmid with high similarity to pEC_Bactec (NCBI acc. no. GU371927) that harbors TEM-1 and CTX-M-15 beta-lactamase genes [17]. The large plasmid of strain 01-09591 appears to be closely related to the IncI1 family plasmid pSERB1 (NCBI acc. no. AY686591) from EAEC strain C1096 [18]. This strain (01-09591) also has a TEM-1 beta-lactamase that is located on a sequence contig carrying a number of genes encoding plasmid functions; we propose that this locus is also carried on the larger plasmid. The smaller plasmid of 01-09591 is an EAEC plasmid containing AAF/III, which is astA-positive, and closely related to the plasmid from EAEC 55989. Vitek® 2 and E-test® resistance testing indicates that both strains (LB226692 and 01-09591) have a TEM-1 phenotype. The ESBL genotype (CTX-M-15) of strain LB226692 was also phenotypically confirmed.

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