Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli.

Analysis of
Collection of
Hemolytic Uremic
Syndrome–
associated
Enterohemorrhagic
Escherichia coli
Alexander Mellmann,* Martina Bielaszewska,*
Robin Köck,*Alexander W. Friedrich,* Angelika
Fruth,† Barbara Middendorf,* Dag Harmsen,‡
M. Alexander Schmidt,§ and Helge Karch*
Multilocus sequence typing of 169 non-O157 entero-
hemorrhagic Escherichia coli (EHEC) isolated from patients
with hemolytic uremic syndrome (HUS) demonstrated 29
different sequence types (STs); 78.1% of these strains clus-
tered in 5 STs. From all STs and serotypes identifi ed, we es-
tablished a reference panel of EHEC associated with HUS
(HUSEC collection).
Enterohemorrhagic Escherichia coli (EHEC) strains are a highly pathogenic subgroup of Shiga toxin–producing
E. coli (STEC) that cause severe human diseases, including
bloody diarrhea and hemolytic uremic syndrome (HUS)
(1). The ability to cause severe human disease differenti-
ates EHEC from other STEC found in the environment that
are less pathogenic or nonpathogenic. E. coli O157:H7 is
the most frequent EHEC implicated as a cause of HUS (2),
but non–O157:H7 EHEC are variably present as the only
pathogens in stools from HUS patients (1,3,4).
A recent phylogenetic analysis of E. coli isolated from
humans and animals in different geographic areas by mul-
tilocus sequence typing (MLST), the current standard for
phylogenetic analyses of bacteria, indicated extensive al-
lelic variations and homolog recombinations in pathogenic
lineages and demonstrated repeated and independent evo-
lution of pathogenic strains (5,6). However, only a limited
number of EHEC associated with HUS have been so inves-
tigated. Therefore, we performed a comprehensive MLST-
based examination of the molecular phylogeny of EHEC
isolated from HUS patients and established a collection of
representative HUS-associated enterohemorrhagic E. coli
(HUSEC) (www.ehec.org).
The Study
From 1996 through 2006, 524 EHEC were isolated as
the only pathogens from fecal samples of epidemiologi-
cally unrelated patients with HUS (1 strain per patient).
The isolation was achieved by using previously described
procedures (7). The isolates were confi rmed as E. coli by
API 20 E (bioMérieux, Marcy l’Etoile, France) and se-
rotyped (8) by using antisera against E. coli O antigens
1–181 and H antigens 1–56. In all nonmotile isolates from
serogroups O26, O103, O111, O145, and O157, fl iC genes
were genotyped (9,10). MLST was performed as described
previously (6) with small modifi cations (11). Phylogenetic
analyses were based on allelic data that used the BURST
algorithm (12) to achieve a more robust interpretation of
the clustering and to reduce the infl uences by the effects
of the recombination, which are widespread in E. coli (6).
In addition, the stringent defi nition of clonal complexes
(CCs), with which strains sharing at least 6 identical alleles
are grouped into the same CC, was applied. The minimum
spanning tree was generated from the allelic profi les by us-
ing Shigella dysenteriae strain M1354 (ST243, by using
data from http://web.mpiib-berlin.mpg.de/mlst/dbs/Ecoli)
as outgroup (online Appendix Figure, available from www.
cdc.gov/EID/content/14/8/1287-appG.htm).
Among 524 EHEC isolated from HUS patients, 355
(67.7%) belonged to serotypes O157:H7/H– (249 were
non–sorbitol-fermenting EHEC O157:H7/H– and 106 were
sorbitol-fermenting EHEC O157:H–), and 169 (32.3%) be-
longed to 34 non-O157 serotypes. Because the phylogeny
of E. coli O157:H7/ H- has been extensively studied and is
well established (5,13), we focused on MLST analysis of
the 169 non-O157 strains, which represent all non-O157
EHEC serotypes from German HUS patients during the
study period. We performed MLST analysis of only a sub-
set of 10 O157 strains as controls.
MLST analysis of 169 non-O157 EHEC isolates
distinguished 29 different sequence types (STs), which
clustered into 10 CCs and 12 singletons (Table 1). The
predominant ST was ST21, which consisted of 43 iso-
lates (25.4% of non-O157 EHEC), followed by ST29 (30
isolates, 17.8%), ST32 (30 isolates, 17.8%), ST17 (15
isolates, 8.9%), and ST16 (14 isolates, 8.3%) (Table 1).
