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. 2004 Mar;42(3):954–962. doi: 10.1128/JCM.42.3.954-962.2004

Phenotypic and Genotypic Analyses of Enterohemorrhagic Escherichia coli O145 Strains from Patients in Germany

Anne-Katharina Sonntag 1, Rita Prager 2, Martina Bielaszewska 1, Wenlan Zhang 1, Angelika Fruth 2, Helmut Tschäpe 2, Helge Karch 1,*
PMCID: PMC356868  PMID: 15004038

Abstract

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome. However, there have been few genetic analyses of this EHEC group. We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002. Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H−). One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains. The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain. Each of the 117 strains with the fliC-RFLPH28 pattern harbored eae γ, whereas the three strains with the fliC-RFLPH25 pattern possessed eae β. Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified. Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles. These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types. The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens.

Enterohemorrhagic Escherichia coli (EHEC) O145 strains were among the first six serogroups isolated from patients with hemolytic-uremic syndrome by Karmali and colleagues (13). Recently, E. coli O145 has emerged as one of the most important non-O157 serogroups associated with diarrhea and hemolytic-uremic syndrome in Europe (1, 4, 6, 7, 8, 34). A large outbreak of EHEC O145 infection has been reported from Japan (15), and this serotype has also been recovered recently in the United States (11). Some EHEC O145 strains isolated from patients possess the H28 flagellar antigen (4), but the majority of these isolates are nonmotile (8). The same observation has been reported for EHEC O145 strains isolated from cattle (9, 21), which are a potential reservoir of these pathogens for humans.

The nonmotility of the majority of EHEC O145 strains isolated from humans and animals complicates classic serotyping and hampers investigations of the population structure of EHEC O145 strains. Moreover, the inability to categorize more thoroughly strains expressing the O145 antigen makes it difficult to study the epidemiology of infections caused by these pathogens, including identifying reservoirs and sources of the infection for humans and modes of transmission. Recently, restriction fragment length polymorphism (RFLP) analysis of the flagellin-encoding (fliC) gene was used to identify and characterize the flagellin-encoding subunit of H antigen (5, 23, 39). This demonstrated that within serogroups O157 and O26, nonmotile strains and those harboring H7 and H11, respectively, belong to a single clone (5, 39). This technique has not yet been applied to E. coli O145.

The defining characteristic of EHEC strains is the production of Shiga toxin (Stx). Two major Shiga toxin types, Stx1 and Stx2, have been differentiated based on toxin neutralization assays (26) and sequence analysis of stx genes (10). In addition, EHEC strains usually cause a particular pathology called attaching and effacing lesions that are mediated in part by the adhesin intimin. This outer membrane protein is encoded by the eae gene within the pathogenicity island locus of enterocyte effacement (37). PCR and sequence analyses demonstrate a growing number of intimin alleles in EHEC strains and enteropathogenic E. coli strains (41). However, only a few alleles are dominant in the major serogroups of EHEC strains. Specifically, intimin γ is found in EHEC O157:H7 and sorbitol-fermenting EHEC O157:H−, EHEC O111:H8, and EHEC O145:H− strains (7, 20). EHEC O26:H11 strains carry intimin β (20, 38), whereas EHEC O103:H2 and O103:H− strains express intimin ɛ (20, 22). Studies investigating the relatedness of multiple EHEC clinical isolates within a single serogroup demonstrated that strains within serogroups O26, O103, and O157 possess a common fliC gene and a single intimin allele (5, 20, 22, 38).

To better characterize EHEC O145 strains, we applied fliC-RFLP to determine the presence of and to characterize the fliC gene in nonmotile strains. In addition, we determined the pulsed-field gel electrophoresis (PFGE) patterns, stx genotypes, eae alleles, plasmid profiles, and distribution of plasmid-encoded putative virulence genes to create a basis for subtyping of these pathogens in epidemiological studies.

MATERIALS AND METHODS

Bacterial strains.

