Amplified fragment length polymorphism
1. Materials and methods
1.1 Bacterial strains, plasmids, media and growth conditions
All strains and plasmids are preserved at the Department of Molecular Biology. The clinical isolates were obtained from several urological departments of hospitals in Bratislava (Slovak Republic), and have previously been subjected to partial characterization of their sensitivity to antibiotics and K2TeO3 (Burian et al. 1990). All bacterial cultures were grown overnight at 37°C in Luria-Bertani (LB) medium. Solid LB broth medium was supplemented with 1,6% agar. Antimicrobial agents were used at the following concentrations: ampicillin 50 μg·ml-1, chloramphenicol 30 μg·ml-1, kanamycin 25 μg·ml-1, tetracycline 25 μg·ml-1, potassium tellurite (Biomark Laboratories Inc.) in ranges of 0,1-5 mmol·l-1. Serotyping of E. coli strain (O:H serotypes) were determined by the antiserum O157; H7 (fi. Denka Seiken, Tokyo, Japan) according to the manufacturer's instructions.
1.2 Amplified fragment length polymorphism (AFLP)
1.2.1 Preparation of AFLP templates
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An adapted AFLP version was performed according to the original technique described previously [Vos et al. 1995; Mikasová et al. 2005a]. An amount of 200 ng of the genomic DNA was digested for 1 hour at 37°C with EcoRI (5 U) and MseI (2 U ) in a10 μl reaction mixture containing 1x T4 DNA ligase buffer (Invitrogen); 0,05 mol·l-1 NaCl and 0,1 mg·ml-1 BSA. Subsequently, 5 μl of the fresh ligation mixture containing 50 pmol each of the EcoRI adapter and MseI adapter (Table 2), and 1 U of T4 DNA ligase were added and the incubation continued for 3 hours. After ligation, the reaction mixture was diluted 20 times with TEAFLP buffer (10 mmol·l-1 Tris·HCl, pH 7,4; 0,1 mmol·l-1 EDTA, pH 8,0) and stored at temperature -20°C.
1.2.2 Amplification of AFLP templates
Polymerase chain reaction was carried out in a GeneAmp 9700 thermal cycler (Applied Biosystems, USA) in a volume of 20 μl, which contained 1x PCR buffer (10 mmol·l-1 Tris·HCl pH 8,8; 50 mmol·l-1 KCl; 1,5 mmol·l-1 MgCl2), 10 pmol of E01-FAM primer (FAM labeled), 10 pmol of M02 primer, 200 μmol·l-1 of each dNTP, 1 U Taq DNA polymerase and 2 μl template DNA (final ligation mixture). For sequence of primers see Table 1. The selective primers extended beyond the adapter and restriction site sequences.The thermocycler program comprised of one initial cycle at 72°C 2 min, followed by 9 cycles (30 s at 94°C, 30 s at 65°C, 90 s at 72°C, the annealing temperature decreased by 1°C with each cycle), 24 cycles (30 s at 94°C, 30 s at 56°C, 90 s at 72°C), and afinal polymerization for 30 min at 60°C.
1.2.3 Analysis of AFLP fingerprints and data evaluation
PCR products were separated on the automatic genetic analyzer ABI Prism 3130 (Applied Biosystems, USA) with LIZ-500 size standard. The AFLP banding patterns were analyzed in the GelCompar II Software (Applied Maths, Belgium). The levels of similarity between AFLP profiles were calculated on the basis of the coefficient of Dice and cluster analysis was generated by UPGMA (Unweighted pair group method using arithmetic average) algorithm.
1.3 Polymerase chain reaction for plasmid replicon typing and molecular detection of iha, pacB and Shiga toxins genes
E. coli strains were examined for the presence of 7 plasmid replicons (Carattoli et al., 2005) and were also surveyed for their possession of iha, pacB and shiga toxin production genes. Primer sequences for plasmid replicons (A/C, FIIA, FIB, I1, Frep, HI1, N) and expected amplicon sizes are listed in Table 3. The specificity of each primer pair, was initially performed on bacterial genomic DNA extracted from the plasmid-free E. coli K-12 MC4100 strain. None of the primers gave positive results on this template.We designed primer pairs to amplify specific virulence genes found in O157 strains (iha, stx1, stx2). Primers for presence of iha gene were based on GenBank accession no. AF126104.2 [Tarr et al., Infect. Immun. 2000]. PCR determinations of presence stx1 and stx2 genes were investigated with primers - designed according to GenBank accesion no NC_004913.1 (stx1) and AP005154.1 (stx2). Primers for pacB part detection, which were based on GenBank accesion no. AF192489.1 (Vilchez et al. 1997, Alonso et al. 2000). Primers were obtained from Operon Corporation (Coralville, IA). Template total DNA for PCR was prepared by the use of DNeasy® Tissue Kit provided by Qiagen. The reaction mixture (final volume 25 μl) contained 50 ng of genomic DNA, primers (10 pmol each), 200 μmol of dNTPs and 1U Taq DNA polymerase in reaction buffer. Appropriate positive and negative controls were included in each assay. PCR products were amplified in conditions as follows: 5 min at 94°C; 30 cycles consisting of denaturation (30 s at 94°C), annealing (30 s at 55°C), elongation (90 s at 72°C); and a final extension of 5 min at 72°C. Amplicons were separated by electrophoresis using a1,5% agarose gel, stained by ethidium bromide and visualized with UV alongside a 1-kb ladder (Fermentas). If an amplicon of the expected size was observed, then an isolate was considered positive for that particular gene.
