Bacterial strains
The reference strains used for production of antisera for O- and H-typing of E. coli were obtained from the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark and are described elsewhere [3]. Origin and serotype data on five additional strains with flagellar type H17 are listed in Table 1. A laboratory collection of 88 E. coli isolates originating from humans and animals was investigated by PCR/RFLP typing for fliC-H4 genes. These strains were previously investigated for the O-types and for production of Shiga-toxins (Stx) and were isolated in different countries between 1941 to 2002 [3, 19, 30] (Table 3). The E. coli K-12 strain JM109 is described elsewhere [23].
Production of E. coli O and H-specific antisera
Rabbit antisera against the different O- and H-antigens of E. coli were prepared according to ∅rskov and ∅rskov [3]. Antisera for typing of flagellar antigens H4 were produced with reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) [3]. Our strain P12b was found to express its fliC encoded H4 antigen and produced flagellar type H4 (this work).
Motility inhibition test
Expression of flagella and swarming of E. coli strains was tested by inoculating bacteria in tubes containing 10 ml portions of swarm-agar (L-broth + 0.3% agar) as described [3]. Inhibition of motility of E. coli strains in the presence of flagellar-specific antiserum (H4 and H48) was tested in swarm-agar containing a 1:600 dilution of the respective antiserum. Cultures which were inhibited for motility were observed over two weeks for possible switch to motility by phase variation.
Serological typing of H-antigens of E. coli
H-serotyping was performed as described [3]. In brief, bacteria were grown in tubes containing 10 ml 0.3% semi-solid LB-agar [23] for two to three passages. Highly motile bacteria were transferred in LB-medium, incubated 6h at 37°C and inactivated by addition of 0.5% formaldehyde in solution. Agglutination reactions were performed in two fold dilutions of 0.5 ml portions of serum in phosphate-buffered saline pH 7.4 (PBS) [22] with 0.5 ml formalized bacteria in glass tubes which were incubated for 2h at 50°C. Agglutination tests were read by eye immediately after incubation as described [3].
PCR-typing of fliC genes
The oligonucleotide primers fliC-1 (5' CAA GTC ATT AAT AC(A/C) AAC AGC C 3') and fliC-2 (5' GAC AT(A/G) TT(A/G) GA(G/A/C) ACT TC(G/C) GT 3') were used for amplification of internal parts of fliC genes present in the E. coli reference strains as described [9]. The PCR was performed for 25 cycles at 94°C for 60 sec, 55°C for 60 sec and 72°C for 120 sec [9]. PCR products of sizes varying between 950 to 2500 bp were obtained with E. coli reference strains for 53 different H-types [3] (Figure 1). Amplified DNA was digested with HhaI and the resulting restriction fragments were compared on 2% agarose gels. Restriction enzymes HpaII and MboI were used for characterization of fliC-H4 specific PCR products. Gel images were stored digitally and analyzed with BioNumerics software, version 2.5 (Applied Maths, Kortrijk, Belgium) for similarity (Dice, complete linkage) (Fig. 1).
Nucleotide sequence analysis of fliC genes
Two primers deduced from the published fliC-H4 sequence (AB028472) were used for the amplification of the entire fliC-H4 coding regions. The PCR was performed for 30 cycles: 30 sec at 94°C, 60 sec at 58.1°C and 90 sec at 72°C with primers fliC-5 (5'-TGA GTG ACC AGA CGA TAA CAG GG-3') and fliC-6 (5'-GGA CGA TTA GTG GGT GAA ATG AGG-3') and yielded a 1243 bp product. PCR products were purified with the QIAquick™ PCR Purification Kit (Qiagen, Hilden, Germany) and used for sequencing. Sequencing reactions were carried out using the dye terminator chemistry (PE Applied Biosystems, Darmstadt, Germany) and separated on an automated DNA sequencer (ABI PRISM® 3100 Genetic Analyzer). The sequences were analysed using the Lasergene software (DNASTAR, Madison,WI, USA) and the Mac Vector software (Oxford Molecular Group, Campell, CA, USA) to assemblings and alignings.
Nucleotide sequence accession numbers
The nucleotide sequence of the genomic region of E. coli strain P12b (O15:H17) with a size of 1234 bp containing the fliC gene for flagellin has been submitted to EMBL data library under accession number AJ515904. The coding sequences of the different fliC genes from the following strains have been assigned the following accession numbers: AJ605764 for strain U1-41 (O5:K4:H4), AJ605765 for strain P7d (O68:H4), AJ605766 for strain C107-74 (O15:H17) and AJ536600 for strain E1541-68 (O154:H4). The origin of the strains is listed in Table 1.
Molecular cloning of fliC gene PCR products
PCR products encompassing the complete coding sequences of the fliC-H4 genes were obtained from genomic DNA prepared as described [9] of E. coli strains U9-41 and P12b using primers fliC-5 and fliC-6. The amplification products were inserted into the vector pLITMUS38 (New England Biolabs, Beverly, MA, USA) digested with EcoRV. The orientation of the the insert PCR products was determined by using commercially available sequencing primers LITMUS forward 28/38 and LITMUS Reverse 28/38 (New England Biolabs).
Immuno electron microscopy (IEM) of E. coli flagellar antigens
Motile cultures of E. coli strains were produced by repeated passage on semi-solid agar followed by growth in L-Broth as described above. Cultures carrying recombinant pLITMUS38 plasmids were grown in the presence of 100 μg/ml ampicillin. IEM was performed using fresh, non-formalized cultures of motile bacteria. For IEM, aliquots of respective bacterial cultures were diluted 1:2 in PBS pH 7.2 and adsorbed onto glow-discharge treated 400 mesh grids coated with Pioloform and carbon (Wacker Chemie, Munich, Germany) [31]. Grids with adsorbed bacteria were preincubated for 30 min at room-temperature with blocking buffer (0.1 % bovine serum albumine (Sigma, Deisenhofen, Germany) in PBS). Rabbit anti-H48- and anti-H4 hyperimmune sera were diluted 1:1000 in blocking buffer. After conditioning, specimens were incubated for 30 min at room temperature on droplets of the specific, unlabelled antibodies. Non-bound antibody was removed by washing the grids twice for 10 min on blocking buffer. Immuno-specifically bound primary antibodies were detected using anti-rabbit-IgG-gold 5 or -gold 10 nm conjugates (British Bio Cell International Ltd, Cardiff, UK). The conjugates were diluted 1:20 in blocking buffer and reacted for 30 min at room temperature. Unbound conjugate was removed by a sequence of washing steps (two times with blocking buffer for 5 min each; once with PBS for 3 min and a final wash with double destilled water for 3 min) at room temperature. Before negative staining with 1 % uranyl acetate (pH 4.0–4.5), the grids were washed rapidly on 4 droplets of double destilled water. The preparations were analyzed with an EM 10 electron microscope (Zeiss-LEO, Oberkochen, Germany) at an accelerating voltage of 80 kV. To look for different antigenic determinants expressed on the flagella of strains JM109 or TPE1978, double immuno-labelling was performed using H48 and H4 antisera sequentially and two anti-rabbit-IgG-gold conjugates with different sized markers for the detection of the bound primary unlabelled rabbit antibody. Both E. coli strains were incubated with rabbit anti-H4 (1:1000) followed by anti-rabbit-IgG-5 nm gold and two washing steps on droplets of blocking buffer for 10 min. The samples were subsequently incubated with anti H48 antibody (1:1000) followed by incubation with anti-rabbit-IgG- 10 nm gold. Removal of surplus conjugate and negative staining were performed as detailed above.