Three E. coli O145:H28, one E. coli O145:H25, and 116 E. coli O145:H− strains isolated from stool samples of patients with hemolytic-uremic syndrome (n = 24), bloody diarrhea (n = 5), or watery diarrhea (n = 91) were investigated (Table 1). The strains were isolated at the Institute of Hygiene and Microbiology, University of Würzburg, Würzburg; at the Institute of Hygiene, University Hospital Münster, Münster; and the Robert Koch Institute, Wernigerode, Germany, during routine diagnostic efforts and epidemiological studies (as reference laboratories) between January 1996 and December 2002. They originated from the analysis of 10,499 stool samples, 732 of which were from patients with hemolytic-uremic syndrome and the remaining 9,767 were from patients with diarrhea. The procedures used for EHEC isolation from stool samples were described previously (6). The EHEC O145 strains were from sporadic cases of infection and from several small outbreaks.

TABLE 1.

Serotypes, fliC-RFLP patterns, and virulence gene profiles of EHEC O145 strains

No. of strains Serotypea fliC-RFLP pattern Chromosomal characteristics
Plasmid-encoded genes
stx eae EHEC-hlyA katP espP etpD
1 O145:H28 H28 1 γ + − − +
1 O145:H28 H28 1 γ + − − −
1 O145:H28 H28 1 γ + − + −
13 O145:H− H28 1 γ + − + −
10 O145:H− H28 1 γ + + + −
67 O145:H− H28 2 γ + − + −
6 O145:H− H28 2 γ + + + −
5 O145:H− H28 2 γ + + − −
3 O145:H− H28 2 γ + − − −
1 O145:H− H28 2 γ + − − +
1 O145:H− H28 2 γ − − + −
2 O145:H− H28 1+2 γ + − + −
1 O145:H− H28 1+2 γ + + + −
3 O145:H− H28 2c γ + − + −
1 O145:H− H28 2c γ + + + −
1 O145:H− H28 2c γ + − − +
1 O145:H25 H25 2 β + − − −
1 O145:H− H25 2 β + − − −
1 O145:H− H25 1+2c β + + + −
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a

H−, nonmotile strains.

Case definition.

Patients with diarrhea had three or more watery stools without visible blood per day. Bloody diarrhea was defined as diarrhea where visible blood was noted in the stool. Hemolytic-uremic syndrome was defined as a case of microangiopathic hemolytic anemia (hematocrit less than 30% with peripheral evidence of intravascular hemolysis), thrombocytopenia (platelet count of less than 150,000/mm3), and renal insufficiency (serum creatinine concentration greater than the upper limit of the normal range for age) (36).

Phenotypic methods.

Isolates were serotyped with antisera against E. coli O antigens 1 to 181 and H antigens 1 to 56, and a microtiter method described by Prager et al. (23). Stx production was tested by a commercial enzyme immunoassay (Ridascreen; R-Biopharm, Darmstadt, Germany). Fermentation of sorbitol was detected on sorbitol MacConkey agar plates after overnight incubation. The enterohemolytic phenotype was sought with blood agar plates containing 5% defibrinated and washed human erythrocytes and 10 mM CaCl2 (28, 30).

PFGE.

PFGE was performed immediately after the isolation of the strains with the procedure described previously (22). Chromosomal DNA of Salmonella braenderup strain H9812 (Centers for Disease Control and Prevention, Atlanta, Ga.) digested with XbaI was used as a molecular size marker. Restriction fragment patterns of genomic DNA were analyzed with the RFLPScan package (Scanalytics CSP, Inc.). Patterns differing in up to four bands were considered related in accordance with the criteria of Tenover et al. (32). Small variations consisting of one to three bands’ difference within related PFGE patterns were regarded as subtypes (32). PFGE patterns were deposited in the national reference library of PFGE patterns (German Pulse-Net) (www.foodborne-net.de /content/e25/e70/). The PFGE patterns of EHEC O157, O26, and O103 strains used for comparison with those of EHEC O145 strains were also from the German Pulse-Net PFGE database.

Plasmid profiles.

Plasmid profiles were determined in freshly isolated EHEC O145 strains with a procedure described by Tietze and Tschäpe (33). Plasmids R27 (169 kb), R100 (90 kb) (Roche Molecular Biochemicals, Mannheim, Germany), and the 54-kb plasmid of strain V517 (17) were used as molecular size markers.

PCR.

