Characterization of Shiga Toxin-Producing Escherichia coli Strains Isolated from Human Patients in Germany over a 3-Year Period

Abstract

We have investigated 677 Shiga toxin-producing Escherichia coli (STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (eae) genes were present in 62.6% of the strains and subtyped into intimins α1, β1, γ1, ɛ, θ, and η. Shiga toxin types 1c and 2d were present only in eae-negative STEC strains, and type 2 was significantly (P < 0.001) more frequent in eae-positive STEC strains. Enterohemorrhagic E. coli hemolysin was associated with 96.2% of the eae-positive strains and with 65.2% of the eae-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with eae-positive STEC were significantly (P < 0.001) more frequent in children under 6 years of age than in other age groups, whereas eae-negative STEC infections dominated in adults. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (fliC) genes in 221 nonmotile STEC strains. Eleven serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 69% of all STEC strains. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins α1 (one serotype), β1 (eight serotypes), ɛ (two serotypes), and η (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.

The association of Shiga (Vero) toxin production in Escherichia coli with human pathogenicity was first described in 1979 (82, 85). However, it was the investigation of an outbreak caused by Shiga toxin-producing E. coli (STEC) O157 which provided the major impetus to study these pathogens (65). In the following years, STEC strains were increasingly isolated from humans with diarrhea and hemolytic-uremic syndrome (HUS) and from farm animals, which serve as a natural reservoir for STEC (52, 86). Today, more than 200 different E. coli O:H serotypes are known to be associated with the production of Shiga toxins (86; K. A. Bettelheim's VTEC table, May 2003 update, www.sciencenet.com.au/vtectable.htm). Certain STEC strains belonging to serogroups O26, O103, O111, O145, and O157 were more frequently isolated from humans with severe diseases such as hemorrhagic colitis and HUS. Accordingly, these highly virulent STEC strains were also designated as enterohemorrhagic E. coli (EHEC) (42, 52). The search for additional virulence markers in these pathogens revealed that most EHEC strains carry a plasmid which encodes a hemolysin (EHEC hemolysin) and the chromosomally located locus of enterocyte effacement (LEE) pathogenicity island (16, 43, 70, 84). The genes carried by the LEE enable the bacteria to produce attaching and effacing lesions in the host intestinal mucosa cells, which increases the virulence of the bacteria for humans (35, 44, 60). Intimate attachment of bacteria to the host cell is mediated by the binding of intimin, the product of the eae gene, to the translocated intimin receptor (80). Nucleotide sequencing of the LEEs from STEC O157 and enteropathogenic E. coli (EPEC) strains revealed differences in the genes coding for intimate attachment of bacteria to the enterocytes (48, 59, 89), and more than 10 genetic variants of the eae gene have been identified in STEC and EPEC strains (32, 34, 55, 77, 88). Some intimin types, such as intimin α, were found to be associated with EPEC, whereas others, such as intimins γ, ɛ, and θ, were found in STEC strains (1, 55, 77, 88). The association between infections with intimin-positive STEC and severe disease in humans was demonstrated previously (13), but it was also shown that intimin is not essential for the virulence of certain STEC strains for humans. Other colonization mechanisms, such as adhesins and pili, were identified in eae-negative strains (58, 75), and certain LEE-negative strains and serotypes of STEC were associated with bloody diarrhea (BD) and HUS (13, 14, 36). These STEC strains might possess other virulence factors which have not yet been characterized.

The virulence of STEC for humans may also be related to the Stx type which is produced by the bacteria. The presence of the stx₂ gene in the infecting strain was previously reported to correlate with severe disease in humans (13), and the administration of purified Stx2, but not of Stx1, was shown to cause HUS in experimentally treated primates (74). A variety of genetic variants of stx₁ and stx₂ were detected by nucleotide sequence analysis of stx genes (27, 30, 56, 61, 73). Some of the stx₁ and stx₂ variants were found to be associated with STEC from sheep (stx_1ox3/stx_1c and stx_2d-ount) (15, 39, 64, 79), pigs (stx_2e) (83), or pigeons (stx_2f) (72). Some genetic variants of stx₁ (stx_1ox3/stx_1c) and stx₂ (stx_2e and stx_2d-ount) are not present in classical EHEC strains but are frequently found in eae-negative STEC strains from patients with uncomplicated diarrhea or asymptomatic infections (23, 24, 25, 39, 61). Other variants, such as stx_2e, were rarely or not (stx_2f) associated with STEC from humans (24).

E. coli strains belonging to serotypes O157:[H7], O145:[H28], O111:[H8], O103:H2, and O26:[H11] are recognized classical EHEC types which occur in different countries worldwide (86; www.sciencenet.com.au/vtectable.htm). Diagnostic tools such as indicator media, O-antigen-agglutinating antisera, and magnetic beads coated with O-antigen-specific capture antibodies were developed for the enrichment and isolation of EHEC strains from fecal, environmental, and food samples. These tools proved to be useful for the detection of some but not all human-pathogenic STEC types (68). New emerging EHEC clones formed by O118:H16 and O121:H19 strains were recently described and were associated with BD and HUS (4, 31, 46, 76). These findings show that the list of human pathogenic STEC types is far from being completed, and further work has to be done to characterize human STEC strains for their serotypes, their virulence markers, and their associations with disease. This was the aim of our study, where we have investigated 677 STEC isolates which were collected between 1997 and 1999 from human patients in Germany.

MATERIALS AND METHODS

Human patients.

