Identification of Sequence Diversity in the Escherichia coli fliC Genes Encoding Flagellar Types H8 and H40 and Its Use in Typing of Shiga Toxin-Producing E. coli O8, O22, O111, O174, and O179 Strains

Abstract

Flagellar type H8 is associated with many strains of pathogenic Shiga toxin-producing Escherichia coli (STEC), such as O8, O22, O111, O174, and O179 strains. Serological typing of the H8 antigen is limited to motile strains only and suffers from cross-reactivity between flagellar H8 and H40 antigens. In order to develop a method useful for typing of motile and nonmotile STEC O111 and other strains, we have analyzed the flagellar antigen (fliC) genes in representative E. coli H8 and H40 types. Two genotypes of the fliC gene encoding H8 (the fliC-H8 gene) were identified. Genotype fliC-H8a was found to be conserved in STEC O111, O174, and O179 strains; and type fliC-H8b was associated with STEC O8 and O22 strains. Sequence variations were also found in the genetically closely related fliC-H40 gene, although the latter was not found to be associated with STEC strains. A PCR was developed for the specific identification of the fliC-H8 and the fliC-H40 genes in motile and nonmotile E. coli strains. Digestion of PCR products with HhaI resulted in restriction fragment length polymorphisms (RFLPs) which were associated with genotypes fliC-H8a and -H8b as well as with genotypes fliC-H40a and -H40b. The fliC-specific PCR/RFLP typing method was suitable for the rapid typing of motile and nonmotile STEC O8, O22, O111, O174, and O179 strains from different sources whose fliC-H8 genotypes were found to be highly conserved. The fliC genotyping method is advantageous over serotyping and is useful for epidemiological investigations and studies of the evolution of STEC clones.

Escherichia coli strains that cause diarrheal disease in humans, such as enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains, are associated with certain serotypes characterized by their O (lipopolysaccharide) and H (flagellar) antigens. Some E. coli serotypes, such as O55:H6 and O157:H7, were found to be associated with EPEC and STEC strains virulent for humans, respectively; and investigations have shown that the major clonal types of EPEC and STEC could be linked to distinct O:H serotypes of their representative strains (25, 38, 39). Because of this, serotyping is used in diagnostic laboratories in many countries for the detection of E. coli strains for pathogenic humans and animals. Major problems with the serological detection of E. coli can arise from serological cross-reactions between E. coli surface antigens, which can cause false-positive results; from spontaneously agglutinating (O rough) strains which are not typeable for their O antigen; and last, but not least, from nonmotile (NM) strains which cannot be typed for their flagellar (H) antigens. Moreover, the high number of typing sera needed for detection of E. coli O groups (O1 to O181) and H types (H1 to H56) limits the use of serotyping as a routine method for nonspecialized laboratories (24, 30).

In order to circumvent the manifold problems related to conventional serotyping, molecular methods which are based on detection of genes encoding the O and H antigens in E. coli were established. Characterization of the E. coli wzx and wzy genes was used for identification of O-antigen specificity in strains pathogenic for humans (11, 35). Molecular H genotyping can be performed by PCR with primers specific for conserved regions of the E. coli fliC (flagellin-encoding) genes. The resulting PCR products are subtyped by digestion with restriction endonucleases, resulting in specific restriction fragment length polymorphism (RFLP) patterns corresponding to fliC genotypes (PCR/RFLP typing) (14, 20). fliC genotyping was found to be particularly useful for characterization of nonmotile STEC strains belonging to the O groups of major STEC strains pathogenic for humans, such as O26, O111, O145, and O157 (2, 36, 40). PCR/RFLP typing of motile and nonmotile STEC strains belonging to the same clonal types has revealed that most of these show identical fliC genotypes, indicating that fliC genotyping is generally applicable as a method for strain characterization (2, 22).

Recent findings have shown that certain flagellar antigen types are dominating within pathogroups of E. coli strains. For example, strains of serotypes H2 and H6 typically belong to clonal lineages of the EPEC 1 and EPEC 2 groups, respectively (25, 38); and H types 8, 21, and 25 are prevalent in many O groups associated with STEC strains (http://www.microbionet.com.au/vtectable.htm) (2). Flagellar type H8 was found to be closely associated with human-pathogenic STEC strains belonging to serogroups O111 (39) as well as with STEC O8, O22, O174, and O179 strains. STEC O111:[H8] strains were identified worldwide as causative agents of bloody diarrhea and hemolytic-uremic syndrome (HUS) in humans (8, 10, 12, 16). STEC O8:H8, O22:H8, O174:[H8], and O179:H8 strains were found frequently in sheep and cattle and, more rarely, in humans with disease (4, 5, 6, 30, 33).