PCRs were performed in the Biometra TGradient 96 cycler (Biometra GmbH, Göttingen, Germany) in 25 μl containing 2.5 μl of bacterial suspension (ca. 104 bacteria), 2.5 μl of 10-fold-concentrated polymerase synthesis buffer Y with 2.0 mM MgCl2, 5 μl of enhancer solution (PEQLAB Biotechnologie, Erlangen, Germany), 200 μM each deoxynucleoside triphosphate, 15 pmol of each primer, and 0.65 U of Taq DNA polymerase (PEQLAB). To detect stx genes, primers KS7 and KS8 (stx1B, stx1cB) and GK3 and GK4 (stx2B, stx2cB) were used as described previously (12, 27). stx1 and stx1c were differentiated by restriction analysis of KS7 and KS8 amplification products with HhaI (40), and stx2 and stx2c were differentiated by restriction analysis of GK3 and GK4 amplification products with HaeIII (24).

eae was detected with primers SK1 and SK2 (27) and further characterized with primer pairs SK1 and LP3 and SK1 and LP4, which amplify eae γ and β, respectively (20). Plasmid genes EHEC-hly, espP, katP, and etpD were detected with primer pairs HlyA1 and HlyA4 (28), esp-A and esp-B (3), wkat-B and wkat-F (2), and D1 and D13R (29), respectively. E. coli O157:H7 strain EDL933 (19) was used as a positive control in PCRs for the detection of stx1, stx2, the eae conserved region, eae γ, and plasmid genes. E. coli O26:H− strain 5720/96 (38) was used as a positive control for eae β.

fliC PCR-RFLP.

The fliC gene was amplified with primers FSa1 [CAA GTC ATT AAT AC(A/C) AAC AGC C] and rFSa1 [GAC AT(A/G) TT(A/G) GA(G/A/C) ACT TC(G/C) GT] (16), and 15 μl of each PCR product was digested with restriction endonuclease HhaI (New England BioLabs, Frankfurt, Germany). Restriction fragments were separated on a 2% (wt/vol) agarose gel and visualized by staining with ethidium bromide.

fliC sequencing.

The fliC PCR amplification products were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced with an automated ABI Prism 3100 Avant genetic analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), PCR and internal primers, and the ABI Prism BigDye terminator ready reaction cycle sequencing kit (Perkin-Elmer Applied Biosystems). Nucleotide sequence analysis was performed with the DNASIS program (Hitachi Software, San Bruno, Calif.) and homology searches were performed with the NCBI GeneBlast (http://www.ncbi.nlm.nih.gov/BLAST/).

Nucleotide sequence accession numbers.

The nucleotide sequences of the fliC genes from EHEC O145 strains 0917/99, 2032/99, 4932/97, and 2839/98 have been entered into the EMBL database library (accession numbers AJ566338 to AJ566341, respectively).

RESULTS

fliC-RFLP and sequence analysis of fliC genes.

PCR with primers FSa1 and rFSa1, which amplify fliC, produced a 1.8-kb product in 117 of the 120 EHEC O145 isolates. These isolates included three motile strains that possessed the H28 antigen by serotyping (Table 1) and 114 nonmotile strains. HhaI-RFLP analysis demonstrated that all 117 strains had an identical fliC restriction pattern (Table 1). In contrast, three remaining strains, one of serotype O145:H25 and two nonmotile EHEC O145 strains, produced a 1.5-kb amplicon in the fliC PCR and displayed a fliC-RFLP pattern that differed from that of the 117 former strains (Table 1). The fliC PCR products and fliC-RFLP patterns of motile strains of serotypes EHEC O145:H28 and O145:H25 and of representative O145:H− strains are demonstrated in Fig. 1.

FIG. 1.

FIG. 1.

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fliC PCR products and fliC-RFLP patterns of EHEC O145 strains. Lane M, molecular size markers (100-bp DNA ladder, Gibco-BRL). In lanes 1 to 4, fliC PCR products of the following EHEC O145 strains (serotypes in parentheses) are shown: lane 1, 0917/99 (O145:H28); lane 2, 2032/99 (O145:H−); lane 3, 4932/97 (O145:H25); lane 4, 2839/98 (O145:H−). Lanes 5 to 11 display the fliC-RFLP patterns of the following strains: lane 5, 0917/99 (O145:H28); lane 6, 2032/99 (O145:H−); lane 7, 0893/98 (O145:H−); lane 8, 3036/99 (O145:H−); lane 9, 4932/97 (O145:H25); lane 10, 2839/98 (O145:H−); lane 11, 03-03027 (O145:H−).