These 5 STs included 78.1% of all HUS-associated non-
O157 EHEC. The remaining 21.9% (n = 37) of the non-
O157 EHEC strains belonged to 24 other STs that com-
prised only 1–3 strains; 14 of these STs were found only
once (Table 1). Among the 10 CCs identifi ed, CC29 was
the most frequent. It comprised 89 strains of 5 STs, cor-
responding to 60.1% of 148 strains that could be assigned
to a CC and to 52.7% of all non-O157 EHEC (Table 1).
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 1287
*Institute for Hygiene and the National Consulting Laboratory on
Hemolytic Uremic Syndrome, Münster, Germany; †Robert Koch
Institute, Wernigerode, Germany; ‡Periodontology, Münster; and
§Center for Molecular Biology of Infl ammation, Münster
DOI: 10.3201/eid1408.071082

After CC29, CC32 and CC20 were the most frequently
identifi ed CCs (Table 1).
The predominant serotypes identifi ed among the 169
non–O157 HUS-associated EHEC were O26:H11/H– (n =
72; 42.6%), O145:H28/H– (n = 32; 18.9%), O111:H8/H– (n
= 14; 8.3%), and O103:H2/H– (n = 14; 8.3%). The nonmo-
tile strains within these serogroups shared the H antigen–
encoding fl iC gene with the motile strains that expressed
the respective H antigen (Table 1). These 8 serotypes to-
gether constituted 132 (78.1%) of the non-O157 EHEC as-
sociated with HUS, whereas the other 37 strains (21.9%)
belonged to 26 different serotypes, 17 of which contained
only a single isolate (Table 1).
The most frequent serotypes including O26:H11/H–,
O103:H2/H–, O111:H8/H–, and O145:H28/H– clustered
into the 5 most prevalent STs (Table 1). However, not
all isolates of the same serotype always belonged to the
same ST (Table 1). One example is serotype O26:H11/H–
(fl iC
H11
), which was the most common non–O157 EHEC
associated with HUS and clustered into 4 STs as single-
locus variants (Table 1). Each of four O rough (OR) strains
(2 OR:H11, and 1 each OR:H2 and OR:H–), none of which
could be successfully serotyped, was matched by its ST to
an O typeable strain, indicating a recent conversion from
the smooth to the rough strain form.
The relationships among members of the different STs
and CCs are demonstrated in the online Appendix Figure.
Within the serogroup O111, 14 isolates belonging to sero-
types O111:H8 and O111:H– (fl iC
H8
) were ST16 (CC29). In
contrast, the EHEC O111:H10 isolate with ST43 (CC10)
shared none of the 7 MLST loci with the O111:H8/H–
strains, indicating that EHEC O111 causing HUS originate
from 2 different clonal sources. Similar differences were
observed between EHEC O145:H25 (ST342)/O145:H–
(fl iC
H25
) (ST659) and O145:H28 (ST32). Whereas ST659 is
a single-locus variant of ST342, both allelic profi les differ
in all loci from ST32.