Many clinical and field isolates of STEC O111 and STEC O174 strains are nonmotile and were typed as O111:[H8] and O174:[H8] by fliC genotyping (2, 17, 33). Previous work has shown that flagellar antigen type H8 is serologically closely related to the E. coli flagellar H40 antigen, leading to serological cross-reactivity between both types of flagella. The use of cross-adsorbed antisera was therefore recommended for serological differentiation between the two flagellar antigens (24). More recently, partial fliC nucleotide sequence data became available for prototype H8 strain Ap 320c (GenBank accession no. AY249993) (37) and H40 strain E 49 (GenBank accession AF517665) (L. M. Perea, unpublished data) and showed high degrees of similarity between the two sequences. In order to develop a specific and commonly applicable method for the typing of STEC strains carrying these closely related H antigens, we have analyzed the complete nucleotide sequences of the fliC genes encoding flagellar types H8 and H40 (the fliC-H8 and fliC-H40 genes, respectively) from E. coli reference strains. We have identified two genetic subtypes within the fliC gene coding for flagellar type H8 which could be associated with different groups of pathogenic STEC strains. A specific PCR/RFLP protocol was developed for the rapid characterization of E. coli H8 and H40 strains.

MATERIALS AND METHODS

Bacteria.

The 200 E. coli wild-type strains whose fliC-H8 and fliC-H40 genes were investigated by PCR/RFLP typing (see below) were from the collection of the Federal Institute for Risk Assessment, Berlin, Germany. The strains belonged to 24 E. coli O serogroups; and some of these strains, mainly the STEC O22:H8, O111:[H8], O174:[H8], and O179:H8 strains, were described previously (2, 15, 17, 33, 34).

The E. coli serotype reference strains for flagellar types H8 and H40 were described by Orskov et al. (24, 26). Serotype reference strains and the prototype STEC O22, O111, O174, and O179 strains which were characterized for their fliC gene sequences and fliC types are listed in Table 1. The E. coli reference strain from this collection, strain H 511, was originally serotyped as O102:H8 (26), was later retyped as O102:H40 (24), and was confirmed to be fliC-H40b according to nucleotide sequence analysis of its fliC gene (this work). Reference strains carrying all other flagellar test antigens (H1 to H56), which were used to control the specificity of the fliC-H8 and fliC-H40 PCRs, were described previously (24, 26).

TABLE 1.

Relevant properties of representative E. coli strains for flagellar types H8 and H40

Strain	Serotype	Reference or sourcea	Virulence propertiesb	fliC genotype	GenBank accession no.
Ap 320c	O2:H8	24c	−	fliC-H8a	AJ865465
CB5778	O111:H8	Human, D, Germany, 1996d	stx1, eae	fliC-H8a	AJ884571
CB6199	O111:NM	Human, HUS, Germany, 1994d	stx2, eae, E-hlyA	fliC-H8a	AM258968
CB1571	O174:NM	Human, D, Germany, 1991d	stx1, E-hlyA	fliC-H8a	AM258969
CB7340	O179:H8	Meat, Germany, 1997d	stx2, E-hlyA	fliC-H8a	AM258970
E 5d	O76:H8	24c	−	fliC-H8a
1792-54	O169:H8	24c	−	fliC-H8a
H 520b	O105:H8	24c	−	fliC-H8a
H 501d	O98:H8	24c	−	fliC-H8a
H 515b	O103:H8	24c	−	fliC-H8b	AJ884569
CB3466	O111:H8	Human, AS, Germany, 1993d	−	fliC-H8b	AJ884570
CB8314	O22:H8	Meat, Germany, 1994d	stx1, stx2, E-hlyA	fliC-H8b	AM258971
E 49	O79:H40	24c	−	fliC-H40a	AJ884568
H 710c	O41:H40	24c	−	fliC-H40a	AM258973
H 511	O102:H40	24c	−	fliC-H40b	AJ865464
CB8532	O124:H40	Dog feces, United Kingdom, 2000d	eae	fliC-H40b	AM258972

^aAS, asymptomatic; D, diarrhea.