The sequences of the fliC PCR products from EHEC O145:H28 strain 0917/99 (GenBank accession number AJ566338) and one of the EHEC O145:H− strains that displayed the fliC-RFLPH28 pattern (strain 2032/99; GenBank accession number AJ566339) were identical. The fliC sequences from EHEC O145:H25 strain 4932/97 (GenBank accession number AJ566340) and from one of two nonmotile strains that displayed the fliC-RFLPH25 pattern (strain 2839/98; GenBank accession number AJ566341) were also identical to each other. The fliC sequences from strains with the fliC-RFLPH28 pattern demonstrated 62% nucleotide sequence homology to those from strains that displayed the fliC-RFLPH25 pattern.

Chromosomal virulence characteristics.

Five different stx genotypes were identified among the 120 EHEC O145 strains, the stx2 genotype being the most prevalent (Table 1). Each of the 120 strains was positive in the PCR with primer pair SK1 and SK2, which amplifies a conserved region of eae. One hundred seventeen of these strains also yielded an amplification product of 2.8 kb with primers SK1 and LP3, demonstrating the presence of eae γ (Table 1). These strains included three motile strains expressing the H28 antigen and 114 nonmotile strains that displayed the fliC-RFLPH28 pattern (Table 1). The three remaining strains, all of which displayed the fliC-RFLPH25 pattern (Fig. 1), yielded a 2.3-kb amplicon with primers SK1 and LP4, demonstrating the presence of eae β (Table 1).

Plasmid characteristics.

Twenty-nine different plasmid profiles were identified among the 120 EHEC O145 strains (Table 2). Seventy-eight (65.0%) of the strains belonged to three plasmid profiles that included the presence of a single plasmid of 90, 82, or 75 kb (Table 2). The remaining 42 strains displayed 26 different plasmid profiles, including mostly combinations of one or two large plasmids with up to three smaller plasmids (Table 2). One of the three EHEC O145 strains which displayed the fliC-RFLPH25 pattern possessed a single 105-kb plasmid. The other two strains harbored a single plasmid of 90 or 82 kb, thus sharing their plasmid profiles with the majority of strains of the fliC-RFLPH28 pattern (Table 2). Six combinations of plasmid-encoded putative virulence genes were detected (Table 1), among which the combination of EHEC-hlyA and espP was the most frequent (Table 1).

TABLE 2.

Plasmid profiles of EHEC O145 strains

Plasmid profile no. Plasmid profile (size of plasmid[s] in kb) Serotypea fliC-RFLP pattern No. of strains
1 90 O145:H− H28 32
2 90 O145:H25 H25 1
3 90; 82 O145:H− H28 2
4 90; 63 O145:H− H28 4
5 90; 51 O145:H− H28 1
6 90; 82; 60 O145:H− H28 1
7 90; 82; 2.7 O145:H− H28 1
8 90; 18; 7; 5.5 O145:H28 H28 1
9 82 O145:H− H28 36
10 82 O145:H− H25 1
11 82; 75 O145:H− H28 2
12 82; 60 O145:H− H28 2
13 82; 2.7 O145:H− H28 1
14 75 O145:H− H28 8
15 75; 60 O145:H− H28 1
16 75; 5.5 O145:H− H28 4
17 75; 57; 5.5 O145:H− H28 1
18 75; 7; 5.5 O145:H− H28 4
19 97; 90 O145:H− H28 3
20 97; 82 O145:H− H28 4
21 97; 72; 6.3 O145:H− H28 1
22 97; 3.4 O145:H− H28 1
23 97; 90; 3.4 O145:H− H28 1
24 105 O145:H− H25 1
25 112; 3.3; 2.7 O145:H28 H28 2
26 120; 54 O145:H− H28 1
27 135; 82 O145:H− H28 1
28 165; 82 O145:H− H28 1
29 165; 82; 5.5 O145:H− H28 1
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a

H−, nonmotile strains.

Virulence gene profiles.

The combination of eae type, stx genotype, and plasmid-encoded putative virulence genes resulted in 17 different virulence gene profiles among the 120 EHEC O145 strains. The most common virulence profile, displayed by 67 strains, included the presence of eae γ, stx2 and EHEC-hly and espP plasmid genes (Table 1).

PFGE.