STEC strains from 677 human patients living in rural and urban areas in different parts of Germany were isolated between 6 January 1997 and 27 December 1999. Only one STEC isolate per patient was taken into the study. The patients' ages were known in 632 (93.5%) cases and were between 5 days and 91 years. The gender was known for 638 (94.2%) of the patients; 296 (46.4%) were male and 342 (53.6%) were female.

Isolation of STEC.

STEC isolates or patients' stools were sent to our laboratory at the Robert Koch Institute for isolation and confirmation of STEC infections. The specimens were obtained from 13 hospitals, 19 public health institutes, and 31 private diagnostic laboratories situated in different parts of Germany. The isolation of STEC from stool and characterization of STEC isolates were performed as previously described (8, 10).

Serotype identification.

The E. coli strains were serotyped for their O (lipopolysaccharide) and H (flagellar) antigens as previously described (54).

E. coli strains representing the new O groups O176 to O181 were kindly provided by Flemming Scheutz (International Escherichia Centre, Statens Serum Institut, Copenhagen, Denmark) and were used for the production of O-antigen-specific antisera (54). The H types of motile STEC strains were analyzed by serotyping using antisera specific for 53 different H antigens (H1 to H56). Nonmotile (NM) STEC strains were investigated for their H-type-specific (fliC) genes by PCR followed by HhaI digestion of fliC PCR products and evaluation of restriction fragment length polymorphism (RFLP) patterns as previously described (7, 45). Reference strains for H types H1 to H56 with characteristic fliC RFLP patterns (54) were used as standard controls for H serotyping and fliC PCR.

Nomenclature of Shiga toxin types.

The nomenclature proposed by Scheutz et al. (67) was used for the designation of Shiga toxin types. This nomenclature presents an updated version of the two currently accepted kinds of nomenclature for Shiga toxins. Accordingly, the indicated stx genotypes (in parentheses) were attributed to the following Shiga toxin types: type 1 (stx₁), type 1c (stx_1-ox3/stx_1c), type 2 (stx₂), type 2c (stx_2c, stx_2d-ount, and stx_2d-OX3a), type 2d (stx_2vha and stx_2vhb), and type 2e (stx_2e and stx_2ev). The toxin types were established with regard to antigenic variations, differences in toxicities, the capacity to be activated, differences in affinities and receptors, and significant differences in DNA and amino acid sequences (67). According to this nomenclature, toxins of Shiga toxin type 2c include the genetically closely related toxins stx_2d-ount (61), stx_2c, (73), and stx_2-OX3a (57). Shiga toxin type 2d is reserved for mucus-activatable toxins (stx_2vha and stx_2vhb) (49), and toxins of type 2e include the porcine edema disease-associated toxins stx_2e and stx_2ev (81). The term Shiga toxin type 1c was introduced for the stx_1c (stx_1-ox3) toxins (15, 87).

Detection of Shiga toxins and subtyping of stx genes.

All E. coli isolates were investigated for cytotoxicity in the Vero cell assay, and Vero cell assay-positive strains were examined with the verocytotoxin-producing E. coli (VTEC)-reverse passive latex agglutination (RPLA) test for the detection of Stx1 (VT1) and Stx2 (VT2) (Denka-Seiken, Tokyo, Japan) (9). The E. coli reference strains used for the detection of stx₁, stx₂, and their genetic variants were previously described (8). Primers KS7 and KS8 were used for the amplification of all genetic variants of the stx₁ gene family (39, 87). Subtyping of the stx_1ox3 (stx_1c) gene variant was performed with primers Lin-up and 1_ox3, which yield an stx_1ox3 gene-specific product of 555 bp (39).

Primers LP43 and LP44 were used for the amplification of all members of the stx₂ gene family (18). The subtyping of stx₂ genes was performed by endonuclease digestion of a 900-bp DNA product, which is obtained by PCR with primers Lin-up and Lin-down, which amplify all variants of the stx family (2, 8). Further identification of genetic variants of stx₂ was performed by PCR and by analysis of the restriction patterns after enzymatic digestion of PCR products as previously described (61). By this process, the following variants of the stx₂ gene could be identified: stx₂, stx_2c, stx_2vha, stx_2vhb, stx_2d-ount, stx_2d-OX3a, stx_2e, and stx_2ev (2, 61).

Dot blot DNA hybridization.

The presence of virulence genes in the STEC isolates was analyzed by dot blot DNA hybridization at high stringency as previously described (7). Gene probes were labeled with digoxigenin-11-dUTP (Roche Diagnostics, Mannheim, Germany).

Detection and subtyping of eae (intimin) genes.

eae genes were detected by dot blot DNA hybridization. The 881-bp eae-specific DNA probe was derived from the STEC O157 strain E32511 (84) by using the universal eae primers SK1 (5′ CCCGAATTCGGCACAAGCATAAGC 3′) and SK2 (5′ CCCGGATCCGTCTCGCCAGTATTCG 3′) (55). The subtyping of eae genes into intimins α, β, γ, and ɛ was performed by PCR with primer SK1 in combination with primers LP2 to LP5. Further subtyping of intimin genes was done by RFLP analysis of PstI-digested PCR products as previously described (55). PCRs for the detection of new intimin variants ζ, η, θ, ι, and κ were performed as recently described (88). The reference strains used for the identification of different intimin variants are listed elsewhere (88).

Hemolytic phenotypes and detection of EHEC hemolysin (EHEC hlyA) sequences.