^bThe strains were investigated for the presence of virulence gene encoding the production of Shiga toxins (stx₁ and stx₂), intimin (eae), and enterohemolysin (E-hlyA). −, absence of virulence genes.

^cThe reference strain used for E. coli serotyping (24) was donated from the International E. coli Reference Laboratory, Copenhagen, Denmark.

^dThis work.

Nonmotile E. coli K-12 strain TPE2273 carries an IS5 insertion-inactivated fliC-H48 gene (32) and was used as the recipient for testing of the expression of the fliC-H8 and fliC-H40 genes cloned on plasmid pGEM-T Easy.

Nucleotide sequencing of complete fliC-H8 and fliC-H40 genes present in E. coli reference strains.

To obtain the complete coding sequence of the fliC-H8 gene, primers FliC-H8.out.1 (5′-AAC TGA TAC TAC TCC TGG TGC CCC-3′) and FliC-H8.out.2 (5′-AAA ACG GTT AGC AAT CGC CTG-3′) were designed from the published partial fliC-H8 gene sequence of strain Ap 320c (AY249993). Primers FliC-H8out.1 (positions 5′-1047 to 1070-3′ in the sequence with GenBank accession no. AY249993) and FliC-H8out.2 (positions 5′-108 to 88-3′ in the sequence with GenBank accession no. AY249993) are located at the ends of the sequence with GenBank accession no. AY249993. A PCR product derived from these primers is obtained only if the DNA sequences adjacent to the 3′ ends of the primers are joined to form a circular DNA molecule. Therefore, total genomic DNA of strains Ap 320c and H 511 was isolated; and aliquots of the DNA were digested with the enzymes BamHI, EcoRI, and HindIII, which have no restriction sites in the sequenced part of the fliC-H8 gene (GenBank accession no. AY249993) of strain Ap 320c. Endonuclease-digested total DNA was purified with Invisorb Spin PCRapid colums (Invitek Gesellschaft für Biotechnik & Biodesign GmbH, Berlin, Germany), and the DNA fragments were ligated with T4 DNA ligase (New England Biolabs, Frankfurt, Germany). The ligated DNA was subjected to PCR with primers FliC-H8out.1 and FliC-H8out.2. The PCR products obtained contain DNA sequences encompassing both ends of the coding sequence and the upstream and the downstream regions of the fliC-H8 gene. The PCR products were purified and analyzed for their nucleotide sequences.

The nucleotide sequence data were used to design forward primer FliC-16 (5′-AAC AGC CCA ATA ACA TCA AGT G-3′) and backward primer FliC-19 (5′-GAC GAT TAG TGG GTG AAA TGA G-3′), which are located upstream and downstream of the fliC-H8 coding sequence, respectively. Primers FliC16 and FliC19 were successfully used for amplification of the coding region and the flanking sequences of the fliC gene present in strain Ap 320c (O2:H8), yielding a product of 1,619 bp. The same primers were also applied for amplification of strains CB5778 (O111:H8), H 515b (O103:H40), CB3466 (O111:H8), E 49 (O79:H40), and H 511 (O102:H40), as listed in Table 1.

The PCR products were purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany), used for sequencing by applying dye terminator chemistry (PE Applied Biosystems, Darmstadt, Germany), and separated on an automated DNA sequencer (ABI PRISM 3100 genetic analyzer; Applied Biosystems, Foster City, CA). The sequences were analyzed with Lasergene software (DNASTAR, Madison, WI) and MacVector software (Oxford Molecular Group, Campbell, CA).

Development of a specific PCR/RFLP method for amplification and subtyping of E. coli fliC-H8 and fliC-H40 genes.

The sequences of primers FliC-H8/40-F (5′-CAT TAA TAC AAA CAG CCT GTC G-3′) and FliC-H8/40-B (5′-TTC GCT AGT TCT GTT GCA TC-3′) were deduced from the fliC-H8 and fliC-H40 DNA sequences with GenBank accession nos. AJ865465, AJ884571, AJ884569, AJ884570, AJ884568, and AJ865464. These primers amplify 741-bp segments of the fliC-H8 and fliC-H40 genes but give no product with any of the other fliC genes of E. coli (see Results). The PCR was performed for 30 cycles at 94°C for 40 s, 62.5°C for 40 s, and 72°C for 60 s. The PCR products were digested with HhaI, which yielded specific restriction profiles for the genetic subtypes found for fliC-H8 and fliC-H40.