Forty-seven PFGE types (designated 1 to 47) were identified among the 120 EHEC O145 strains. The PFGE patterns contained between 12 and 18 bands of between 1,130 and 50 kb. Within 10 of the PFGE types, one to four subtypes could be distinguished that differed from the major PFGE type by one to three bands. In Fig. 2, 77 representative strains are shown. Their banding patterns demonstrated more than 80% similarity. No significant differences were observed between PFGE patterns of the strains displaying fliC-RFLPH28 and fliC-RFLPH25 (Fig. 2). When the PFGE patterns of 15 randomly selected EHEC O145 strains were compared to those of EHEC O157:H7, EHEC O26:H11, and EHEC O103:H2, the EHEC O145 strains belonged to a distinct cluster that was only distantly related to each of the clusters comprising strains of the other three EHEC serogroups (Fig. 3).

FIG. 2.

FIG. 2.

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Cluster analysis of EHEC O145 strains derived from PFGE data. Strain 03-03027 (the last at the bottom) belongs to the H25 fliC-RFLP; all the other strains display the H28 fliC-RFLP. Designations of clusters are given at the left.

FIG. 3.

FIG. 3.

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Genetic distance between EHEC strains of serogroups O145, O26, O103, and O157 as calculated on the basis of their PFGE patterns.

Analysis of PFGE clusters.

Seven clusters of strains that belonged to identical PFGE types were identified among the EHEC O145 strains investigated (Fig. 2, Table 3). The largest cluster consisted of several subclusters comprised of the closely related strains of PFGE type 7 and its subtypes 7a, 7b, 7c, and 7d (Fig. 2, Table 3). In three of these seven PFGE clusters (1, 6, and 10), an epidemiological association between the strains was demonstrated (Table 3). Each of the other clusters contained strains that were isolated in different years and/or in different parts of Germany (Table 3) and thus had no apparent epidemiological linkage. Strains within each of the three PFGE clusters that contained epidemiologically associated strains (clusters 1, 6, and 10) were indistinguishable by the other subtyping methods, including fliC-RFLP, virulence gene profiles, and plasmid profiles (Table 3). In contrast, strains within the four PFGE clusters that contained strains without apparent epidemiological linkages shared, with a single exception (cluster 5), identical virulence profiles, but most of them could be further discriminated by their plasmid profiles (Table 3). If strains with identical plasmid profiles occurred, they belonged to different PFGE subtypes within PFGE type 7 or, except for two strains, were isolated in different years.

TABLE 3.

Discrimination of strains within PFGE clusters by other subtyping methods

PFGE cluster no.a Strain no.b Distancec Epidemiological associationd Serotype fliC- RFLP eae stx genotype EHEC- hly katP espP etpD Size of plasmid(s) (kb)
1 02-08211 A Siblings O145:H− H28 γ stx2 + + + − 97; 90
02-08209 A O145:H− H28 γ stx2 + + + − 97; 90
5 02-07574 C NAEA O145:H− H28 γ stx2 + − + − 90
02-07076 C O145:H− H28 γ stx2 + + + − 90; 82; 2.7
6 99-09136 A Members of the same family O145:H− H28 γ stx2 + − − − 90
99-09025 A O145:H− H28 γ stx2 + − − − 90
7a 02-05482 B NAEA O145:H− H28 γ stx2 + − + − 90; 82
02-07583 B O145:H− H28 γ stx2 + − + − 97; 90; 3.4
7b 02-04447 C NAEA O145:H− H28 γ stx2 + − + − 90; 82
02-05658 C O145:H− H28 γ stx2 + − + − 75
03-02478 C O145:H− H28 γ stx2 + − + − 90
02-11508 C O145:H− H28 γ stx2 + − + − 90
7c 01-04033 C NAEA O145:H− H28 γ stx2 + − + − 82
03-03009 C O145:H− H28 γ stx2 + − + − 90
7d 01-05410 B NAEA O145:H− H28 γ stx2 + − + − 82
03-01219 B O145:H− H28 γ stx2 + − + − 90
7 02-02879 C NAEA O145:H− H28 γ stx2 + − + − 82; 2.7
02-09128 C O145:H− H28 γ stx2 + − + − 90
01-01612 C O145:H− H28 γ stx2 + − + − 90
02-11117 C O145:H− H28 γ stx2 + − + − 90
9 03-02868 C NAEA O145:H− H28 γ stx2 + − + − 90
02-02289 C O145:H− H28 γ stx2 + − + − 82
10 01-08447 A Kindergarten outbreak O145:H− H28 γ stx2 + − + − 82
01-08445 A O145:H− H28 γ stx2 + − + − 82
01-08767 A O145:H− H28 γ stx2 + − + − 82
01-08766 A O145:H− H28 γ stx2 + − + − 82
01-08450 A O145:H− H28 γ stx2 + − + − 82
11 01-11921 A NAEA O145:H− H28 γ stx1 + + + − 165; 82; 5.5
99-08292 A O145:H− H28 γ stx1 + + + − 75; 5.5
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a