All STEC strains were grown on enterohemolysin agar (Oxoid, Wesel, Germany), which contains washed sheep blood (9). The plates were incubated at 37°C and were examined for hemolysis after 3 h of incubation (indicating alpha-hemolysin) and after overnight incubation (indicating EHEC hemolysin) (9). The reference strains for the different E. coli hemolysins were used as previously described (70). Primers EHA1 and EHA2 were used for the detection of the EHEC hlyA gene and for preparation of the 1,551-bp EHEC hlyA-specific gene probe (69). STEC strains showing an alpha-hemolytic phenotype were investigated for the presence of an alpha-hemolysin gene (α-hlyA) by PCR. The PCR for the detection of the α-hlyA gene was developed on the basis of published nucleotide sequences (GenBank accession number M10133) by using McVector software (Oxford Molecular Group). The oligonucleotides hlyA-F16 (5′ CAGTCCTCATTACCCAGCAAC 3′) and hlyA-B14 (5′ ACAGACCCCTTGTCCTGAAC 3′) were selected as common primers for the α-hlyA gene. The PCR was run for 30 cycles (94°C for 40 s, 52.6°C for 40 s, and 72°C for 40 s) and yielded a 355-bp amplification product from the α-hlyA gene.

EaggEC-specific gene sequences.

Enteroaggregative E. coli (EaggEC)-specific DNA sequences were detected by dot blot DNA hybridization. A 765-bp PCR product from the EaggEC plasmid pCVD432 (3) was obtained with primers pCVD432/Start and pCVD432/Stop (71) and used as a gene probe. E. coli strains 17-2 and HS were used as positive and negative controls, respectively (3).

RESULTS

Serological diversity of human STEC isolates.

The 677 human STEC strains were typed into 55 E. coli O groups and 24 different H types. All together, 74 different O:H combinations (O:H serotypes) were found among the 677 strains (Tables 1 and 2). Forty O groups were associated with only one H type, and 15 O groups (O1, O5, O68, O77, O88, O91, O113, O115, O118, O123, O125, O146, O156, O174, and O177) were associated with multiple H types (Table 3). A rough lipopolysaccharide (O rough) was found in 86 (12.7%) of the STEC strains, and 12 (1.8%) strains showed O antigens which were not typeable (Ont), with antisera specific for O1 to O181 (Tables 1 and 2).

TABLE 1.

Intimin-positive STEC strains

Serotypea	Toxin type(s)b	Intimin type	No. of strains (n = 424)
O5:H−	1	β1	3
O5:H11c	1	β1	1
O26:[H11]d	1, 2, 1+2	β1	87
O43:[H30]c	2e	β1	1
O68:H11c	1	β1	1
O68:H25c	2c	β1	3
O77:H11c	1+2	β1	1
O103:H2d	1, 2	ɛ	38
O109:H25c	1	η	2
O111:[H8]d	1, 2, 1+2	θ	28
O118:H16d	1, 2, 1+2	β1	15
O121:[H19]	2	ɛ	5
O123:H2c	1	ɛ	1
O123:H11c	1	β1	2
O145:[H28]d	1, 2, 1+2	γ1	24
O150:H−	1	β1	1
O156:H25	1	η	2
O157:[H7]d	1, 1+2, 2c, 2+2c	γ1	169
O177:[H7]c	1	α1	2
O177:H11c	1	β1	1
O177:[H25]c	2+2c	β1	4
Ont:H2	2c	ɛ	1
Ont:H25	2	η	3
Orough:H−	1	β1	2
Orough:H2	1	ɛ	14
Orough:H7	1+2	γ1	3
Orough:H8d	1, 2	θ	2
Orough:H11	1	β1	1
Orough:H16	1	ɛ	1
Orough:H19	2	ɛ	1
Orough:H25	1	η	1
Orough:[H25]	2+2c	ɛ	2
Orough:[H28]	1	γ1	1
Orough:[H33]	1	θ	1

^aO:H serotypes of STEC strains. An H type in brackets indicates the presence of NM strains, which were analyzed for their fliC type by PCR as described in Materials and Methods.

^bThe toxin types of strains were determined on the basis of their stx genotypes as described above. Toxin types which were present in combination in all strains belonging to a given serotype are added together (for example, 1+2), and toxin types which were found in different combinations in strains belonging to one serotype are listed separately (for example, 1, 2).

^cSTEC strain belonging to an O:H type which was not previously described to occur in humans.

^dSerotypes of STEC strains showing differences in their Shiga toxin types as follows: O26:[H11], toxin types 1 (n = 65 strains), 2 (n = 14), and 1+2 (n = 8); O103:H2, toxin types 1 (n = 37) and 2 (n = 1); O111:[H8], toxin types 1 (n = 21), 2 (n = 1), and 1+2 (n = 6); O118:H16, toxin types 1 (n = 13), 2 (n = 1), and 1+2 (n = 1):O145:[H28], toxin types 2 (n = 18), 1 (n = 4), and 1 + 2 (n = 2); O157:[H7], toxin types 1 (n = 2), 2 (n = 43), 1+2c (n = 57), 2+2c (n = 39), and 2c (n = 23); Ont:H2, toxin types 1c (n = 1) and 1+2 (n = 1); and Orough:H8, toxin types 1 (n = 1) and 1+2 (n = 1).

TABLE 2.