Molecular cloning of fliC gene PCR products.

PCR products encompassing the complete coding sequences of the fliC-H8 and fliC-H40 genes were amplified from the genomic DNA of E. coli strains Ap 320c, CB5778, H 515b, CB3466, E 49, and H 511 by using primers FliC-16 and FliC-19 (see above). The amplification products were inserted into the pGM-T Easy vector by using a PCR cloning vector system (Promega, Mannheim, Germany). Recombinant plasmids carrying the inserted PCR products were transformed into nonmotile (fliC-H48::IS5) E. coli K-12 strain TPE2273, which carries an insertional mutation in its fliC gene (32). Selection was carried out on LB agar supplemented with ampicillin (100 μg/ml), and the strains were tested for motility in tubes containing motility medium (L broth plus 0.3% agar), as described previously (32). Motile recombinant strains were tested for expression of the cloned flagellar antigens by H serotyping and for carriage of recombinant fliC genes by PCR with primers FliC-H8/40-F andFliC-H8/40-B, followed by HhaI digestion of the PCR products.

Nucleotide sequence accession numbers.

The nucleotide sequences of the complete coding regions of the fliC genes representing flagellar types H8 and H40 were submitted to the EMBL data library under the following accession numbers: AJ865465 for strain Ap 320c, AJ884571 for strain CB5778, AJ884569 for strain H 515b, AJ884570 for strain CB3466, AJ884568 for strain E 49, and AJ865464 for strain H 511. All sequences have a length of 1,479 bp and encompass the complete fliC gene (Table 1). In order to confirm the specificity of the newly developed PCR/RFLP typing system for fliC-H8 and fliC-H40, we have determined the nucleotide sequences of PCR products obtained from representative wild-type E. coli strains obtained with primers FliC-H8/40-F and FliC-H8/40-B (EMBL accession numbers AM258968 to AM258973) (Table 1).

RESULTS

Diversity of fliC genotypes in E. coli H8 and H40 strains.

Flagellar antigen type H8 is frequently found in STEC strains belonging to different O serogroups, such as O8, O22, O111, O174, and O179 strains (2, 27, 30). Flagellar types H8 and H40 are serologically closely related (13, 24), and partial nucleotide sequences of the fliC genes present in the E. coli reference strains for flagellar antigen type H8 (strain Ap 320c; GenBank accession no. AY249993) (37) and H40 (strain E 49; GenBank accession no. AF517665) were found to be highly similar. It was previously shown that the fliC genotypes of nonmotile derivatives of STEC O111 and O174 were similar to those of motile O111:H8 and O174:H8 strains, respectively (2, 33). The association of flagellar type H8 strains with strains of the STEC group and the similarity between flagellar types H8 and H40 prompted us to investigate the genetic relationships between the fliC genes present in E. coli H8 and H40 strains and their association with STEC strains. For this purpose, we have investigated the fliC genotypes of E. coli wild-type and reference strains expressing flagellar antigen H8 or H40 by a fliC-specific PCR with primers FliC-1 and FliC-2, as described previously (3). All strains showed PCR products of 1,385 bp (data not shown). Subtyping of the fliC PCR products was performed by digestion with HhaI, and analysis of the restriction fragments generated revealed the presence of four different restriction fragment (RFLP) patterns; two of these (called fliC-H8a and fliC-H8b) were associated with flagellar type H8, and two (called fliC-H40a and fliC-H40b) were associated with flagellar type H40 (Fig. 1; Table 2). Because of these findings, we became interested in a further analysis of the fliC genes present in E. coli strains showing different subtypes and further investigated the six representative strains listed in Fig. 1 for their complete fliC nucleotide sequences. The fliC-H8 and -H40 genotypes were detected in 200 E. coli strains belonging to 24 O serogroups, as well as in the O-rough and the O-untypeable strains which were investigated (Table 2).

FIG. 1.

