Up to 17 epidemiologically unrelated EHEC O157:H7/H^– isolates per year obtained during 1987–2008 were randomly selected from the strain collection of the Institute of Hygiene and the National Consulting Laboratory on HUS, University Hospital Münster, Germany. All 202 O157 strains (61 of which were SF EHEC O157:H^–) were isolated from humans, including patients with HUS (145), bloody diarrhea (12), or diarrhea without visible blood (40), and asymptomatic carriers (5) during epidemiologic investigations. Isolates were obtained from areas throughout Germany. Procedures used for detecting and isolating EHEC O157 from stool samples were described (26,27). Isolates were confirmed as E. coli by the API 20 E test (bioMérieux, Marcy l’Etoile, France) and serotyped by using antisera against E. coli O antigens 1–181 and H antigens 1–56 (28). Subtyping of fliC genes in nonmotile isolates by using HhaI restriction fragment length polymorphism of amplicons obtained with primers FSa1 and rFSa1 (29,30) confirmed the presence of fliCH7 in all isolates. EHEC O157:H7 strain EDL933 (31,32) was used as a reference strain in all analyses.

Strains were grown overnight on Columbia blood agar (Heipha; Eppelheim, Germany) at 37°C. A loop of a fresh culture was suspended in 100 μL of Chelex-100 solution (Bio-Rad, Hercules, CA, USA) and vortexed briefly. After boiling and thorough mixing, samples were centrifuged and DNA-containing supernatants were stored at –20°C until use. To calibrate sequencer-specific variation of fragment length, the exact number of repeats of reference strain O157:H7 EDL933 was initially determined in silico on the basis of its genome sequence (reference sequences NC_002655 [chromosome] and NC_007414 [plasmid]; National Center for Biotechnology Information [NCBI], Bethesda, MD, USA) by using Tandem Repeats Finder software (33). Subsequently, the length of the in silico–determined repeats was subtracted from the fragment length of each respective VNTR locus generated in 8 independent capillary electrophoresis runs of strain EDL933 to determine the offset (primer plus VNTR-flanking regions). This locus-specific offset was then used to calculate the correct number of repeats of unknown isolates. Fragments for MLVA typing were generated in 2 multiplex PCRs comprising either VNTR loci 3, 9, 25, and 34 (multiplex 1) or VNTR loci 17, 19, 36, and 37 (multiplex 2) (Table A1), according to the current PulseNet MLVA protocol for E. coli O157 (23).

PCR amplification was performed in a reaction mixture of 10 μL containing 5 μL of Type-it Multiplex Master Mix (QIAGEN, Hilden, Germany), ≈30 ng of DNA template, and VNTR-specific primers for each of the 4 VNTR loci. Concentration, primer sequences, and respective dyes used are shown in Table A1. PCRs were performed and prepared for subsequent analysis on sequencers in accordance with the manufacturer’s instructions (Table A1). PCR products were diluted 1:10 with water purified by high performance liquid chromatography, and 1.0 μL of diluted DNA was mixed with 13.7 μL of HiDi formamide (Applied Biosystems, Foster City, CA, USA) and 0.3 μL of GeneScan-600 LIZ Size Standard (Applied Biosystems) as internal lane size standard. Before fragment sizing in the ABI Prism 3130xl Genetic Analyzer System (Applied Biosystems), samples were incubated for 5 min at 95°C and immediately frozen at –20°C for >3 min to denature the DNA.

If a VNTR locus was not detected during fragment analysis, reactions were repeated by using singleplex reactions with minor modifications to amplify the specific locus. In that particular instance, the primer concentration was increased to 0.2 µmol/L, annealing temperatures were reduced to 55°C, and the extension time was tripled to amplify larger fragments because of possible insertion sequence element transposition or other genetic events. Subsequently, fragments were characterized by using standard agarose gel electrophoresis. If the fragment was larger than the usual range of fragment sizes of the corresponding VNTR, the PCR product was sequenced.