Intimin-negative STEC strains

Serotypea	Toxin type(s)b	No. of strains (n = 253)
O1:H7	1	1
O1:H20	1	2
O2:H27	2	1
O4:H10	1c+2c	1
O6:H12c	1c	1
O11:H48c	1	1
O15:H8	2c	3
O22:H8d	1c+2c, 1c, 2, 2c	4
O27:H30	2c	1
O28:H25	2	1
O50:[H8]c	1c+2c	1
O54:H25c	1c+2c	1
O55:H9c	1	1
O60:[H4]c	2e	1
O68:H18c	2c	1
O71:[H12]	1c+2c	1
O73:H18c	2	1
O76:[H19]d	1c, 1c+2c	15
O77:H18	1+2	1
O79:H14c	1c	1
O88:H8c,d	1, 2c	2
O88:H25c,d	1, 2	2
O91:H10	1+2	1
O91:[H14]d	1, 1+2c	37
O91:[H21]d	1+2d, 2d, 1+2e	6
O104:[H21]	1+2	2
O105:H18	1+2	3
O113:[H4]d	1c, 2c	17
O113:[H21]d	1+2d, 2+2d, 2d	3
O114:H4	1	1
O115:H10	1	3
O115:[H44]	2c	1
O116:[H21]	2	1
O117:H7	2	1
O118:H12	2c	1
O125:[H19]c	1c+2c	1
O125:H10c	1c	1
O128:H2d	1c+2c, 2c	23
O132:[H19]c	1c	1
O146:H21d	1c, 2c, 1c+2c	15
O146:H28	2c	7
O148:H8c	2d	1
O153:H25	2	1
O154:[H31]c	1	1
O156:H21c	1+2c	1
O163:[H19]d	1, 2+2d	3
O166:H28d	1c, 1c+2c	4
O174:H2	1+2	2
O174:H16c	2c	1
O174:H21	2d	2
O178:H7	1	1
O179:H8c	2	1
O181:H16c	1c+2c	1
Ont:H2	1c, 1+2	2
Ont:H10	2e	1
Ont:[H19]	2c+2e	2
Ont:[H21]	2c+2d	1
Ont:[H28]	1+2	1
Ont:H31	1	1
Orough:[H2]d	1+2c, 1c+2c, 1, 2c	13
Orough:[H4]d	1, 1c+2c	2
Orough:[H7]	1c+2c	1
Orough:H8d	1, 1c, 1c+2c	3
Orough:[H10]	1c+2c	1
Orough:H12	2c	1
Orough:[H14]d	1, 1c, 1+2c, 1c+2c	27
Orough:[H19]d	1c, 2c, 1c+2c	4
Orough:H28d	1c, 2c	3
Orough:[H45]	1	1
Orough:H56	2c	1

^aO:H serotypes of STEC strains. An H type in brackets indicates the presence of NM strains, which were analyzed for their fliC type by PCR as described in Materials and Methods.

^bThe toxin types of strains were determined on the basis of their stx genotypes as described above. Toxin types which were present in combination in all strains belonging to a given serotype are added together (for example, 1+2), and toxin types which were found in different combinations in strains belonging to one serotype are listed separately (for example, 1, 2).

^cSTEC strain belonging to an O:H type which was not previously described to occur in humans.

^dSerotypes of STEC strains showing differences in their Shiga toxin types as follows: O22:H8, toxin types 1c (n = 1 strain), 1c+2c (n = 1), 2c (n = 1), and 2 (n = 1); O76:[H19], toxin types 1c (n = 5) and 1c+2c (n = 10); O88:H8, toxin types 1 (n = 1) and 2c (n = 1); O88:H25, toxin types 1 (n = 1) and 2 (n = 1); O91:[H14], toxin types 1 (n = 30) and 1+2c (n = 7); O91:[H21], toxin types 2d (n = 4), 1+2d (n = 1), and 1+2e (n = 1); O113:[H4], toxin types 1c (n = 3), 2c (n = 1), and 1c+2c (n = 13); O113:[H21], toxin types 1+2d (n = 1), 2d (n = 1), and 2+2d (n = 1); O128:H2, toxin types 1c+2c (n = 21) and 2c (n = 2); O146:H21, toxin types 1c+2c (n = 10), 2c (n = 3), and 1c (n = 2); O163:[H19], toxin types 1 (n = 1) and 2+2d (n = 2); O166:H28, toxin types 1c (n = 1) and 1c+2c (n = 3); Ont:H2, toxin types 1c (n = 1) and 1+2 (n = 1); Orough:H2, toxin types 1 (n = 1), 1c (n = 2), 1+2c (n = 3), 1c+2c (n = 6), and 2c (n = 1); Orough:[H4], toxin types 1 (n = 1) and 1c+2c (n = 1); Orough:H8, toxin types 1 (n = 1), 1c (n = 1), and 1c+2c (n = 1); Orough:[H14], toxin type 1 (n = 11), 1c (n = 2), 1+2c (n = 10), and 1c+2c (n = 4); Orough:[H19], toxin types 1c (n = 2) and 1c+2c (n = 2); and Orough:H28, toxin types 1c (n = 1) and 2c (n = 2).

TABLE 3.

Association of H-antigen types with STEC

H type	Associated O-antigen group(s)a	No. of strains
H2	O103, O123, O128, O174, Ont, Orough	94
H4	O60, O113, O114, Orough	21
H7	O1, O117, O157, O177, O178, Orough	178
H8	O15, O22, O50, O88, O111, O148, O179, Orough	45
H9	O55	1
H10	O4, O91, O115, O125, Ont, Orough	8
H11	O5, O26, O68, O77, O123, O177, Orough	94
H12	O6, O71, O118, Orough	4
H14	O79, O91, Orough	65
H16	O118, O174, O181, Orough	18
H18	O68, O73, O77, O105	6
H19	O76, O121, O125, O132, O163, Ont, Orough	32
H20	O1	2
H21	O91, O104, O113, O116, O146, O156, O174, Ont	31
H25	O28, O54, O68, O88, O109, O153, O156, O177, Ont, Orough	22
H27	O2	1
H28	O145, O146, O166, Ont, Orough	40
H30	O27, O43	2
H31	O154, Ont	2
H33	Orough	1
H44	O115	1
H45	Orough	1
H48	O11	1
H56	Orough	1
H−	O5, O150, Orough	6

^aBold indicates O groups associated with eae-positive STEC.