The combination of MLST analysis and serotyping en-
abled us to establish the HUSEC collection. This collection
comprises 41 EHEC isolated from HUS patients in Germa-
ny, which includes all 36 EHEC serotypes (O157 and non-
O157) isolated from HUS patients and all 31 STs identifi ed
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1288 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008
Table 1. Clonal complexes, sequence types, and serotypes of non-O157 EHEC from patients with hemolytic uremic syndrome*
CC No. strains (%)† ST No. strains (%)† Serotype‡ (no. strains)§
29 89 (52.7) ST21 43 (25.4) O26:H11/H– (fliCH11) (41), OR:H11 (1), Ont:Hnt (1)
ST29 30 (17.8) O26:H11/H– (fliCH11) (29), OR:H11 (1)
ST27 1 (0.6) O26:H11
ST396 1 (0.6) O26:H11
ST16 14 (8.3) O111:H8/H– (fliCH8)
32 32 (18.9) ST32 30 (17.8) O145:H28/H– (fliCH28)
ST137 2 (1.2) O145:H– (fliCH28)
20 16 (9.5) ST17 15 (8.9) O103:H2/H– (fliCH2) (14), OR:H2 (1)
ST20 1 (0.6) O119:H2
10 3 (1.8) ST43 1 (0.6) O111:H10
ST330 2 (1.2) Ont:H–
11 3 (1.8) ST335 3 (1.8) O55:H7
40 1 (0.6) ST40 1 (0.6) O112:H–
69 1 (0.6) ST69 1 (0.6) O73:H18
101 1 (0.6) ST101 1 (0.6) O55:Hnt
155 1 (0.6) ST56 1 (0.6) O113:H21
469 1 (0.6) ST679 1 (0.6) O163:H19
NA 2 (1.2) ST25 2 (1.2) O128:H2
NA 2 (1.2) ST678 2 (1.2) O104:H4
NA 2 (1.2) ST655 2 (1.2) O121:H19
NA 1 (0.6) ST329 1 (0.6) O136:Hnt
NA 3 (1.8) ST342 2 (1.2) O145:H25/H– (fliCH25)
ST659¶ 1 (0.6) O145:H– (fliCH25)
NA 1 (0.6) ST677 1 (0.6) O174:H21
NA 1 (0.6) ST39 1 (0.6) O70:H8
NA 1 (0.6) ST675 1 (0.6) O76:H19
NA 3 (1.8) ST442 3 (1.8) O91:H21
NA 3 (1.8) ST306 3 (1.8) O98:H– (2), OR:H– (1)
NA 2 (0.6) ST672 2 (1.2) O104:H21 (1), Ont:H21 (1)
*CC, clonal complex; ST, sequence type; EHEC, enterohemorrhagic Escherichia coli; HUS, hemolytic uremic syndrome; NA, not assigned.
†% of strains of a CC and ST among all 169 non-O157 EHEC isolated from HUS patients.
‡H–, nonmotile; OR, O rough (autoagglutinable strain); nt, not typeable by the E. coli O and H antisera used.
§Number of strains of the serotype that belonged to the respective ST; if no number is given, all strains of the serotype belonged to the respective ST.
¶ST659 is a single-locus variant of ST342.

HUS-associated Escherichia coli
within these serotypes (Table 2). The strains included in
this HUSEC collection were reserotyped and characterized
for their stx genotypes and the presence of the eae gene
(Table 2). Phenotypic characteristics and additional proper-
ties such as putative virulence determinants are available at
www.EHEC.org.
Conclusions
Most (81.1%) of the non-O157 EHEC clustered into 3
CCs and belonged to a limited number of serotypes. These
strains were recovered independently from different re-
gions in Germany over an 11-year period. For the remain-
ing strains, epidemiologic support is not as strong, and the
clonal analysis demonstrated that their chromosomal back-
grounds are highly divergent from those of CC29, CC32,
and CC20. In 14 STs, we have only 1 isolate. In these cases,
excluding concurrent or recent infection by E. coli O157
serologically is even more important. This exclusion was
not always possible because patients’ serum for the investi-
gation of immunoglobulin M anti-O157 lipopolysaccharide
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 1289
Table 2. Strains of the HUSEC collection representing all serotypes of HUS-associated EHEC strains isolated in Germany, 1996–
2006*
Strain Original Year of isolation Serotype ST (CC) eae stx1 stx2†
HUSEC001 05-946 2005 O111:H10 43 (10) – – 2
HUSEC002 5152/97 1997 Ont:H– 330 (10) – – 2
HUSEC003 6334/96 1996 O157:H7 11 (11) + – 2
HUSEC004 3072/96 1996 O157:H– 11 (11) + – 2