Numbers of PFGE clusters correspond to the PFGE types of the isolates.

b

The first two numbers in the strain number designate the year of isolation.

c

A, same city of origin; B, cities of origin located <50 miles from each other; C, cities of origin located >50 miles from each other.

d

NAEA, no apparent epidemiological association (with the other strains in the PFGE cluster).

Comparison of the non-PFGE typing methods for their potential to discriminate between the strains belonging to the seven PFGE clusters (Table 3) demonstrated no discriminatory power for the eae typing and fliC-RFLP, which each identified only a single type among these strains. stx genotyping and virulence gene profile typing differentiated two and four types, respectively, and were not able to discriminate between epidemiologically related and epidemiologically unrelated strains. Plasmid profiling distinguished 10 different types; plasmid profiles were identical in epidemiologically associated strains but could mostly discriminate between epidemiologically unrelated strains that belonged to the same PFGE cluster.

The algorithm employed for the discrimination of epidemiologically related and unrelated EHEC O145 isolates and the levels at which these methods are feasible are demonstrated in Fig. 4.

FIG. 4.

FIG. 4.

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Algorithm used for subtyping of EHEC O145 isolates.

Phenotypes.

All 120 EHEC O145 strains produced Stx, as detected by enzyme immunoassay. Also, each of the 119 EHEC O145 strains that possessed the EHEC-hly gene produced EHEC hemolysin, as evidenced by an enterohemolytic phenotype on blood agar with washed human erythrocytes. All strains fermented sorbitol when grown on Sorbitol MacConkey agar plates.

DISCUSSION

The prototype EHEC strain is E. coli O157:H7, which is the most prevalent serotype worldwide (18). However, several non-O157 serotypes have recently emerged in addition to EHEC O157:H7 as important causes of human diseases. These include EHEC O26:H11//H−, O103:H2, O111:H8/H−, and O145:H28/H− (1, 4, 8, 34). In this study, we applied fliC-RFLP typing to EHEC O145 strains isolated over 7 years from patients throughout Germany and characterized the virulence profiles of these emerging pathogens. The fliC-RFLP analysis demonstrated two different patterns, one of which was shared by three O145:H28 and almost all nonmotile strains, and the other of which was common to the O145:H25 strain and the two remaining nonmotile strains. The fliC-RFLPH28 pattern was associated with eae γ, whereas the fliC-RFLPH25 pattern was associated with eae β. The occurrence of two different fliC-RFLP patterns and two different eae types suggests that, in contrast to EHEC strains of serogroups O157 (5), O26 (38), and O103 (22), EHEC strains expressing the O145 antigen actually belong to at least two different clonal lineages. However, strains of these two lineages demonstrated more than 80% homology in their PFGE patterns.

As previously shown for each of the EHEC serogroups O26 (30, 38), O103 (22, 30), and O111 (30), the EHEC O145 strains demonstrate unique profiles of their putative virulence genes that differ from those of EHEC strains of the other major non-O157 serogroups. Specifically, five different stx genotypes were identified among the 120 EHEC O145 strains (Table 1), but only three of them, including stx1, stx2 and stx1 and stx2 were previously found in EHEC O26, O103, and O111 strains (22, 30, 38). The other two stx genotypes found in EHEC O145 strains in the present study (Table 1) have not been observed among non-O157 EHEC strains of the above three serogroups (22, 30, 38). Moreover, the most prevalent combination of plasmid-encoded genes identified in EHEC O145 strains, EHEC-hly and espP (Table 1), has not been found in EHEC strains of any of the serogroups O26, O103, or O111 (22, 30, 38). Also, most of the EHEC O145 strains differ from EHEC strains of serogroups O26 and O103 with respect to the eae type (20, 22).