Forty-one (55.4%) of the 74 STEC-associated O:H serotypes were each represented by only one STEC isolate. On the other hand, 11 serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 468 (69%) of all STEC strains from this study (Tables 1 and 2). The H type was put in brackets when NM strains were present in a given O group, and NM strains were analyzed for their H type by a fliC-specific PCR (see above). Only six (0.9%) of the STEC strains were NM and yielded no amplification product with the fliC-specific PCR; these strains were classified as H antigen negative (H⁻).

Characterization of Shiga toxins.

The 677 STEC isolates were shown to produce cytotoxins in the Vero cell toxicity test (9). The production of Stx1 and Stx2 was examined with the VTEC-RPLA assay, and the stx genotypes of the strains were determined by PCR as described above.

The production of Stx1 and the presence of stx₁ or stx_1c variant genes were found in 475 (70.1%) of the strains. Production of Stx2 was detected in 353 (52.1%) of the strains which were positive in a PCR directed to stx₂ and stx₂ variants (primers LP43 and LP44). In addition, stx₂ genes were detected in 82 STEC strains which were negative for Stx2 production as determined by the VTEC-RPLA. These 82 strains harbored either stx_2c, stx_2d-ount, stx_2d-OX3a, stx_2e, or stx_2ev genes. All together, 435 (64.3%) of the 677 STEC strains carried stx₂ or stx₂ variant gene sequences.

The subtyping of stx₁ and stx₂ genes resulted in the detection of two genotypes (stx₁ and stx_1c) among Stx1 strains and of eight genotypes (stx₂, stx_2c, stx_2d-ount, stx_2d-OX3a, stx_2vha, stx_2vhb, stx_2e, and stx_2ev) among Stx2 strains. According to the nomenclature, the STEC strains were grouped into Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e), which were present alone or in different combinations in the STEC strains (Tables 1 and 2). Intimin-positive and intimin-negative STEC strains differed in their Shiga toxin types. Toxin types 1c and 2d were found only in eae-negative STEC strains. Toxin type 2 was associated with 85.5% of eae-positive and with 14.4% of eae-negative strains (Table 4). Toxin type 2c (genotype stx_2c, stx_2d-ount, or stx_2d-OX3a) was present in both eae-positive (44.4%) and eae-negative (55.6%) strains, and the genetic variants stx_2d-ount and stx_2d-OX3a were found only in eae-negative strains. Toxin type 2d (stx_2vha and stx_2vhb) was detected in 14 STEC strains belonging to serotypes O91:[H21], O113:[H21], O148:H8, O163:[H19], O174:H21, and Ont:[H21]. Toxin type 2e (genotypes stx_2e and stx_2ev) was present only in six strains belonging to serotypes O43:[H30], O60:[H4], O91:H21, Ont:H10, and Ont:[H19] (Tables 2 and 4).

TABLE 4.

Association of Shiga toxin types with the eae gene in human STEC strains

Shiga toxin type	eae-positive STEC strains (n = 424)	No. of serotypesa	eae-negative STEC strains (n = 253)	No. of serotypesa	Total no. of STEC strains (%)	Significance (P value by χ2 test)b
1	258	26	99	27	357 (52.7)	<0.001
1c	0	0	104	24	104 (15.4)	<0.001
2	154	14	26	6	180 (26.6)	<0.001
2c	106	5	133	34	239 (35.3)	NS
2d	0	0	14	6	14 (2.1)	<0.001
2e	1	1	5	4	6 (0.9)	NS

^aIncluding Ont and O rough strains.

^bNS, not significant.

Characterization of intimin (eae) genes.

eae genes were detected in 424 (62.6%) of the STEC strains by DNA hybridization and by PCR. The eae-positive STEC strains could be subtyped for their intimins by PCR with specific primers as previously described (55, 88). Six intimin types, namely, α1 (2 strains), β1 (123 strains), γ1 (197 strains), ɛ (63 strains), θ (31 strains), and η (8 strains), were detected (Table 1). The intimin subtypes were found to be associated with distinct O:H serotypes of STEC as well as with Ont (intimins η and γ1) and O rough (intimins β1, γ1, ɛ, η, and θ) strains. Intimin β1 was the most widespread among STEC and was found in strains belonging to 12 different serotypes. In contrast, intimins α1, γ1, ɛ, η, and θ were associated with fewer serotypes (Table 1). However, these serotypes were isolated most frequently and included O157:[H7] (γ1), O103:H2 (ɛ), and O111:[H8] (θ).

Hemolytic phenotype and association with EHEC hemolysin (EHEC hlyA).

Hemolytic activity was detectable in 576 (85.1%) of the 677 STEC strains. An alpha-hemolytic phenotype was found for two STEC strains (0.3%), and an enterohemolytic phenotype was found for 574 STEC strains (84.8%). The remaining 101 strains showed no hemolysis and were distributed over 30 different O:H types and Ont and O rough strains (data not shown).

The EHEC hlyA gene was present in 573 of the 574 STEC strains that had an enterohemolytic phenotype and in eight strains which showed no hemolytic activity. The EHEC hlyA gene was absent in both strains that had an alpha-hemolytic phenotype (Ont:H10 and Orough:H45), and these were positive for an α-hlyA gene. One of the hemolytic strains (Ont:H31) was negative for both α-hlyA and EHEC hlyA sequences and may produce a different type of hemolysin. An enterohemolytic phenotype and the presence of the EHEC hlyA gene were associated with 408 eae-positive (96.2%) and 165 (65.2%) eae-negative STEC strains.