HUSEC005 2907/97 1997 O55:H7 335 (11) + – 2
HUSEC006 5376/99 1999 O157:H– 587 (11) + – 2
HUSEC007 7382/96 1996 O103:H2 17 (20) + – 2
HUSEC008 2791/97 1997 O103:H– 17 (20) + – 2
HUSEC009 6833/96 1996 OR:H2 17 (20) + – 2
HUSEC010 1805/00/A 2000 O119:H2 20 (20) + 1 –
HUSEC011 2516/00 2000 O111:H8 16 (29) + 1 2
HUSEC012 6037/96 1996 O111:H– 16 (29) + 1 2
HUSEC013 2245/98 1998 O26:H11 21 (29) + 1 –
HUSEC014 5080/97 1997 O26:H– 21 (29) + 1 2
HUSEC015 126814/98 1998 OR:H11 21 (29) + 1 2
HUSEC016 5028/97 1997 Ont:Hnt 21 (29) + 1 –
HUSEC017 3319/99 1999 O26:H11 27 (29) + 1 2
HUSEC018 1530/99 1999 O26:H11 29 (29) + – 2
HUSEC019 1588/98 1998 OR:H11 29 (29) + 1 –
HUSEC020 3271/00 2000 O26:H11 396 (29) + – 2
HUSEC021 0488/99 1999 O145:H28 32 (32) + – 2
HUSEC022 4557/99 1999 O145:H– 137 (32) + – 2
HUSEC023 1169/97/1 1997 O112:H– 40 (40) – – 2dact
HUSEC024 2996/96 1996 O73:H18 69 (69) – – 2dact
HUSEC025 06-05009 2006 O55:Hnt 101 (101) – 1 –
HUSEC026 99-09355 1999 O113:H21 56 (155) – – 2dact
HUSEC027 03-07727 2003 O163:H19 679 (469) – – 2dact
HUSEC028 03-06687 2003 O128:H2 25 (NA) – 1c 2d
HUSEC029 4256/99 1999 O70:H8 39 (NA) + – 2
HUSEC030 05-03519 2005 O98:H– 306 (NA) – 1 –
HUSEC031 7792/96 1996 OR:H– 306 (NA) + 1 –
HUSEC032 2441/98 1998 O136:Hnt 329 (NA) – 1c 2
HUSEC033 4392/97 1997 O145:H25 342 (NA) + – 2
HUSEC034 3332/99 1999 O91:H21 442 (NA) – 1 2+2dact
HUSEC035 1529/98 1998 O121:H19 655 (NA) + – 2
HUSEC036 2839/98 1998 O145:H– 659 (NA) + 1 2c
HUSEC037 02-03885 2002 O104:H21 672 (NA) – 1 2+2dact
HUSEC038 3356/97/B 1997 Ont:H21 672 (NA) – 1 2dact
HUSEC039 3651/96 1996 O76:H19 675 (NA) – 1c –
HUSEC040 220/00 2000 O174:H21 677 (NA) – – 2c
HUSEC041 01-09591 2001 O104:H4 678 (NA) – – 2
*HUSEC, hemolytic uremic syndrome–associated enterohemorrhagic Escherichia coli; EHEC, enterohemorrhagic E. coli. For each serotype, the
multilocus sequence type (ST) and the corresponding clonal complex (CC) are given in accordance to the E. coli multilocus sequence typing website
(http://web.mpiib-berlin.mpg.de/mlst/dbs/Ecoli). Furthermore, the presence (+, present; – absent) of the intimin gene (eae), the Shiga toxin gene (stx), and
its subtype(s) are specified. nt, not typeable by the O and H antisera used; H–, nonmotile; OR, O rough (autoagglutinable strain); NA, not assigned.
†2dact, stx2d-activatable.

antibodies is frequently not available. However, at least
some of these strains might represent emerging clones in
the human population, such as O111:H10 (10), O113:H21
(14), and O121:H19 (15). Thus, strains of these serotypes
included in our HUSEC collection can be used in future
studies as a reference to compare EHEC isolated in other
countries from HUS patients. This would allow timely dis-
covery of the emergence of new non-O157 clones associ-
ated with HUS and the virulence traits that they contain
(www.ehec.org).
Acknowledgment
We are grateful to Phillip I. Tarr for extensive discussions of
the manuscript.
This study was supported by grants from the Federal Min-
istry of Education and Research Network Zoonoses and the Net-
work European Research ERA–NET PathoGenoMics.
Dr Mellmann is a consultant for hygiene and infectious dis-
eases at the University Hospital Münster. His professional inter-
ests include molecular identifi cation and epidemiology of EHEC
and other bacterial pathogens.
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Address for correspondence: Alexander Mellmann, Institute for Hygiene,
University Hospital Münster, Robert Koch Str 41, 48149 Münster,
Germany; email: mellmann@uni-muenster.de
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1290 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008