Figure 1.
Events timeline of German EHEC O104:H4 outbreak.
Major events relating to the outbreak epidemiology (below arrow) and those relating to genomic elucidation efforts (above arrow) are noted separately in the graph. Lines within the arrow indicate single day progression, with the date noted every 5th day. Events span from early May 2011 to early June 2011. Times are noted in Central European Time (CET). Abbreviations: BfR = Bundesinstitut für Risikobewertung (Federal Institute for Risk Assessment, Germany), BGI = Beijing Genomics Institute (People's Republic of China), ECDC = European Center for Disease Prevention and Control (Sweden), HPA = Health Protection Agency (United Kingdom), HUS = hemolytic uremic syndrome, LT = Life Technologies Group, PGM™ = Ion Torrent Personal Genome Machine™, RKI = Robert Koch Institute (Germany), ST = multilocus sequence type, UKE = University Hospital Hamburg (Germany), UKM = University Hospital Muenster (Germany), WGS = whole genome sequencing.
Figure 2.
Optical Map similarity cluster of German EHEC O104:H4 outbreak.
De novo whole genome optical maps from EHEC O104:H4 outbreak strains (‘LB’ prefix) and historical O104:H4 and O104:H21 strains (01-09591 and 02-03885) were created using the Argus™ Optical Mapping System with the NcoI restriction enzyme. An in silico genomic map of the reference strain 55989 was created in MapSolver™ by importing genomic sequence data from NCBI (acc. no. NC_011478) and applying the NcoI restriction pattern. Optical maps and in silico maps were compared using the default MapSolver™ parameters and clustered using UPGMA based on the resulting pairwise distance metrics. Scale represents percent difference. Strain name, serotype, country, city, and year of isolation are indicated.
Figure 3.
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.
Figure 4.
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.
Figure 5.
Evolutionary model of the origin of the German EHEC O104:H4 outbreak strain.
Evolutionary model of the current outbreak strain (LB226692) and the historical strain (01-09591) from 2001 based on whole chromosomal and plasmid data. Numbers on connecting lines indicate the number of loci that differ between the strains as determined by analysis of 1,144 core genome genes. The genes of the PCR test for differentiation of the HUSEC041 complex (stx2: red; terD: blue; rfbO104: green; fliCH4: yellow), [16] for antibiotic resistance (orange: TEM-1 and CTX-M-15), and for the differentiation of EAEC plasmids [5] (astA: white) are colored. The order of plasmid acquisition and loss were arbitrarily chosen in the illustration as the exact sequence of events is not known. Year of isolation for each strain is noted in the lower left corner of each rectangle.
Figure 6.
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).