RFLP patterns of E. coli H8 and H40 strains obtained by HhaI digestion of PCR products obtained with common fliC primers FliC-1 and FliC-2 (1,385-bp product). The HhaI-digested fliC PCR products (obtained with primers FliC-1 and FliC-2) of E. coli H8 and H40 strains were electrophoretically separated on 2% agarose. Lanes: M: 100-bp molecular size ladder, 1, Ap 320c (O2:H8, pattern fliC-H8a); 2, CB5778 (O111:H8, fliC-H8a); 3, H 515b (O103:H8, fliC-H8b); 4, CB3466 (O111:H8, fliC-H8b); 5, E 49 (O79:H40, fliC-H40a); 6, H 511 (O102:H40, fliC-H40b). The sizes of the HhaI fragments generated are listed in Table 2. Restriction fragments smaller than 50 bp (Table 2) as well as fragments with very similar molecular sizes were not visibly separated on the 2% agarose gel.

TABLE 2.

PCR/HhaI RFLP typing scheme for identification of fliC-H8 and fliC-H40 genotypes

fliC genotype (prototype strain)	Sizes of DNA fragments (bp) obtained by enzymatic digestion with HhaI of the PCR products obtained with primersa:
	Common fliC PCR primers	fliC-H8- and -H40-specific PCR primers
fliC-H8a (Ap 320c)	307, 289, 147, 144, 115, 104, 103, 99, 39, 18, 18, 2	289, 110, 104, 103, 76, 39, 18, 2
fliC-H8b (H 515b)	307, 289, 219, 147, 144, 103, 99, 39, 18, 18, 2	289, 214, 103, 76, 39, 18, 2
fliC-H40a (E49)	325, 289, 285, 142, 115, 105, 104, 18, 2	289, 142, 110, 104, 76, 39, 18, 2
fliC-H40b (H511)	325, 289, 285, 219, 142, 105, 18, 2	289, 214, 142, 76, 39, 18, 2

^aHhaI-digested PCR products were separated on 2% agarose gels. The presence or absence of HhaI restriction fragments indicated (in boldface and underlined) was used as a major element to discriminate the different subtypes of the fliC-H8 and the fliC-H40 genes. Fragments indicated in italics are calculated from nucleotide sequence data and could not be separated well by the agarose gel electrophoresis conditions used (Fig. 1 and 2). The common fliC PCR primers were primers FliC-1 and FliC-2 (1,385-bp product) (Fig. 1). The fliC-H8- and -H40-specific PCR primers were primers FliC-H8/40-F and FliC-H8/40-B (741-bp product) (Fig. 2). The following O groups of E. coli were found to be associated with the genotypes: for fliC-H8a, O2, O3, O86, O76, O88, O111, O112, O118, O152, O174, and O179; for fliC-H8b, O8, O22, O111, and O146; for fliC-H40a: O41, O70, O79, O90, and O153; and for fliC-H40b, O4, O71, O102, O117, O124, and O127.

Nucleotide sequence analysis of fliC genes present in flagellar type E. coli H8 and H40 strains.

The published nucleotide sequences of the fliC genes of flagellar type H8 reference strain Ap 320c (GenBank accession no. AY249993; 1,383 bp) and of the flagellar type H40 reference strain E 49 (GenBank accession no. AF517665; 1,366 bp) cover only a part of the coding regions. In order to obtain the complete coding sequences of the fliC genes present in the flagellar type H8 and H40 strains, we have designed two primers (primers FliC-8.out.1 and FliC-8.out.2) to amplify the adjacent regions at the left and the right ends of the published sequence for fliC-H8 in strain Ap 320c (GenBank accession no. AY249993), as described in Material and Methods. Nucleotide sequencing of the PCR products obtained with primers FliC-8.out.1 and FliC-8.out.2 allowed us to design primers FliC-16 and FliC-19, which were used for amplification of the entire fliC-coding region of Ap 320c (O2:H8). Primers FliC-16 and FliC-19 were suitable for amplification of the fliC genes present in all fliC-H8 and fliC-H40 strains investigated and were used for amplification of the fliC genes from strains CB5778 (O111:H8), H 515b (O103:H8), CB3466 (O111:H8); E 49 (O79:H40), and H 511 (O102:H40). The lengths of the fliC-coding regions of strain Ap 320c and of all other strains were 1,476 bp.