After fragment analysis, corresponding peak data were examined by using GeneMapper 4.0 software (Applied Biosystems) to calculate the repeat number for each VNTR locus on the basis of fragment length. Partial repeats were rounded to the closest repeat number in accordance with the current Centers for Disease Control and Prevention (CDC) (Atlanta, GA, USA) MLVA O157 protocol (23). If >1 amplicon for a specific VNTR locus was detected and the size difference matched >1 repeat lengths (so-called stutter peaks), the one with the highest fluorescence level was used to calculate the repeat number. A null allele was assigned if either no amplicon was detected or agarose gel electrophoresis data showed an amplicon of a size that was beyond the usual range of fragment size of the specific VNTR locus. Corresponding alleles were designated as –2. In the hypothetical situation in which an amplicon without the repeat region was detected, it was designated as –1 (8). Null alleles were also included in the overall number of alleles in a specific VNTR locus.

Index of diversity (ID) (34) and typeability were calculated by using EpiCompare 1.0 software (Ridom GmbH, Würzburg, Germany). A minimum spanning tree (MST) was generated by using SeqSphere software 0.9 β (Ridom GmbH). All MLVA profiles that differed at <2 alleles were grouped as an MLVA cluster. To determine the cluster-defining profile of clusters containing >2 MLVA profiles, the MST priority rule (that the profile with the highest number of single locus variants is chosen) was applied.

Significance of associations of MLVA profiles or clusters comprising >4 strains with clinical outcome (HUS vs. non-HUS) were calculated by using a χ² test with Yates correction (EpiInfo 6 software; CDC) when appropriate. p values <0.05 were considered significant.

The 202 EHEC O157 strains showed 141 MLVA profiles. Of these profiles, MLVA profile 4/8/-2/2/3/9/-2/5 was the most common and was present in 30 isolates. In contrast, 122 profiles were detected only once. Detailed characteristics of the different VNTR loci identified in this study are shown in Table 1. The number of alleles for the VNTRs ranged from 6 (VNTR-25) to 22 (VNTR-3). Calculation of the ID resulted in values from 0.66 (VNTR-34) to 0.9 (VNTR-9). Half of the VNTR loci (VNTR-3, VNTR-9, VNTR-36, and VNTR-37) showed null alleles with different frequencies ranging from 2.0% (VNTR-3 and VNTR-37) to 44.1% (VNTR-36).

To determine whether amplification failure caused by mutations in primer-binding regions or by complete deletions of the VNTR region were the reason for these null alleles or the insertion of fragments such as mobile genetic elements resulted in larger (and therefore by capillary electrophoresis) undetectable fragments, the respective fragments were analyzed by using standard gel electrophoresis. In some cases, large fragments (>1.3 kb) were detected. Sequence analysis and an NCBI nucleotide BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of randomly selected samples indicated the presence of insertion sequence elements of the IS3 family. The typeability of different VNTR loci ranged from 55.9% to 100% (Table 1). Null alleles were present in 48.5% of the strains (98/202). The overall ID of all MLVA profiles was 0.98.

Comparison of MLVA profiles assembled 136 of the 202 strains (67.3%) into clusters sharing >6 of the 8 VNTR loci. This grouping resulted in 19 MLVA clusters consisting of 2–61 isolates comprising 81 MLVA profiles. The remaining 66 strains (32.7%), which had 60 MLVA profiles, could not be associated with any cluster (Table 2). Strains within clusters consisting of >4 MLVA profiles were isolated over a period >7 years. The 61 strains of cluster 1 were isolated during 1988–2008. Among these strains, the cluster-defining MLVA profile 4/8/-2/2/3/9/-2/5 was the most common profile. It included 30 (14.9%) of 202 strains isolated during 1988–2008. The profile of the same cluster (cluster 1) with the second highest number of strains differed only in VNTR-37 with an additional repeat and included 5.0% of the strains (10/202). The corresponding strains were isolated during 1995–2008 (Table 2; Figure).

All 61 strains in cluster 1 were SF EHEC O157:H^–. The remaining 141 strains did not ferment sorbitol (Table 2, Figure). The phylogenetic relationship of 202 EHEC O157 strains based on the 141 MLVA profiles is shown in an MST in the Figure. The reference strain EDL933, which is also included in the MST, shares its MLVA profile with 4 isolates from Germany obtained during 2007–2008.

To determine whether there was an association between MLVA profiles or clusters and the ability of these strains to cause HUS, we performed a significance test. The 2 most common MLVA profiles were significantly associated with HUS (p = 0.023). Testing for specific clusters resulted in a significant association of HUS with cluster 1 (p = 0.009) (Figure).