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Characterization and determination of uropathogenic clinical isolates of E. coli were performed prior to the molecular typing analysis.
To interpret the evolution of virulence and pathogenic mechanisms, we studied 13 strains representing non-pathogenic collection strains (E. coli K-12 MC4100, E. coli O83:K24:H31), clinical uropathogenic E. coli isolates (shown in Table 1), the primary food-borne pathogen enterohaemorrhagic E. coli O157:H7 and E. coli J53 plasmid Mip233 (Plasmid IncHI3 Scr Te Phi PacB). The clonality of strains was measured and analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), thereafter compatible adapters were ligated to the ends of the cleaved DNA fragments. The total pool of the resulted DNA fragments were then amplified by the PCR using selective primers. One of the primers was labeled with a fluorescent dye (FAM), which enabled amplified fragments to be sized automatically on an automated DNA sequencer. In each AFLP profile, 50-70 DNA fragments of 50-500 bp were detected. The reproducibility of typing was evaluated by a repeated testing of E. coli K-12 in every AFLP experiment, the coefficients of Dice in parallel analyses reached 98,4-100%. Within AFLP profiles (DNA fingerprints), a great variability was observed, the similarity among strains reached values 42,4-100%. The dendrogram showing the percentage of relative similarity between strains was constructed by an average-linkage method of clustering UPGMA using the Gel-Compar II software (Fig. 1). Strains were clustered into 5 main groups (cluster A, B, C, D, E) at the similarity level of 81% and subsequently into 11 subgroups containing greater similarity. From mentioned clusters, the cluster ´D´ included most of analyzed strains (E. coli O83:K24:H31, E. coli KL44, E. coli KL39, E. coli KL36, E. coli KL26), which were clinical UPEC without ter genes with the exception of nonpathogenic E. coli O83:K24:H31. If the lineage of relatedness was shifted to 91%, the main clusters were divided into several subgroups (Fig. 2). The most similarity by AFLP method was observed within pair of UPEC with no ter genes detected, in the concrete 93% similarity between E. coli KL30 and E. coli KL25 (involved in ´B´ cluster, ´B1´ subgroup) and 92,9% between E. coli KL44 and E. coli KL39 (involved in ´D´ cluster, ´D1´ subgroup). Regarding to uropathogenic strains E. coli KL6, E. coli KL53 and E. coli O157:H7, that are characteristic by presence of tellurite resistance genes, the clustering was as follows. UPEC KL6 was classified in claster ´B´ (subgroup ´B2´). Within the frame of cluster ´C´ were identified EHEC O157:H7 (belonged to subgroup ´C1´) and UPEC KL53 (belonged to subgroup ´C2´). On the basis of AFLP profile analysis was defined relatedness between strains E. coli KL6 and E. coli O157:H7 to 76,4%, E. coli KL6 and E. coli KL53 to 75,3%, E. coli O157:H7 and E. coli KL53 to 81,4%. In consideration of a relatively relatedness between strains with ter genes, which are component of several uropathogenic E. coli, there is assumption for current formation of pathogenic strain by horizontal gene transfer, for example by transferring of the large conjugative plasmid pTE53 originated in UPEC KL53 or EHEC O157:H7 TAI-like genomic islands using/on several mobile elements...
Because the Shiga toxin production has been linked to virulence of E. coli O157:H7 (EHEC) strains, we examined strains for distribution of Shiga toxin genes (stx1 and stx2), widely disseminated in the E. coli population and associated with lambdoid bacteriophages. Although stx1 gene was not occurred in analysed strains, the stx2 gene was found in 2 strains - E. coli O157:H7 and E. coli KL53. These strains were simultaneously present in the same AFLP typing cluster ´C´, with common relatedness 81,4%.
Tarr et al. (2000) determined the recently acquired E. coli chromosomal island TAI (Tellurite resistance and Adherence confering Island) with a novel bacterial adherence-conferring gene iha and its conserved structure. The product of iha gene is the IrgA homologue adhesin similar to iron-regulated gene A (irgA) of Vibrio cholerae. In spite of ahigh homology of ter operon in E. coli O157:H7 and analysed UPEC bearing ter genes, there was no iha gene determined in E. coli KL53 and E. coli KL6. The results of the examination of Shiga toxin like type and presence of virulence associated genes are shown for each strain in Table 4.
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