EaggEC-specific DNA sequences.

None of the 677 VTEC strains from this study was positive for DNA hybridization with the EaggEC gene probe, which was previously reported to react with some STEC O111 strains (51).

Relationship between STEC type and clinical status of a patient.

Clinical data were provided for 608 patients (89.8%). The patients were divided into six groups according to their clinical status at the period of sampling (Table 5). A majority (59.2%) suffered from nonbloody diarrhea, 14.3% of patients had bloody diarrhea (BD), and HUS was present in 3.5% of the patients. Abdominal pain without diarrhea was reported for 8.7% of the patients. A group of asymptomatic excreters (11.0%) was formed from patients who had recovered from diarrhea or were sampled in control investigations. A few patients (3.3%) were reported to have diseases other than diarrhea or HUS.

TABLE 5.

Association between clinical signs and the presence of an eae gene in the infecting STEC strain

Clinical sign	No. of patientsa (%)	No. of STEC strains that were		Significance (P value by χ2 test)f
		eae positive	eae negative
Asymptomatic	67 (11.0)	36	31	NS
Other disease	20 (3.3)	10	10	NS
Abdominal pain	53 (8.7)	12	41	<0.001
Diarrhea	360 (59.2)	240	120	<0.001
BD	87 (14.3)	74b	13c	<0.001
HUS	21 (3.5)	20d	1e	<0.001
Total	608 (100)	392	216

^aOnly patients with known clinical histories (n = 608).

^bSerotypes (with numbers of strains in parentheses): O26:[H11] (9), O103:H2 (4); O111:[H8] (5); O118:H16 (2), O121:H19 (2), O145:[H28] (5), O157:[H7] (45), Orough:H7 (1); and Orough:H8 (1).

^cSerotypes (with numbers of strains in parentheses): O22:H8 (1), O73:H18 (1), O77:H18 (1), O88:H25 (1), O91:[H14] (1), O115:H10 (1), O148:H8 (1), O174:H2 (2), O174:H21 (1), O181:H16 (1), Orough:H2 (1), and Orough:[H14] (1).

^dSerotypes (with numbers of strains in parentheses): O26:[H11] (4), O103:H2 (1), O118:H16 (1), O145:[H28] (3), O157:[H7] (9), Orough:H7 (1), and Orough:H11 (1).

^eSerotype O105:H18, one strain.

^fNS, not significant.

Severe disease, such as BD and HUS, was significantly (P value of <0.001 by χ² test) associated with eae-positive STEC. Classical EHEC strains (O26:[H11], O103:H2, O111:[H8], O145:[H28], and O157:[H7]) predominated in patients with HUS (81.0%) and BD (78.0%). Strains belonging to the new EHEC types O118:[H16] and O121:H19 were isolated from four cases of BD (4.6%) and from one case of HUS (4.8%) (Table 5). eae-positive STEC strains were also found to predominate in patients with nonbloody diarrhea, whereas eae-negative STEC strains were frequent in patients with abdominal pain, and classical EHEC serotypes were isolated in only 13.0% of these cases.

A close relation was found between the patient's age and the presence of an eae gene in the STEC isolate (Table 6). The mean age of all 632 patients with known clinical history was 16 years 3 months. Patients infected with eae-positive STEC (n = 393) were younger (mean age, 8 years 3 months) than patients infected with eae-negative STEC (n = 239) (mean age, 29 years 4 months). STEC infections in children up to 6 years of age were significantly (P < 0.001) associated with eae-positive STEC isolates, which were found in 311 (88.1%) of the 353 STEC-infected children of this age group. In the group of patients older than 6 years, eae-negative STEC infections were significantly (P < 0.001) more frequent than in older age groups (Table 6). Thus, 197 (70.6%) of the 279 STEC strains isolated from patients older than 6 years did not carry an eae gene.

TABLE 6.

Patients' ages and STEC types (eae)

Age group (yr)	No. of patients	No. of patients with eae-negative STEC	No. of patients with eae-positive STEC
0-1	45	10	35
1-2	138	13	125
2-3	71	6	65
3-4	53	7	46
4-5	21	4	17
5-6	25	2	23
6-7	21	13	8
7-8	14	8	6
8-9	13	6	7
9-10	14	6	8
10-20	34	22	12
20-30	35	30	5
30-40	47	36	11
40-50	31	28	3
50-60	25	18	7
60-70	19	12	7
70-80	19	14	5
80-92	7	4	3
All	632	239b	393a

^aMean age, 8 years 3 months.

^bMean age, 29 years 4 months.

DISCUSSION

Studies from different countries have shown that humans can be infected with a large spectrum of serologically different STEC types (86; www.sciencenet.com.au/vtectable.htm). In an earlier study, we analyzed 89 human non-O157 STEC strains which were isolated in 1996 (10). As a result, eae-positive O118:H16 strains were identified as emerging EHEC strains in Germany, and some eae-negative STEC strains belonging to serogroups O91, O128, and O146 were frequently found among human clinical isolates. These findings encouraged us to examine larger numbers of human STEC strains in order to characterize the STEC strains associated with human infections in more detail and also to detect possible new STEC types.