Analysis of the nucleotide sequences of the E. coli H8 and H40 reference strains confirmed the presence of subtypes fliC-H8a and fliC-H8b, as well as subtypes fliC-H40a and fliC-H40b, which were detected by RFLP analysis of the HhaI-digested products obtained by PCR with FliC-1 and FliC-2 (Fig. 1). The differences between the analyzed fliC-H8 and fliC-H40 genes at the nucleotide sequence and the amino acid levels are summarized in Tables 3 and 4, respectively.

TABLE 3.

Similarities and divergences after ClustalW alignment of the fliC-coding nucleotide sequences of strains Ap 320c, CB5778, CB3466, H 515b, E 49, and H 511

Strain (GenBank accession no., fliC genotype)	% Similarity or divergencea
	Ap 320c	CB5778	CB3466	H 515b	E 49	H 511
Ap 320c (AJ865465, fliC-H8a)		100.0	99.1	99.1	95.2	94.2
CB5778 (AJ884571, fliC-H8a)	0.0		99.1	99.1	95.2	94.2
CB3466 (AJ884570, fliC-H8b)	0.9	0.9		100.0	94.5	94.3
H 515b (AJ884569, fliC-H8b)	0.9	0.9	0.0		94.5	94.3
E 49 (AJ884568, fliC-H40a)	5.0	5.0	5.8	5.8		98.5
H 511 (AJ865464, fliC-H40b)	6.1	6.1	6.0	6.0	1.5

^aThe values above and below the blank diagonal space are the percentages of similarity and divergence, respectively.

TABLE 4.

Similarities and divergences after alignment of FliC protein sequences of the strains Ap 320c, CB5778, CB3466, H 515b, E 49, and H 511

Strain (GenBank accession no., fliC genotype)	% Similarity or divergencea
	Ap 320c	CB5778	CB3466	H 515b	E 49	H 511
Ap 320c (AJ865465, fliC-H8a)		100.0	100.0	100.0	94.9	94.7
CB5778 (AJ884571, fliC-H8a)	0.0		100.0	100.0	94.9	94.7
CB3466 (AJ884570, fliC-H8b)	0.0	0.0		100.0	94.9	94.7
H 515b (AJ884569, fliC-H8b)	0.0	0.0	0.0		94.9	94.7
E 49 (AJ884568, fliC-H40a)	5.3	5.3	5.3	5.3		99.2
H 511 (AJ865464, fliC-H40b)	5.5	5.5	5.5	5.5	0.8

^aThe values above and below the blank diagonal space are the percentages of similarity and divergence, respectively.

The previously published partial fliC-H8 sequence of Ap 320c (GenBank accession no. AY249993) was identical to the corresponding part of our sequence with GenBank accession no. AJ865465 (Ap 320c), while another partial fliC-H40 sequence of strain E 49 (O79:H40) (GenBank accession no. AF517665 [unpublished data]) differed from our sequence with GenBank accession no. AJ884568 (E 49) at four positions over a total length of 1,365 bp.

Development of a PCR specific for fliC-H8 and -H40 genes and genetic subtyping of STEC strains.

The sequences of primers FliC-H8/40-F (5′ CAT TAA TAC AAA CAG CCT GTC G-3′) and FliC-H8/40-B (5′-TTC GCT AGT TCT GTT GCA TC-3′) were deduced from the fliC-H8 and fliC-H40 DNA sequences with GenBank accession nos. AJ865465, AJ884571, AJ884569, AJ884570, AJ884568, and AJ865464. These primers amplify a 741-bp segment of the fliC-H8a and fliC-H8b genes and the fliC-H40a and fliC-H40b genes but did not give a product with any of the reference strains expressing other flagellar (H1 to H56) antigens of E. coli, as tested in this work (data not shown). HhaI digestion of the PCR products obtained with these primers yielded specific patterns (Fig. 2) corresponding to the fliC-H8 and fliC-H40 genotypes, as summarized in Table 2. The PCR with primer FliC-H8/40 was used for identification and genetic subtyping of the E. coli wild-type and reference strains used for serotyping of E. coli, as described by Orskov et al. (24). The results are summarized in Tables 2 and 5. Within the STEC group, the fliC-H8a type was found to be closely associated with STEC O111:[H8], O174:[H8], and O179:H8 strains; and the fliC-H8b genotype was found to be closely associated with STEC O8:H8 and O22:H8 strains as well as with single isolates of Shiga toxin-negative E. coli. The fliC-H8b genotype was additionally found in four E. coli O111:H8 strains which were negative for the stx and eae genes (prototype strain CB3466, GenBank accession no. AJ884570) and in single non-STEC strains belonging to other O serogroups.

