Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC)

Abstract

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).

Fig. 1

Comparisons of enterobacteria phage VT2phi_272 with the corresponding genomic region of E. coli GOS1 and E. coli strain 55989. Analysis was performed by employing the ACT software tool (Sanger Institute, http://www.sanger.ac.uk). The relationship between each pair of sequences are depicted. Similar coding sequences are indicated by red-colored lines. The stx genes are boxed

Fig. 2

Phylogenetic analysis of completely sequenced E. coli strains based on multilocus sequence typing. The phylogenetic analysis was conducted with MEGA 5.05 (Tamura et al. 2011). The resulting Maximum Likelihood tree illustrates the close relationship of the German outbreak strain (red dot) to EAEC 55989 (black dot). The pathotype of each E. coli strain is indicated in front of the strain name (see below for abbreviations). Bootstrap values were calculated from 100 resamplings. Bootstrap values below 50 were not shown. The following E. coli strains were used in the analysis: entero-aggregative E. coli (EAEC) 042 (FN554766), uropathogenic E. coli (UPEC) 536 (CP000247), EAEC 55989 (CU928145), commensal non-pathogenic E. coli (NPEC) ABU83972 (CP001671), avian pathogenic E. coli (APEC) O1 (CP000468), lab B strain BL21(DE3) (AM946981), lab B strain REL606 (CP000819), industrial production strain KO11 (CP002516), enteropathogenic E. coli (EPEC) CB9615 (CP001846), UPEC CFT073 (AE014075), EPEC E2348/69 (FM180568), enterotoxigenic E. coli (ETEC) E24377A (CP000800), commensal ED1a (CU928162), ETEC H10407 (FN649414), commensal HS (CP000802), commensal IAI1 (CU928160), UPEC IAI39 (CU928164), meningitis-associated E. coli (MNEC) IHE3034 (CP001969), commensal strain K-12 substrain ATCC 8739/Crooks (CP000946), lab strain K-12 substrain BW2952 (CP001396), lab strain K-12 substrain DH1 (CP001637), lab strain K-12 substrain DH10B (CP000948), lab strain K-12 substrain MG1655 (U00096), lab strain K-12 substrain W3110 (AP009048), adherent-invasive E. coli (AIEC) LF82 (CU651637), AIEC NRG 857C (CP001855), EHEC O103:H2 12009 (AP010958), EHEC O111:H- 11128 (AP010960), EHEC O157:H7 EC4115 (CP001164), EHEC O157:H7 EDL933 (AE005174), EHEC O157:H7 Sakai (BA000007), EHEC O157:H7 TW14359 (CP001368), EHEC O26:H11 11368 (AP010953), MNEC S88 (CU928161), commensal SE11 (AP009240), commensal SE15 (AP009378), environmental strain SECEC SMS-3-5 (CP000970), AIEC UM146 (CP002167), UPEC UMN026 (CU928163), porcine ETEC UMNK88 (CP002729), UPEC UTI89 (CP000243), and lab strain W (CP002185). Escherichia fergusonii ATCC 35469 was used as outgroup (CU928158)

Fig. 3

Linear comparison of E. coli pEC_Bactec plasmid with corresponding GOS1 and GOS2 contigs. The top map represents the pEC_Bactec plasmid (GU371927.1), the resistance genes are highlighted in pink, IS-elements/transposases in yellow, plasmid replication/stabilization genes in blue, the tra operon in orange, pil operon in brown, and remaining genes in gray. The scale is in base pairs. All maps were done with GenVision software (http://www.dnastar.com/t-products-genvision.aspx)

Fig. 4

Comparison of GOS1 and GOS2 genes with two different pAA-type plasmids. The two outermost rings represent maps of a 55989p and b p042 from strain E. coli 55989 and E. coli 042, respectively. Virulence factors and selected important genes are highlighted and colored. The second and the third rings represent presence (colored) or absence (gray) of GOS1 and GOS2 orthologs. The inner rings represent the GC contents of the plasmids

Fig. 5

Proposed scheme of the origin of the new E. coli pathotype—EAHEC