The characterization of clonal types in E. coli populations by multilocus enzyme electrophoresis and by multilocus nucleotide sequencing has shown that the O:H serotype is a good indicator for the identification of strains belonging to distinct clonal groups (17, 22). However, many EHEC and STEC isolates are phenotypically NM (www.sciencenet.com.au/vtectable.htm) and therefore cannot be grouped into distinct O:H serotypes. In order to detect the H types of NM STEC strains, we have characterized the fliC genes in these strains by PCR-RFLP typing. This method has previously been shown to be suitable for the grouping of STEC O91:NM and O128:NM strains into serotypes O91:[H14] and O128:[H2], respectively, and the close relationship between motile and NM strains belonging to the same serotype was confirmed by pulsed-field gel electrophoresis typing (79). The contribution of molecular H-antigen typing for the identification of STEC serotypes is emphasized by the facts that 221 (32.6%) of the strains from our study were phenotypically NM and that only 6 of these were negative in the fliC PCR and were classified as H antigen negative. High numbers of NM strains were also detected in other studies of EHEC O26, O111, O145, and O157 (13, 24) and STEC O91, O113, and O174 (old designation, OX3) strains (28, 62, 63). In our study, NM and motile strains belonging to the EHEC O-antigen groups O26, O103, O111, O118, O121, O145, and O157 could be assigned to single O:H types by fliC genotyping (Table 1). NM STEC strains belonging to O-antigen groups which were associated with more than one H-antigen type (O91, O113, O115, O125, and O177) could be typed accordingly (Table 2). Twenty-four of 53 known E. coli H types (54) were found in the STEC strains, and 15 H types were associated with strains belonging to more than one O-antigen group (Table 3). Only 10 H types (H2, H7, H8, H11, H16, H19, H25, H28, H30, and H33) were linked with the 424 eae-positive STEC strains from our study, and these were distributed over 21 O groups and Ont and O rough strains. These findings indicate that the determination of the fliC type may be a useful diagnostic approach for the detection and characterization of STEC strains.

In order to search for new STEC types which are not yet known to occur in humans, we used a reference list which summarizes published data on the serotypes and origins of non-O157 STEC strains (www.sciencenet.com.au/vtectable.htm). By this list, we could identify 41 STEC strains belonging to 31 different serotypes which were not previously described as human STEC (Tables 1 and 2). Some of these “new” O:H types were already reported to be STEC strains from animals, and it was shown that certain STEC serotypes are closely associated with some animal host species (12, 26, 29, 41, 53, 78, 79). Based on these reports, we made an estimate about the possible animal source of the STEC strains from our study. Most of the eae-positive human STEC strains listed in Table 1 belong to serotypes which are closely associated with cattle. The eae-negative STEC strains were more diverse in their relations to animal hosts. Serotypes O91:[H14], O128:H2, and O146:H21, which represent about 30% of the human eae-negative STEC strains (Table 2), were reported to be associated with sheep (5, 12, 20, 41, 78, 79), whereas others, such as O22:H8, O113:[H4], and O113:[H21], are common in cattle (29, 63). Animal and human STEC strains which belong to the same serotype were found to be similar in their virulence markers, and the transmission of STEC from animals to humans has been reported (86). According to these findings, cattle and sheep represent an important source of STEC types which were frequently isolated from humans in our study.

Previous studies have shown that the virulence of STEC for humans may be related to the type of Shiga toxin which is produced by the bacteria. Of the different Shiga toxins, Stx2 (stx₂) was found to be related with high virulence and was significantly associated with STEC strains from BD and HUS patients (13, 24). Similar findings were made in our study, as toxin type 2 was present in 20 of 21 STEC strains from HUS patients (Table 5). Moreover, toxin type 2 (stx₂) was found more frequently in eae-positive STEC strains than in eae-negative STEC strains (Tables 1, 2, and 4). The combination of the stx₂ and eae genes was found to be significant in another study, which was performed on STEC strains of different origins and serotypes (13).

In contrast, toxin type 1c (genotype stx_1ox3/stx_1c) STEC strains were shown to be closely associated with sheep (15, 39, 78, 79). In our study, toxin type 1c was present in 41% of the eae-negative STEC strains and spread over 24 different serotypes. Toxin type 1c was often present in combination with toxin type 2c (stx_2d-ount or stx_2d-OX3a) but not with toxin type 2 (Table 2). STEC strains with toxin type 1c and/or 2c from our study were frequently isolated from patients with milder disease (abdominal pain or nonbloody diarrhea), corresponding to previously published results (24, 25, 61). The lower virulence of these STEC strains for humans may be explained by the lack of the attaching and effacing property and of Stx2; both factors were reported to be major virulence attributes of STEC strains causing severe disease in humans (13, 24).

A mucus elastase-activatable variant of Stx2 called stx_2d (stx_2vha and stx_2vhb) was previously described for an STEC strain of serotype O91:H21 (40, 49). Toxin type 2d-positive STEC strains of serotypes O91:H10, O91:H21, O174:NM, and O174:H21 were isolated from patients with BD and HUS (30, 49, 62), and a linkage was found between severe gastrointestinal disease in patients and the presence of stx_2d (31). In our study, we could identify two known (O91:[H21] and O174:H21) and four new (O113:[H21], O148:H8, O163:[H19], and Ont:[H21]) serotypes of toxin 2d strains (Table 2). Two of these serotypes (O148:H8 and O174:H21) were from patients with BD.

It was reported that toxin type 2e strains are rarely isolated from humans and cause milder disease (24). Similar findings were made in our study (Table 4). The production of Stx2e is characteristic for porcine STEC strains, which cause edema disease in pigs (26, 83). However, none of the strains from our study belonged to typical porcine serotypes, indicating that these are not as important as human pathogens.