FIG. 2.

RFLP patterns of E. coli H8 and H40 strains obtained by HhaI digestion of PCR products obtained with fliC-H8- and fliC-H40-specific primers FliC-H8/40-F and FliC-H8/40-B (741-bp product). The HhaI-digested fliC PCR products (obtained with FliC-H8/40-F and FliC-H8/40-B) of E. coli H8 and H40 strains were electrophoretically separated on 2% agarose: Lanes: M, 100-bp molecular size ladder; 1, Ap 320c (O2:H8, pattern fliC-H8a); 2, H 515b (O103:H8, fliC-H8b); 3, CB5778 (O111:H8, fliC-H8a); 4, CB3466 (O111:H8, fliC-H8b); 5, H 511 (O102:H40, fliC-H40b) 6, CB8532 (O124:H40, fliC-H40b, 7) E 49 (O79:H40, fliC-H40a); 8, CB6199 (O111:NM, fliC-H8a). Restriction fragments smaller than 50 bp (Table 2) as well as fragments with very similar molecular sizes (lanes 1, 3, 7, and 8) were not visibly separated on the 2% agarose gel.

TABLE 5.

Association of fliC-H8a and fliC-H8b genotypes with major STEC groups

STEC serotype	fliC type	No. of strains	Sources, countries of origin (no. of strains)
O111:[H8]	H8a	85	Human, Germany (73), Brazil (1), and United States (3); cattle, Germany (3), Switzerland (1), and Brazil (4)
O174:[H8]	H8a	25	Human, Germany (2); sheep, Norway (15) and Germany (5); cheese, France (3)
O179:[H8]	H8a	16	Human, Germany (2); cattle, Germany (3), Norway (5), and France (6)
O22:[H8]	H8b	14	Human, Germany (5); cattle, Germany (1), Norway (5), and France (1); cheese, France (2)
O8:H8	H8b	6	Human, Germany (3); cattle, Uganda (1) and France (1); cheese, France (1)

The fliC-H40a and fliC-H40b genotypes were found to be associated with a relatively few E. coli strains from humans and animals, and none of these strains was found to belong to the STEC group (Table 2).

Cloning and expression of complete fliC-H8 and fliC-H40 genes in nonmotile (fliC::IS5) E. coli K-12 strain TPE2273.

In order to examine the functional expression of the fliC-H8 and fliC-H40 genes which were characterized in this work, we cloned the amplified (with primers FliC-16 and FliC-19) fliC genes of strains Ap 320c, CB5778, H 515b, CB3466, E 49, and H 511 on plasmid pGM-T Easy. Recombinant plasmids were transformed into nonmotile E. coli K-12 strain TPE2273 (fliC-H48::IS5), as described in Materials and Methods. Motile recombinant strains were selected on motility medium containing ampicillin and were obtained for all fliC-H8 and fliC-H40 variants from the parental strains listed above. These were investigated serologically for their H antigens, and their respective H types were confirmed. The fliC genotypes of the recombinant strains were investigated and confirmed by PCR with primers FliC-H8/40-F and FliC-H8/40-B and corresponded to the patterns obtained with the parental strains (data not shown).

DISCUSSION

Serotyping of E. coli O and H antigens has been shown to be suitable for identification of the major clonal types of human pathogenic STEC strains, such as O157 strains and others. Investigation of serotypes is important in investigations of outbreaks and for studying STEC reservoirs and the spread and emergence of new STEC types. The characterization of the O-antigen type only is not sufficient enough to identify potential STEC strains (31). Only some of the strains belonging to STEC O groups O26, O111, and O157 were shown to produce Shiga toxins (17, 18, 19). On the other hand, it was found that Shiga toxin production is closely associated with strains belonging to certain O:H serotypes, such as O26:H11, O111:H8, and O157:H7 (28, 39, 40). However, H serotyping of STEC strains is often limited by a lack of specific typing sera and by the high number of nonmotile STEC strains, which cannot be analyzed for their H antigens (1, 21, 40). In order to overcome these problems, molecular methods for the typing of the fliC genes, which encode the characteristic H11 and H7 antigens in STEC O26 and O157, were developed and found to be suitable for characterization of motile and nonmotile STEC O26 and O157 strains (36, 40).