Although 47 different PFGE patterns occurred among the EHEC O145 strains, seven clusters of strains that demonstrated highly related PFGE patterns were identified (Fig. 2, Table 3). One of these clusters contained strains isolated during an outbreak in a kindergarten, and two others included strains isolated from members of the same family. Strains within each of these three clusters had identical fliC-RFLP, virulence gene, and plasmid profiles. In contrast, no obvious epidemiological linkages could be demonstrated between strains within any of the other four clusters; these strains were isolated from patients throughout Germany and within a range of up to 2 years. Some strains within these four PFGE clusters could be further discriminated either by their virulence profiles (cluster 5) or by their plasmid profiles (clusters 7, 9, and 11). Therefore, these clusters cannot be considered parts of diffuse outbreaks of EHEC O145 infection, as it might appear if only PFGE analysis was performed. This suggests that in addition to PFGE, which forms the basis for national molecular subtyping networks for food-borne disease surveillance such as PulseNet (31) and German Pulse-Net, additional subtyping methods may be useful for the final decision about the occurrence of an outbreak. Although plasmid profiling proved in our study to have a high discriminatory potential, this method is cumbersome and needs further interlaboratory standardization. Therefore, phage typing, which has been well established as a rapid subtyping method for EHEC O157 strains (14) and EHEC O103 strains (22), should be explored as an additional technique to subtype EHEC serogroup O145 strains.

The data reported in this study have important practical implications. First, because nearly all (96.6%) of the O145 clinical isolates were nonmotile, they could not be H typed. Thus, the fliC-RFLP procedure is of particular importance in these strains. It allows determination of the H type of an isolate in 48 h and therefore can be particularly useful in epidemiological investigations when prompt information about the H type is needed. As demonstrated in previous studies, the fliC-RFLP approach is reliable, rapid, and easy to perform for determining the H types of E. coli clinical isolates (5, 23, 39). Second, comparison of the discriminatory power of the subtyping methods for EHEC O145 strains demonstrated that PFGE was the most discriminatory method for the identification of epidemiologically associated strains. This finding is in agreement with the observation reported by others for EHEC O157 strains (31) and O26 strains (35) and for other enteric pathogens (25, 31). However, because interlaboratory comparisons of PFGE data require strict adherence to standardized experimental protocols, using appropriate standard equipment and software (31), this method is limited to specialized or national reference laboratories. Therefore, a practical recommendation for a clinical laboratory when an outbreak is suspected based on an increased frequency of the isolation of EHEC O145 strains is to perform subytping of the isolates with a combination of fliC-RFLP, eae typing, stx subtyping, and determination of plasmid-encoded genes. If these characteristics are identical, further typing of the isolates in a reference laboratory by PFGE is necessary to confirm an outbreak. Based on the above data and presently available technology, we propose that an algorithm as illustrated in Fig. 4 be employed for the subtyping of E. coli O145 strains. Third, despite our observation that the three EHEC O145 strains with the H25 fliC-RFLP pattern represented a minority within the 120 human O145 EHEC strains isolated from patients in Germany during the 7-year study period, each of these three strains originated from a hemolytic-uremic syndrome patient. Therefore, subtyping of EHEC O145 clinical isolates in prospective studies with the procedures developed in this study offers a suitable approach to further evaluate the association between this clonal lineage and the clinical outcome of the infection.

In conclusion, we demonstrate that, in contrast to the other major EHEC serogroups, EHEC O145 serogroup contains strains of two different fliC-RFLP types and intimin types. Presently, one of these clonal lineages, characterized by fliC-RFLPH28 and intimin γ, is mostly associated with human disease in Germany. The extensive genetic diversity of such strains provides a basis for their molecular subtyping in clinical and epidemiological studies.

Acknowledgments

This study was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) Project Network of Competence Pathogenomics Alliance “Functional genomic research on enterohaemorrhagic, enteropathogenic and enteroaggregative Escherichia coli (EHEC, EPEC, EAEC),” Project Group “Schmidt/Karch, Universitätsklinikum Münster” (BD number 119523), by grants from BMBF Verbundprojekt, Forschungsnetzwerk “Emerging food-borne pathogens in Germany” no. 01KI 9903 and no. 01KI 9901, and by a grant from the Fonds der Chemischen Industrie (FCI).

We thank Phillip I. Tarr (Washington University School of Medicine, St. Louis, Mo.) for critical reading of the manuscript and helpful discussions and Ursula Keckevoet (Münster) and Gerlinde Bartel and Barbara Knüppel (Wernigerode) for excellent technical assistance.

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