Intimins η and α were previously not described as being associated with human STEC (55, 77). In this study, we have detected intimin η in eight STEC strains with flagellar type H25 (O109:H25, O156:H25, Ont:H25, and Orough:H25) which were positive for EHEC hlyA and, except in Ont:H25 (toxin type 2) strains, for toxin type 1 (Table 1). Intimin η was recently described to occur in bovine STEC strains which belonged to serotypes other than those of our human STEC isolates (33, 38). Intimin α was previously detected in EPEC but not in STEC strains (7, 32, 55) and was detected in our study in two strains representing a new STEC serotype, O177:[H7] (toxin type 1, EHEC hlyA negative).

Intimin β is reported to be the most frequent type of intimin in EPEC and STEC strains from humans and animals, and its presence is associated with multiple E. coli serotypes (1, 7, 19, 55). In this study, intimin β1 was detected in 123 STEC strains and 12 serotypes. Intimin β1 was found as a characteristic trait of all STEC strains with flagellar type H11 (Tables 1 and 3). Fourteen intimin β1-positive STEC strains belonging to eight serotypes (O5:H11, O43:[H30], O68:H11, O68:H25, O77:H11, O123:H11, O177:H11, and O177:[H25]) were identified as new groups of human STEC strains in this study.

Intimin ɛ was first described for EHEC O103:H2 strains (55) and more recently for emerging EHEC O121:H19 strains (31, 76). Apart from EHEC O103 and O121 strains, we have detected intimin ɛ in two new groups of STEC O123:H2 and Ont:H2 strains (Table 1). Intimin ɛ was also found in many STEC strains which were O rough; 14 of these were positive for flagellar type H2, toxin type 1, and EHEC hlyA and resembled strains of the EHEC O103:H2 clone. Characteristic combinations between H types, toxin types, and EHEC hlyA and intimin types were found in other STEC O rough strains, which may indicate that these strains originated from classical EHEC O157:H7 and O111:H8 strains (Table 1).

EHEC hemolysin, which causes an enterohemolytic phenotype on blood agar, was detected in many STEC strains of different origins (12, 13, 28, 29, 69) and was found to be significantly associated with eae-positive STEC strains belonging to classical and emerging EHEC types (13, 28, 46, 69, 76). Similar findings were made in our study, where EHEC hemolysin was detected in 96.2% of the eae-positive STEC strains. In contrast, alpha-hemolysin, which is known as a characteristic trait of porcine STEC strains (26), was detected in only two (0.3%) of the human STEC strains. The presence of toxin type 2e in the two alpha-hemolytic human STEC strains indicates that these strains could have originated from pigs.

It was previously reported that aggregative adherence and EaggEC-specific DNA sequences were found in STEC O111:H2 strains from HUS patients in France (51). EaggEC-specific DNA sequences were not found in any of the 677 STEC strains from our study, indicating that this pathotype is not common among STEC strains from Germany.

Ninety-four (87.0%) of 108 STEC strains which were isolated from patients with BD or HUS belonged to EHEC-related O groups and/or carried virulence markers (intimin, EHEC hlyA, and Stx2) which were previously associated with severe disease in humans (Table 5). Thirteen strains from patients with BD and one strain from a patient with HUS did not belong to classical EHEC serotypes and were negative for intimin (Table 5). These 14 strains were distributed over 13 serotypes, and some of these (O22:H8, O105:H18, O174:H2, and O174:H21) were already described as isolates from patients with BD or HUS (11, 23, 66). Our findings support previous studies indicating that certain serotypes of eae-negative STEC strains may cause severe disease (14, 23, 37, 63). The pathomechanism by which these atypical EHEC strains cause disease is not well known. Toxin types 2 and 2d may contribute to the virulence of atypical EHEC strains. In our study, toxin type 2 was found in 7 of 14 eae-negative strains (50%) from patients with BD and HUS but only in 19 (9.4%) of 202 eae-negative strains from all other patients, and two patients with BD were infected with toxin type 2d strains.

The search for new STEC types in a large group of human patients resulted in the detection of 41 strains and 31 serotypes which have not been described before as human STEC. Nineteen strains distributed over 11 serotypes represented new types of eae-positive STEC, and 16 of these expressed EHEC hemolysin; both properties are virulence attributes of classical EHEC strains (52). None of these strains were from patients with BD or HUS. Toxin type 2, which is associated with increased virulence of STEC, was found in only five of these strains belonging to serotypes O77:H11 and O177:[H25] (Table 1). The small number of cases which involved infection with the new types of eae-positive STEC strains does not permit an estimate of the virulence potential of these strains.

We had previously reported that infections with eae-positive STEC are associated with young age but that eae-negative isolates are more frequently isolated from adult patients (5). These findings were confirmed (P < 0.001) in the present study, which was performed on a larger number of isolates. Protective immunity to intimin may be acquired in early childhood due to infections with eae-positive EPEC and STEC strains (7, 21, 47), and this may explain why these strains are less frequently isolated from adults. On the other hand, adults are principally more exposed to STEC strains from nonhuman sources due to occupational contact with animals, food, and the environment, and the majority of STEC strains from these sources are negative for intimin (6, 20, 29, 50). This may explain the high frequency of infections with eae-negative STEC strains in adults. Severe disease such as BD or HUS was more frequent in young patients, which corresponds to the virulence attributes of their STEC isolates.

Our study shows that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains can be identified by subtyping of virulence genes and by serotyping of new O-antigen groups, including O175 to O181. The fliC PCR allowed the determination of H-antigen types in 221 (32.6%) STEC strains which were phenotypically NM. The finding of Ont strains in this study (Tables 1 and 2) suggests that further O types need to be designated.