In this work, we have developed a specific PCR for detection of the fliC-H8 gene as a characteristic trait of STEC O111 and other STEC strains pathogenic for humans. The genetic subtypes fliC-H8a and fliC-H8b could be associated with STEC O8, O22, O111, O174, and O179 strains, which were isolated at different times from different geographical locations and from different sources. Our data show that the respective fliC-H8 genotypes are highly conserved in strains belonging to these STEC groups and thus can be used as targets for diagnostic and epidemiological identification of bacterial isolates. Within in the group of E. coli O111 strains, four non-STEC strains that showed the fliC-H8b genotype were identified. These strains were lacking Shiga toxin- and intimin-encoding genes, suggesting that they are genetically unrelated to the STEC O111:[H8a] group.

Variation in the fliC sequence was already described for the fliC-H7 gene. Genetic subtypes of the fliC-H7 gene were attributed to different clonal groups of E. coli, including O55:H7 and O157:H7, and specific PCRs were developed for their detection (29, 36). Similar findings were made in this work for the fliC-H8 gene. Besides the association found between fliC-H8 subtypes and certain STEC groups, the molecular analysis of the E. coli fliC-H8 and fliC-H40 genes allows a clear distinction between flagellar antigen types H8 and H40 to be made on the basis of their genotypes. Because of their antigenic similarity, the serological differentiation between flagellar types H8 and H40 causes difficulties, which can lead to the misinterpretation of typing results (24, 26). Here, we showed that the E. coli fliC-H40 type is not associated with major STEC groups, and we were able to characterize the reference strains for flagellar serotypes H8 and H40 according to variations in the sequences of the fliC-encoding genes.

Data from partial sequence analysis of the fliC genes present in E. coli reference strains for H8 and H40 indicated that the H40 reference strain carries a fliC-H8 gene, suggesting that the H40 antigen is not encoded by fliC in E. coli (37). In order to investigate this possibility, we have cloned the complete coding sequences of the fliC genes present in fliC-H8 and fliC-H40 reference strains in the fliC insertion mutant strain TPE2273 and have investigated the recombinant strains for expression of the fliC-H8 and fliC-H40 antigens. In this way we could show that all genetic types of fliC-H8 and fliC-H40 produce functional flagella that show serological specificity to their respective H types. These results indicate that both H types H8 and H40 are encoded by the fliC gene in E. coli. Interestingly, there was no difference in the FliC protein sequences encoded by fliC-H8a and fliC-H8b. In contrast, small differences in the amino acid compositions of the flagellins of the flagellar type H40a (strain E 49) and H40b (strain H 511) strains were found (99.2% similarity), and their amino acid compositions were found to be less related to that of flagellar type H8 (94.7 to 94.9% similarity).

More than 200 serotypes of E. coli are associated with the production of Shiga toxins, which presents a major problem for the identification of STEC strains pathogenic for humans. By investigating 677 STEC isolates from patients in Germany, we found the strains distributed over 55 O serogroups and 24 H types; however, 11 serotypes were detected, and among those serotype O111:[H8] accounted for 69% of all STEC isolates (2). STEC O111:[H8] strains were listed together with a few other groups, such as O26, O103, and O113, which cause hemorrhagic diseases such as bloody diarrhea and HUS in humans and which have caused outbreaks of infections in different countries (23). Among the non-O157 STEC strains, STEC O111 strains accounted for most cases of HUS and were also the cause of three of seven non-O157 STEC outbreaks reported in the United States (8). In Australia, STEC O111 was found to be among the most important STEC type pathogenic for humans (12), and STEC O111 was reported to cause outbreaks and HUS in different European countries (7, 9, 16). Many of the clinical isolates of STEC O111 were found to be nonmotile or were not investigated for their flagellar types. The fliC-H8- and -H40-specific PCR/RFLP typing procedure developed in this work will be useful for the identification and characterization of types and outbreak strains of STEC O111 and of emerging STEC types, such as O174 and O179 (30), that are isolated from humans, animals, and food in different countries.